U.S. patent application number 13/239584 was filed with the patent office on 2014-10-30 for process for the production of fine chemicals.
This patent application is currently assigned to Metanomics GmbH. The applicant listed for this patent is Astrid Blau, Beate Kamlage, Ralf Looser, Gunnar Plesch, Piotr Puzio, Oliver Schmitz, Birgit Wendel. Invention is credited to Astrid Blau, Beate Kamlage, Ralf Looser, Gunnar Plesch, Piotr Puzio, Oliver Schmitz, Birgit Wendel.
Application Number | 20140325709 13/239584 |
Document ID | / |
Family ID | 36589176 |
Filed Date | 2014-10-30 |
United States Patent
Application |
20140325709 |
Kind Code |
A1 |
Plesch; Gunnar ; et
al. |
October 30, 2014 |
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Abstract
The present invention relates to a process for the production of
fine chemicals in a microorganism, a plant cell, a plant, a plant
tissue or in one or more parts thereof. The present invention
relates further to a process for the control of the production of
fine chemicals in a microorganism, a plant cell, a plant, a plant
tissue or in one or more parts thereof. The invention furthermore
relates to nucleic acid molecules, polypeptides, nucleic acid
constructs, vectors, antisense molecules, antibodies, host cells,
plant tissue, propagation material, harvested material, plants,
microorganisms as well as agricultural compositions and to their
use.
Inventors: |
Plesch; Gunnar; (Potsdam,
DE) ; Puzio; Piotr; (Mariakerke (gent), BE) ;
Blau; Astrid; (Stahnsdorf, DE) ; Looser; Ralf;
(Berlin, DE) ; Wendel; Birgit; (Berlin, DE)
; Kamlage; Beate; (Berlin, DE) ; Schmitz;
Oliver; (Dallgow-Doberitz, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Plesch; Gunnar
Puzio; Piotr
Blau; Astrid
Looser; Ralf
Wendel; Birgit
Kamlage; Beate
Schmitz; Oliver |
Potsdam
Mariakerke (gent)
Stahnsdorf
Berlin
Berlin
Berlin
Dallgow-Doberitz |
|
DE
BE
DE
DE
DE
DE
DE |
|
|
Assignee: |
Metanomics GmbH
Berlin
DE
|
Family ID: |
36589176 |
Appl. No.: |
13/239584 |
Filed: |
September 22, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11644015 |
Dec 21, 2006 |
8541208 |
|
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13239584 |
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PCT/EP05/07080 |
Jun 29, 2005 |
|
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11644015 |
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PCT/EP05/13673 |
Dec 19, 2005 |
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PCT/EP05/07080 |
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60801017 |
May 17, 2006 |
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Current U.S.
Class: |
800/298 ; 252/1;
426/531; 435/252.2; 435/29; 435/320.1; 435/41; 435/419; 435/6.1;
435/6.11; 435/69.1; 530/300; 530/387.1; 536/23.1 |
Current CPC
Class: |
C12P 1/00 20130101; C12Q
1/68 20130101; C12P 13/06 20130101; C12Q 1/025 20130101; C07K
14/395 20130101; C07K 14/245 20130101; C12Q 1/6813 20130101; C12Y
101/05002 20130101; C12P 13/08 20130101; C12N 15/8243 20130101 |
Class at
Publication: |
800/298 ; 435/41;
536/23.1; 435/320.1; 435/419; 435/69.1; 530/300; 530/387.1;
435/252.2; 435/29; 435/6.11; 435/6.1; 252/1; 426/531 |
International
Class: |
C12P 1/00 20060101
C12P001/00; C12Q 1/68 20060101 C12Q001/68; C12Q 1/02 20060101
C12Q001/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 2, 2004 |
EP |
04015608.5 |
Jul 15, 2004 |
EP |
04016615.9 |
Aug 5, 2004 |
EP |
04018543.1 |
Aug 23, 2004 |
EP |
04105689.6 |
Aug 27, 2004 |
EP |
04105535.1 |
Nov 3, 2004 |
EP |
04026007.7 |
Nov 3, 2004 |
EP |
04026008.5 |
Nov 4, 2004 |
EP |
04026056.4 |
Nov 4, 2004 |
EP |
04026057.2 |
Dec 3, 2004 |
EP |
04028670.0 |
Dec 3, 2004 |
EP |
04028671.8 |
Dec 17, 2004 |
EP |
04106931.1 |
Dec 18, 2004 |
EP |
04030100.4 |
Dec 18, 2004 |
EP |
04030101.2 |
Dec 22, 2004 |
EP |
04030391.9 |
Dec 23, 2004 |
EP |
04107024.4 |
Dec 28, 2004 |
EP |
04107025.1 |
Jan 10, 2005 |
EP |
05100166.7 |
Jan 26, 2005 |
EP |
05100704.5 |
Mar 14, 2005 |
EP |
05101970.1 |
Apr 20, 2005 |
EP |
05103164.9 |
Apr 22, 2005 |
EP |
05103283.7 |
Apr 22, 2005 |
EP |
05103449.4 |
Apr 22, 2005 |
EP |
05103455.1 |
Apr 27, 2005 |
EP |
05103428.8 |
May 25, 2005 |
EP |
05104479.0 |
May 25, 2005 |
EP |
05104496.4 |
May 27, 2005 |
EP |
05104781.9 |
May 30, 2005 |
EP |
05104630.8 |
Jun 1, 2005 |
EP |
05104761.1 |
Jun 2, 2005 |
EP |
05104811.4 |
Jun 2, 2005 |
EP |
05104818.9 |
Jun 3, 2005 |
EP |
05104874.2 |
Jun 6, 2005 |
EP |
05105001.1 |
Jun 8, 2005 |
EP |
05105021.9 |
Jun 8, 2005 |
EP |
05105028.4 |
Jun 10, 2005 |
EP |
05105345.2 |
Jun 13, 2005 |
EP |
05105136.5 |
Jun 17, 2005 |
EP |
05105401.3 |
Jun 17, 2005 |
EP |
05105405.4 |
Jun 17, 2005 |
EP |
05105406.2 |
Jun 21, 2005 |
EP |
05105508.5 |
Jun 21, 2005 |
EP |
05105510.1 |
Jun 22, 2005 |
EP |
05105570.5 |
Jun 22, 2005 |
EP |
05105571.3 |
Jun 22, 2005 |
EP |
05105575.4 |
Jun 23, 2005 |
EP |
05105624.0 |
Jun 23, 2005 |
EP |
05105643.0 |
Jun 27, 2005 |
EP |
05105992.1 |
Jun 27, 2005 |
EP |
05105993.9 |
Claims
1. A process for the production of a fine chemical as indicated in
Table II and/or XII, column 6, which comprises (a) increasing or
generating the activity of a protein as indicated in Table II
and/or XII, application no. 0 to 45, columns 5 or 7, or a
functional equivalent thereof in a non-human organism, or in one or
more parts thereof; and (b) growing the organism under conditions
which permit the production of the respective fine chemical in said
organism.
2. A process for the production of a fine chemical as indicated in
Table II and/or XII, column 6, comprising increasing or generating
in an organism or a part thereof the expression of at least one
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: a) a nucleic acid molecule encoding a
polypeptide as indicated in Table II and/or XII, application no. 0
to 45, columns 5 or 7, or a fragment thereof, which confers an
increase in the amount of the respective fine chemical in an
organism or a part thereof; b) a nucleic acid molecule comprising a
nucleic acid molecule as indicated in Table I and/or XI,
application no. 0 to 45, columns 5 or 7; c) a nucleic acid molecule
whose sequence can be deduced from a polypeptide sequence encoded
by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; d) a nucleic acid molecule which encodes a polypeptide
which has at least 90% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; e) a nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under stringent hybridization conditions and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; f) a nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table III and/or
XIII, application no. 0 to 45, columns 7, and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; g) a nucleic acid molecule encoding a
polypeptide which is isolated with the aid of monoclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (f) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; h) a
nucleic acid molecule encoding a polypeptide comprising a consensus
sequence as indicated in Table IV and/or XIV, application no. 0 to
45, columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and i) a
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (h) or with a fragment thereof having at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (k) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof. or comprising a sequence
which is complementary thereto.
3. The process of claim 1, comprising recovering the free or bound
respective fine chemical.
4. The process of claim 2, comprising the following steps: (a)
selecting an organism or a part thereof expressing a polypeptide
encoded by the nucleic acid molecule characterized in claim 2; (b)
mutagenizing the selected organism or the part thereof; (c)
comparing the activity or the expression level of said polypeptide
in the mutagenized organism or the part thereof with the activity
or the expression of said polypeptide of the selected organisms or
the part thereof; (d) selecting the mutated organisms or parts
thereof, which comprise an increased activity or expression level
of said polypeptide compared to the selected organism or the part
thereof; (e) optionally, growing and cultivating the organisms or
the parts thereof; and (f) recovering, and optionally isolating,
the free or bound respective fine chemical produced by the selected
mutated organisms or parts thereof.
5. The process of claim 2, wherein the expression of said nucleic
acid molecule is increased or generated transiently or stably.
6. An isolated nucleic acid molecule comprising a nucleic acid
molecule selected from the group consisting of: a) a nucleic acid
molecule encoding a polypeptide as indicated in Table II and/or
XII, application no. 0 to 45, columns 5 or 7, or a fragment
thereof, which confers an increase in the amount of the respective
fine chemical as indicated in the corresponding column 6 in an
organism or a part thereof; b) a nucleic acid molecule comprising a
nucleic acid molecule as indicated in Table I and/or XI,
application no. 0 to 45, columns 5 or 7; c) a nucleic acid molecule
whose sequence can be deduced from a polypeptide sequence encoded
by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; d) a nucleic acid molecule which encodes a polypeptide
which has at least 90% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; e) a nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under stringent hybridization conditions and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; f) a nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table III and/or
XIII, application no. 0 to 45, columns 7, and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; g) a nucleic acid molecule encoding a
polypeptide which is isolated with the aid of monoclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (f) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; h) a
nucleic acid molecule encoding a polypeptide comprising a consensus
sequence as indicated in Table IV and/or XIV, application no. 0 to
45, columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and i) a
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (h) or with a fragment thereof having at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (h) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof, whereby the nucleic acid
molecule distinguishes over the sequence as indicated in Table I
and/or XI, application no. 0 to 45, columns 5 or 7, by one or more
nucleotides.
7. An isolated nucleic acid molecule comprising a nucleic acid
molecule selected from the group of nucleic acid molecules as
defined in claim 2 part a) to i).
8. A nucleic acid construct which confers the expression of the
nucleic acid molecule of claim 6, comprising one or more regulatory
elements.
9. A nucleic acid construct which confers the expression of the
nucleic acid molecule of claim 7, comprising one or more regulatory
elements.
10. A vector comprising the nucleic acid molecule as claimed in
claim 6.
11. A vector comprising the nucleic acid molecule as claimed in
claim 7.
12. A host cell, which has been transformed stably or transiently
with the nucleic acid molecule as claimed in claim 6.
13. A host cell, which has been transformed stably or transiently
with the nucleic acid molecule as claimed in claim 7.
14. A process for producing a polypeptide, comprising expressing
the polypeptide in a host cell as claimed in claim 12.
15. A process for producing a polypeptide, comprising expressing
the polypeptide in a host cell as claimed in claim 13.
16. A polypeptide encoded by the nucleic acid molecule as claimed
in claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table II and/or XII, application no. 0 to 45, columns
5 or 7, by one or more amino acids.
17. A polypeptide encoded by the nucleic acid molecule as claimed
in claim 7.
18. An antibody, which binds specifically to the polypeptide as
claimed in claim 16.
19. An antibody, which binds specifically to the polypeptide as
claimed in claim 17.
20. A plant tissue, propagation material, harvested material or a
plant comprising the host cell as claimed in claim 12 wherein the
host cell is a plant cell or an Agrobacterium.
21. A plant tissue, propagation material, harvested material or a
plant comprising the host cell as claimed in claim 13 wherein the
host cell is a plant cell or an Agrobacterium.
22. A method for screening for agonists and antagonists of the
activity of a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof comprising: (a)
contacting cells, tissues, plants or microorganisms which express
the polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof with a candidate compound
or a sample comprising a plurality of compounds under conditions
which permit the expression of the polypeptide; (b) assaying the
respective fine chemical level or the polypeptide expression level
in the cell, tissue, plant or microorganism or the media the cell,
tissue, plant or microorganisms is cultured or maintained in; and
(c) identifying an agonist or antagonist by comparing the measured
fine chemical level or polypeptide expression level with a standard
fine chemical or polypeptide expression level measured in the
absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist.
23. A method for screening for agonists and antagonists of the
activity of a polypeptide encoded by the nucleic acid molecule of
claim 7 conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof comprising: (a)
contacting cells, tissues, plants or microorganisms which express
the polypeptide encoded by the nucleic acid molecule of claim 7
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof with a candidate compound
or a sample comprising a plurality of compounds under conditions
which permit the expression of the polypeptide; (b) assaying the
respective fine chemical level or the polypeptide expression level
in the cell, tissue, plant or microorganism or the media the cell,
tissue, plant or microorganisms is cultured or maintained in; and
(c) identifying an agonist or antagonist by comparing the measured
fine chemical level or polypeptide expression level with a standard
fine chemical or polypeptide expression level measured in the
absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist.
24. A process for the identification of a compound conferring
increased fine chemical production in a plant or microorganism,
comprising the steps: (a) culturing a plant cell or tissue or
microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; (b) identifying if the
compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout
system.
25. A process for the identification of a compound conferring
increased fine chemical production in a plant or microorganism,
comprising the steps: (a) culturing a plant cell or tissue or
microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 7 conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 7
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; (b) identifying if the
compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout
system.
26. A method for the identification of a gene product conferring an
increase in the respective fine chemical production in a cell,
comprising the following steps: (a) contacting the nucleic acid
molecules of a sample, which can contain a candidate gene encoding
a gene product conferring an increase in the respective fine
chemical after expression with the nucleic acid molecule of claim
6; (b) identifying the nucleic acid molecules, which hybridise
under relaxed stringent conditions with the nucleic acid molecule
of claim 6; (c) introducing the candidate nucleic acid molecules in
host cells appropriate for producing the respective fine chemical;
(d) expressing the identified nucleic acid molecules in the host
cells; (e) assaying the respective fine chemical level in the host
cells; and (f) identifying nucleic acid molecule and its gene
product which expression confers an increase in the respective fine
chemical level in the host cell after expression compared to the
wild type.
27. A method for the identification of a gene product conferring an
increase in the respective fine chemical production in a cell,
comprising the following steps: (a) contacting the nucleic acid
molecules of a sample, which can contain a candidate gene encoding
a gene product conferring an increase in the respective fine
chemical after expression with the nucleic acid molecule of claim
7; (b) identifying the nucleic acid molecules, which hybridise
under relaxed stringent conditions with the nucleic acid molecule
of claim 7; (c) introducing the candidate nucleic acid molecules in
host cells appropriate for producing the respective fine chemical;
(d) expressing the identified nucleic acid molecules in the host
cells; (e) assaying the respective fine chemical level in the host
cells; and (f) identifying nucleic acid molecule and its gene
product which expression confers an increase in the respective fine
chemical level in the host cell after expression compared to the
wild type.
28. A method for the identification of a gene product conferring an
increase in the respective fine chemical production in a cell,
comprising the following steps: (a) identifying in a data bank
nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the respective fine chemical amount or level in an organism or a
part thereof after expression, and which are at least 90% homolog
to the nucleic acid molecule of claim 6; (b) introducing the
candidate nucleic acid molecules in host cells appropriate for
producing the respective fine chemical; (c) expressing the
identified nucleic acid molecules in the host cells; (d) assaying
the respective fine chemical level in the host cells; and (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the respective fine chemical
level in the host cell after expression compared to the wild
type.
29. A method for the identification of a gene product conferring an
increase in the respective fine chemical production in a cell,
comprising the following steps: (a) identifying in a data bank
nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the respective fine chemical amount or level in an organism or a
part thereof after expression, and which are at least 90% homolog
to the nucleic acid molecule of claim 7; (b) introducing the
candidate nucleic acid molecules in host cells appropriate for
producing the respective fine chemical; (c) expressing the
identified nucleic acid molecules in the host cells; (d) assaying
the respective fine chemical level in the host cells; and (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the respective fine chemical
level in the host cell after expression compared to the wild
type.
30. A method for the production of an agricultural composition
comprising the steps of the method of claim 24 and formulating the
compound identified in claim 24 in a form acceptable for an
application in agriculture.
31. A method for the production of an agricultural composition
comprising the steps of the method of claim 25 and formulating the
compound identified in claim 25 in a form acceptable for an
application in agriculture.
32. A composition produced by the method of claim 30, wherein the
composition is a food or feed composition.
33. A composition produced by the method of claim 31, wherein the
composition is a food or feed composition.
34. A process for the control of the production of fine chemicals
comprising (a) increasing or generating the activity of one or more
b0019, b0050, b0057, b0112, b0124, b0138, b0149, b0161, b0175,
b0196, b0251, b0252, b0255, b0376, b0462, b0464, b0486, b0577,
b0651, b0695, b0730, b0828, b0847, b0849, b0880, b0970, b0986,
b1097, b1284, b1318, b1343, b1360, b1463, b1693, b1708, b1736,
b1738, b1829, b1886, b1896, b1926, b1961, b2023, b2078, b2095,
b2211, b2239, b2307, b2414, b2426, b2478, b2489, b2491, b2507,
b2553, b2576, b2597, b2599, b2664, b2699, b2703, b2710, b2753,
b2796, b2822, b3064, b3074, b3116, b3129, b3160, b3166, b3169,
b3172, b3231, b3256, b3260, b3430, b3457, b3462, b3578, b3619,
b3644, b3684, b3767, b3791, b3919, b3926, b3936, b3938, b3966,
b3983, b4004, b4054, b4063, b4074, b4122, b4129, b4139, b4232,
b4239, b4327, b4346, b4401, YAL049C, YBL015W, YBR084W, YBR089C-A,
YBR184W, YBR204C, YCL038C, YCR012W, YCR059C, YDL127W, YDR271C,
YDR316W, YDR447C, YDR513W, YEL045C, YEL046C, YER152C, YER156C,
YER173W, YER174C, YFL019C, YFL050C, YFL053W, YFR007W, YFR042W,
YGL205W, YGL237C, YGR101W, YGR104C, YGR261C, YHR072W, YHR072W-A,
YHR130C, YHR189W, YHR201C, YIL150C, YJL055W, YJL072C, YJL099W,
YKL132C, YKR057W, YLL013C, YLR082C, YLR089C, YLR224W, YLR255C,
YLR375W, YOR024W, YOR044W, YOR084W, YOR245C, YOR317W, YOR344C,
YOR350C, YPL099C, YPL268W, YPRO24W, YPR138C and/or YPR172W and/or
b0021, b0043, b0134, b0186, b0186, b0328, b0677, b0734, b0763,
b0895, b0895, b1054, b1183, b1217, b1249, b1292, b1874, b2110,
b2696, b2901, b3025, b3091, b3335, b3709, b3825, b3924, b4101,
b4113, b4242, b4359, YGL005C and/or YML005W protein(s) or homologs
thereof, having the sequence of a polypeptide encoded by a
corresponding nucleic acid molecule indicated in Table I, columns 5
or 7 or indicated in Table V columns 5 or 7, in a non-human
organism or in one or more parts thereof; and (b) growing the
organism under conditions which permit the production of the fine
chemical in said organism in a metabolic profile as indicated in
Table X and/or IX, wherein, in the metabolic profile, a numerical
value greater than "1" represents an increase of a metabolite
content and a numerical value less than "1" represents a decrease
of a metabolite content compared to the wild type cell,
microorganism, plant cell, plant, plant tissue or one or more parts
thereof, and no number in Table X means a numerical value of "1"
concerning the metabolite profile which is essentially identical to
the metabolite profile of the wild type.
35. A process for the control of the production of fine chemicals
comprising (a) increasing or generating the activity of one or more
b0019, b0050, b0057, b0112, b0124, b0138, b0149, b0161, b0175,
b0196, b0251, b0252, b0255, b0376, b0462, b0464, b0486, b0577,
b0651, b0695, b0730, b0828, b0847, b0849, b0880, b0970, b0986,
b1097, b1284, b1318, b1343, b1360, b1463, b1693, b1708, b1736,
b1738, b1829, b1886, b1896, b1926, b1961, b2023, b2078, b2095,
b2211, b2239, b2307, b2414, b2426, b2478, b2489, b2491, b2507,
b2553, b2576, b2597, b2599, b2664, b2699, b2703, b2710, b2753,
b2796, b2822, b3064, b3074, b3116, b3129, b3160, b3166, b3169,
b3172, b3231, b3256, b3260, b3430, b3457, b3462, b3578, b3619,
b3644, b3684, b3767, b3791, b3919, b3926, b3936, b3938, b3966,
b3983, b4004, b4054, b4063, b4074, b4122, b4129, b4139, b4232,
b4239, b4327, b4346, b4401, YAL049C, YBL015W, YBR084W, YBR089C-A,
YBR184W, YBR204C, YCL038C, YCR012W, YCR059C, YDL127W, YDR271C,
YDR316W, YDR447C, YDR513W, YEL045C, YEL046C, YER152C, YER156C,
YER173W, YER174C, YFL019C, YFL050C, YFL053W, YFR007W, YFR042W,
YGL205W, YGL237C, YGR101W, YGR104C, YGR261C, YHR072W, YHR072W-A,
YHR130C, YHR189W, YHR201C, YIL150C, YJL055W, YJL072C, YJL099W,
YKL132C, YKR057W, YLL013C, YLR082C, YLR089C, YLR224W, YLR255C,
YLR375W, YOR024W, YOR044W, YOR084W, YOR245C, YOR317W, YOR344C,
YOR350C, YPL099C, YPL268W, YPRO24W, YPR138C and/or YPR172W and/or
b0021, b0043, b0134, b0186, b0186, b0328, b0677, b0734, b0763,
b0895, b0895, b1054, b1183, b1217, b1249, b1292, b1874, b2110,
b2696, b2901, b3025, b3091, b3335, b3709, b3825, b3924, b4101,
b4113, b4242, b4359, YGL005C and/or YML005W protein(s) or homologs
thereof, having the sequence of a polypeptide encoded by a
corresponding nucleic acid molecule indicated in Table I, columns 5
or 7, or indicated in Table V columns 5 or 7, in a non-human
organism or in one or more parts thereof and (b) growing the
organism under conditions which permit the production of fine
chemicals in defined ratios in said organism resulting in a defined
metabolic profile.
36. A process for the control of the production of fine chemicals
comprising expressing in an organism or a part thereof at least one
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: a) a nucleic acid molecule encoding a
polypeptide having a sequence as indicated in Table II, columns 5
or 7, or indicated in Table VI, or selected from the group
consisting of b0019, b0050, b0057, b0112, b0124, b0138, b0149,
b0161, b0175, b0196, b0251, b0252, b0255, b0376, b0462, b0464,
b0486, b0577, b0651, b0695, b0730, b0828, b0847, b0849, b0880,
b0970, b0986, b1097, b1284, b1318, b1343, b1360, b1463, b1693,
b1708, b1736, b1738, b1829, b1886, b1896, b1926, b1961, b2023,
b2078, b2095, b2211, b2239, b2307, b2414, b2426, b2478, b2489,
b2491, b2507, b2553, b2576, b2597, b2599, b2664, b2699, b2703,
b2710, b2753, b2796, b2822, b3064, b3074, b3116, b3129, b3160,
b3166, b3169, b3172, b3231, b3256, b3260, b3430, b3457, b3462,
b3578, b3619, b3644, b3684, b3767, b3791, b3919, b3926, b3936,
b3938, b3966, b3983, b4004, b4054, b4063, b4074, b4122, b4129,
b4139, b4232, b4239, b4327, b4346, b4401, YAL049C, YBL015W,
YBR084W, YBR089C-A, YBR184W, YBR204C, YCL038C, YCR012W, YCR059C,
YDL127W, YDR271C, YDR316W, YDR447C, YDR513W, YEL045C, YEL046C,
YER152C, YER156C, YER173W, YER174C, YFL019C, YFL050C, YFL053W,
YFR007W, YFR042W, YGL205W, YGL237C, YGR101W, YGR104C, YGR261C,
YHR072W, YHR072W-A, YHR130C, YHR189W, YHR201C, YIL150C, YJL055W,
YJL072C, YJL099W, YKL132C, YKR057W, YLL013C, YLR082C, YLR089C,
YLR224W, YLR255C, YLR375W, YOR024W, YOR044W, YOR084W, YOR245C,
YOR317W, YOR344C, YOR350C, YPL099C, YPL268W, YPRO24W, YPR138C and
YPR172W and/or b0021, b0043, b0134, b0186, b0186, b0328, b0677,
b0734, b0763, b0895, b0895, b1054, b1183, b1217, b1249, b1292,
b1874, b2110, b2696, b2901, b3025, b3091, b3335, b3709, b3825,
b3924, b4101, b4113, b4242, b4359, YGL005C and/or YML005W, or a
fragment thereof, which confers an increase or a decrease in the
amount of the respective fine chemical as shown in Table X and/or
indicated in Table IX in an organism or a part thereof; b) a
nucleic acid molecule comprising a nucleic acid molecule having a
sequence as indicated in Table I, columns 5 or 7, and encoding a
polypeptide as defined in (a) and named in Table X and/or IX and/or
a sequence as indicated in Table V columns 5 or 7, c) a nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as result
of the degeneracy of the genetic code and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; d) a nucleic acid molecule encoding a polypeptide which
has at least 90% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase or decrease in the amount of the respective
fine chemical in an organism or a part thereof; e) a nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under stringent hybridization conditions and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; f) a nucleic acid molecule encoding a
polypeptide, the polypeptide being derived by substituting,
deleting and/or adding one or more amino acids of the amino acid
sequence of the polypeptide encoded by the nucleic acid molecules
(a) to (d) and conferring an increase or decrease in the amount of
the respective fine chemical in an organism or a part thereof; g) a
nucleic acid molecule encoding a fragment or an epitope of a
polypeptide which is encoded by one of the nucleic acid molecules
of (a) to (e) and conferring an increase or decrease in the amount
of the respective fine chemical in an organism or a part thereof;
h) a nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers pairs having a
sequence as indicated in Table III, columns 7, and corresponding to
a polypeptide as defined in (a) and named in Table X and/or IX,
and/or primers pairs as indicated in Table VII, and conferring an
increase or decrease in the amount of the respective fine chemical
in an organism or a part thereof; i) a nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h) and conferring an increase or decrease in
the amount of the respective fine chemical in an organism or a part
thereof; j) a nucleic acid molecule which encodes a polypeptide
comprising the consensus sequence having a sequences as indicated
in Table IV, columns 7, and corresponding to a polypeptide as
defined in (a) and named in Table X and/or IX, and/or the consensus
sequence having a sequences as indicated in Table VIII, columns 7
and conferring an increase or decrease in the amount of the
respective fine chemical in an organism or a part thereof; k) a
nucleic acid molecule comprising one or more of the nucleic acid
molecules encoding the amino acid sequence of a polypeptide
comprising a domain of a polypeptide indicated in Table II, columns
5 or 7, and as defined in (a) and named in Table X, and/or as
indicated in Table VI columns 5 or 7, and conferring an increase or
decrease in the amount of the respective fine chemical in an
organism or a part thereof; and l) a nucleic acid molecule which is
obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment of at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (k) and
conferring an increase or decrease in the amount of the respective
fine chemical in an organism or a part thereof; or which comprises
a sequence which is complementary thereto.
37. The process of claim 34, wherein one or more fine chemicals are
isolated.
38. A host cell which exhibits a metabolic profile according to any
of the column as depicted in Table X and/or to any line as depicted
in Table IX.
39. A process for the identification of a compound conferring a
metabolic profile as shown in Table X or in Table IX in a cell,
plant or microorganism, comprising the steps: (a) culturing a plant
cell or tissue or microorganism or maintaining a plant expressing a
polypeptide encoded by the nucleic acid molecule of claim 36 a)
conferring an increase or decrease in the amount of the respective
fine chemicals in an organism or a part thereof and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 42 a) conferring an increase or decrease in the
amount of the respective fine chemicals in an organism or a part
thereof; and (b) identifying if the compound is an effective
agonist by detecting the presence or absence or increase of a
signal produced by said readout system.
40. A method for the identification of a gene product conferring an
increase or decrease in fine chemical production in a cell
according to the metabolic profile disclosed in Table X and/or in
Table IX, comprising the following steps: (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase or decrease in
one or more fine chemicals according to Table X and/or Table IX
after expression with the nucleic acid molecule of claim 36 a); (b)
identifying the nucleic acid molecules, which hybridize under
relaxed stringent conditions with the nucleic acid molecule of
claim 36 a); (c) introducing the candidate nucleic acid molecules
in host cells appropriate for producing one or more fine chemicals
according to Table X; (d) expressing the identified nucleic acid
molecules in the host cells; (e) assaying the level of one or more
fine chemicals according to Table X and/or Table IX in the host
cells; and (f) identifying a nucleic acid molecule and its gene
product which expression confers an increase or decrease in the
level of one or more fine chemicals according to Table X and/or
Table IX in the host cell after expression compared to the wild
type.
41. A method for the identification of a gene product conferring an
increase or decrease in fine chemical production in a cell
according to the metabolic profile disclosed in Table X and/or
Table IX in a cell, comprising the following steps: (a) identifying
in a data bank nucleic acid molecules of an organism; which can
contain a candidate gene encoding a gene product conferring an
increase or decrease in the level of one or more fine chemicals
according to Table X and/or Table IX in an organism or a part
thereof after expression, and which are at least 90% homologous to
the nucleic acid molecule of claim 42 a); (b) introducing the
candidate nucleic acid molecules in host cells appropriate for
producing a metabolic profile according to Table X and/or Table IX;
(c) expressing the identified nucleic acid molecules in the host
cells; (d) assaying the metabolic profile in the host cells; and
(e) identifying a nucleic acid molecule and its gene product which
expression confers the desired metabolic profile according to Table
X and/or Table IX in the host cell after expression compared to the
wild type.
42. A process for the production of a composition of fine chemicals
comprising (a) increasing or generating the activity of one or more
b0019, b0050, b0057, b0112, b0124, b0138, b0149, b0161, b0175,
b0196, b0251, b0252, b0255, b0376, b0462, b0464, b0486, b0577,
b0651, b0695, b0730, b0828, b0847, b0849, b0880, b0970, b0986,
b1097, b1284, b1318, b1343, b1360, b1463, b1693, b1708, b1736,
b1738, b1829, b1886, b1896, b1926, b1961, b2023, b2078, b2095,
b2211, b2239, b2307, b2414, b2426, b2478, b2489, b2491, b2507,
b2553, b2576, b2597, b2599, b2664, b2699, b2703, b2710, b2753,
b2796, b2822, b3064, b3074, b3116, b3129, b3160, b3166, b3169,
b3172, b3231, b3256, b3260, b3430, b3457, b3462, b3578, b3619,
b3644, b3684, b3767, b3791, b3919, b3926, b3936, b3938, b3966,
b3983, b4004, b4054, b4063, b4074, b4122, b4129, b4139, b4232,
b4239, b4327, b4346, b4401, YAL049C, YBL015W, YBR084W, YBR089C-A,
YBR184W, YBR204C, YCL038C, YCR012W, YCR059C, YDL127W, YDR271C,
YDR316W, YDR447C, YDR513W, YEL045C, YEL046C, YER152C, YER156C,
YER173W, YER174C, YFL019C, YFL050C, YFL053W, YFR007W, YFR042W,
YGL205W, YGL237C, YGR101W, YGR104C, YGR261C, YHR072W, YHR072W-A,
YHR130C, YHR189W, YHR201C, YIL150C, YJL055W, YJL072C, YJL099W,
YKL132C, YKR057W, YLL013C, YLR082C, YLR089C, YLR224W, YLR255C,
YLR375W, YOR024W, YOR044W, YOR084W, YOR245C, YOR317W, YOR344C,
YOR350C, YPL099C, YPL268W, YPRO24W, YPR138C and/or YPR172W and/or
b0021, b0043, b0134, b0186, b0186, b0328, b0677, b0734, b0763,
b0895, b0895, b1054, b1183, b1217, b1249, b1292, b1874, b2110,
b2696, b2901, b3025, b3091, b3335, b3709, b3825, b3924, b4101,
b4113, b4242, b4359, YGL005C and/or YML005W protein(s) or homologs
thereof, having the sequence of a polypeptide encoded by a
corresponding nucleic acid molecule indicated in Table I, columns 5
or 7 or indicated in Table V columns 5 or 7, in a non-human
organism or in one or more parts thereof; and (b) growing the
organism under conditions which permit production of a composition
of fine chemicals in said organism in a relative ratio as indicated
in Table X and/or IX, wherein a numerical value greater than "1"
stands for an increase of a metabolite content, a numerical value
less than "1" stands for a decrease of a metabolite content,
compared to the wild type cell, microorganism, plant cell, plant,
plant tissue or one or more parts thereof and no number in Table X
means a numerical value of "1" concerning the metabolite profile,
which is essentially identical to the metabolite profile of the
wild type, wherein said composition is a biological
composition.
43. A biological composition of fine chemicals in a defined ratio
produced by the process of claim 42.
44. A method of diagnosis of the level of expression of the nucleic
acid molecule of claim 36 a) by (a) measuring the metabolic content
in a microorganism, a plant cell, a plant, a plant tissue or in one
or more parts thereof; and (b) comparing the metabolic content with
the relative metabolic profile as depicted in Table X and/or IX.
Description
RELATED APPLICATIONS
[0001] The instant application is a divisional application of U.S.
application Ser. No. 11/644,015, which is a continuation-in-part
application of International Patent Application No. PCT/EP
2005/007080, filed Jun. 29, 2005 and of International Patent
Application No. PCT/EP 2005/013673, filed Dec. 19, 2005, and claims
benefit to U.S. Provisional Application No. 60/801,017, filed May
17, 2006. International Patent Application No. PCT/EP 2005/007080
and International Patent Application No. PCT/EP 2005/013673 claim
the benefit of European Application No. 04030101.2, filed Dec. 18,
2004, European Application No. 04106931.1, filed Dec. 17, 2005,
European Application No. 04030391.9, filed Dec. 22, 2004, European
Application No. 05100166.7, filed Jan. 10, 2005, European
Application No. 05103449.4, filed Apr. 22, 2005, European
Application No. 04107024.4, filed Dec. 23, 2004, European
Application No. 04030100.4, filed Dec. 18, 2004, European
Application No. 05101970.1, filed Mar. 14, 2005, European
Application No. 04107025.1, filed Dec. 28, 2004, European
Application No. 05104781.9, filed May 27, 2005, European
Application No. 05100704.5, filed Jan. 26, 2005, European
Application No. 05103283.7, filed Apr. 22, 2005, European
Application No. 05103455.1, filed Apr. 22, 2005, European
Application No. 05103164.9, filed Apr. 20, 2005, European
Application No. 05103428.8, filed Apr. 27, 2005, European
Application No. 05104479.0, filed May 25, 2005, European
Application No. 05104496.4, filed May 25, 2005, European
Application No. 05105001.1, filed Jun. 6, 2005, European
Application No. 05104874.2, filed Jun. 3, 2005, European
Application No. 05105345.2, filed Jun. 10, 2005, European
Application No. 05104630.8, filed May 30, 2005, European
Application No. 05104761.1, filed Jun. 1, 2005, European
Application No. 05104811.4, filed Jun. 2, 2005, European
Application No. 05104818.9, filed Jun. 2, 2005, European
Application No. 05105021.9, filed Jun. 8, 2005, European
Application No. 05105028.4, filed Jun. 8, 2005, European
Application No. 05105136.5, filed Jun. 13, 2005, European
Application No. 05105993.9, filed Jun. 27, 2005, European
Application No. 05105508.5, filed Jun. 21, 2005, European
Application No. 05105575.4, filed Jun. 22, 2005, European
Application No. 05105510.1, filed Jun. 21, 2005, European
Application No. 05105401.3, filed Jun. 17, 2005, European
Application No. 05105405.4, filed Jun. 17, 2005, European
Application No. 05105992.1, filed Jun. 27, 2005, European
Application No. 05105570.5, filed Jun. 22, 2005, European
Application No. 05105406.2, filed Jun. 17, 2005, European
Application No. 05105624.0, filed Jun. 23, 2005, European
Application No. 05105643.0, filed Jun. 23, 2005, and European
Application No. 05105571.3, filed Jun. 22, 2005. Additionally,
International Patent Application PCT/EP 2005/007080 also claims
benefit to European Application No. 04015608.5, filed Jul. 2, 2004,
European Application No. 04016615.9, filed Jul. 15, 2004, European
Application No. 04018543.1, filed Aug. 5, 2004, European
Application No. 04105689.6, filed Aug. 23, 2004, European
Application No. 04105535.1, filed Aug. 27, 2004, European
Application No. 04026008.5, filed Nov. 3, 2004, European
Application No. 04026007.7, filed Nov. 3, 2004, European
Application No. 04026057.2, filed Nov. 4, 2004, European
Application No. 04026056.4, filed Nov. 4, 2004, European
Application No. 04028670.0, filed Dec. 3, 2004, and European
Application No. 04028671.8, filed Dec. 3, 2004. The entire content
of the above-referenced patent applications are incorporated herein
by this reference in their entirety.
[0002] [0001.0.0.0] The present invention relates to a process for
the production of a fine chemical in a microorgansm, a plant cell,
a plant, a plant tissue or in one or more parts thereof. The
invention furthermore relates to nucleic acid molecules,
polypeptides, nucleic acid constructs, vectors, antisense
molecules, antibodies, host cells, plant tissue, propagation
material, harvested material, plants, microorganisms as well as
agricultural compositions and to their use.
SUBMISSION ON COMPACT DISC
[0003] The contents of the following submission on compact discs
are incorporated herein by reference in its entirety: two copies of
the Sequence Listing (COPY 1 and COPY 2) and a computer readable
form copy of the Sequence Listing (CRF COPY), all on compact disc,
each containing: file name: date recorded: Sep. 20, 2011, size:
.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1 depicts a vector map of vector EG073qcz (SEQ ID NO:
68240).
[0005] FIG. 2 depicts a vector map of vector EG065qcz (SEQ ID NO:
68241).
[0006] FIG. 3 depicts a vector map of the binary vector for corn
transformation pMME0607 (SEQ ID NO: 68242).
[0007] FIG. 4A-F depicts the sequences of SEQ ID NO: 68240, SEQ ID
NO: 68241, and SEQ ID NO: 68242.
[0008] [0002.0.0.0] Amino acids are used in many branches of
industry, including the food, animal feed, cosmetics,
pharmaceutical and chemical industries. Amino acids such as
D,L-methionine, L-lysine or L-threonine are used in the animal feed
industry. The essential amino acids valine, leucine, isoleucine,
lysine, threonine, methionine, tyrosine, phenylalanine and
tryptophan are particularly important for the nutrition of humans
and a number of livestock species. Glycine, L-methionine and
tryptophan are all used in the pharmaceutical industry. Glutamine,
valine, leucine, isoleucine, histidine, arginine, proline, serine
and alanine are used in the pharmaceutical and cosmetics
industries. Threonine, tryptophan and D,L-methionine are widely
used feed additives (Leuchtenberger, W. (1996) Amino
acids--technical production and use, pp. 466-502 in Rehm et al.,
(Ed.) Biotechnology vol. 6, chapter 14a, VCH Weinheim). Moreover,
amino acids are suitable for the chemical industry as precursors
for the synthesis of synthetic amino acids and proteins, such as
N-acetylcysteine, S-carboxymethyl-L-cysteine,
(S)-5-hydroxytryptophan and other substances described in Ullmann's
Encyclopedia of Industrial Chemistry, vol. A2, pp. 57-97, VCH
Weinheim, 1985.
[0009] [0003.0.0.0] Over one million tons of amino acids are
currently produced annually; their market value amounts to over 2.5
billion US dollars. They are currently produced by four competing
processes: Extraction from protein hydrolysates, for example
L-cystine, L-leucine or L-tyrosine, chemical synthesis, for example
of D-, L-methionine, conversion of chemical precursors in an enzyme
or cell reactor, for example L-phenylalanine, and fermentative
production by growing, on an industrial scale, bacteria which have
been developed to produce and secrete large amounts of the desired
molecule in question. An organism, which is particularly suitable
for this purpose is Corynebacterium glutamicum, which is used for
example for the production of L-lysine or L-glutamic acid. Other
amino acids which are produced by fermentation are, for example,
L-threonine, L-tryptophan, L-aspartic acid and L-phenylalanine.
[0010] [0004.0.0.0] The biosynthesis of the natural amino acids in
organisms capable of producing them, for example bacteria, has been
characterized thoroughly; for a review of the bacterial amino acid
biosynthesis and its regulation, see Umbarger, H. E. (1978) Ann.
Rev. Biochem. 47: 533-606.
[0011] [0005.0.0.0] It is known that amino acids are produced by
fermentation of strains of coryneform bacteria, in particular
Corynebacterium glutamicum. Due to their great importance, the
production processes are constantly being improved. Process
improvements can relate to measures regarding technical aspects of
the fermentation, such as, for example, stirring and oxygen supply,
or the nutrient media composition, such as, for example, the sugar
concentration during fermentation, or to the work-up to give the
product, for example by ion exchange chromatography, or to the
intrinsic performance properties of the microorganism itself.
Bacteria from other genera such as Escherichia or Bacillus are also
used for the production of amino acids. A number of mutant strains,
which produce an assortment of desirable compounds from the group
of the sulfur-containing fine chemicals, have been developed via
strain selection. The performance properties of said microorganisms
are improved with respect to the production of a particular
molecule by applying methods of mutagenesis, selection and mutant
selection. Methods for the production of methionine have also been
developed. In this manner, strains are obtained which are, for
example, resistant to antimetabolites, such as, for example, the
methionine analogues .alpha.-methylmethionine, ethionine,
norleucine, N-acetylnorleucine, S-trifluoromethylhomocysteine,
2-amino-5-heprenoitic acid, selenomethionine, methionine
sulfoximine, methoxine, 1-aminocyclopentanecarboxylic acid or which
are auxotrophic for metabolites with regulatory importance and
which produce sulfur-containing fine chemicals such as, for
example, L-methionine. However, such processes developed for the
production of methionine have the disadvantage that their yields
are too low for being economically exploitable and that they are
therefore not yet competitive with regard to chemical
synthesis.
[0012] [0006.0.0.0] Zeh (Plant Physiol., Vol. 127, 2001: 792-802)
describes increasing the methionine content in potato plants by
inhibiting threonine synthase by what is known as antisense
technology. This leads to a reduced threonine synthase activity
without the threonine content in the plant being reduced. This
technology is highly complex; the enzymatic activity must be
inhibited in a very differentiated manner since otherwise
auxotrophism for the amino acid occurs and the plant will no longer
grow.
[0013] [0007.0.0.0] U.S. Pat. No. 5,589,616 teaches the production
of higher amounts of amino acids in plants by overexpressing a
monocot storage protein in dicots. WO 96/38574, WO 97/07665, WO
97/28247, U.S. Pat. No. 4,886,878, U.S. Pat. No. 5,082,993 and U.S.
Pat. No. 5,670,635 are following this approach. That means in all
the aforementioned intellectual property rights different proteins
or polypeptides are expressed in plants. Said proteins or
polypeptides should function as amino acid sinks. Other methods for
increasing amino acids such as lysine are disclosed in WO 95/15392,
WO 96/38574, WO 89/11789 or WO 93/19190. In this cases special
enzymes in the amino acid biosynthetic pathway such as the
diphydrodipicolinic acid synthase are deregulated. This leads to an
increase in the production of lysine in the different plants.
Another approach to increase the level of amino acids in plants is
disclosed in EP-A-0 271 408. EP-A-0 271 408 teaches the mutagenesis
of plant and selection afterwards with inhibitors of certain
enzymes of amino acid biosynthetic pathway.
[0014] [0008.0.0.0] Methods of recombinant DNA technology have also
been used for some years to improve Corynebacterium strains
producing L-amino acids by amplifying individual amino acid
biosynthesis genes and investigating the effect on amino acid
production.
[0015] [0009.0.0.0] As described above, the essential amino acids
are necessary for humans and many mammals, for example for
livestock. L-methionine is important as methyl group donor for the
biosynthesis of, for example, choline, creatine, adrenaline, bases
and RNA and DNA, histidine, and for the transmethylation following
the formation of S-adenosylmethionine or as a sulfhydryl group
donor for the formation of cysteine. Moreover, L-methionine appears
to have a positive effect in depression.
[0016] [0010.0.0.0] Improving the quality of foodstuffs and animal
feeds is an important task of the food-and-feed industry. This is
necessary since, for example, certain amino acids, which occur in
plants are limited with regard to the supply of mammals. Especially
advantageous for the quality of foodstuffs and animal feeds is as
balanced as possible an amino acid profile since a great excess of
an amino acid above a specific concentration in the food has no
further positive effect on the utilization of the food since other
amino acids suddenly become limiting. A further increase in quality
is only possible via addition of further amino acids, which are
limiting under these conditions. The targeted addition of the
limiting amino acid in the form of synthetic products must be
carried out with extreme caution in order to avoid amino acid
imbalance. For example, the addition of an essential amino acid
stimulates protein digestion, which may cause deficiency situations
for the second or third limiting amino acid, in particular. In
feeding experiments, for example casein feeding experiments, the
additional provision of methionine, which is limiting in casein,
has revealed the fatty degeneration of liver, which could only be
alleviated after the additional provision of tryptophan.
[0017] [0011.0.0.0] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add a plurality of amino acids
in a balanced manner to suit the organism.
[0018] [0012.0.0.0] It is an object of the present invention to
develop an inexpensive process for the synthesis of L-methionine.
L-methionine is with lysine or threonine (depending on the
organism) one of the two amino acids which are most frequently
limiting
[0019] [0013.0.0.0] It was now found that this object is achieved
by providing the process according to the invention described
herein and the embodiments characterized in the claims.
[0020] [0014.0.0.0] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is methionine Accordingly, in
the present invention, the term "the fine chemical" as used herein
relates to "methadone". Further, in another embodiment the term
"the fine chemicals" as used herein also relates to compositions of
fine chemicals comprising methionine.
[0021] [0015.0.0.0] In one embodiment, the term "the fine chemical"
or "the respective fine chemical" means L-methionine. Throughout
the specification the term "the fine chemical" or "the respective
fine chemical" means methionine, preferably L-methionine, its
salts, ester or amids in free form or bound to proteins. In a
preferred embodiment, the term "the fine chemical" means
L-methionine in free form or its salts or bound to proteins. In one
embodiment, the term "the fine chemical" and the term "the
respective fine chemical" mean at least one chemical compound with
an activity of the above mentioned fine chemical.
[0022] [0016.0.0.0] Accordingly, the present invention relates to a
process comprising [0023] (a) increasing or generating the activity
of one or more YLR375W, YBL015w, YER173w, YOR084w and/or b1829
and/or b4232, b0464, b1343, b2414, and/or b2762 protein(s) or of a
protein having the sequence of a polypeptide encoded by a nucleic
acid molecule indicated in Table I, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 in a non-human organism or in one or more
parts thereof and [0024] (b) growing the organism under conditions
which permit the production of the fine chemical, thus, methionine
or a fine chemicals comprising methionine, in said organism.
Accordingly, the present invention relates to a process for the
production of a fine chemical comprising [0025] (a) increasing or
generating the activity of one or more proteins having the activity
of a protein indicated in Table II, column 3, lines 1 to 5 and/or
lines 334 to 338 or having the sequence of a polypeptide encoded by
a nucleic acid molecule indicated in Table I, column 5 or 7, lines
1 to 5 and/or lines 334 to 338, in a non-human organism in one or
more parts thereof and [0026] (b) growing the organism under
conditions which permit the production of the fine chemical, in
particular methionine.
[0027] [0016.1.0.0] ./.
[0028] [0017.0.0.0] Comprises/comprising and grammatical variations
thereof when used in this specification are to be taken to specify
the presence of stated features, integers, steps or components or
groups thereof, but not to preclude the presence or addition of one
or more other features, integers, steps, components or groups
thereof. The term "Table I" used in this specification is to be
taken to specify the content of Table I A and Table I B. The term
"Table II" used in this specification is to be taken to specify the
content of Table II A and Table II B. The term "Table I A" used in
this specification is to be taken to specify the content of Table I
A. The term "Table I B" used in this specification is to be taken
to specify the content of Table I B. The term "Table II A" used in
this specification is to be taken to specify the content of Table
II A. The term "Table II B" used in this specification is to be
taken to specify the content of Table II B. In one preferred
embodiment, the term "Table I" means Table I B. In one preferred
embodiment, the term "Table II" means Table II B.
[0029] [0018.0.0.0] Preferably, this process further comprises the
step of recovering the fine chemical, which is synthesized by the
organism from the organism and/or from the culture medium used for
the growth or maintenance of the organism. The term "recovering"
means the isolation of the fine chemical in different purities,
that means on the one hand harvesting of the biological material,
which contains the fine chemical without further purification and
on the other hand purities of the fine chemical between 5% and 100%
purity, preferred purities are in the range of 10% and 99%. In one
embodiment, the purities are 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95% or 99%.
[0030] [0019.0.0.0] Advantageously the process for the production
of the fine chemical leads to an enhanced production of the fine
chemical. The terms "enhanced" or "increase" mean at least a 10%,
20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or
100%, more preferably 150%, 200%, 300%, 400% or 500% higher
production of the fine chemical in comparison to the reference as
defined below, e.g. that means in comparison to an organism without
the aforementioned modification of the activity of a protein having
the activity of a protein indicated in Table II, column 3, lines 1
to 5 and/or lines 334 to 338 or encoded by nucleic acid molecule
indicated in Table I, columns 5 or 7, lines 1 to 5 and/or lines 334
to 338.
[0031] [0020.0.0.0] Surprisingly it was found, that the transgenic
expression of at least one of the Saccharomyces cerevisiae
protein(s) indicated in Table II, Column 3, lines 1 to 4 and/or at
least one of the Escherichia coli K12 protein(s) indicated in Table
II, Column 3, line 5 and/or lines 334 to 338 in Arabidopsis
thaliana conferred an increase in the methionine content of the
transformed plants.
[0032] [0021.0.0.0] In accordance with the invention, the term
"organism" as understood herein relates always to a non-human
organism, in particular to an animal or plant organism or to a
microorganism. Further, the term "animal" as understood herein
relates always to a non-human animal.
[0033] In accordance with the invention it is known to the skilled
that anionic compounds such as acids are present in aqueous
solutions in an equilibrium between the acid and its salts
according to the pH present in the respective compartment of the
cell or organism and the pK of the acid. Depending on the strength
of the acid (pK) and the pH the salt or the free acid are
predominant. Thus, the term "the fine chemical", the term "the
respective fine chemical", or the term "acid" or the use of a
denomination referring to a neutralized anionic compound relates to
the anionic form as well as the neutralised status of that compound
according to the milieu of the aqueous solution in which they are
present.
[0034] [0022.0.0.0] The sequence of YLR375w from Saccharomyces
cerevisiae has been published in Johnston, Nature 387 (6632 Suppl),
87-90, 1997, and Goffeau, Science 274 (5287), 546-547, 1996, and
its activity is being "involved in pre-tRNA slicing and in uptake
of branched-chain amino acids; YLR375wp". Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product "involved in pre-tRNA slicing and in uptake of
branched-chain amino skids" from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of methionine, in particular for increasing the
amount of methionine in free or bound form in an organism or a part
thereof, as mentioned.
[0035] The sequence of YBL015w from Saccharomyces cerevisiae has
been published in Goffeau, Science 274 (5287), 546-547, 1996, and
in Feldmann, EMBO J., 13, 5795-5809, 1994 and its activity is being
defined as an "Mannose-containing glycoprotein which binds
concanavalin A; Ach1p". In another reference, the activity is
described as "acetyl-CoA hydrolase". Accordingly, in one
embodiment, the process of the present invention comprises the use
of a "Mannose-containing glycoprotein which binds concanavalin A;
Ach1p" from Saccharomyces cerevisiae or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
methionin, in particular for increasing the amount of methionine in
free or bound form in an organism or a part thereof, as mentioned.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a "Acetyl-CoA hydrolase" from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of methionin, in
particular for increasing the amount of methionine in free or bound
form in an organism or a part thereof, as mentioned.
[0036] The sequence of YER173w from Saccharomyces cerevisiae has
been published in Dietrich, Nature 387 (6632 Suppl), 78-81, 1997,
and Goffeau, Science 274 (5287), 546-547, 1996, and its activity is
being defined as an "Checkpoint protein, involved in the activation
of the DNA damage and meiotic pachytene checkpoints; subunit of a
clamp loader that loads Rad17p-Mec3p-Dc1p onto DNA, homolog of the
human and S. pompe Rad17 protein; Rad24p". Accordingly, in one
embodiment, the process of the present invention comprises the use
of a "Checkpoint protein, involved in the activation of the DNA
damage and meiotic pachytene checkpoints" or its "subunit of a
clamp loader that loads Rad17p-Mec3p-Dc1p onto DNA" or a Rad24p
from Saccharomyces cerevisiae or a Rad17 protein or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of methionin, in particular for increasing the amount of
methionine in free or bound form in an organism or a part thereof,
as mentioned.
[0037] The sequence of YOR084w from Saccharomyces cerevisiae has
been published in Dujon, Nature 387 (6632 Suppl), 98-102, 1997, and
Goffeau, Science 274 (5287), 546-547, 1996, and its activity is
being defined as a putative lipase of the peroxisomal matrix.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a putative lipase of the peroxisomal
matrix" from Saccharomyces cerevisiae or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
methionin, in particular for increasing the amount of methionine in
free or bound form in an organism or a part thereof, as
mentioned.
[0038] The sequence of b1829 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a heat shock protein. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a "heat shock protein" from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
methionin, in particular for increasing the amount of methionine in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of a htpX heat shock protein is increased or generated,
e.g. from E. coli or a homolog thereof. Homologs are also for
example the htpX heat shock protein is also annotated as having a
protease activity. Accordingly, in one embodiment, in the process
of the present invention the activity of a protease, preferably of
a heat shock protease, more preferred of a htpX protease or its
homolog is increased for the production of the fine chemical,
meaning of methionin, in particular for increasing the amount of
methionine in free or bound form in an organism or a part thereof,
as mentioned.
[0039] The sequence of b0464 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a "transcriptional repressor for
multidrug efflux pump (TetR/AcrR family)". Accordingly, in one
embodiment, the process of the present invention comprises the use
of a "transcriptional repressor for multidrug efflux pump
(TetR/AcrR family)" from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
methionine, in particular for increasing the amount of methionine
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a protein of the superfamily "probable
transcription repressor mtrr", is increased or generated,
preferably having activity in transcriptional control and/or DNA
binding, e.g. from E. coli or a homolog thereof. Accordingly, in
one embodiment, in the process of the present invention the
activity of a "transcriptional repressor for multidrug efflux pump
(TetR/AcrR family)" or its homolog is increased for the production
of the fine chemical, meaning of methionine, in particular for
increasing the amount of methionine in free or bound form in an
organism or a part thereof, as mentioned.
[0040] The sequence of b1343 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as an ATP-dependent RNA helicase,
stimulated by 23S rRNA. Accordingly, in one embodiment, the process
of the present invention comprises the use of an "ATP-dependent RNA
helicase, stimulated by 23S rRNA" from E. coli or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
of methionine, in particular for increasing the amount of
methionine in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a protein having an activity in rRNA
processing or translation is increased or generated, e.g. from E.
coli or a homolog thereof. Accordingly, in one embodiment, in the
process of the present invention the activity of a ATP-dependent
RNA helicase, stimulated by 23S rRNA or its homolog is increased
for the production of the fine chemical, meaning of methionine, in
particular for increasing the amount of methionine in free or bound
form in an organism or a part thereof, as mentioned.
[0041] The sequence of b2414 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a subunit of cysteine synthase A and
O-acetylserine sulfhydrolase A, PLP-dependent enzyme. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a "subunit of cysteine synthase A and O-acetylserine
sulfhydrolase A, PLP-dependent enzyme" from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of methionine, in particular for increasing the amount of
methionine in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a protein of the superfamily "threonine
dehydratase", preferably having an activity in amino acid
biosynthesis, biosynthesis of the cysteine-aromatic group,
degradation of amino acids of the cysteine-aromatic group, nitrogen
and sulfur utilization biosynthesis of the aspartate family,
degradation of amino acids of the aspartate group, biosynthesis of
sulfuric acid and L-cysteine derivatives, biosynthesis of secondary
products derived from primary amino acids, biosynthesis of
secondary products derived from glycine, L-serine and L-alanine,
pyridoxal phosphate binding, more preferred having an "subunit of
cysteine synthase A and O-acetylserine sulfhydrolase A,
PLP-dependent enzyme"-activity is increased or generated, e.g. from
E. coli or a homolog thereof. Accordingly, in one embodiment, in
the process of the present invention the activity of a "subunit of
cysteine synthase A and O-acetylserine sulfhydrolase A,
PLP-dependent enzyme" or its homolog is increased for the
production of the fine chemical, meaning of methionine, in
particular for increasing the amount of methionine in free or bound
form in an organism or a part thereof, as mentioned.
[0042] The sequence of b2762 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a 3'-phosphoadenosine
5'-phosphosulfate (PAPS) reductase. Accordingly, in one embodiment,
the process of the present invention comprises the use of a
"3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductase" from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of methionine, in particular for
increasing the amount of methionine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a protein of the
superfamily "3'-phosphoadenosine 5'-phosphosulfate reductase",
preferably having an activity in biosynthesis of cysteine, nitrogen
and sulfur utilization, amino acid biosynthesis more preferred
having an "3'-phosphoadenosine 5'-phosphosulfate (PAPS)
reductase"-activity is increased or generated, e.g. from E. coli or
a homolog thereof. Accordingly, in one embodiment, in the process
of the present invention the activity of a "3'-phosphoadenosine
5'-phosphosulfate (PAPS) reductase" or its homolog is increased for
the production of the fine chemical, meaning of methionine, in
particular for increasing the amount of methionine in free or bound
form in an organism or a part thereof, as mentioned. The sequence
of b4232 from Escherichia coli K12 has been published in Blattner,
Science 277(5331), 1453-1474, 1997, and its activity is being
defined as a fructose-1,6-bisphosphatase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a "fructose-1,6-bisphosphatase" from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of methionine, in particular for increasing the amount of
methionine in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a protein of the superfamily
"fructose-bisphosphatase", preferably having an activity in
C-compound and carbohydrate metabolism, C-compound and carbohydrate
utilization, energy, glycolysis and gluconeogenesis, plastid,
photosynthesis, more preferred having an
"fructose-1,6-bisphosphatase"-activity, is increased or generated,
e.g. from E. coli or a homolog thereof. Accordingly, in one
embodiment, in the process of the present invention the activity of
a "fructose-1,6-bisphosphatase" or its homolog is increased for the
production of the fine chemical, meaning of methionine, in
particular for increasing the amount of methionine in free or bound
form in an organism or a part thereof, as mentioned.
[0043] Homologues (=homologs) of the present gene products can be
derived from any organisms as long as the homologue confers the
herein mentioned activity, in particular, confers an increase in
the fine chemical amount or content. Further, in the present
invention, the term "homologue" relates to the sequence of an
organism having the highest sequence homology to the herein
mentioned or listed sequences of all expressed sequences of said
organism.
[0044] However, the person skilled in the art knows, that,
preferably, the homologue has said the--fine-chemical-increasing
activity and, if known, the same biological function or activity in
the organism as at least one of the protein(s) indicated in Table
I, Column 3, lines 1 to 5 and/or lines 334 to 338, e.g. having the
sequence of a polypeptide encoded by a nucleic acid molecule
comprising the sequence indicated in indicated in Table I, Column 5
or 7, lines 1 to 5 and/or lines 334 to 338.
[0045] In one embodiment, the homolog of any one of the
polypeptides indicated in Table II, lines 1 to 4 is a homolog
having the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms and being derived from an Eukaryot. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 5 and/or lines 334 to 338 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from bacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 1 to 4 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in an organisms or part
thereof, and being derived from Fungi. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 5
and/or lines 334 to 338 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or part
thereof and being derived from Proteobacteria. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
1 to 4 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof and
being derived from Ascomycota. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 5 and/or lines
334 to 338 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or part thereof, and
being derived from Gammaproteobacteria. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 1
to 4 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or part thereof, and
being derived from Saccharomycotina. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, line 5 and/or
lines 334 to 338 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or part
thereof, and being derived from Enterobacteriales. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 1 to 4 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or a part
thereof, and being derived from Saccharomycetes. In one embodiment,
the homolog of the a polypeptide indicated in Table II, column 3,
line 5 and/or lines 334 to 338 is a homolog having the same or a
similar activity, in particular an increase of activity confers an
increase in the content of the fine chemical in the organisms or
part thereof, and being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 1 to 4 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms, and being
derived from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 5 and/or lines
334 to 338 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof,
and being derived from Escherichia. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 1 to 4 is a
homolog having the same or a similar activity, in particular an
increase of activity confers an increase in the content of the fine
chemical in the organisms or a part thereof, and being derived from
Saccharomycetaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 1 to 4 is a homolog having
the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms or a part thereof, and being derived from
Saccharomycetes.
[0046] [0023.1.0.0] Homologs of the polypeptides polypeptide
indicated in Table II, column 3, lines 1 to 4 may be the
polypeptides encoded by the nucleic acid molecules polypeptide
indicated in Table I, column 7, lines 1 to 4 or may be the
polypeptides indicated in Table II, column 7, lines 1 to 4.
Homologs of the polypeptides polypeptide indicated in Table II,
column 3, line 5 and/or lines 334 to 338 may be the polypeptides
encoded by the nucleic acid molecules polypeptide indicated in
Table I, column 7, line 5 and/or lines 334 to 338 or may be the
polypeptides indicated in Table II, column 7, lines 5 and/or lines
334 to 338.
[0047] [0024.0.0.0] Further homologs of are described herein
below.
[0048] [0025.0.0.0] In accordance with the invention, a protein or
polypeptide has the "activity of an protein of the invention", or
of a protein as used in the invention, e.g. a protein having the
activity of a protein indicated in Table II, column 3, lines 1 to 5
and/or lines 334 to 338 if its de novo activity, or its increased
expression directly or indirectly leads to an increased methionine,
preferably L-methionine level in the organism or a part thereof,
preferably in a cell of said organism. In a preferred embodiment,
the protein or polypeptide has the above-mentioned additional
activities of a protein indicated in Table II, column 3, lines 1 to
5 and/or lines 334 to 338. During the specification the activity or
preferably the biological activity of such a protein or polypeptide
or an nucleic acid molecule or sequence encoding such protein or
polypeptide is identical or similar if it still has the biological
or enzymatic activity of any one of the proteins indicated in Table
II, column 3, lines 1 to 5 and/or lines 334 to 338, i.e. if it has
at least 10% of the original enzymatic activity, preferably 20%,
particularly preferably 30%, most particularly preferably 40% in
comparison to an any one of the proteins indicated in Table II,
column 3, lines 1 to 4 of Saccharomyces cerevisiae and/or any one
of the proteins indicated in Table II, column 3, line 5 and/or
lines 334 to 338 of E. coli K12.
[0049] [0025.1.0.0] In one embodiment, the polypeptide of the
invention or the polypeptide used in the method of the invention
confers said activity, e.g. the increase of the fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary distant to the organism in which it is
expressed. For example origin and expressing organism are derived
from different families, orders, classes or phylums.
[0050] [0025.2.0.0] In one embodiment, the polypeptide of the
invention or the polypeptide used in the method of the invention
confers said activity, e.g. the increase of the fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[0051] [0026.0.0.0] The terms "increased", "rose", "extended",
"enhanced", "improved" or "amplified" relate to a corresponding
change of a property in an organism, a part of an organism such as
a tissue, seed, root, leave, flower etc. or in a cell and are
interchangeable. Preferably, the overall activity in the volume is
increased or enhanced in cases if the increase or enhancement is
related to the increase or enhancement of an activity of a gene
product, independent whether the amount of gene product or the
specific activity of the gene product or both is increased or
enhanced or whether the amount, stability or translation efficacy
of the nucleic acid sequence or gene encoding for the gene product
is increased or enhanced. The terms "reduction", "decrease" or
"deletion" relate to a corresponding change of a property in an
organism, a part of an organism such as a tissue, seed, root,
leave, flower etc. or in a cell. Preferably, the overall activity
in the volume is reduced, decreased or deleted in cases if the
reduction, decrease or deletion is related to the reduction,
decrease or deletion of an activity of a gene product, independent
whether the amount of gene product or the specific activity of the
gene product or both is reduced, decreased or deleted or whether
the amount, stability or translation efficacy of the nucleic acid
sequence or gene encoding for the gene product is reduced,
decreased or deleted.
[0052] [0027.0.0.0] The terms "increase" or "decrease" relate to a
corresponding change of a property an organism or in a part of an
organism, such as a tissue, seed, root, leave, flower etc. or in a
cell. Preferably, the overall activity in the volume is increased
in cases the increase relates to the increase of an activity of a
gene product, independent whether the amount of gene product or the
specific activity of the gene product or both is increased or
generated or whether the amount, stability or translation efficacy
of the nucleic acid sequence or gene encoding for the gene product
is increased.
[0053] [0028.0.0.0] Under "change of a property" it is understood
that the activity, expression level or amount of a gene product or
the metabolite content is changed in a specific volume relative to
a corresponding volume of a control, reference or wild type,
including the de novo creation of the activity or expression.
[0054] [0029.0.0.0] The terms "increase" or "decrease" include the
change or the modulation of said property in only parts of the
subject of the present invention, for example, the modification can
be found in compartment of a cell, like a organelle, or in a part
of a plant, like tissue, seed, root, leave, flower etc. but is not
detectable if the overall subject, i.e. complete cell or plant, is
tested. Preferably, the increase or decrease is found cellular,
thus the term "increase of an activity" or "increase of a
metabolite content" relates to the cellular increase compared to
the wild type cell. However, the terms increase or decrease as used
herein also include the change or modulation of a property in the
whole organism as mentioned.
[0055] [0030.0.0.0] Accordingly, the term "increase" or "decrease"
means that the specific activity of an enzyme, preferably the
amount of a compound or metabolite, e.g. of a polypeptide, a
nucleic acid molecule or of the respective fine chemical of the
invention or an encoding mRNA or DNA, can be increased or decreased
in a volume.
[0056] [0031.0.0.0] The terms "wild type", "control" or "reference"
are exchangeable and can be a cell or a part of organisms such as
an organelle or a tissue, or an organism, in particular a
microorganism or a plant, which was not modified or treated
according to the herein described process according to the
invention. Accordingly, the cell or a part of organisms such as an
organelle or a tissue, or an organism, in particular a
microorganism or a plant used as wild type, control or reference
corresponds to the cell, organism or part thereof as much as
possible and is in any other property but in the result of the
process of the invention as identical to the subject matter of the
invention as possible. Thus, the wild type, control, or reference
is treated identically or as identical as possible, saying that
only conditions or properties might be different which do not
influence the quality of the tested property.
[0057] [0032.0.0.0] Preferably, any comparison is carried out under
analogous conditions. The term "analogous conditions" means that
all conditions such as, for example, culture or growing conditions,
assay conditions (such as buffer composition, temperature,
substrates, pathogen strain, concentrations and the like) are kept
identical between the experiments to be compared.
[0058] [0033.0.0.0] The "reference", "control", or "wild type" is
preferably a subject, e.g. an organelle, a cell, a tissue, an
organism, in particular a plant or a microorganism, which was not
modified or treated according to the herein described process of
the invention and is in any other property as similar to the
subject matter of the invention as possible. The reference,
control, or wild type is in its genome, transcriptome, proteome or
meta-bolome as similar as possible to the subject of the present
invention. Preferably, the term "reference-" "control-" or "wild
type-"-organelle, .cndot.cell, .cndot.tissue or -organism, in
particular plant or microorganism, relates to an organelle, cell,
tissue or organism, in particular plant or microorganism, which is
nearly genetically identical to the organelle, cell, tissue or
organism, in particular microorganism or plant, of the present
invention or a part thereof preferably 95%, more preferred are 98%,
even more preferred are 99.00%, in particular 99.10%, 99.30%,
99.50%, 99.70%, 99.90%, 99.99%, 99, 999% or more. Most preferable
the "reference", "control", or "wild type" is a subject, e.g. an
organelle, a cell, a tissue, an organism, which is genetically
identical to the organism, cell or organelle used according to the
process of the invention except that the responsible or activity
conferring nucleic acid molecules or the gene product encoded by
them are amended, manipulated, exchanged or introduced according to
the inventive process.
[0059] [0034.0.0.0] Preferably, the reference, control or wild type
differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. as result of
an increase in the level of the nucleic acid molecule of the
present invention or an increase of the specific activity of the
polypeptide of the invention or the polypeptide used in the method
of the invention. E.g., it differs by or in the expression level or
activity of an protein having the activity of a protein as
indicated in Table II, column 3, lines 1 to 5 and/or lines 334 to
338 or being encoded by a nucleic acid molecule indicated in Table
I, column 5, lines 1 to 5 and/or lines 334 to 338 or its homologs,
e.g. as indicated in Table I, column 7, lines 1 to 5 and/or lines
334 to 338, its biochemical or genetical causes and therefore shows
the increased amount of the fine chemical.
[0060] [0035.0.0.0] In case, a control, reference or wild type
differing from the subject of the present invention only by not
being subject of the process of the invention can not be provided,
a control, reference or wild type can be an organism in which the
cause for the modulation of an activity conferring the increase of
the fine chemical or expression of the nucleic acid molecule as
described herein has been switched back or off, e.g. by knocking
out the expression of responsible gene product, e.g. by antisense
inhibition, by inactivation of an activator or agonist, by
activation of an inhibitor or antagonist, by inhibition through
adding inhibitory antibodies, by adding active compounds as e.g.
hormones, by introducing negative dominant mutants, etc. A gene
production can for example be knocked out by introducing
inactivating point mutations, which lead to an enzymatic activity
inhibition or a destabilization or an inhibition of the ability to
bind to cofactors etc.
[0061] [0036.0.0.0] Accordingly, preferred reference subject is the
starting subject of the present process of the invention.
Preferably, the reference and the subject matter of the invention
are compared after standardization and normalization, e.g. to the
amount of total RNA, DNA, or Protein or activity or expression of
reference genes, like housekeeping genes, such as ubiquitin, actin
or ribosomal proteins.
[0062] [0037.0.0.0] A series of mechanisms exists via which a
modification of a protein, e.g. the polypeptide of the invention or
the polypeptide used in the method of the invention can directly or
indirectly affect the yield, production and/or production
efficiency of the fine chemical.
[0063] [0038.0.0.0] For example, the molecule number or the
specific activity of the polypeptide or the nucleic acid molecule
may be increased. Larger amounts of the fine chemical can be
produced if the polypeptide or the nucleic acid of the invention is
expressed de novo in an organism lacking the activity of said
protein. However, it is also possible to increase the expression of
the gene which is naturally present in the organisms, for example
by amplifying the number of gene(s), by modifying the regulation of
the gene, or by increasing the stability of the corresponding mRNA
or of the corresponding gene product encoded by the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention, or by introducing homologous genes from
other organisms which are differently regulated, e.g. not feedback
sensitive.
[0064] [0039.0.0.0] This also applies analogously to the combined
increased expression of the nucleic acid molecule of the present
invention or its gene product with that of further enzymes or
regulators of the biosynthesis pathways of the respective fine
chemical, e.g. which are useful for the synthesis of the respective
fine chemicals.
[0065] [0040.0.0.0] The increase, decrease or modulation according
to this invention can be constitutive, e.g. due to a stable
permanent transgenic expression or to a stable mutation in the
corresponding endogenous gene encoding the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention or to a modulation of the expression or of the
behaviour of a gene conferring the expression of the polypeptide of
the invention or the polypeptide used in the method of the
invention, or transient, e.g. due to an transient transformation or
temporary addition of a modulator such as a agonist or antagonist
or inducible, e.g. after transformation with a inducible construct
carrying the nucleic acid molecule of the invention or the nucleic
acid molecule used in the method of the invention under control of
a inducible promoter and adding the inducer, e.g. tetracycline or
as described herein below.
[0066] [0041.0.0.0] The increase in activity of the polypeptide
amounts in a cell, a tissue, a organelle, an organ or an organism
or a part thereof preferably to at least 5%, preferably to at least
20% or at to least 50%, especially preferably to at least 70%, 80%,
90% or more, very especially preferably are to at least 200%, most
preferably are to at least 500% or more in comparison to the
control, reference or wild type.
[0067] [0042.0.0.0] The specific activity of a polypeptide encoded
by a nucleic acid molecule of the present invention or of the
polypeptide of the present invention can be tested as described in
the examples. In particular, the expression of a protein in
question in a cell, e.g. a plant cell or a microorganism and the
detection of an increase the respective fine chemical level in
comparison to a control is an easy test and can be performed as
described in the state of the art.
[0068] [0043.0.0.0] The term "increase" includes, that a compound
or an activity is introduced into a cell de novo or that the
compound or the activity has not been detectable before, in other
words it is "generated".
[0069] [0044.0.0.0] Accordingly, in the following, the term
"increasing" also comprises the term "generating" or "stimulating".
The increased activity manifests itself in an increase of the fine
chemical.
[0070] [0045.0.0.0] In one embodiment, in case the activity of the
Saccharomyces cerevisiae protein YLR375W or its homologs, e.g. as
indicated in Table II, columns 5 or 7, line 1, is increased;
preferably, an increase of the fine chemical between 110% and 300%
or more is conferred.
[0071] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YBL015w or an acetyl-CoA hydrolase, or its
homologs, e.g. as indicated in Table II, columns 5 or 7, line 2, is
increased; preferably, the increase of the fine chemical between
110% and 300% or more is conferred.
[0072] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YER173w or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 3, e.g. a checkpoint protein,
involved in the activation of the DNA damage and meiotic pychtene
checkpoints; subunit of a clamp loader that loads
Rad17p-Mec3p-Ddc1p onto DNA or Rad24p or its homologs, e.g. the
human or S. pombe Rad17 is increased; preferably, the increase of
the fine chemical between 110% and 200% or more is conferred.
[0073] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YOR084w or an putative Lipase of the peroxisomal
matrix or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 4, is increased; preferably, the increase of the fine
chemical between 110% and 350% or more is conferred.
[0074] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1829 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 5, is increased, e.g. the activity of a
protease is increased, preferably, the activity of a heat shock
protein is increased, more preferred the activity of a htpX protein
or its homolog is increased; preferably, the increase of the fine
chemical between 110% and 400% or more is conferred.
[0075] In one embodiment, in case the activity of the Escherichia
coli K12 protein b4232 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 338, is increased, e.g. the activity of a
fructose-bisphosphatase-superfamily-protein is increased,
preferably, the activity a protein involved in C-compound and
carbohydrate metabolism, C-compound and carbohydrate utilization,
ENERGY, glycolysis and gluconeogenesis, plastid, and/or
photosynthesis is increased, more preferred the activity of a
fructose-1,6-bisphosphatase or its homolog is increased.
Preferably, the increase of the fine chemical around 20% or more is
conferred.
[0076] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0464 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 334, is increased, e.g. the activity of a
probable transcription repressor mtrr superfamily-protein is
increased, preferably, the activity a protein involved in
transcriptional control, and/or DNA binding is increased, more
preferred the activity of a transcriptional repressor for multidrug
efflux pump (TetR/AcrR family) or its homolog is increased
preferably, an increase of the respective fine chemical around
between 35% and 366% or more is conferred. In one embodiment, in
case the activity of the Escherichia coli K12 protein b1343 or its
homologs, e.g. as indicated in Table II, columns 5 or 7, line 335,
is increased, e.g. the activity of a protein involved in rRNA
processing and/or translation is increased, preferred the activity
of a ATP-dependent RNA helicase, stimulated by 23S rRNA or its
homolog is increased. Preferably, an increase of the respective
fine chemical around between 38% and 51% or more is conferred.
[0077] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2414 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 336, is increased, e.g. the activity of a
protein of the threonine dehydratase-superfamily is increased
preferably the activity of a protein involved in amino acid
biosynthesis, biosynthesis of the cysteine-aromatic group,
degradation of amino acids of the cysteine-aromatic group, nitrogen
and sulfur utilizationbiosynthesis of the aspartate family,
degradation of amino acids of the aspartate group, biosynthesis of
sulfuric acid and L-cysteine derivatives, biosynthesis of secondary
products derived from primary amino acids, biosynthesis of
secondary products derived from glycine, L-serine and L-alanine,
pyridoxal phosphate binding is increased, preferred the activity of
a subunit of cysteine synthase A and O-acetylserine sulfhydrolase
A, PLP-dependent enzyme or its homolog is increased. Preferably, an
increase of the respective fine chemical around between 37% and 75%
or more is conferred.
[0078] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2762 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 337, is increased, e.g. the activity of a
3'-phosphoadenosine 5'-phosphosulfate reductase-superfamily-protein
is increased, preferably, the activity a protein involved in
C-compound and carbohydrate metabolism, C-compound and carbohydrate
utilization, ENERGY, glycolysis and gluconeogenesis, plastid,
and/or photosynthesis is increased, more preferred the activity of
a fructose-1,6-bisphosphatase or its homolog is increased.
Preferably, the increase of the fine chemical around 20% or more is
conferred.
[0079] [0046.0.0.0] In one embodiment, in case the activity of the
Saccharomyces cerevisiae protein YLR375W or its homologs is
increased, preferably, an increase of the fine chemical and of
shikimic acid is conferred.
[0080] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YBL015w or its homologs, e.g. an Ach1p, is
increased, preferably, an increase of the fine chemical and of a
further amino acid, e.g. alanine is conferred.
[0081] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YER173w or its homologs, e.g. a checkpoint
protein, involved in the activation of the DNA damage and meiotic
pychtene checkpoints; subunit of a clamp loader that loads
Rad17p-Mec3p-Ddc1p onto DNA or Rad24p or its homologs, e.g. the
human or S. pombe Rad17 is increased, preferably, an increase of
the fine chemical and of a further amino acid, e.g. leucine, is
conferred.
[0082] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YOR084w or a putative lipase of the peroxisomal
matrix or its homologs is increased, preferably, an increase of the
fine chemical and of beta-sitosterol is conferred.
[0083] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1829 or its homologs is increased, e.g. the
activity of a protease is increased, preferably, the activity of a
heat shock protein is increased, more preferred the activity of a
htpX protein or its homolog is increased, preferably, an increase
of the fine chemical and of a further amino acid, e.g.
phenylalanine, is conferred.
[0084] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0464 or its homologs is increased, e.g. the
activity of a transcriptional repressor for multidrug efflux pump
(TetR/AcrR family) or its homolog is increased, preferably in an
increase of the fine chemical and of a further amino acid is
conferred.
[0085] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1343 or its homologs is increased, e.g. the
activity of a ATP-dependent RNA helicase, stimulated by 23S rRNA is
increased or its homolog is increased, preferably, an increase of
the fine chemical and of a further amino acid is conferred.
[0086] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2414 or its homologs is increased, e.g. the
activity of a subunit of cysteine synthase A and O-acetylserine
sulfhydrolase A, PLP-dependent enzyme is increased.
[0087] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2762 or its homologs is increased, e.g. the
activity of a 3'-phosphoadenosine 5'-phosphosulfate (PAPS)
reductase or its homolog is increased, preferably, an increase of
the fine chemical and of a further amino acid is conferred.
[0088] In one embodiment, in case the activity of the Escherichia
coli K12 protein b4232 or its homologs is increased, e.g. the
activity of a ructose-1,6-bisphosphatase or its homolog is
increased, preferably, an increase of the fine chemical and of a
further amino acid is conferred.
[0089] [0047.0.0.0] In this context, the respective fine chemical
amount in a cell, preferably in a tissue, more preferred in a
organism as a plant or a microorganism or part thereof, is
increased by 3% or more, especially preferably are 10% or more,
very especially preferably are more than 30% and most preferably
are 70% or more, such as 100%, 300% or 500%.
[0090] [0048.0.0.0] The respective fine chemical can be contained
in the organism either in its free form and/or bound to proteins or
polypeptides or mixtures thereof. Accordingly, in one embodiment,
the amount of the free form in a cell, preferably in a tissue, more
preferred in a organism as a plant or a microorganism or part
thereof, is increased by 3% or more, especially preferably are 10%
or more, very especially preferably are more than 30% and most
preferably are 70% or more, such as 100%, 300% or 500%.
Accordingly, in an other embodiment, the amount of the bound the
respective fine chemical in a cell, preferably in a tissue, more
preferred in a organism as a plant or a microorganism or part
thereof, is increased by 3% or more, especially preferably are 10%
or more, very especially preferably are more than 30% and most
preferably are 70% or more, such as 100%, 300% or 500%.
[0091] [0049.0.0.0] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
preferably has the structure of the polypeptide described herein,
in particular of a polypeptides comprising a consensus sequence as
indicated in Table IV, columns 7, line 1 to 5 or lines 334 to 338
or of a polypeptide as indicated in Table II, columns 5 or 7, line
1 to 5 or lines 334 to 338 or the functional homologues thereof as
described herein, or of a polypeptide which is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by a nucleic acid
molecule as indicated in Table I, columns 5 or 7, line 1 to 5 or
lines 334 to 338 or its herein described functional homologues and
has the herein mentioned activity.
[0092] [0050.0.0.0] For the purposes of the present invention, the
terms "L-methionine", "methionine", "homocysteine",
"S-adenosylmethionine" and "threonine" also encompass the
corresponding salts, such as, for example, methionine hydrochloride
or methionine sulfate. Preferably the terms methionine or threonine
are intended to encompass the terms L-methionine or
L-threonine.
[0093] [0051.0.0.0] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g. fine chemical compositions. Depending
on the choice of the organism used for the process according to the
present invention, for example a microorganism or a plant,
compositions or mixtures of various fine chemicals, e.g. comprising
further distinct amino acids, fatty acids, vitamins, hormones,
sugars, lipids, etc. can be produced.
[0094] [0052.0.0.0] The term "expression" refers to the
transcription and/or translation of a codogenic gene segment or
gene. As a rule, the resulting product is an mRNA or a protein.
However, expression products can also include functional RNAs such
as, for example, antisense, nucleic acids, tRNAs, snRNAs, rRNAs,
RNAi, siRNA, ribozymes etc. Expression may be systemic, local or
temporal, for example limited to certain cell types, tissues organs
or time periods.
[0095] [0053.0.0.0] In one embodiment, the process of the present
invention comprises one or more of the following steps: [0096] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention or the nucleic acid molecule or the
polypeptide used in the method of the invention, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 1 to 5 or lines 334 to 338 or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 1 to
5 or lines 334 to 338, having herein-mentioned the fine
chemical-increasing activity; [0097] b) stabilizing a mRNA
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, e.g. of a polypeptide having
an activity of a protein as indicated in Table II, column 3, lines
1 to 5 or lines 334 to 338 or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 1 to 5 or lines 334 to
338, or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned methionine increasing activity; [0098] c)
increasing the specific activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention or of the polypeptide of the present
invention or the nucleic acid molecule or polypeptide used in the
method of the invention, having herein-mentioned methionine
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, line 1 to 5 or lines
334 to 338, or its homologs activity, e.g. as indicated in Table
II, columns 5 or 7, line 1 to 5 or lines 334 to 338, or decreasing
the inhibitory regulation of the polypeptide of the invention or
the polypeptide used in the method of the invention; [0099] d)
generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention or of the polypeptide
of the invention or the polypeptide used in the method of the
invention having herein-mentioned methionine increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, line 1 to 5 or lines 334 to 338, or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
line 1 to 5 or lines 334 to 338,; [0100] e) stimulating activity of
a protein conferring the increased expression of a protein encoded
by the nucleic acid molecule of the present invention or a
polypeptide of the present invention having herein-mentioned
methionine increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 1
to 5 or lines 334 to 338, or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 1 to 5 or lines 334 to
338, by adding one or more exogenous inducing factors to the
organism or parts thereof; [0101] f) expressing a transgenic gene
encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned methionine increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 1 to 5 or lines 334 to 338, or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 1 to
5 or lines 334 to 338; [0102] g) increasing the copy number of a
gene conferring the increased expression of a nucleic acid molecule
encoding a polypeptide encoded by the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention or the polypeptide of the invention or the polypeptide
used in the method of the invention having herein-mentioned
methionine increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 1
to 5 or lines 334 to 338, or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 1 to 5 or lines 334 to
338; [0103] h) Increasing the expression of the endogenous gene
encoding the polypeptide of the invention or the polypeptide used
in the method of the invention, e.g. a polypeptide having an
activity of a protein as indicated in Table II, column 3, line 1 to
5 or lines 334 to 338, or its homologs activity, e.g. as indicated
in Table II, columns 5 or 7, line 1 to 5 or lines 334 to 338, by
adding positive expression or removing negative expression
elements, e.g. homologous recombination can be used to either
introduce positive regulatory elements like for plants the 35S
enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; [0104]
i) Modulating growth conditions of an organism in such a manner,
that the expression or activity of the gene encoding the protein of
the invention or the protein itself is enhanced for example
microorganisms or plants can be grown under a higher temperature
regime leading to an enhanced expression of heat shock proteins,
e.g. the heat shock protein of the invention, which can lead an
enhanced the fine chemical production; and/or [0105] j) selecting
of organisms with especially high activity of the proteins of the
invention from natural or from mutagenized resources and breeding
them into the target organisms, e.g. the elite crops.
[0106] [0054.0.0.0] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of methionine after
increasing the expression or activity of the encoded polypeptide or
having the activity of a polypeptide having an activity of a
protein as indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 or its homologs activity, e.g. as indicated
in Table II, columns 5 or 7, lines 1 to 5 and/or lines 334 to
338.
[0107] [0055.0.0.0] In general, the amount of mRNA or polypeptide
in a cell or a compartment of a organism correlates with the amount
of encoded protein and thus with the overall activity of the
encoded protein in said volume. Said correlation is not always
linear, the activity in the volume is dependent on the stability of
the molecules or the presence of activating or inhibiting
co-factors. Further, product and educt inhibitions of enzymes are
well known and described in Textbooks, e.g. Stryer,
Biochemistry.
[0108] [0056.0.0.0] In general, the amount of mRNA, polynucleotide
or nucleic acid molecule in a cell or a compartment of an organism
correlates with the amount of encoded protein and thus with the
overall activity of the encoded protein in said volume. Said
correlation is not always linear, the activity in the volume is
dependent on the stability of the molecules, the degradation of the
molecules or the presence of activating or inhibiting co-factors.
Further, product and educt inhibitions of enzymes are well known,
e.g. Zinser et al. "Enzyminhibitoren"/Enzyme inhibitors".
[0109] [0057.0.0.0] The activity of the abovementioned proteins
and/or polypeptide encoded by the nucleic acid molecule of the
present invention can be increased in various ways. For example,
the activity in an organism or in a part thereof, like a cell, is
increased via increasing the gene product number, e.g. by
increasing the expression rate, like introducing a stronger
promoter, or by increasing the stability of the mRNA expressed,
thus increasing the translation rate, and/or increasing the
stability of the gene product, thus reducing the proteins decayed.
Further, the activity or turnover of enzymes can be influenced in
such a way that a reduction or increase of the reaction rate or a
modification (reduction or increase) of the affinity to the
substrate results, is reached. A mutation in the catalytic centre
of an polypeptide of the invention or the polypeptide used in the
method of the invention, e.g. as enzyme, can modulate the turn over
rate of the enzyme, e.g. a knock out of an essential amino acid can
lead to a reduced or completely knock out activity of the enzyme,
or the deletion or mutation of regulator binding sites can reduce a
negative regulation like a feedback inhibition (or a substrate
inhibition, if the substrate level is also increased). The specific
activity of an enzyme of the present invention can be increased
such that the turn over rate is increased or the binding of a
co-factor is improved. Improving the stability of the encoding mRNA
or the protein can also increase the activity of a gene product.
The stimulation of the activity is also under the scope of the term
"increased activity".
[0110] [0058.0.0.0] Moreover, the regulation of the abovementioned
nucleic acid sequences may be modified so that gene expression is
increased. This can be achieved advantageously by means of
heterologous regulatory sequences or by modifying, for example
mutating, the natural regulatory sequences which are present. The
advantageous methods may also be combined with each other.
[0111] [0059.0.0.0] In general, an activity of a gene product in an
organism or part thereof, in particular in a plant cell, a plant,
or a plant tissue or a part thereof or in a microorganism can be
increased by increasing the amount of the specific encoding mRNA or
the corresponding protein in said organism or part thereof. "Amount
of protein or mRNA" is understood as meaning the molecule number of
polypeptides or mRNA molecules in an organism, a tissue, a cell, or
a cell compartment. "Increase" in the amount of a protein means the
quantitative increase of the molecule number of said protein in an
organism, a tissue, a cell or a cell compartment or part
thereof--for example by one of the methods described herein
below--in comparison to a wild type, control or reference.
[0112] [0060.0.0.0] The increase in molecule number amounts
preferably to at least 1%, preferably to more than 10%, more
preferably to 30% or more, especially preferably to 50%, 70% or
more, very especially preferably to 100%, most preferably to 500%
or more. However, a de novo expression is also regarded as subject
of the present invention.
[0113] [0061.0.0.0] A modification, i.e. an increase or decrease,
can be caused by endogenous or exogenous factors. For example, an
increase in activity in an organism or a part thereof can be caused
by adding a gene product or a precursor or an activator or an
agonist to the media or nutrition or can be caused by introducing
said subjects into a organism, transient or stable.
[0114] [0062.0.0.0] In one embodiment the increase in the amount of
the fine chemical in the organism or a part thereof, e.g. in a
cell, a tissue, a organ, an organelle etc., is achieved by
increasing the endogenous level of the polypeptide of the invention
or the polypeptide used in the method of the invention.
Accordingly, in an embodiment of the present invention, the present
invention relates to a process wherein the gene copy number of a
gene encoding the polynucleotide or nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention as herein described is increased. Further, the endogenous
level of the polypeptide of the invention or the polypeptide used
in the method of the invention as described can for example be
increased by modifying the transcriptional or translational
regulation of the polypeptide.
[0115] [0063.0.0.0] In one embodiment the amount of the fine
chemical in the organism or part thereof can be increase by
targeted or random mutagenesis of the endogenous genes of the
invention. For example homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. In addition gene conversion like methods
described by Kochevenko and Willmitzer (Plant Physiol. 2003 May;
132(1): 174-84) and citations therein can be used to disrupt
repressor elements or to enhance to activity of positive regulatory
elements.
[0116] Furthermore positive elements can be randomly introduced in
(plant) genomes by T-DNA or transposon mutagenesis and lines can be
screened for, in which the positive elements has be integrated near
to a gene of the invention, the expression of which is thereby
enhanced. The activation of plant genes by random integrations of
enhancer elements has been described by Hayashi et al., 1992
(Science 258:1350-1353) or Weigel et al., 2000 (Plant Physiol. 122,
1003-1013) and others citied therein. Reverse genetic strategies to
identify insertions (which eventually carrying the activation
elements) near in genes of interest have been described for various
cases e.g. Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290);
Sessions et al., 2002 (Plant Cell 2002, 14, 2985-2994); Young et
al., 2001, (Plant Physiol. 2001, 125, 513-518); Koprek et al., 2000
(Plant J. 2000, 24, 253-263); Jeon et al., 2000 (Plant J. 2000, 22,
561-570); Tissier et al., 1999 (Plant Cell 1999, 11, 1841-1852);
Speulmann et al., 1999 (Plant Cell 1999, 11, 1853-1866). Briefly
material from all plants of a large T-DNA or transposon mutagenized
plant population is harvested and genomic DNA prepared. Then the
genomic DNA is pooled following specific architectures as described
for example in Krysan et al., 1999 (Plant Cell 1999, 11,
2283-2290). Pools of genomics DNAs are then screened by specific
multiplex PCR reactions detecting the combination of the
insertional mutagen (e.g. T-DNA or Transposon) and the gene of
interest. Therefore PCR reactions are run on the DNA pools with
specific combinations of T-DNA or transposon border primers and
gene specific primers. General rules for primer design can again be
taken from Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290)
Rescreening of lower levels DNA pools lead to the identification of
individual plants in which the gene of interest is disrupted by the
insertional mutagen. The enhancement of positive regulatory
elements or the disruption or weaking of negative regulatory
elements can also be achieved through common mutagenesis
techniques: The production of chemically or radiation mutated
populations is a common technique and known to the skilled worker.
Methods for plants are described by Koorneef et al. 1982 and the
citations therein and by Lightner and Caspar in "Methods in
Molecular Biology" Vol 82. These techniques usually induce
pointmutations that can be identified in any known gene using
methods such as tilling (Colbert et al. 2001).
[0117] Accordingly, the expression level can be increased if the
endogenous genes encoding a polypeptide conferring an increased
expression of the polypeptide of the present invention, in
particular genes comprising the nucleic acid molecule of the
present invention, are modified via homologous recombination,
tilling approaches or gene conversion
[0118] [0064.0.0.0] Regulatory sequences can be operatively linked
to the coding region of an endogenous protein and control its
transcription and translation or the stability or decay of the
encoding mRNA or the expressed protein. In order to modify and
control the expression, promoter, UTRs, splicing sites, processing
signals, polyadenylation sites, terminators, enhancers, repressors,
post transcriptional or posttranslational modification sites can be
changed, added or amended for example, the activation of plant
genes by random integrations of enhancer elements has been
described by Hayashi et al., 1992 (Science 258:1350-1353) or Weigel
et al., 2000 (Plant Physiol. 122, 1003-1013) and others citied
therein. For example, the expression level of the endogenous
protein can be modulated by replacing the endogenous promoter with
a stronger transgenic promoter or by replacing the endogenous 3'UTR
with a 3'UTR, which provides more stability without amending the
coding region. Further, the transcriptional regulation can be
modulated by introduction of an artificial transcription factor as
described in the examples. Alternative promoters, terminators and
UTR are described below.
[0119] [0065.0.0.0] The activation of an endogenous polypeptide
having above-mentioned activity, of the polypeptide of the
invention or the polypeptide used in the method of the invention,
e.g. conferring the increase of the respective fine chemical after
increase of expression or activity can also be increased by
introducing a synthetic transcription factor, which binds close to
the coding region of an endogenous polypeptide of the invention or
the polypeptide used in the method of the invention- or used in the
process of the invention or its endogenous homolog-encoding gene
and the synthetic transcription factor activates its transcription.
A chimeric zinc finger protein can be construed, which comprises a
specific DNA-binding domain and an activation domain as e.g. the
VP16 domain of Herpes Simplex virus. The specific binding domain
can bind to the regulatory region of the endogenous protein coding
region. The expression of the chimeric transcription factor in a
organism, in particular in a plant, leads to a specific expression
of an endogenous polypeptid of the invention or used in the process
of the invention, in particular a plant homolog thereof, see e.g.
in WO01/52620, Oriz, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99,
13290 or Guan, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99,
13296.
[0120] [0066.0.0.0] In one further embodiment of the process
according to the invention, organisms are used in which one of the
abovementioned genes, or one of the abovementioned nucleic acids,
is mutated in a way that the activity of the encoded gene products
is less influenced by cellular factors, or not at all, in
comparison with the unmutated proteins. For example, well known
regulation mechanism of enzymic activity are substrate inhibition
or feed back regulation mechanisms. Ways and techniques for the
introduction of substitutions, deletions and additions of one or
more bases, nucleotides or amino acids of a corresponding sequence
are described herein below in the corresponding paragraphs and the
references listed there, e.g. in Sambrook et al., Molecular
Cloning, Cold Spring Habour, N.Y., 1989. The person skilled in the
art will be able to identify regulation domains and binding sites
of regulators by comparing the sequence of the nucleic acid
molecule of the present invention or the expression product thereof
with the state of the art by computer software means which comprise
algorithms for the identifying of binding sites and regulation
domains or by introducing into a nucleic acid molecule or in a
protein systematically mutations and assaying for those mutations
which will lead to an increased specific activity or an increased
activity per volume, in particular per cell.
[0121] [0067.0.0.0] It is therefore advantageously to express in an
organism a nucleic acid molecule of the invention or the nucleic
acid molecule used in the method of the invention or a polypeptide
of the invention or the polypeptide used in the method of the
invention derived from a evolutionary distantly related organism,
as e.g. using a prokaryotic gene in an eukaryotic host, as in these
cases the regulation mechanism of the host cell may not weaken the
activity (cellular or specific) of the gene or its expression
product
[0122] [0068.0.0.0] The mutation is introduced in such a way that
the production of the amino acids is not adversely affected.
[0123] [0069.0.0.0] Less influence on the regulation of a gene or
its gene product is understood as meaning a reduced regulation of
the enzymatic activity leading to an increased specific or cellular
activity of the gene or its product. An increase of the enzymatic
activity is understood as meaning an enzymatic activity, which is
increased by at least 10%, advantageously at least 20, 30 or 40%,
especially advantageously by at least 50, 60 or 70% in comparison
with the starting organism. This leads to an increased productivity
of the desired respective fine chemical(s).
[0124] [0070.0.0.0] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention or the polypeptide of the invention or the
polypeptide used in the method of the invention as described below,
for example the nucleic acid construct mentioned below, into an
organism alone or in combination with other genes, it is possible
not only to increase the biosynthetic flux towards the end product,
but also to increase, modify or create de novo an advantageous,
preferably novel metabolites composition in the organism, e.g. an
advantageous amino acid composition comprising a higher content of
(from a viewpoint of nutritional physiology limited) respective
fine chemicals, in particular amino acids, likewise the fine
chemical.
[0125] [0071.0.0.0] Preferably the composition further comprises
higher amounts of metabolites positively affecting or lower amounts
of metabolites negatively affecting the nutrition or health of
animals or humans provided with said compositions or organisms of
the invention or parts thereof. Likewise, the number or activity of
further genes which are required for the import or export of
nutrients or metabolites, including amino acids or its precursors,
required for the cell's biosynthesis of amino acids may be
increased so that the concentration of necessary or relevant
precursors, cofactors or intermediates within the cell(s) or within
the corresponding storage compartments is increased. Owing to the
increased or novel generated activity of the polypeptide of the
invention or the polypeptide used in the method of the invention or
owing to the increased number of nucleic acid sequences of the
invention and/or to the modulation of further genes which are
involved in the biosynthesis of the amino acids, e.g. by increasing
the activity of enzymes synthesizing precursors or by destroying
the activity of one or more genes which are involved in the
breakdown of the amino acids, it is possible to increase the yield,
production and/or production efficiency of amino acids in the host
organism, such as the plants or the microorganisms.
[0126] [0072.0.0.0] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous sulfur-containing compounds, which contain at
least one sulfur atom bound covalently. Examples of such compounds
are, in addition to methionine, homocysteine, S-adenosylmethionine,
cysteine, advantageously methionine and S-adenosylmethionine.
[0127] [0073.0.0.0] Accordingly, in one embodiment, the process
according to the invention relates to a process which comprises:
[0128] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [0129] b) increasing an activity of a
polypeptide of the invention or the polypeptide used in the method
of the invention or a homolog thereof, e.g. as indicated in Table
II, columns 5 or 7, line 1 to 5, or of a polypeptide being encoded
by the nucleic acid molecule of the present invention and described
below, i.e. conferring an increase of the respective fine chemical
in the organism, preferably in a microorganism, a non-human animal,
a plant or animal cell, a plant or animal tissue or a plant, [0130]
c) growing the organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a
plant, under conditions which permit the production of the
respective fine chemical in the organism, preferably the
microorganism, the plant cell, the plant tissue or the plant; and
[0131] d) if desired, recovering, optionally isolating, the free
and/or bound the respective fine chemical and, optionally further
free and/or bound amino acids synthesized by the organism, the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue or the plant.
[0132] [0074.0.0.0] The organism, in particular the microorganism,
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant is advantageously grown in such a way that it
is not only possible to recover, if desired isolate the free or
bound the respective fine chemical or the free and bound the fine
chemical but as option it is also possible to produce, recover and,
if desired isolate, other free or/and bound amino acids, in
particular lysine. Galili et al., Transgenic Res., 200, 9, 2,
137-144 describes that the heterologous expression of a bacterial
gene for the amino acid biosynthesis confers the increase of free
as well as of protein-bound amino acids.
[0133] [0075.0.0.0] After the above-described increasing (which as
defined above also encompasses the generating of an activity in an
organism, i.e. a de novo activity), for example after the
introduction and the expression of the nucleic acid molecules of
the invention or described in the methods or processes according to
the invention, the organism according to the invention,
advantageously, a microorganism, a non-human animal, a plant, plant
or animal tissue or plant or animal cell, is grown and subsequently
harvested.
[0134] [0076.0.0.0] Suitable organisms or host organisms
(transgenic organism) for the nucleic acid molecule used according
to the invention and for the inventive process, the nucleic acid
construct or the vector (both as described below) are, in
principle, all organisms which are capable of synthesizing the
respective fine chemical, and which are suitable for the
activation, introduction or stimulation genes. Examples which may
be mentioned are plants, microorganisms such as fungi, bacteria,
yeasts, alga or diatom, transgenic or obtained by site directed
mutagenesis or random mutagenesis combined with specific selection
procedures. Preferred organisms are those which are naturally
capable of synthesizing the respective fine chemical in substantial
amounts, like fungi, yeasts, bactria or plants. In principle,
transgenic animals, for example Caenorhabditis elegans, are also
suitable as host organisms.
[0135] [0077.0.0.0] In the event that the transgenic organism is a
microorganism, such as a eukaryotic organism, for example a fungus,
an alga, diatom or a yeast in particular a fungus, alga, diatom or
yeast selected from the families Chaetomiaceae, Choanephoraceae,
Cryptococcaceae, Gun ninghamellaceae, Demetiaceae, Moniliaceae,
Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae,
Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae,
Sporobolomycetaceae Tuberculariaceae, Adelotheciaceae, Dinophyceae,
Ditrichaceae or Prasinophyceae, or a prokaryotic organism, for
example a bacterium or blue alga, in particular a bacterium from
the families Actinomycetaceae, Bacillaceae, Brevibacteriaceae,
Corynebacteriaceae, Enterobacteriacae, Gordoniaceae, Nocardiaceae,
Micrococcaceae, Mycobacteriaceae, Pseudomonaceae, Rhizobiaceae or
Streptomycetaceae, this microorganism is grown on a solid or in a
liquid medium which is known to the skilled worker and suits the
organism. After the growing phase, the organisms can be
harvested.
[0136] [0078.0.0.0] The microorganisms or the recovered, and if
desired isolated, respective fine chemical can then be processed
further directly into foodstuffs or animal feeds or for other
applications, for example according to the disclosures made in
EP-B-0 533 039 or EP-A-0 615 693, which are expressly incorporated
herein by reference. The fermentation broth or fermentation
products can be purified in the customary manner by extraction and
precipitation or via ion exchangers and other methods known to the
person skilled in the art and described herein below. Products of
these different work-up procedures are amino acids or amino acid
compositions which still comprise fermentation broth and cell
components in different amounts, advantageously in the range of
from 0 to 99% by weight, preferably below 80% by weight, especially
preferably between below 50% by weight.
[0137] [0079.0.0.0] Preferred microorganisms are selected from the
group consisting of Chaetomiaceae such as the genera Chaetomium
e.g. the species Chaetomidium fimeti; Choanephoraceae such as the
genera Blakeslea, Choanephora e.g. the species Blakeslea trispora,
Choanephora cucurbitarum or Choanephora infundibulifera var.
cucurbitarum; Cryptococcaceae such as the genera Candida,
Crytococcus, Rhodotorula, Torulopsis e.g. the species Candida
albicans, Candida albomarginata, Candida antarctica, Candida
bacarum, Candida bogoriensis, Candida boidinii, Candida bovina,
Candida brumptii, Candida cacaoi, Candida cariosilignicola, Candida
catenulata, Candida chalmersii, Candida ciferrii, Candida
cylindracea, Candida edax, Candida ernobii, Candida famata, Candida
freyschussii, Candida friedrichii, Candida glabrata, Candida
guilliermondii, Candida haemulonii, Candida humicola, Candida
inconspicua, Candida ingens, Candida intermedia, Candida kefyr,
Candida krusei, Candida lactiscondensi, Candida lambica, Candida
lipolyfica, Candida lusitaniae, Candida macedoniensis, Candida
magnoliae, Candida membranaefaciens, Candida mesenterica, Candida
multigemmis, Candida mycoderma, Candida nemodendra, Candida
nitratophila, Candida norvegensis, Candida norvegica, Candida
parapsilosis, Candida pelliculosa, Candida pe/tata, Candida pini,
Candida pseudotropicalis, Candida pulcherrima, Candida punicea,
Candida pustula, Candida ravautii, Candida reukaufii, Candida
rugosa, Candida sake, Candida silvicola, Candida solani, Candida
sp., Candida spandovensis, Candida succiphila, Candida tropicalis,
Candida utilis, Candida valida, Candida versatilis, Candida vini,
Candida zeylanoides, Cryptococcus albidus, Cryptococcus curvatus,
Cryptococcus flavus, Cryptococcus humicola, Cryptococcus
hungaricus, Cryptococcus kuetzingii, Cryptococcus laurentii,
Cryptococcus macerans, Cryptococcus neoformans, Cryptococcus
terreus, Cryptococcus uniguttulatus, Rhodotorula acheniorum,
Rhodotorula bacarum, Rhodotorula bogoriensis, Rhodotorula flava,
Rhodotorula glutinis, Rhodotorula macerans, Rhodotorula minuta,
Rhodotorula mucilaginosa, Rhodotorula pilimanae, Rhodotorula
pustula, Rhodotorula rubra, Rhodotorula tokyoensis, Torulopsis
colliculosa, Torulopsis dattila or Torulopsis neoformans;
Cunninghamellaceae such as the genera Cunninghamella e.g. the
species Cunninghamella blakesleeana, Cunninghamella echinulata,
Cunninghamella echinulata var. elegans, Cunninghamella elegans or
Cunninghamella homothallica; Demetiaceae such as the genera
Alternaria, Bipolaris, Cercospora, Chalara, Cladosporium,
Curvularia, Exophilia, Helicosporium, Helminthosporium, Orbimyces,
Philalophora, Pithomyces, Spilocaea, Thielaviopsis, Wangiella e.g.
the species Curvularia affinis, Curvularia clavata, Curvularia
fallax, Curvularia inaequalis, Curvularia indica, Curvularia
lunata, Curvularia pallescens, Curvularia verruculosa or
Helminthosporium sp.; Moniliaceae such as the genera Arthrobotrys,
Aspergillus, Epidermophyton, Geotrichum, Gliocladium, Histoplasma,
Microsporum, Monilia, Oedocephalum, Oidium, Penicillium,
Trichoderma, Trichophyton, Thrichoteclum, Verticillium e.g. the
species Aspergillus aculeatus, Aspergillus albus, Aspergillus
alliaceus, Aspergillus asperescens, Aspergillus awamori,
Aspergillus candidus, Aspergillus carbonarius, Aspergillus carneus,
Aspergillus chevalieri, Aspergillus chevalieri var. intermedius,
Aspergillus clavatus, Aspergillus ficuum, Aspergillus flavipes,
Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus,
Aspergillus giganteus, Aspergillus humicola, Aspergillus
intermedius, Aspergillus japonicus, Aspergillus nidulans,
Aspergillus niger, Aspergillus niveus, Aspergillus ochraceus,
Aspergillus oryzae, Aspergillus ostianus, Aspergillus parasiticus,
Aspergillus parasiticus var. globosus, Aspergillus penicillioides,
Aspergillus phoenicis, Aspergillus rugulosus, Aspergillus
sclerotiorum, Aspergillus sojae var. gymnosardae, Aspergillus
sydowii, Aspergillus tamarii, Aspergillus terreus, Aspergillus
terricola, Aspergillus toxicarius, Aspergillus unguis, Aspergillus
ustus, Aspergillus versicolor, Aspergillus vitricolae, Aspergillus
wentii, Penicillium adametzi, .cndot.Penicillium albicans,
Penicillium arabicum, Penicillium arenicola, Penicillium
argillaceum, Penicillium arvense, Penicillium asperosporum,
.cndot.Penicillium aurantiogriseum, .cndot.Penicillium avellaneum,
.cndot.Penicillium baamense, .cndot.Penicillium bacillisporum,
.cndot.Penicillium brasilianum, .cndot.Penicillium brevicompactum,
.cndot.Penicillium camemberti, .cndot.Penicillium canadense,
.cndot.Penicillium canescens, .cndot.Penicillium caperatum,
.cndot.Penicillium capsulatum, .cndot.Penicillium caseicolum,
.cndot.Penicillium chrysogenum, .cndot.Penicillium citreonigrum,
.cndot.Penicillium citrinum, .cndot.Penicillium claviforme,
.cndot.Penicillium commune, .cndot.Penicillium corylophilum,
.cndot.Penicillium corymbiferum, .cndot.Penicillium crustosum,
.cndot.Penicillium cyclopium, .cndot.Penicillium daleae,
.cndot.Penicillium decumbens, .cndot.Penicillium dierckxii,
.cndot.Penicillium digitatum, .cndot.Penicillium digitatum var.
latum, .cndot.Penicillium divaricatum, .cndot.Penicillium diversum,
.cndot.Penicillium duclauxii, .cndot.Penicillium echinosporum,
.cndot.Penicillium expansum, .cndot.Penicillium fellutanum,
.cndot.Penicillium frequentans, .cndot.Penicillium funiculosum,
.cndot.Penicillium glabrum, .cndot.Penicillium gladioli,
.cndot.Penicillium griseofulvum, .cndot.Penicillium hirsutum,
.cndot.Penicillium hispanicum, .cndot.Penicillium islandicum,
.cndot.Penicillium italicum, .cndot.Penicillium italicum var.
avellaneum, .cndot.Penicillium janczewskii, .cndot.Penicillium
janthinellum, .cndot.Penicillium japonicum, .cndot.Penicillium
lavendulum, .cndot.Penicillium lilacinum, .cndot.Penicillium
lividum, .cndot.Penicillium martensii, .cndot.Penicillium
megasporum, .cndot.Penicillium miczynskii, .cndot.Penicillium
nalgiovense, .cndot.Penicillium nigricans,
.cndot..cndot.Penicillium notatum, .cndot.Penicillium ochrochloron,
.cndot.Penicillium odoratum, .cndot.Penicillium oxalicum,
.cndot.Penicillium paraherquei, .cndot.Penicillium patulum,
.cndot.Penicillium pinophilum, .cndot.Penicillium piscarium,
.cndot.Penicillium pseudostromaticum, .cndot.Penicillium puberulum,
.cndot.Penicillium purpurogenum, .cndot.Penicillium raciborskii,
.cndot.Penicillium roqueforti, .cndot.Penicillium rotundum,
.cndot.Penicillium rubrum, .cndot.Penicillium sacculum,
.cndot.Penicillium simplicissimum, .cndot.Penicillium sp.,
Penicillium spinulosum, Penicillium steckii, Penicillium
stoloniferum, Penicillium striatisporum, Penicillium striatum,
Penicillium tardum, Penicillium thomii, Penicillium turbatum,
Penicillium variabile, Penicillium vermiculatum, Penicillium
vermoesenii, Penicillium verrucosum, Penicillium verrucosum var.
corymbiferum, Penicillium verrucosum var. cyclopium, Penicillium
verruculosum, Penicillium vinaceum, Penicillium violaceum,
Penicillium viridicatum, Penicillium vulpinum, Trichoderma hamatum,
Trichoderma harzianum, Trichoderma koningii, Trichoderma
longibrachiatum, Trichoderma polysporum, Trichoderma reesei,
Trichoderma virens or Trichoderma viride; Mortierellaceae such as
the genera Mortierella e.g. the species Mortierella isabellina,
Mortierella polycephala, Mortierella ramanniana, Mortierella
vinacea or Mortierella zonata; Mucoraceae such as the genera
Actinomucor, Mucor, Phycomyces, Rhizopus, Zygorhynchus e.g. the
species Mucor amphibiorum, Mucor circinelloides f. circinelloides,
Mucor circinelloides var. griseocyanus, Mucor flavus, Mucor fuscus,
Mucor griseocyanus, Mucor heterosporus, Mucor hiemalis, Mucor
hiemalis f. hiemalis, Mucor inaequisporus, Mucor indicus, Mucor
javanicus, Mucor mucedo, Mucor mucilagineus, Mucor piriformis,
Mucor plasmaticus, Mucor plumbeus, Mucor racemosus, Mucor racemosus
f. racemosus, Mucor racemosus f. sphaerosporus, Mucor rouxianus,
Mucor Mucor sinensis, Mucor sp., Mucor spinosus, Mucor
tuberculisporus, Mucor variisporus, Mucor variosporus, Mucor
wosnessenskii, Phycomyces blakesleeanus, Rhizopus achlamydosporus,
Rhizopus arrhizus, Rhizopus chinensis, Rhizopus delemar, Rhizopus
formosaensis, Rhizopus japonicus, Rhizopus javanicus, Rhizopus
microsporus, Rhizopus microsporus var. chinensis, Rhizopus
microsporus var. oligosporus, Rhizopus microsporus var.
rhizopodiformis, Rhizopus nigricans, Rhizopus niveus, Rhizopus
oligosporus, Rhizopus oryzae, Rhizopus pygmaeus, Rhizopus
rhizopodiformis, Rhizopus semarangensis, Rhizopus sontii, Rhizopus
stolonifer, Rhizopus thermosus, Rhizopus tonkinensis, Rhizopus
tritici or Rhizopus usamii; Pythiaceae such as the genera Phytium,
Phytophthora e.g. the species Pythium debaryanum, Pythium
intermedium, Pythium irregulars, Pythium megalacanthum, Pythium
paroecandrum, Pythium sylvaticum, Pythium ultimum, Phytophthora
cactorum, Phytophthora cinnamomi, Phytophthora citricola,
Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora
drechsieri, Phytophthora erythroseptica, Phytophthora lateralis,
Phytophthora megasperma, Phytophthora nicotianae, Phytophthora
nicotianae var. parasitica, Phytophthora palmivora, Phytophthora
parasitica or Phytophthora syringae; Sacharomycetaceae such as the
genera Hansenula, Pichia, Saccharomyces, Saccharomycodes, Yarrowia
e.g. the species Hansenula anomala, Hansenula californica,
Hansenula canadensis, Hansenula capsulata, Hansenula ciferrii,
Hansenula glucozyma, Hansenula henricii, Hansenula hoistii,
Hansenula minuta, Hansenula nonfermentans, Hansenula philodendri,
Hansenula polymorpha, Hansenula saturnus, Hansenula subpelliculosa,
Hansenula wickerhamii, Hansenula wingei, Pichia alcoholophila,
Pichia angusta, Pichia anomala, Pichia bispora, Pichia burtonii,
Pichia canadensis, Pichia capsulata, Pichia carsonii, Pichia
cellobiosa, Pichia ciferrii, Pichia farinosa, Pichia fermentans,
Pichia finiandica, Pichia glucozyma, Pichia guilliermondii, Pichia
haplophila, Pichia henricii, Pichia holstii, Pichia jadinii, Pichia
lindnerii, Pichia membranaefaciens, Pichia methanolica, Pichia
minuta var. minuta, Pichia minuta var. nonfermentans, Pichia
norvegensis, Pichia ohmeri, Pichia pastoris, Pichia philodendri,
Pichia pini, Pichia polymorpha, Pichia quercuum, Pichia
rhodanensis, Pichia sargentensis, Pichia stipitis, Pichia
strasburgensis, Pichia subpelliculosa, Pichia toietana, Pichia
trehalophila, Pichia vini, Pichia xylosa, Saccharomyces aceti,
Saccharomyces bailii, Saccharomyces bayanus, Saccharomyces
bisporus, Saccharomyces capensis, Saccharomyces carlsbergensis,
Saccharomyces cerevisiae, Saccharomyces cerevisiae var.
ellipsoideus, Saccharomyces chevalieri, Saccharomyces deibrueckii,
Saccharomyces diastaticus, Saccharomyces drosophilarum,
Saccharomyces eiegans, Saccharomyces ellipsoideus, Saccharomyces
fermentati, Saccharomyces florentinus, Saccharomyces fragilis,
Saccharomyces heterogenicus, Saccharomyces hienipiensis,
Saccharomyces inusitatus, Saccharomyces italicus, Saccharomyces
kluyveri, Saccharomyces krusei, Saccharomyces iactis, Saccharomyces
marxianus, Saccharomyces microellipsoides, Saccharomyces montanus,
Saccharomyces norbensis, Saccharomyces oieaceus, Saccharomyces
paradoxus, Saccharomyces pastorianus, Saccharomyces pretoriensis,
Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces uvarum,
Saccharomycodes ludwigii or Yarrowia lipolytica; Saprolegniaceae
such as the genera Saprolegnia e.g. the species Saprolegnia ferax;
Schizosacharomycetaceae such as the genera Schizosaccharomyces e.g.
the species Schizosaccharomyces japonicus var. japonicus,
Schizosaccharomyces japonicus var. versatilis, Schizosaccharomyces
malidevorans, Schizosaccharomyces octosporus, Schizosaccharomyces
pombe var. malidevorans or Schizosaccharomyces pombe var. pombe;
Sodariaceae such as the genera Neurospora, Sordaria e.g. the
species Neurospora africana, Neurospora crassa, Neurospora
intermedia, Neurospora sitophila, Neurospora tetrasperma, Sordaria
fimicola or Sordaria macrospora; Tuberculariaceae such as the
genera Epicoccum, Fusarium, Myrothecium, Sphacelia, Starkeyomyces,
Tubercularia e.g. the species Fusarium acuminatum, Fusarium
anthophilum, Fusarium aquaeductuum, Fusarium aquaeductuum var.
medium, Fusarium avenaceum, Fusarium buharicum, Fusarium
camptoceras, Fusarium cerealis, Fusarium chlamydosporum, Fusarium
ciliatum, Fusarium coccophilum, Fusarium coeruleum, Fusarium
concolor, Fusarium crookwellense, Fusarium culmorum, Fusarium
dimerum, Fusarium diversisporum, Fusarium equiseti, Fusarium
equiseti var. bullatum, Fusarium eumartii, Fusarium flocciferum,
Fusarium fujikuroi, Fusarium graminearum, Fusarium graminum,
Fusarium heterosporum, Fusarium incamatum, Fusarium inflexum,
Fusarium javanicum, Fusarium lateritium, Fusarium lateritium var.
majus, Fusarium longipes, Fusarium melanochlorum, Fusarium
merismoides, Fusarium merismoides var. chlamydosporale, Fusarium
moniliforme, Fusarium moniliforme var. anthophilum, Fusarium
moniliforme var. subglutinans, Fusarium nivale, Fusarium nivale
var. majus, Fusarium oxysporum, Fusarium oxysporum f. sp. aechmeae,
Fusarium oxysporum f. sp. cepae, Fusarium oxysporum f. sp.
conglutinans, Fusarium oxysporum f. sp. cucumerinum, Fusarium
oxysporum f. sp. cyclaminis, Fusarium oxysporum f. sp. dianthi,
Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum f. sp.
melonis, Fusarium oxysporum f. sp. passiflorae, Fusarium oxysporum
f. sp. pisi, Fusarium oxysporum f. sp. tracheiphilum, Fusarium
oxysporum f. sp. tuberosi, Fusarium oxysporum f. sp. tulipae,
Fusarium oxysporum f. sp. vasinfectum, Fusarium pallidoroseum,
Fusarium poae, Fusarium proliferatum, Fusarium proliferatum var.
minus, Fusarium redolens, Fusarium redolens f. sp. dianthi,
Fusarium reticulatum, Fusarium roseum, Fusarium sacchari var.
elongatum, Fusarium sambucinum, Fusarium sambucinum var. coeruleum,
Fusarium semitectum, Fusarium semitectum var. majus, Fusarium
solani, Fusarium solani f. sp. pisi, Fusarium sporotrichioides,
Fusarium sporotrichioides var. minus, Fusarium sublunatum, Fusarium
succisae, Fusarium sulphureum, Fusarium tabacinum, Fusarium
tricinctum, Fusarium udum, Fusarium ventricosum, Fusarium
verticillioides, Fusarium xylarioides or Fusarium zonatum;
Sporobolomycetaceae such as the genera Bullera, Sporobolomyces,
Itersonilia e.g. the species Sporobolomyces holsaticus,
Sporobolomyces odorus, Sporobolomyces puniceus, Sporobolomyces
salmonicolor, Sporobolomyces singularis or Sporobolomyces tsugae;
Adelotheciaceae such as the genera e.g. the species Physcomitrella
patens; Dinophyceae such as the genera Crypthecodinium,
Phaeodactylum e.g. the species Crypthecodinium cohnii or
Phaeodactylum tricornutum; Ditrichaceae such as the genera
Ceratodon, Pleuridium, Astomiopsis, Ditrichum, Philibertiella,
Ceratodon, Distichium, Skottsbergia e.g. the species Ceratodon
antarcticus, Ceratodon purpureus, Ceratodon purpureus ssp.
convolutes or Ceratodon purpureus ssp. stenocarpus; Prasinophyceae
such as the genera
Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella,
Ostreococcus e.g. the species Nephroselmis olivacea, Prasinococcus
capsulatus, Scherffelia dubia, Tetraselmis chui, Tetraselmis
suecica, Mantoniella squamata or Ostreococcus tauri;
Actinomycetaceae such as the genera Actinomyces, Actinobaculum,
Arcanobacterium, Mobiluncus e.g. the species Actinomyces bemardiae,
Actinomyces bovis, Actinomyces bowdenii, Actinomyces canis,
Actinomyces cardiffensis, Actinomyces catuli, Actinomyces
coleocanis, Actinomyces denticolens, Actinomyces europaeus,
Actinomyces funkei, Actinomyces georgiae, Actinomyces gerencseriae,
Actinomyces hordeovulneris, Actinomyces howellii, Actinomyces
humiferus, Actinomyces hyovaginalis, Actinomyces israelii,
Actinomyces marimammalium, Actinomyces meyeri, Actinomyces
naeslundii, Actinomyces nasicola, Actinomyces neuii subsp.
anitratus, Actinomyces neuii subsp. neuii, Actinomyces
odontolyticus, Actinomyces oricola, Actinomyces pyogenes,
Actinomyces radicidentis, Actinomyces radingae, Actinomyces
slackii, Actinomyces suimastitidis, Actinomyces suis, Actinomyces
turicensis, Actinomyces urogenitalis, Actinomyces vaccimaxillae,
Actinomyces viscosus, Actinobaculum schaalii, Actinobaculum suis,
Actinobaculum urinale, Arcanobacterium bemardiae, Arcanobacterium
haemolyticum, Arcanobacterium hippocoleae, Arcanobacterium phocae,
Arcanobacterium pluranimalium, Arcanobacterium pyogenes, Mobiluncus
curtisii subsp. curtisii Mobiluncus curtisii subsp. holmesii or
Mobiluncus mulieris; Bacillaceae such as the genera Amphibacillus,
Anoxybacillus, Bacillus, Exiguobacterium, Gracilibacillus,
Holobacillus, Saccharococcus, Salibacillus, Virgibacillus e.g. the
species Amphibacillus fermentum, Amphibacillus tropicus,
Amphibacillus xylanus, Anoxybacillus flavithermus, Anoxybacillus
gonensis, Anoxybacillus pushchinoensis, Bacillus acidocaldarius,
Bacillus acidoterrestris, Bacillus aeolius, Bacillus agaradhaerens,
Bacillus agri, Bacillus alcalophilus, Bacillus alginolyticus,
Bacillus alvei, Bacillus amyloliquefaciens, Bacillus amylolyticus,
Bacillus aneurinilyticus, Bacillus aquimaris, Bacillus
arseniciselenatis, Bacillus atrophaeus, Bacillus azotofixans,
Bacillus azotoformans, Bacillus badius, Bacillus barbaricus,
Bacillus benzoevorans, Bacillus borstelensis, Bacillus brevis,
Bacillus carboniphilus, Bacillus centrosporus, Bacillus cereus,
Bacillus chitinolyticus, Bacillus chondroitinus, Bacillus
choshinensis, Bacillus circulans, Bacillus clarkii, Bacillus
clausii, Bacillus coagulans, Bacillus cohnii, Bacillus
curdlanolyticus, Bacillus cycloheptanicus, Bacillus decolorationis,
Bacillus dipsosauri, Bacillus edaphicus, Bacillus ehimensis,
Bacillus endophyticus, Bacillus fastidiosus, Bacillus firmus,
Bacillus flexus, Bacillus formosus, Bacillus fumarioli, Bacillus
funiculus, Bacillus fusiformis, Bacillus sphaericus subsp.
fusiformis, Bacillus galactophilus, Bacillus globisporus, Bacillus
globisporus subsp. marinus, Bacillus glucanolyticus, Bacillus
gordonae, Bacillus halmapalus, Bacillus haloalkaliphilus, Bacillus
halodenitrificans, Bacillus halodurans, Bacillus halophilus,
Bacillus horikoshii, Bacillus horti, Bacillus infernos, Bacillus
insolitus, Bacillus jeotgali, Bacillus kaustophilus, Bacillus
kobensis, Bacillus krulwichiae, Bacillus laevolacticus, Bacillus
larvae, Bacillus laterosporus, Bacillus lautus, Bacillus
lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus
luciferensis, Bacillus macerans, Bacillus macquariensis, Bacillus
marinus, Bacillus marisflavi, Bacillus marismortui, Bacillus
megaterium, Bacillus methanolicus, Bacillus migulanus, Bacillus
mojavensis, Bacillus mucilaginosus, Bacillus mycoides, Bacillus
naganoensis, Bacillus nealsonii, Bacillus neidei, Bacillus niacini,
Bacillus okuhidensis, Bacillus oleronius, Bacillus pabuli, Bacillus
pallidus, Bacillus pantothenticus, Bacillus parabrevis, Bacillus
pasteurii, Bacillus peoriae, Bacillus polymyxa, Bacillus popilliae,
Bacillus pseudalcaliphilus, Bacillus pseudofirmus, Bacillus
pseudomycoides, Bacillus psychrodurans, Bacillus psychrophilus,
Bacillus psychrosaccharolyticus, Bacillus psychrotolerans, Bacillus
pulvifaciens, Bacillus pumilus, Bacillus pycnus, Bacillus reuszeri,
Bacillus salexigens, Bacillus schlegelii, Bacillus
selenitireducens, Bacillus silvestris, Bacillus simplex, Bacillus
siralis, Bacillus smithii, Bacillus sonorensis, Bacillus
sphaericus, Bacillus sporothermodurans, Bacillus
stearothermophilus, Bacillus subterraneus, Bacillus subtilis subsp.
spizizenii, Bacillus subtilis subsp. subtilis, Bacillus
thermantarcticus, Bacillus thermoaerophilus, Bacillus
thermoamylovorans, Bacillus thermoantarcticus, Bacillus
thermocatenulatus, Bacillus thermocloacae, Bacillus
thermodenitrificans, Bacillus thermoglucosidasius, Bacillus
thermoleovorans, Bacillus thermoruber, Bacillus thermosphaericus,
Bacillus thiaminolyticus, Bacillus thuringiensis, Bacillus tusciae,
Bacillus validus, Bacillus vallismortis, Bacillus vedderi, Bacillus
vulcani, Bacillus weihenstephanensis, Exiguobacterium acetylicum,
Exiguobacterium antarcticum, Exiguobacterium aurantiacum,
Exiguobacterium undae, Gracilibacillus dipsosauri, Gracilibacillus
halotolerans, Halobacillus halophilus, Halobacillus karajensis,
Halobacillus litoralis, Halobacillus salinus, Halobacillus
trueperi, Saccharococcus caldoxylosilyticus, Saccharococcus
thermophilus, Salibacillus marismortui, Salibacillus salexigens,
Virgibacillus carmonensis, Virgibacillus marismortui, Virgibacillus
necropolis, Virgibacillus pantothenticus, Virgibacillus picturae,
Virgibacillus proomii or Virgibacillus salexigens,
Brevibacteriaceae such as the genera Brevibacterium e.g. the
species Brevibacterium acetylicum, Brevibacterium albidum,
Brevibacterium ammoniagenes, Brevibacterium avium, Brevibacterium
casei, Brevibacterium citreum, Brevibacterium divaricatum,
Brevibacterium epidermidis, Brevibacterium fermentans,
Brevibacterium frigoritolerans, Brevibacterium halotolerans,
Brevibacterium imperiale, Brevibacterium incertum, Brevibacterium
iodinum, Brevibacterium linens, Brevibacterium liquefaciens,
Brevibacterium lutescens, Brevibacterium luteum, Brevibacterium
lyticum, Brevibacterium mcbrellneri, Brevibacterium otitidis,
Brevibacterium oxydans, Brevibacterium paucivorans, Brevibacterium
protophormiae, Brevibacterium pusillum, Brevibacterium saperdae,
Brevibacterium stationis, Brevibacterium testaceum or
Brevibacterium vitaeruminis; Corynebacteriaceae such as the genera
Corynebacterium e.g. the species Corynebacterium accolens,
Corynebacterium afermentans subsp. afermentans, Corynebacterium
afermentans subsp. lipophilum, Corynebacterium ammoniagenes,
Corynebacterium amycolatum, Corynebacterium appendicis,
Corynebacterium aquilae, Corynebacterium argentoratense,
Corynebacterium atypicum, Corynebacterium aurimucosum,
Corynebacterium auris, Corynebacterium auriscanis, Corynebacterium
betae, Corynebacterium beticola, Corynebacterium bovis,
Corynebacterium callunae, Corynebacterium camporealensis,
Corynebacterium capitovis, Corynebacterium casei, Corynebacterium
confusum, Corynebacterium coyleae, Corynebacterium cystitidis,
Corynebacterium durum, Corynebacterium efficiens, Corynebacterium
equi, Corynebacterium falsenii, Corynebacterium fascians,
Corynebacterium felinum, Corynebacterium flaccumfaciens,
Corynebacterium flavescens, Corynebacterium freneyi,
Corynebacterium glaucum, Corynebacterium glucuronolyticum,
Corynebacterium glutamicum, Corynebacterium Corynebacterium ilicis,
Corynebacterium imitans, Corynebacterium insidiosum,
Corynebacterium iranicum, Corynebacterium jeikeium, Corynebacterium
kroppenstedtii, Corynebacterium kutscheri, Corynebacterium Ilium,
Corynebacterium lipophiloflavum, Corynebacterium macginleyi,
Corynebacterium mastitidis, Corynebacterium matruchotii,
Corynebacterium michiganense, Corynebacterium michiganense subsp.
tessellarius, Corynebacterium minutissimum, Corynebacterium
mooreparkense, Corynebacterium mucifaciens, Corynebacterium
mycetoides, Corynebacterium nebraskense, Corynebacterium oortii,
Corynebacterium paurometabolum, Corynebacterium phocae,
Corynebacterium pilosum, Corynebacterium poinsettiae,
Corynebacterium propinquum, Corynebacterium pseudodiphtheriticum,
Corynebacterium pseudotuberculosis, Corynebacterium pyogenes,
Corynebacterium rathayi, Corynebacterium renale, Corynebacterium
riegelii, Corynebacterium seminale, Corynebacterium sepedonicum,
Corynebacterium simulans, Corynebacterium singulare,
Corynebacterium sphenisci, Corynebacterium spheniscorum,
Corynebacterium striatum, Corynebacterium suicordis,
Corynebacterium sundsvallense, Corynebacterium terpenotabidum,
Corynebacterium testudinoris, Corynebacterium thomssenii,
Corynebacterium tritici, Corynebacterium ulcerans, Corynebacterium
urealyticum, Corynebacterium variabile, Corynebacterium
vitaeruminis or Corynebacterium xerosis; Enterobacteriacae such as
the genera Alterococcus, Arsenophonus, Brenneria, Buchnera,
Budvicia, Buttiauxella, Calymmatobacterium, Cedecea, Citrobacter,
Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella,
Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella, Moellerella,
Morganella, Obesumbacterium, Pantoea, Pectobacterium, Photorhabdus,
Plesiomonas, Pragia, Proteus, Providencia, Rahnella,
Saccharobacter, Salmonella, Shigella, Serratia, Sodalis, Tatumella,
Trabulsiella, Wigglesworthia, Xenorhabdus, Yersinia and Yokenella
e.g. the species Arsenophonus nasoniae, Brenneria alni, Brenneria
nigrifluens, Brenneria quercina, Brenneria rubrifaciens, Brenneria
salicis, Budvicia aquatica, Buttiauxella agrestis, Buttiauxella
brennerae, Buttiauxella ferragutiae, Buttiauxella gaviniae,
Buttiauxella izardii, Buttiauxella noackiae, Buttiauxella
warmboldiae, Cedecea davisae, Cedecea lapagei, Cedecea neteri,
Citrobacter amalonaticus, Citrobacter diversus, Citrobacter
freundii, Citrobacter genomospecies, Citrobacter gillenii,
Citrobacter intermedium, Citrobacter koseri, Citrobacter murliniae,
Citrobacter sp., Edwardsiella hoshinae, Edwardsiella ictaluri,
Edwardsiella tarda, Erwinia alni, Erwinia amylovora, Erwinia
ananatis, Erwinia aphidicola, Erwinia billingiae, Erwinia
cacticida, Erwinia cancerogena, Erwinia camegieana, Erwinia
carotovora subsp. atroseptica, Erwinia carotovora subsp.
betavasculorum, Erwinia carotovora subsp. odorifera, Erwinia
carotovora subsp. wasabiae, Erwinia chrysanthemi, Erwinia
cypripedii, Erwinia dissolvens, Erwinia herbicola, Erwinia
mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia
nimipressuralis, Erwinia persicina, Erwinia psidii, Erwinia
pyrifoliae, Erwinia quercina, Erwinia rhapontici, Erwinia
rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwinia
tracheiphila, Erwinia uredovora, Escherichia adecarboxylata,
Escherichia anindolica, Escherichia aurescens, Escherichia blattae,
Escherichia coli, Escherichia coli var. communior, Escherichia
coli-mutabile, Escherichia fergusonii, Escherichia hermannii,
Escherichia sp., Escherichia vulneris, Ewingella americana, Hafnia
alvei, Klebsiella aerogenes, Klebsiella edwardsii subsp. atlantae,
Klebsiella ornithinolytica, Klebsiella oxytoca, Klebsiella
planticola, Klebsiella pneumoniae, Klebsiella pneumoniae subsp.
pneumoniae, Klebsiella sp., Klebsiella terrigena, Klebsiella
trevisanii, Kluyvera ascorbata, Kluyvera citrophila, Kluyvera
cochleae, Kluyvera cryocrescens, Kluyvera georgiana, Kluyvera
noncitrophila, Kluyvera sp., Leclercia adecarboxylata, Leminorella
grimontii, Leminorella richardii, Moellerella wisconsensis,
Morganella morganii, Morganella morganii subsp. morganii,
Morganella morganii subsp. Obesumbaterium proteus, Pantoea
agglomerans, Pantoea ananatis, Pantoea citrea, Pantoea dispersa,
Pantoea punctata, Pantoea stewartii subsp. stewartii, Pantoea
terrea, Pectobacterium atrosepticum, Pectobacterium carotovorum
subsp. atrosepticum, Pectobacterium carotovorum subsp. carotovorum,
Pectobacterium chrysanthemi, Pectobacterium cypripedii,
Photorhabdus asymbiotica, Photorhabdus luminescens, Photorhabdus
luminescens subsp. akhurstii, Photorhabdus luminescens subsp.
laumondii, Photorhabdus luminescens subsp. luminescens,
Photorhabdus sp., Photorhabdus temperata, Plesiomonas shigelloides,
Pragia fontium, Proteus hauseri, Proteus ichthyosmius, Proteus
inconstans, Proteus mirabilis, Proteus morganii, Proteus
myxofaciens, Proteus penneri, Proteus rettgeri, Proteus
shigelloides, Proteus vulgaris, Providencia alcalifaciens,
Providencia friedericiana, Providencia heimbachae, Providencia
rettgeri, Providencia rustigianii, Providencia stuartii, Rahnella
aquatilis, Salmonella abony, Salmonella arizonae, Salmonella
bongori, Salmonella choleraesuis subsp. arizonae, Salmonella
choleraesuis subsp. bongori, Salmonella choleraesuis subsp.
cholereasuis, Salmonella choleraesuis subsp. diarizonae, Salmonella
choleraesuis subsp. houtenae, Salmonella choleraesuis subsp.
indica, Salmonella choleraesuis subsp. salamae, Salmonella
daressalaam, Salmonella enterica subsp. houtenae, Salmonella
enterica subsp. salamae, Salmonella enteritidis, Salmonella
gallinarum, Salmonella heidelberg, Salmonella panama, Salmonella
senftenberg, Salmonella typhimurium, Serratia entomophila, Serratia
ficaria, Serratia fonticola, Serratia grimesii, Serratia
liquefaciens, Serratia marcescens, Serratia marcescens subsp.
marcescens, Serratia marinorubra, Serratia odorifera, Serratia
plymouthensis, Serratia plymuthica, Serratia proteamaculans,
Serratia proteamaculans subsp. quinovora, Serratia quinivorans,
Serratia rubidaea, Shigella boydii, Shigella flexneri, Shigella
paradysenteriae, Shigella sonnei, Tatumella ptyseos, Xenorhabdus
beddingii, Xenorhabdus bovienii, Xenorhabdus luminescens,
Xenorhabdus nematophila, Xenorhabdus nematophila subsp. beddingii,
Xenorhabdus nematophila subsp. bovienii, Xenorhabdus nematophila
subsp. poinarii or Xenorhabdus poinarii; Gordoniaceae such as the
genera Gordonia, Skermania e.g. the species Gordonia aichiensis,
Gordonia alkanivorans, Gordonia amarae, Gordonia amicalis, Gordonia
bronchialis, Gordonia desulfuricans, Gordonia hirsuta, Gordonia
hydrophobica, Gordonia namibiensis, Gordonia nitida, Gordonia
paraffinivorans, Gordonia polyisoprenivorans, Gordonia rhizosphera,
Gordonia rubripertincta, Gordonia sihwensis, Gordonia sinesedis,
Gordonia sputi, Gordonia terrae or Gordonia westfalica;
Micrococcaceae such as the genera Micrococcus, Arthrobacter,
Kocuria, Nesterenkonia, Renibacterium, Rothia, Stomatococcus e.g.
the species Micrococcus agilis, Micrococcus antarcticus,
Micrococcus halobius, Micrococcus kristinae, Micrococcus luteus,
Micrococcus lylae, Micrococcus nishinomiyaensis, Micrococcus
roseus, Micrococcus sedentarius, Micrococcus varians, Arthrobacter
agilis, Arthrobacter albus, Arthrobacter atrocyaneus, Arthrobacter
aurescens, Arthrobacter chlorophenolicus, Arthrobacter citreus,
Arthrobacter creatinolyticus, Arthrobacter crystallopoietes,
Arthrobacter cumminsii, Arthrobacter duodecadis, Arthrobacter
flavescens, Arthrobacter flavus, Arthrobacter gandavensis,
Arthrobacter globiformis, Arthrobacter histidinolovorans,
Arthrobacter ilicis, Arthrobacter koreensis, Arthrobacter luteolus,
Arthrobacter methylotrophus, Arthrobacter mysorens, Arthrobacter
nasiphocae, Arthrobacter nicotianae, Arthrobacter nicotinovorans,
Arthrobacter oxydans, Arthrobacter pascens, Arthrobacter
picolinophilus, Arthrobacter polychromogenes, Arthrobacter
protophormiae, Arthrobacter psychrolactophilus, Arthrobacter
radiotolerans, Arthrobacter ramosus, Arthrobacter rhombi,
Arthrobacter roseus, Arthrobacter siderocapsulatus, Arthrobacter
simplex, Arthrobacter sulfonivorans, Arthrobacter sulfureus,
Arthrobacter
terregens, Arthrobacter tumescens, Arthrobacter uratoxydans,
Arthrobacter ureafaciens, Arthrobacter variabilis, Arthrobacter
viscosus, Arthrobacter woluwensis, Kocuria erythromyxa, Kocuria
kristinae, Kocuria palustris, Kocuria polaris, Kocuria rhizophila,
Kocuria rosea, Kocuria varians, Nesterenkonia halobia,
Nesterenkonia lacusekhoensis, Renibacterium salmoninarum, Rothia
amarae, Rothia dentocariosa, Rothia mucilaginosa, Rothia nasimurium
or Stomatococcus mucilaginosus; Mycobacteriaceae such as the genera
Mycobacterium e.g. the species Mycobacterium africanum,
Mycobacterium agri, Mycobacterium aichiense, Mycobacterium alvei,
Mycobacterium asiaticum, Mycobacterium aurum, Mycobacterium
austroafricanum, Mycobacterium bohemicum, Mycobacterium botniense,
Mycobacterium brumae, Mycobacterium chelonae subsp. abscessus,
Mycobacterium chitae, Mycobacterium chlorophenolicum, Mycobacterium
chubuense, Mycobacterium confluentis, Mycobacterium cookii,
Mycobacterium diemhoferi, Mycobacterium doricum, Mycobacterium
duvalii, Mycobacterium fallax, Mycobacterium farcinogenes,
Mycobacterium flavescens, Mycobacterium frederiksbergense,
Mycobacterium gadium, Mycobacterium gilvum, Mycobacterium gordonae,
Mycobacterium hassiacum, Mycobacterium hibemiae, Mycobacterium
hodleri, Mycobacterium holsaticum, Mycobacterium komossense,
Mycobacterium lacus, Mycobacterium madagascariense, Mycobacterium
mageritense, Mycobacterium montefiorense, Mycobacterium
moriokaense, Mycobacterium murale, Mycobacterium neoaurum,
Mycobacterium nonchromogenicum, Mycobacterium obuense,
Mycobacterium palustre, Mycobacterium parafortuitum, Mycobacterium
peregrinum, Mycobacterium phlei, Mycobacterium pinnipedii,
Mycobacterium poriferae, Mycobacterium pulveris, Mycobacterium
rhodesiae, Mycobacterium shottsii, Mycobacterium sphagni,
Mycobacterium terrae, Mycobacterium thermoresistibile,
Mycobacterium tokaiense, Mycobacterium triviale, Mycobacterium
tusciae or Mycobacterium vanbaalenii; Nocardiaceae such as the
genera Nocardia, Rhodococcus e.g. the species Nocardia abscessus,
Nocardia africana, Nocardia amarae, Nocardia asteroides, Nocardia
autotrophica, Nocardia beijingensis, Nocardia brasiliensis,
Nocardia brevicatena, Nocardia caishijiensis, Nocardia calcarea,
Nocardia carnea, Nocardia cellulans, Nocardia cerradoensis,
Nocardia coeliaca, Nocardia corynebacterioides, Nocardia
crassostreae, Nocardia cummidelens, Nocardia cyriacigeorgica,
Nocardia farcinica, Nocardia flavorosea, Nocardia fluminea,
Nocardia globerula, Nocardia hydrocarbonoxydans, Nocardia ignorata,
Nocardia mediterranei, Nocardia nova, Nocardia orientalis, Nocardia
otitidis-caviarum, Nocardia otitidiscaviarum, Nocardia paucivorans,
Nocardia petroleophila, Nocardia pinensis, Nocardia
pseudobrasiliensis, Nocardia pseudovaccinii, Nocardia purls,
Nocardia restricta, Nocardia rugosa, Nocardia salmonicida, Nocardia
saturnea, Nocardia seriolae, Nocardia soli, Nocardia sulphurea,
Nocardia transvalensis, Nocardia uniformis, Nocardia vaccinii,
Nocardia veterana or Nocardia vinacea; Pseudomonaceae such as the
genera Azomonas, Azotobacter, Cellvibrio, Chryseomonas,
Flaviomonas, Lampropedia, Mesophilobacter, Morococcus, Oligella,
Pseudomonas, Rhizobacter, Rugamonas, Serpens, Thermoleophilum,
Xylophilus e.g. the species Azomonas agilis, Azomonas insignis,
Azomonas macrocytogenes, Azotobacter agilis, Azotobacter agilis
subsp. armeniae, Azotobacter armeniacus, Azotobacter beijerinckii,
Azotobacter chroococcum, Azotobacter indicum, Azotobacter
macrocytogenes, Azotobacter miscellum, Azotobacter nigricans subsp.
nigricans, Azotobacter paspali, Azotobacter salinestris,
Azotobacter sp., Azotobacter vinelandii, Flavimonas oryzihabitans,
Mesophilobacter marinus, Oligella urethralis, Pseudomonas
acidovorans, Pseudomonas aeruginosa, Pseudomonas agarici,
Pseudomonas alcaligenes, Pseudomonas aminovorans, Pseudomonas
amygdali, Pseudomonas andropogonis, Pseudomonas anguilliseptica,
Pseudomonas antarctica, Pseudomonas antimicrobica, Pseudomonas
antimycetica, Pseudomonas aptata, Pseudomonas arvilla, Pseudomonas
asplenii, Pseudomonas atlantica, Pseudomonas atrofaciens,
Pseudomonas aureofaciens, Pseudomonas avellanae, Pseudomonas
azelaica, Pseudomonas azotocolligans, Pseudomonas balearica,
Pseudomonas barkeri, Pseudomonas bathycetes, Pseudomonas
beijerinckii, Pseudomonas brassicacearum, Pseudomonas brenneri,
Pseudomonas butanovora, Pseudomonas carboxydoflava, Pseudomonas
carboxydohydrogena, Pseudomonas carboxydovorans, Pseudomonas
carrageenovora, Pseudomonas caryophylli, Pseudomonas cepacia,
Pseudomonas chloritidismutans, Pseudomonas chlororaphis,
Pseudomonas cichorii, Pseudomonas citronellolis, Pseudomonas
cocovenenans, Pseudomonas compransoris, Pseudomonas congelans,
Pseudomonas coronafaciens, Pseudomonas corrugata, Pseudomonas
dacunhae, Pseudomonas delafieldii, Pseudomonas delphinii,
Pseudomonas denitrificans, Pseudomonas desmolytica, Pseudomonas
diminuta, Pseudomonas doudoroffii, Pseudomonas echinoides,
Pseudomonas elongata, Pseudomonas extorquens, Pseudomonas
extremorientalis, Pseudomonas facilis, Pseudomonas ficuserectae,
Pseudomonas flava, Pseudomonas flavescens, Pseudomonas fluorescens,
Pseudomonas fragi, Pseudomonas frederiksbergensis, Pseudomonas
fulgida, Pseudomonas fuscovaginae, Pseudomonas gazotropha,
Pseudomonas gladioli, Pseudomonas glathei, Pseudomonas glumae,
Pseudomonas graminis, Pseudomonas halophila, Pseudomonas helianthi,
Pseudomonas huttiensis, Pseudomonas hydrogenothermophila,
Pseudomonas hydrogenovora, Pseudomonas indica, Pseudomonas
indigofera, Pseudomonas iodinum, Pseudomonas kilonensis,
Pseudomonas lachrymans, Pseudomonas lapsa, Pseudomonas lemoignei,
Pseudomonas lemonnieri, Pseudomonas lundensis, Pseudomonas luteola,
Pseudomonas maltophilia, Pseudomonas marginalis, Pseudomonas
marginata, Pseudomonas marina, Pseudomonas meliae, Pseudomonas
mendocina, Pseudomonas mesophilica, Pseudomonas mixta, Pseudomonas
monteilii, Pseudomonas morsprunorum, Pseudomonas multivorans,
Pseudomonas natriegens, Pseudomonas nautica, Pseudomonas
nitroreducens, Pseudomonas oleovorans, Pseudomonas oryzihabitans,
Pseudomonas ovalis, Pseudomonas oxalaticus, Pseudomonas palleronii,
Pseudomonas paucimobilis, Pseudomonas phaseolicola, Pseudomonas
phenazinium, Pseudomonas pickettii, Pseudomonas pisi, Pseudomonas
plantarii, Pseudomonas plecoglossicida, Pseudomonas poae,
Pseudomonas primulae, Pseudomonas proteolytica, Pseudomonas
pseudoalcaligenes, Pseudomonas pseudoalcaligenes subsp. konjaci,
Pseudomonas pseudoalcaligenes subsp. pseudoalcaligenes, Pseudomonas
pseudoflava, Pseudomonas putida, Pseudomonas putida var. naraensis,
Pseudomonas putrefaciens, Pseudomonas pyrrocinia, Pseudomonas
radiora, Pseudomonas reptilivora, Pseudomonas rhodesiae,
Pseudomonas rhodos, Pseudomonas riboflavina, Pseudomonas rubescens,
Pseudomonas rubrisubalbicans, Pseudomonas ruhlandii, Pseudomonas
saccharophila, Pseudomonas savastanoi, Pseudomonas savastanoi pvar.
glycinea, Pseudomonas savastanoi pvar. phaseolicola, Pseudomonas
solanacearum, Pseudomonas sp., Pseudomonas spinosa, Pseudomonas
stanieri, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas
syringae pvar. aptata, Pseudomonas syringae pvar. atrofaciens,
Pseudomonas syringae pvar. coronafaciens, Pseudomonas syringae
pvar. delphinii, Pseudomonas syringae pvar. glycinea, Pseudomonas
syringae pvar. helianthi, Pseudomonas syringae pvar. lachrymans,
Pseudomonas syringae pvar. lapsa, Pseudomonas syringae pvar.
morsprunorum, Pseudomonas syringae pvar. phaseolicola, Pseudomonas
syringae pvar. primulae, Pseudomonas syringae pvar. syringae,
Pseudomonas syringae pvar. tabaci, Pseudomonas syringae pvar.
tomato, Pseudomonas syringae subsp. glycinea, Pseudomonas syringae
subsp. savastanoi, Pseudomonas syringae subsp. syringae,
Pseudomonas syzygii, Pseudomonas tabaci, Pseudomonas
taeniospiralis, Pseudomonas testosterone, Pseudomonas
thermocarboxydovorans, Pseudomonas thermotolerans, Pseudomonas
thivervalensis, Pseudomonas tomato, Pseudomonas trivialis,
Pseudomonas veronii, Pseudomonas vesicularis, Pseudomonas
viridiflava, Pseudomonas viscogena, Pseudomonas woodsii,
Rhizobacter dauci, Rhizobacter daucus or Xylophilus ampelinus;
Rhizobiaceae such as the genera Agrobacterium, Carbophilus,
Chelatobacter, Ensifer, Rhizobium, Sinorhizobium e.g. the species
Agrobacterium atlanticum, Agrobacterium ferrugineum, Agrobacterium
gelatinovorum, Agrobacterium larrymoorei, Agrobacterium meteori,
Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium
rubi, Agrobacterium stellulatum, Agrobacterium tumefaciens,
Agrobacterium vitis, Carbophilus carboxidus, Chelatobacter
heintzii, Ensifer adhaerens, Ensifer arboris, Ensifer fredii,
Ensifer kostiensis, Ensifer kummerowiae, Ensifer medicae, Ensifer
meliloti, Ensifer saheli, Ensifer terangae, Ensifer xinjiangensis,
Rhizobium ciceri Rhizobium etli, Rhizobium fredii, Rhizobium
galegae, Rhizobium gallicum, Rhizobium giardinii, Rhizobium
hainanense, Rhizobium huakuii, Rhizobium huautlense, Rhizobium
indigoferae, Rhizobium japonicum, Rhizobium leguminosarum,
Rhizobium loessense, Rhizobium loti, Rhizobium lupini, Rhizobium
mediterraneum, Rhizobium meliloti, Rhizobium mongolense, Rhizobium
phaseoli, Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium
rubi, Rhizobium sullae, Rhizobium tianshanense, Rhizobium trifolii,
Rhizobium tropici, Rhizobium undicola, Rhizobium vitis,
Sinorhizobium adhaerens, Sinorhizobium arboris, Sinorhizobium
fredii, Sinorhizobium kostiense, Sinorhizobium kummerowiae,
Sinorhizobium medicae, Sinorhizobium meliloti, Sinorhizobium
morelense, Sinorhizobium saheli or Sinorhizobium xinjiangense;
Streptomycetaceae such as the genera Kitasatosprora, Streptomyces,
Streptoverticillium e.g. the species Streptomyces abikoensis,
Streptomyces aburaviensis, Streptomyces achromogenes subsp.
achromogenes, Streptomyces achromogenes subsp. rubradiris,
Streptomyces acid iscabies, Streptomyces acrimycini, Streptomyces
aculeolatus, Streptomyces afghaniensis, Streptomyces alanosinicus,
Streptomyces albaduncus, Streptomyces albiaxialis, Streptomyces
albidochromogenes, Streptomyces albidoflavus, Streptomyces
albireticuli, Streptomyces albofaciens, Streptomyces alboflavus,
Streptomyces albogriseolus, Streptomyces albolongus, Streptomyces
alboniger, Streptomyces albospinus, Streptomyces albosporeus subsp.
albosporeus, Streptomyces albosporeus subsp. labilomyceticus,
Streptomyces alboverticillatus, Streptomyces albovinaceus,
Streptomyces alboviridis, Streptomyces albulus, Streptomyces albus
subsp. albus, Streptomyces albus subsp. pathocidicus, Streptomyces
almquistii, Streptomyces althioticus, Streptomyces amakusaensis,
Streptomyces ambofaciens, Streptomyces aminophilus, Streptomyces
anandii, Streptomyces anthocyanicus, Streptomyces antibioticus,
Streptomyces antimycoticus, Streptomyces anulatus, Streptomyces
arabicus, Streptomyces ardus, Streptomyces arenae, Streptomyces
argenteolus, Streptomyces armeniacus, Streptomyces asiaticus,
Streptomyces asterosporus, Streptomyces atratus, Streptomyces
atroaurantiacus, Streptomyces atroolivaceus, Streptomyces
atrovirens, Streptomyces aurantiacus, Streptomyces aurantiogriseus,
Streptomyces aureocirculatus, Streptomyces aureofaciens,
Streptomyces aureorectus, Streptomyces aureoversilis, Streptomyces
aureoverticillatus, Streptomyces aureus, Streptomyces avellaneus,
Streptomyces avermectinius, Streptomyces avermitilis, Streptomyces
avidinii, Streptomyces azaticus, Streptomyces azureus, Streptomyces
baamensis, Streptomyces bacillaris, Streptomyces badius,
Streptomyces baldaccii, Streptomyces bambergiensis, Streptomyces
beijiangensis, Streptomyces bellus, Streptomyces bikiniensis,
Streptomyces biverticillatus, Streptomyces blastmyceticus,
Streptomyces bluensis, Streptomyces bobili, Streptomyces
bottropensis, Streptomyces brasiliensis, Streptomyces bungoensis,
Streptomyces cacaoi subsp. asoensis, Streptomyces cacaoi subsp.
cacaoi, Streptomyces caelestis, Streptomyces caeruleus,
Streptomyces califomicus, Streptomyces calvus, Streptomyces
canaries, Streptomyces candidus, Streptomyces canescens,
Streptomyces cangkringensis, Streptomyces caniferus, Streptomyces
canus, Streptomyces capillispiralis, Streptomyces capoamus,
Streptomyces carpaticus, Streptomyces carpinensis, Streptomyces
catenulae, Streptomyces caviscabies, Streptomyces cavourensis
subsp. cavourensis, Streptomyces cavourensis subsp.
washingtonensis, Streptomyces cellostaticus, Streptomyces
celluloflavus, Streptomyces cellulolyticus, Streptomyces
cellulosae, Streptomyces champavatii, Streptomyces chartreuses,
Streptomyces chattanoogensis, Streptomyces chibaensis, Streptomyces
chrestomyceticus, Streptomyces chromofuscus, Streptomyces chryseus,
Streptomyces chrysomallus subsp. chrysomallus, Streptomyces
chrysomallus subsp. fumigatus, Streptomyces cinereorectus,
Streptomyces cinereoruber subsp. cinereoruber, Streptomyces
cinereoruber subsp. fructofermentans, Streptomyces cinereospinus,
Streptomyces cinereus, Streptomyces cinerochromogenes, Streptomyces
cinnabarinus, Streptomyces cinnamonensis, Streptomyces cinnamoneus,
Streptomyces cinnamoneus subsp. albosporus, Streptomyces
cinnamoneus subsp. cinnamoneus, Streptomyces cinnamoneus subsp.
lanosus, Streptomyces cinnamoneus subsp. sparsus, Streptomyces
cirratus, Streptomyces ciscaucasicus, Streptomyces
citreofluorescens, Streptomyces clavifer, Streptomyces
clavuligerus, Streptomyces cochleatus, Streptomyces coelescens,
Streptomyces coelicoflavus, Streptomyces coelicolor, Streptomyces
coeruleoflavus, Streptomyces coeruleofuscus, Streptomyces
coeruleoprunus, Streptomyces coeruleorubidus, Streptomyces
coerulescens, Streptomyces collinus, Streptomyces colombiensis,
Streptomyces corchorusii, Streptomyces costaricanus, Streptomyces
cremeus, Streptomyces crystallinus, Streptomyces curacoi,
Streptomyces cuspidosporus, Streptomyces cyaneofuscatus,
Streptomyces cyaneus, Streptomyces cyanoalbus, Streptomyces
cystargineus, Streptomyces daghestanicus, Streptomyces diastaticus
subsp. ardesiacus, Streptomyces diastaticus subsp. diastaticus,
Streptomyces diastatochromogenes, Streptomyces distallicus,
Streptomyces djakartensis, Streptomyces durhamensis, Streptomyces
echinatus, Streptomyces echinoruber, Streptomyces ederensis,
Streptomyces ehimensis, Streptomyces endus, Streptomyces
enissocaesilis, Streptomyces erumpens, Streptomyces erythraeus,
Streptomyces erythrogriseus, Streptomyces eurocidicus, Streptomyces
europaeiscabiei, Streptomyces eurythermus, Streptomyces exfoliates,
Streptomyces felleus, Streptomyces fervens, Streptomyces fervens
subsp. fervens, Streptomyces fervens subsp. melrosporus,
Streptomyces filamentosus, Streptomyces filipinensis, Streptomyces
fimbriatus, Streptomyces fimicarius, Streptomyces finlayi,
Streptomyces flaveolus, Streptomyces flaveus, Streptomyces
flavidofuscus, Streptomyces flavidovirens, Streptomyces
flavisderoticus, Streptomyces flavofungini, Streptomyces
flavofuscus, Streptomyces flavogriseus, Streptomyces flavopersicus,
Streptomyces flavotricini, Streptomyces flavovariabilis,
Streptomyces flavovirens, Streptomyces flavoviridis, Streptomyces
flocculus, Streptomyces floridae, Streptomyces fluorescens,
Streptomyces fradiae, Streptomyces fragilis, Streptomyces
fulvissimus, Streptomyces fulvorobeus, Streptomyces fumanus,
Streptomyces fumigatiscleroticus, Streptomyces galbus, Streptomyces
galilaeus, Streptomyces gancidicus, Streptomyces gardneri,
Streptomyces gelaticus, Streptomyces geysiriensis, Streptomyces
ghanaensis, Streptomyces gibsonii, Streptomyces glaucescens,
Streptomyces glaucosporus, Streptomyces glaucus, Streptomyces
globisporus
subsp. caucasicus, Streptomyces globisporus subsp. flavofuscus,
Streptomyces globisporus subsp. globisporus, Streptomyces globosus,
Streptomyces glomeratus, Streptomyces glomeroaurantiacus,
Streptomyces gobitricini, Streptomyces goshikiensis, Streptomyces
gougerotii, Streptomyces graminearus, Streptomyces graminofaciens,
Streptomyces griseinus, Streptomyces griseoaurantiacus,
Streptomyces griseobrunneus, Streptomyces griseocarneus,
Streptomyces griseochromogenes, Streptomyces griseoflavus,
Streptomyces griseofuscus, Streptomyces griseoincarnatus,
Streptomyces griseoloalbus, Streptomyces griseolosporeus,
Streptomyces griseolus, Streptomyces griseoluteus, Streptomyces
griseomycini, Streptomyces griseoplanus, Streptomyces griseorubens,
Streptomyces griseoruber, Streptomyces griseorubiginosus,
Streptomyces griseosporeus, Streptomyces griseostramineus,
Streptomyces griseoverticillatus, Streptomyces griseoviridis,
Streptomyces griseus subsp. alpha, Streptomyces griseus subsp.
cretosus, Streptomyces griseus subsp. griseus, Streptomyces griseus
subsp. solvifaciens, Streptomyces hachijoensis, Streptomyces
halstedii, Streptomyces hawaiiensis, Streptomyces heliomycini,
Streptomyces helvaticus, Streptomyces herbaricolor, Streptomyces
hiroshimensis, Streptomyces hirsutus, Streptomyces humidus,
Streptomyces humiferus, Streptomyces hydrogenans, Streptomyces
hygroscopicus subsp. angustmyceticus, Streptomyces hygroscopicus
subsp. decoyicus, Streptomyces hygroscopicus subsp. glebosus,
Streptomyces hygroscopicus subsp. hygroscopicus, Streptomyces
hygroscopicus subsp. ossamyceticus, Streptomyces iakyrus,
Streptomyces indiaensis, Streptomyces indigoferus, Streptomyces
indonesiensis, Streptomyces intermedius, Streptomyces inusitatus,
Streptomyces ipomoeae, Streptomyces janthinus, Streptomyces
javensis, Streptomyces kanamyceticus, Streptomyces kashmirensis,
Streptomyces kasugaensis, Streptomyces katrae, Streptomyces
kentuckensis, Streptomyces kifunensis, Streptomyces kishiwadensis,
Streptomyces kunmingensis, Streptomyces kurssanovii, Streptomyces
labedae, Streptomyces laceyi, Streptomyces ladakanum, Streptomyces
lanatus, Streptomyces lateritius, Streptomyces laurentii,
Streptomyces lavendofoliae, Streptomyces lavendulae subsp.
grasserius, Streptomyces lavendulae subsp. lavendulae, Streptomyces
lavenduligriseus, Streptomyces lavendulocolor, Streptomyces levis,
Streptomyces libani subsp. libani, Streptomyces libani subsp.
rufus, Streptomyces lienomycini, Streptomyces lilacinus,
Streptomyces limosus, Streptomyces lincolnensis, Streptomyces
lipmanii, Streptomyces litmocidini, Streptomyces lomondensis,
Streptomyces longisporoflavus, Streptomyces longispororuber,
Streptomyces longisporus, Streptomyces longwoodensis, Streptomyces
lucensis, Streptomyces luridiscabiei, Streptomyces luridus,
Streptomyces lusitanus, Streptomyces luteireticuli, Streptomyces
luteogriseus, Streptomyces luteosporeus, Streptomyces
luteoverticillatus, Streptomyces lydicus, Streptomyces macrosporus,
Streptomyces malachitofuscus, Streptomyces malachitospinus,
Streptomyces malaysiensis, Streptomyces mashuensis, Streptomyces
massasporeus, Streptomyces matensis, Streptomyces mauvecolor,
Streptomyces mediocidicus, Streptomyces mediolani, Streptomyces
megasporus, Streptomyces melanogenes, Streptomyces
melanosporofaciens, Streptomyces mexicanus, Streptomyces
michiganensis, Streptomyces microflavus, Streptomyces
minutiscleroticus, Streptomyces mirabilis, Streptomyces
misakiensis, Streptomyces misionensis, Streptomyces mobaraensis,
Streptomyces monomycini, Streptomyces morookaensis, Streptomyces
murinus, Streptomyces mutabilis, Streptomyces mutomycini,
Streptomyces naganishii, Streptomyces narbonensis, Streptomyces
nashvillensis, Streptomyces netropsis, Streptomyces neyagawaensis,
Streptomyces niger, Streptomyces nigrescens, Streptomyces
nigrifaciens, Streptomyces nitrosporeus, Streptomyces
niveiciscabiei, Streptomyces niveoruber, Streptomyces niveus,
Streptomyces noboritoensis, Streptomyces nodosus, Streptomyces
nogalater, Streptomyces nojiriensis, Streptomyces noursei,
Streptomyces novaecaesareae, Streptomyces ochraceisderoticus,
Streptomyces odorifer, Streptomyces olivaceiscleroticus,
Streptomyces olivaceoviridis, Streptomyces olivaceus, Streptomyces
ofivochromogenes, Streptomyces olivomycini, Streptomyces
olivoreticuli, Streptomyces olivoreticuli subsp. cellulophilus,
Streptomyces olivoreticuli subsp. olivoreticuli, Streptomyces
olivoverticillatus, Streptomyces olivoviridis, Streptomyces
omiyaensis, Streptomyces orinoci, Streptomyces pactum, Streptomyces
paracochleatus, Streptomyces paradoxus, Streptomyces
parvisporogenes, Streptomyces parvulus, Streptomyces parvus,
Streptomyces peucetius, Streptomyces phaeochromogenes, Streptomyces
phaeofaciens, Streptomyces phaeopurpureus, Streptomyces
phaeoviridis, Streptomyces phosalacineus, Streptomyces pilosus,
Streptomyces platensis, Streptomyces plicatus, Streptomyces
pluricolorescens, Streptomyces polychromogenes, Streptomyces
poonensis, Streptomyces praecox, Streptomyces prasinopilosus,
Streptomyces prasinosporus, Streptomyces prasinus, Streptomyces
prunicolor, Streptomyces psammoticus, Streptomyces
pseudoechinosporeus, Streptomyces pseudogriseolus, Streptomyces
pseudovenezuelae, Streptomyces pulveraceus, Streptomyces puniceus,
Streptomyces puniciscabiei, Streptomyces purpeofuscus, Streptomyces
purpurascens, Streptomyces purpureus, Streptomyces
purpurogeneiscleroticus, Streptomyces racemochromogenes,
Streptomyces rameus, Streptomyces ramulosus, Streptomyces
rangoonensis, Streptomyces recifensis, Streptomyces
rectiverticillatus, Streptomyces rectiviolaceus, Streptomyces
regensis, Streptomyces resistomycificus, Streptomyces
reticuliscabiei, Streptomyces rhizosphaericus, Streptomyces rimosus
subsp. paromomycinus, Streptomyces rimosus subsp. rimosus,
Streptomyces rishiriensis, Streptomyces rochei, Streptomyces
roseiscleroticus, Streptomyces roseodiastaticus, Streptomyces
roseoflavus, Streptomyces roseofulvus, Streptomyces roseolilacinus,
Streptomyces roseolus, Streptomyces roseosporus, Streptomyces
roseoverticillatus, Streptomyces roseoviolaceus, Streptomyces
roseoviridis, Streptomyces rubber, Streptomyces rubiginosohelvolus,
Streptomyces rubiginosus, Streptomyces rubrogriseus, Streptomyces
rutgersensis subsp. castelarensis, Streptomyces rutgersensis subsp.
rutgersensis, Streptomyces salmonis, Streptomyces sampsonii,
Streptomyces sanglieri, Streptomyces sannanensis, Streptomyces
sapporonensis, Streptomyces scabiei, Streptomyces sclerotialus,
Streptomyces scopiformis, Streptomyces seoulensis, Streptomyces
septatus, Streptomyces setae, Streptomyces setonii, Streptomyces
showdoensis, Streptomyces sindenensis, Streptomyces sioyaensis,
Streptomyces somaliensis, Streptomyces sparsogenes, Streptomyces
spectabilis, Streptomyces speibonae, Streptomyces speleomycini,
Streptomyces spheroids, Streptomyces spinoverrucosus, Streptomyces
spiralis, Streptomyces spiroverticillatus, Streptomyces
spitsbergensis, Streptomyces sporocinereus, Streptomyces
sporoclivatus, Streptomyces spororaveus, Streptomyces
sporoverrucosus, Streptomyces stelliscabiei, Streptomyces
stramineus, Streptomyces subrutilus, Streptomyces sulfonofaciens,
Streptomyces sulphurous, Streptomyces syringium, Streptomyces
tanashiensis, Streptomyces tauricus, Streptomyces tendae,
Streptomyces termitum, Streptomyces thermoalcalitolerans,
Streptomyces thermoautotrophicus, Streptomyces
thermocarboxydovorans, Streptomyces thermocarboxydus, Streptomyces
thermocoprophilus, Streptomyces thermodiastaticus, Streptomyces
thermogriseus, Streptomyces thermolineatus, Streptomyces
thermonitrificans, Streptomyces thermospinosisporus, Streptomyces
thermoviolaceus subsp. apingens, Streptomyces thermoviolaceus
subsp. thermoviolaceus, Streptomyces thermovulgaris, Streptomyces
thioluteus, Streptomyces torulosus, Streptomyces toxytricini,
Streptomyces tricolor, Streptomyces tubercidicus, Streptomyces
tuirus, Streptomyces turgidiscabies, Streptomyces umbrinus,
Streptomyces variabilis, Streptomyces variegates, Streptomyces
varsoviensis, Streptomyces vastus, Streptomyces venezuelae,
Streptomyces vinaceus, Streptomyces vinaceusdrappus, Streptomyces
violaceochromogenes, Streptomyces violaceolatus, Streptomyces
violaceorectus, Streptomyces violaceoruber, Streptomyces
violaceorubidus, Streptomyces violaceus, Streptomyces
violaceusniger, Streptomyces violarus, Streptomyces violascens,
Streptomyces violatus, Streptomyces violens, Streptomyces virens,
Streptomyces virginiae, Streptomyces viridiflavus, Streptomyces
viridiviolaceus, Streptomyces viridobrunneus, Streptomyces
viridochromogenes, Streptomyces viridodiastaticus, Streptomyces
viridosporus, Streptomyces vitaminophileus, Streptomyces
vitaminophilus, Streptomyces wedmorensis, Streptomyces werraensis,
Streptomyces willmorei, Streptomyces xanthochromogenes,
Streptomyces xanthocidicus, Streptomyces xantholiticus,
Streptomyces xanthophaeus, Streptomyces yatensis, Streptomyces
yerevanensis, Streptomyces yogyakartensis, Streptomyces
yokosukanensis, Streptomyces yunnanensis, Streptomyces
zaomyceticus, Streptoverticillium abikoense, Streptoverticillium
albireticuli, Streptoverticillium alboverticillatum,
Streptoverticillium album, Streptoverticillium ardum,
Streptoverticillium aureoversale, Streptoverticillium aureoversile,
Streptoverticillium baldaccii, Streptoverticillium biverticillatum,
Streptoverticillium blastmyceticum, Streptoverticillium cinnamoneum
subsp. albosporum, Streptomyces cinnamoneus subsp. albosporus,
Streptoverticillium cinnamoneum subsp. cinnamoneum,
Streptoverticillium cinnamoneum subsp. lanosum, Streptoverticillium
cinnamoneum subsp. sparsum, Streptoverticillium distallicum,
Streptoverticillium ehimense, Streptoverticillium eurocidicum,
Streptoverticillium fervens subsp. fervens, Streptoverticillium
fervens subsp. melrosporus, Streptoverticillium flavopersicum,
Streptoverticillium griseocarneum, Streptoverticillium
griseoverticillatum, Streptoverticillium hachijoense,
Streptoverticillium hiroshimense, Streptoverticillium kashmirense,
Streptoverticillium kentuckense, Streptoverticillium kishiwadense,
Streptoverticillium ladakanum, Streptoverticillium
lavenduligriseum, Streptoverticillium lilacinum,
Streptoverticillium luteoverticillatum, Streptoverticillium
mashuense, Streptoverticillium mobaraense, Streptoverticillium
morookaense, Streptoverticillium netropsis, Streptoverticillium
olivomycini, Streptomyces olivomycini, Streptoverticillium
olivoreticuli subsp. cellulophilum, Streptoverticillium
olivoreticuli subsp. olivoreticuli, Streptoverticillium
olivoreticulum, Streptoverticillium olivoreticulum subsp.
cellulophilum, Streptoverticillium olivoverticillatum,
Streptoverticillium orinoci, Streptoverticillium parvisporogenes,
Streptoverticillium parvisporogenum, Streptoverticillium
rectiverticillatum, Streptoverticillium reticulum subsp.
protomycicum, Streptoverticillium roseoverticillatum,
Streptoverticillium salmonis, Streptoverticillium sapporonense,
Streptoverticillium septatum, Streptoverticillium syringium,
Streptoverticillium thioluteum, Streptoverticillium verticillium
subsp. quantum, Streptoverticillium verticillium subsp.
tsukushiense or Streptoverticillium viridoflavum.
[0138] [0080.0.0.0] Particular preferred strains are strains
selected from the group consisting of Bacillaceae,
Brevibacteriaceae, Corynebacteriaceae, Nocardiaceae,
Mycobacteriaceae, Streptomycetaceae, Enterobacteriaceae such as
Bacillus circulans, Bacillus subtilis, Bacillus sp., Brevibacterium
albidum, Brevibacterium album, Brevibacterium cerinum,
Brevibacterium flavum, Brevibacterium glutamigenes, Brevibacterium
iodinum, Brevibacterium ketoglutamicum, Brevibacterium
lactofermentum, Brevibacterium linens, Brevibacterium roseum,
Brevibacterium saccharolyticum, Brevibacterium sp., Corynebacterium
acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium
ammoniagenes, Corynebacterium glutamicum (=Micrococcus glutamicum),
Corynebacterium melassecola, Corynebacterium sp., Nocardia
rhodochrous (Rhodococcus rhodochrous), Mycobacterium rhodochrous,
Streptomyces lividans and Escherichia coli especially Escherichia
coli K12.
[0139] [0081.0.0.0] In addition particular preferred strains are
strains selected from the group consisting of Cryptococcaceae,
Saccharomycetaceae, Schizosaccharo-mycetacease such as the genera
Candida, Hansenula, Pichia, Saccharomyces and Schizosaccharomyces
preferred are strains selected from the group consisting of the
species Rhodotorula rubra, Rhodotorula glutinis, Rhodotorula
graminis, Yarrowia lipolytica, Sporobolomyces salmonicolor,
Sporobolomyces shibatanus, Saccharomyces cerevisiae, Candida
boidinii, Candida bombicola, Candida cylindracea, Candida
parapsilosis, Candida rugosa, Candida tropicalis, Pichia
methanolica and Pichia pastoris.
[0140] [0082.0.0.0] Anacardiaceae such as the genera Pistacia,
Mangifera, Anacardium e.g. the species Pistacia vera [pistachios,
Pistazie], Mangifer indica [Mango] or Anacardium occidentale
[Cashew]; Asteraceae such as the genera Calendula, Carthamus,
Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta,
Tagetes, Valeriana e.g. the species Calendula officinalis
[Marigold], Carthamus tinctorius [safflower], Centaurea cyanus
[cornflower], Cichorium intybus [blue daisy], Cynara scolymus
[Artichoke], Helianthus annus [sunflower], Lactuca sativa, Lactuca
crispa, Lactuca esculenta, Lactuca scariola L. ssp. sativa, Lactuca
scariola L. var. integrata, Lactuca scariola L. var. integrifolia,
Lactuca sativa subsp. romana, Locusta communis, Valeriana locusta
[lettuce], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia
[Marigold]; Apiaceae such as the genera Daucus e.g. the species
Daucus carota [carrot]; Betulaceae such as the genera Corylus e.g.
the species Corylus avellana or Corylus colurna [hazelnut];
Boraginaceae such as the genera Borago e.g. the species Borago
officinalis [borage]; Brassicaceae such as the genera Brassica,
Melanosinapis, Sinapis, Arabadopsis e.g. the species Brassica
napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape],
Sinapis arvensis Brassica juncea, Brassica juncea var. juncea,
Brassica juncea var. crispifolia, Brassica juncea var. foliosa,
Brassica nigra, Brassica sinapioides, Melanosinapis communis
[mustard], Brassica oleracea [fodder beet] or Arabidopsis thaliana;
Bromeliaceae such as the genera Anana, Bromelia e.g. the species
Anana comosus, Ananas ananas or Bromelia comosa [pineapple];
Caricaceae such as the genera Carica e.g. the species Carica papaya
[papaya]; Cannabaceae such as the genera Cannabis e.g. the species
Cannabis sative [hemp], Convolvulaceae such as the genera Ipomea,
Convolvulus e.g. the species Ipomoea batatus, Ipomoea pandurata,
Convolvulus batatas, Convolvulus tiliaceus, Ipomoea fastigiata,
Ipomoea tiliacea, Ipomoea triloba or Convolvulus panduratus [sweet
potato, Man of the Earth, wild potato], Chenopodiaceae such as the
genera Beta, i.e. the species Beta vulgaris, Beta vulgaris var.
altissima, Beta vulgaris var. Vulgaris, Beta maritima, Beta
vulgaris var. perennis, Beta vulgaris var. conditiva or Beta
vulgaris var. esculenta [sugar beet]; Cucurbitaceae such as the
genera Cucubita e.g. the species Cucurbita maxima, Cucurbita mixta,
Cucurbita pepo or Cucurbita moschata [pumpkin, squash];
Elaeagnaceae such as the genera Elaeagnus e.g. the species Olea
europaea [olive]; Ericaceae such as the genera Kalmia e.g. the
species Kalmia latifolia, Kalmia angustifolia, Kalmia microphylla,
Kalmia polifolia, Kalmia occidentalis, Cistus chamaerhodendros or
Kalmia lucida [American laurel, broad-leafed laurel, calico bush,
spoon wood, sheep laurel, alpine laurel, bog laurel, western
bog-laurel, swamp-laurel]; Euphorbiaceae such as the genera
Manihot, Janipha, Jatropha, Ricinus e.g. the species Manihot
utilissima, Janipha manihot, Jatropha manihot, Manihot aipil,
Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot
esculenta [manihot, arrowroot, tapioca, cassava] or Ricinus
communis [castor bean, Castor Oil Bush, Castor Oil Plant, Palma
Christi, Wonder Tree]; Fabaceae such as the genera Pisum, Albizia,
Cathormion, Feuillea, Inga, Pithecolobium, Acacia, Mimosa,
Medicajo, Glycine, Dolichos, Phaseolus, Soja e.g. the species Pisum
sativum, Pisum arvense, Pisum humile [pea], Albizia berteriana,
Albizia julibrissin, Albizia lebbeck, Acacia berteriana, Acacia
littoralis, Albizia berteriana, Albizzia berteriana, Cathormion
berteriana, Feuillea berteriana, Inga fragrans, Pithecellobium
berterianum, Pithecellobium fragrans, Pithecolobium berterianum,
Pseudalbizzia berteriana, Acacia julibrissin, Acacia nemu, Albizia
nemu, Feuilleea julibrissin, Mimosa julibrissin, Mimosa speciosa,
Sericanrda julibrissin, Acacia lebbeck, Acacia macrophylla, Albizia
lebbek, Feuilleea lebbeck, Mimosa lebbeck, Mimosa speciosa [bastard
logwood, silk tree, East Indian Walnut], Medicago sativa, Medicago
falcata, Medicago varia [alfalfa] Glycine max Dolichos soja,
Glycine gracilis, Glycine hispida, Phaseolus max, Soja hispida or
Soja max [soybean]; Geraniaceae such as the genera Pelargonium,
Cocos, Oleum e.g. the species Cocos nucifera, Pelargonium
grossularioides or Oleum cocois [coconut]; Gramineae such as the
genera Saccharum e.g. the species Saccharum officinarum;
Juglandaceae such as the genera Juglans, Wallia e.g. the species
Juglans regia, Juglans ailanthifolia, Juglans sieboldiana, Juglans
cinerea, Wallia cinerea, Juglans bixbyi, Juglans californica,
Juglans hindsii, Juglans intermedia, Juglans jamaicensis, Juglans
major, Juglans microcarpa, Juglans nigra or Wallia nigra [walnut,
black walnut, common walnut, persian walnut, white walnut,
butternut, black walnut]; Lauraceae such as the genera Persea,
Laurus e.g. the species laurel Laurus nobilis [bay, laurel, bay
laurel, sweet bay], Persea americana Persea americana, Persea
gratissima or Persea persea [avocado]; Leguminosae such as the
genera Arachis e.g. the species Arachis hypogaea [peanut]; Linaceae
such as the genera Linum, Adenolinum e.g. the species Linum
usitatissimum, Linum humile, Linum austriacum, Linum bienne, Linum
angustifolium, Linum catharticum, Linum flavum, Linum grandiflorum,
Adenolinum grandiflorum, Linum lewisii, Linum narbonense, Linum
perenne, Linum perenne var. lewisii, Linum pratense or Linum
trigynum [flax, linseed]; Lythrarieae such as the genera Punica
e.g. the species Punica granatum [pomegranate]; Malvaceae such as
the genera Gossypium e.g. the species Gossypium hirsutum, Gossypium
arboreum, Gossypium barbadense, Gossypium herbaceum or Gossypium
thurberi [cotton]; Musaceae such as the genera Musa e.g. the
species Musa nana, Musa acuminata, Musa paradisiaca, Musa spp.
[banana]; Onagraceae such as the genera Camissonia, Oenothera e.g.
the species Oenothera biennis or Camissonia brevipes [primrose,
evening primrose]; Palmae such as the genera Elacis e.g. the
species Elacis guineensis [oil plam]; Papaveraceae such as the
genera Papaver e.g. the species Papaver orientale, Papaver rhoeas,
Papaver dubium [poppy, oriental poppy, corn poppy, field poppy,
shirley poppies, field poppy, long-headed poppy, long-pod poppy];
Pedaliaceae such as the genera Sesamum e.g. the species Sesamum
indicum [sesame]; Piperaceae such as the genera Piper, Artanthe,
Peperomia, Steffensia e.g. the species Piper aduncum, Piper
amalago, Piper angustifolium, Piper auritum, Piper betel, Piper
cubeba, Piper longum, Piper nigrum, Piper retrofractum, Artanthe
adunca, Artanthe elongata, Peperomia elongata, Piper elongatum,
Steffensia elongata. [Cayenne pepper, wild pepper]; Poaceae such as
the genera Hordeum, Secale, Avena, Sorghum, Andropogon, Holcus,
Panicum, Oryza, Zea, Triticum e.g. the species Hordeum vulgare,
Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum
distichon Hordeum aegiceras, Hordeum hexastichon, Hordeum
hexastichum, Hordeum irregulare, Hordeum sativum, Hordeum secalinum
[barley, pearl barley, foxtail barley, wall barley, meadow barley],
Secale cereale [rye], Avena sativa, Avena fatua, Avena byzantina,
Avena fatua var. sativa, Avena hybrida [oat], Sorghum bicolor,
Sorghum halepense, Sorghum saccharatum, Sorghum vulgare, Andropogon
drummondii, Holcus bicolor, Holcus sorghum, Sorghum aethiopicum,
Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum, Sorghum
dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense,
Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghum
subglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcus
halepensis, Sorghum miliaceum millet, Panicum militaceum [Sorghum,
millet], Oryza sativa, Oryza latifolia [rice], Zea mays [corn,
maize] Triticum aestivum, Triticum durum, Triticum turgidum,
Triticum hybernum, Triticum macha, Triticum sativum or Triticum
vulgare [wheat, bread wheat, common wheat], Proteaceae such as the
genera Macadamia e.g. the species Macadamia intergrifolia
[macadamia]; Rubiaceae such as the genera Coffea e.g. the species
Cofea spp., Coffea arabica, Coffea canephora or Coffea liberica
[coffee]; Scrophulariaceae such as the genera Verbascum e.g. the
species Verbascum blattaria, Verbascum Verbascum densiflorum,
Verbascum lagurus, Verbascum longifolium, Verbascum lychnitis,
Verbascum nigrum, Verbascum olympicum, Verbascum phlomoides,
Verbascum phoenicum, Verbascum pulverulentum or Verbascum thapsus
[mullein, white moth mullein, nettle-leaved mullein, dense-flowered
mullein, silver mullein, long-leaved mullein, white mullein, dark
mullein, greek mullein, orange mullein, purple mullein, hoary
mullein, great mullein]; Solanaceae such as the genera Capsicum,
Nicotiana, Solanum, Lycopersicon e.g. the species Capsicum annuum,
Capsicum annuum var. glabriusculum, Capsicum frutescens [pepper],
Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata,
Nicotiana attenuata, Nicotiana glauca, Nicotiana langsdorffii,
Nicotiana obtusifolia, Nicotiana quadrivalvis, Nicotiana repanda,
Nicotiana rustica, Nicotiana sylvestris [tobacco], Solanum
tuberosum [potato], Solanum melongena [egg-plant] (Lycopersicon
esculentum, Lycopersicon lycopersicum., Lycopersicon pyriforme,
Solanum integrifolium or Solanum lycopersicum [tomato];
Sterculiaceae such as the genera Theobroma e.g. the species
Theobroma cacao [cacao]; Theaceae such as the genera Camellia e.g.
the species Camellia sinensis) [tea].
All abovementioned organisms can in princible also function as host
organisms.
[0141] [0083.0.0.0] Particular preferred plants are plants selected
from the group consisting of Asteraceae such as the genera
Helianthus, Tagetes e.g. the species Helianthus annus [sunflower],
Tagetes lucida, Tagetes erecta or Tagetes tenuifolia [Marigold],
Brassicaceae such as the genera Brassica, Arabadopsis e.g. the
species Brassica napus, Brassica rapa ssp. [canola, oilseed rape,
turnip rape] or Arabidopsis thaliana. Fabaceae such as the genera
Glycine e.g. the species Glycine max, Soja hispida or Soja max
[soybean] (wobei ich nicht sicher bin, ob es Soja max uberhaupt
gibt, die hei.beta.t eigentlich Glycine max). Linaceae such as the
genera Linum e.g. the species Linum usitatissimum, [flax, linseed];
Poaceae such as the genera Hordeum, Secale, Avena, Sorghum, Oryza,
Zea, Triticum e.g. the species Hordeum vulgare [barley]; Secale
cereale [rye], Avena sativa, Avena fatua, Avena byzantina, Avena
fatua var. sativa, Avena hybrida [oat], Sorghum bicolor [Sorghum,
millet], Oryza sativa, Oryza latifolia [rice], Zea mays [corn,
maize] Triticum aestivum, Triticum durum, Triticum turgidum,
Triticum hybernum, Triticum macha, Triticum sativum or Triticum
vulgare [wheat, bread wheat, common wheat]; Solanaceae such as the
genera Solanum, Lycopersicon e.g. the species Solanum tuberosum
[potato], Lycopersicon esculentum, Lycopersicon lycopersicum.,
Lycopersicon pyriforme, Solanum integrifolium or Solanum
lycopersicum [tomato].
[0142] [0084.0.0.0] All abovementioned organisms can in princible
also function as host organisms.
[0143] [0085.0.0.0] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [0144] a) a nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 1 to 5 and/or lines 334
to 338, or a derivative thereof, or [0145] b) a genetic regulatory
element, for example a promoter, which is functionally linked to
the nucleic acid sequence as indicated in Table I, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338, or a derivative thereof, or
[0146] c) (a) and (b) is/are not present in its/their natural
genetic environment or has/have been modified by means of genetic
manipulation methods, it being possible for the modification to be,
by way of example, a substitution, addition, deletion, inversion or
insertion of one or more nucleotide. "Natural genetic environment"
means the natural chromosomal locus in the organism of origin or
the presence in a genomic library. In the case of a genomic
library, the natural, genetic environment of the nucleic acid
sequence is preferably at least partially still preserved. The
environment flanks the nucleic acid sequence at least on one side
and has a sequence length of at least 50 bp, preferably at least
500 bp, particularly preferably at least 1000 bp, very particularly
preferably at least 5000 bp.
[0147] [0086.0.0.0] The use of the nucleic acid sequence according
to the invention or of the nucleic acid construct according to the
invention for the generation of transgenic plants is therefore also
subject matter of the invention.
[0148] [0087.0.0.0] The respective fine chemical, which is
synthesized in the organism, in particular the microorganism, the
cell, the tissue or the plant, of the invention can be isolated if
desired. Depending on the use of the respective fine chemical,
different purities resulting from the purification may be
advantageous as will be described herein below.
[0149] [0088.0.0.0] In an advantageous embodiment of the invention,
the organism takes the form of a plant whose amino acid content is
modified advantageously owing to the nucleic acid molecule of the
present invention expressed. This is important for plant breeders
since, for example, the nutritional value of plants for monogastric
animals is limited by a few essential amino acids such as lysine,
threonine or methionine.
[0150] [0088.1.0.0] In one embodiment, after an activity of a
polypeptide of the present invention or used in the process of the
present invention has been increased or generated, or after the
expression of a nucleic acid molecule or polypeptide according to
the invention has been generated or increased, the transgenic plant
generated can be grown on or in a nutrient medium or else in the
soil and subsequently harvested.
[0151] [0089.0.0.0] The plants or parts thereof, e.g. the leaves,
roots, flowers, and/or stems and/or other harvestable material as
described below, can then be used directly as foodstuffs or animal
feeds or else be further processed. Again, the amino acids can be
purified further in the customary manner via extraction and
precipitation or via ion exchangers and other methods known to the
person skilled in the art and described herein below. Products
which are suitable for various applications and which result from
these different processing procedures are amino acids or amino acid
compositions which can still comprise further plant components in
different amounts, advantageously in the range of from 0 to 99% by
weight, preferably from below 90% by weight, especially preferably
below 80% by weight. The plants can also advantageously be used
directly without further processing, e.g. as feed or for
extraction.
[0152] [0090.0.0.0] The chemically pure respective fine chemical or
chemically pure compositions comprising the respective fine
chemical may also be produced by the process described above. To
this end, the respective fine chemical or the compositions are
isolated in the known manner from an organism according to the
invention, such as the microorganisms, non-human animal or the
plants, and/or their culture medium in which or on which the
organisms had been grown. These chemically pure respective fine
chemical or said compositions are advantageous for applications in
the field of the food industry, the cosmetics industry or the
pharmaceutical industry.
[0153] [0091.0.0.0] Thus, the content of plant components and
preferably also further impurities is as low as possible, and the
abovementioned respective fine chemical is obtained in as pure form
as possible. In these applications, the content of plant components
advantageously amounts to less than 10%, preferably 1%, more
preferably 0.1%, very especially preferably 0.01% or less.
[0154] [0092.0.0.0] Accordingly, the respective fine chemical
produced by the present invention is at least 0.1% by weight pure,
preferably more than 1% by weight pure, more preferred 10% by
weight pure, even more preferred are more than 50, 60, 70 or 80% by
weight purity, even more preferred are more than 90 weight-%
purity, most preferred are 95% by weight, 99% by weight or
more.
[0155] [0093.0.0.0] In this context, the amount of the respective
fine chemical in a cell of the invention may be increased according
to the process of the invention by at least a factor of 1.1,
preferably at least a factor of 1.5; 2; or 5, especially preferably
by at least a factor of 10 or 30, very especially preferably by at
least a factor of 50, in comparison with the wild type, control or
reference. Preferably, said increase is found a tissue, more
preferred in an organism or in a harvestable part thereof.
[0156] [0094.0.0.0] In principle, the respective fine chemicals
produced can be increased in two ways by the process according to
the invention. The pool of free respective fine chemicals, in
particular of the free respective fine chemical, and/or the content
of protein-bound respective fine chemicals, in particular of the
protein-bound respective fine chemical may advantageously be
increased.
[0157] [0095.0.0.0] It may be advantageous to increase the pool of
free amino acids in the transgenic organisms by the process
according to the invention in order to isolate high amounts of the
pure respective fine chemical.
[0158] [0096.0.0.0] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid, which functions as a
sink for the desired amino acid for example methionine, lysine or
threonine in the organism is useful to increase the production of
the respective fine chemical (see U.S. Pat. No. 5,589,616, WO
96/38574, WO 97/07665, WO 97/28247, U.S. Pat. No. 4,886,878, U.S.
Pat. No. 5,082,993 and U.S. Pat. No. 5,670,635). Galili et al.,
Transgenic Res. 2000 showed, that enhancing the synthesis of
threonine by a feed back insensitive aspartate kinase did not lead
only to in increase in free threonine but also in protein bound
threonine.
[0159] [0097.0.0.0] In may also be advantageous to increase the
content of the protein-bound respective fine chemical.
[0160] [0098.0.0.0] In a preferred embodiment, the respective fine
chemical (methionine) and/or threonine are produced in accordance
with the invention and, if desired, are isolated. The production of
further amino acids such as lysine and of amino acid mixtures by
the process according to the invention is advantageous.
[0161] [0099.0.0.0] In the case of the fermentation of
microorganisms, the abovementioned amino acids may accumulate in
the medium and/or the cells. If microorganisms are used in the
process according to the invention, the fermentation broth can be
processed after the cultivation. Depending on the requirement, all
or some of the biomass can be removed from the fermentation broth
by separation methods such as, for example, centrifugation,
filtration, decanting or a combination of these methods, or else
the biomass can be left in the fermentation broth. The fermentation
broth can subsequently be reduced, or concentrated, with the aid of
known methods such as, for example, rotary evaporator, thin-layer
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. This concentrated fermentation broth can
subsequently be processed by lyophilization, spray drying, spray
granulation or by other methods.
[0162] [0100.0.0.0] To purify an amino acid, a product-containing
fermentation broth from which the biomass has been separated may be
subjected to chromatography with a suitable resin such as ion
exchange resin for example anion or cation exchange resin,
hydrophobic resin or hydrophilic resin for example epoxy resin,
polyurethane resin or polyacrylamid resin, or resin for separation
according to the molecular weight of the compounds for example
polyvinyl chloride homopolymer resin or resins composed for example
of polymers of acrylic acid, crosslinked with polyalkenyl ethers or
divinyl glycol such as Carbopol.RTM., Pemulen.RTM. and Noveon.RTM..
If necessary these chromatography steps may be repeated using the
same or other chromatography resins. The skilled worker is familiar
with the choice of suitable chromatography resins and their most
effective use. The purified product may be concentrated by
filtration or ultrafiltration and stored at a temperature, which
ensures the maximum stability of the product.
[0163] [0101.0.0.0] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[0164] [0102.0.0.0] Amino acids can for example be detected
advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55). Amino acids can be extracted with hot water. After
filtration the extracts are diluted with water containing 20 mg/mL
sodium acide. The separation and detection of the amino acids is
performed using an anion exchange column and an electrochemical
detector. Technical details can be taken from Y. Ding et al., 2002,
Direct determination of free amino acids and sugars in green tea by
anion-exchange chromatography with integrated pulsed amperometric
detection, J Chromatogr A, (2002) 982; 237-244, or e.g. from Karchi
et al., 1993, Plant J. 3: 721-727; Matthews M J, 1997 (Lysine,
threonine and methionine biosynthesis. In BK Singh, ed, Plant Amino
Acids: Biochemistry and Biotechnology. Dekker, New York, pp
205-225; H Hesse and R Hoefgen. (2003) Molecular aspects of
methionine biosynthesis. TIPS 8(259-262.
[0165] [0103.0.0.0] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [0166] a) nucleic acid molecule encoding, preferably
at least the mature form, of a polypeptide having a sequence as
indicated in Table II, columns 5 or 7, lines 1 to 5 and/or lines
334 to 338, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [0167] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table I, columns 5 or 7, lines 1 to 5 and/or lines
334 to 338; [0168] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [0169] d) nucleic
acid molecule encoding a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [0170] e) nucleic acid molecule which hybridizes with
a nucleic acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [0171]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [0172] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to [0173] (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [0174] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers pairs
having a sequence as indicated in Table III, columns 7, lines 1 to
5 and/or lines 334 to 338, and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[0175] i) nucleic acid molecule encoding a polypeptide which is
isolated, e.g. from an expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (h), preferably to (a) to (c), and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [0176] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequences as indicated in Table IV, columns 7,
lines 1 to 5 and/or lines 334 to 338, and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [0177] k) nucleic acid molecule comprising one or more of
the nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of a polypeptide indicated in Table
II, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338, and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; and [0178] l) nucleic
acid molecule which is obtainable by screening a suitable library
under stringent conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k), preferably to
(a) to (c), or with a fragment of at least 15 nt, preferably 20 nt,
30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k), preferably to (a) to (c), and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which comprises a
sequence which is complementary thereto.
[0179] [0103.1.0.0] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 1 to 5 and/or
lines 334 to 338, by one or more nucleotides. In one embodiment,
the nucleic acid molecule used in the process of the invention does
not consist of the sequence shown in indicated in Table I A,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338: In one
embodiment, the nucleic acid molecule used in the process of the
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I A, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II A, columns 5 or 7, lines 1 to 5 and/or lines
334 to 338.
[0180] [0103.1.0.0] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338, by one or more nucleotides. In one
embodiment, the nucleic acid molecule used in the process of the
invention does not consist of the sequence shown in indicated in
Table I B, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338: In
one embodiment, the nucleic acid molecule used in the process of
the invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I B, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II B, columns 5 or 7, lines 1 to 5 and/or lines
334 to 338.
[0181] [0104.0.0.0] In one embodiment, the nucleic acid molecule of
the invention or used in the process of the invention distinguishes
over the sequence indicated in Table I, columns 5 or 7, lines 1 to
5 and/or lines 334 to 338, by one or more nucleotides. In one
embodiment, the nucleic acid molecule of the invention or the
nucleic acid used in the process of the invention does not consist
of the sequence shown in indicated in Table I, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338: In one embodiment, the
nucleic 99% identical to a sequence indicated in Table I, columns 5
or 7, lines 1 to 5 and/or lines 334 to 338. In another embodiment,
the nucleic acid molecule does not encode a polypeptide of a
sequence indicated in Table II, columns 5 or 7, lines 1 to 5 and/or
lines 334 to 338.
[0182] [0105.0.0.0] Unless otherwise specified, the terms
"polynucleotides", "nucleic acid" and "nucleic acid molecule" are
interchangeably in the present context. Unless otherwise specified,
the terms "peptide", "polypeptide" and "protein" are
interchangeably in the present context. The term "sequence" may
relate to polynucleotides, nucleic acids, nucleic acid molecules,
peptides, polypeptides and proteins, depending on the context in
which the term "sequence" is used. The terms "gene(s)",
"polynucleotide", "nucleic acid sequence", "nucleotide sequence",
or "nucleic acid molecule(s)" as used herein refers to a polymeric
form of nucleotides of any length, either ribonucleotides or
deoxyribonucleotides. The terms refer only to the primary structure
of the molecule.
[0183] [0106.0.0.0] Thus, The terms "gene(s)", "polynucleotide",
"nucleic acid sequence", "nucleotide sequence", or "nucleic acid
molecule(s)" as used herein include double- and single-stranded DNA
and RNA. They also include known types of modifications, for
example, methylation, "caps", substitutions of one or more of the
naturally occurring nucleotides with an analog. Preferably, the DNA
or RNA sequence of the invention comprises a coding sequence
encoding the herein defined polypeptide.
[0184] [0107.0.0.0] A "coding sequence" is a nucleotide sequence,
which is transcribed into mRNA and/or translated into a polypeptide
when placed under the control of appropriate regulatory sequences.
The boundaries of the coding sequence are determined by a
translation start codon at the 5'-terminus and a translation stop
codon at the 3'-terminus. A coding sequence can include, but is not
limited to mRNA, cDNA, recombinant nucleotide sequences or genomic
DNA, while introns may be present as well under certain
circumstances.
[0185] [0108.0.0.0] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 1 to 5 and/or lines 334
to 338, nucleic acid molecules which are derived from a amino acid
sequences as indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 or from polypeptides comprising the
consensus sequence as indicated in Table IV, columns 7, lines 1 to
5 and/or lines 334 to 338, or their derivatives or homologues
encoding polypeptides with the enzymatic or biological activity of
a polypeptide as indicated in Table II, column 3, 5 or 7, lines 1
to 5 and/or lines 334 to 338 or e.g. conferring a increase of the
respective fine chemical after increasing its expression or
activity are advantageously increased in the process according to
the invention.
[0186] [0109.0.0.0] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemical produced in the process
according to the invention.
[0187] [0110.0.0.0] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide of the invention or
the polypeptide used in the method of the invention or used in the
process of the invention, e.g. of a protein as indicated in Table
II, column 5, lines 1 to 5 and/or lines 334 to 338 or being encoded
by a nucleic acid molecule indicated in Table I, column 5, lines 1
to 5 and/or lines 334 to 338 or of its homologs, e.g. as indicated
in Table II, column 7, lines 1 to 5 and/or lines 334 to 338, can be
determined from generally accessible databases.
[0188] [0111.0.0.0] Those, which must be mentioned, in particular
in this context are general gene databases such as the EMBL
database (Stoesser G. et al., Nucleic Acids Res 2001, Vol. 29,
17-21), the GenBank database (Benson D. A. et al., Nucleic Acids
Res 2000, Vol. 28, 15-18), or the PIR database (Barker W. C. et
al., Nucleic Acids Res. 1999, Vol. 27, 39-43). It is furthermore
possible to use organism-specific gene databases for determining
advantageous sequences, in the case of yeast for example
advantageously the SGD database (Cherry J. M. et al., Nucleic Acids
Res. 1998, Vol. 26, 73-80) or the MIPS database (Mewes H. W. et
al., Nucleic Acids Res. 1999, Vol. 27, 44-48), in the case of E.
coli the GenProtEC database
(http://web.bham.ac.uk/bcm4ght6/res.html), and in the case of
Arabidopsis the TAIR-database (Huela, E. et al., Nucleic Acids Res.
2001 Vol. 29(1), 102-5) or the MIPS database.
[0189] [0112.0.0.0] The nucleic acid molecules used in the process
according to the invention take the form of isolated nucleic acid
sequences, which encode polypeptides with an activity of a
polypeptide as indicated in Table I, column 3, lines 1 to 5 and/or
lines 334 to 338 or having the sequence of a polypeptide as
indicated in Table II, columns 5 and 7, lines 1 to 5 and/or lines
334 to 338 and conferring an increase of the respective fine
chemical.
[0190] [0113.0.0.0] The nucleic acid sequence(s) used in the
process for the production of the respective fine chemical in
transgenic organisms originate advantageously from an eukaryote but
may also originate from a prokaryote or an archebacterium, thus it
can derived from e.g. a microorganism, an animal or a plant.
[0191] [0114.0.0.0] For the purposes of the invention, as a rule
the plural is intended to encompass the singular and vice
versa.
[0192] [0115.0.0.0] In order to improve the introduction of the
nucleic acid sequences and the expression of the sequences in the
transgenic organisms, which are used in the process, the nucleic
acid sequences are incorporated into a nucleic acid construct
and/or a vector. In addition to the herein described sequences
which are used in the process according to the invention, further
nucleic acid sequences, advantageously of biosynthesis genes of the
respective fine chemical produced in the process according to the
invention, may additionally be present in the nucleic acid
construct or in the vector and may be introduced into the organism
together. However, these additional sequences may also be
introduced into the organisms via other, separate nucleic acid
constructs or vectors.
[0193] [0116.0.0.0] Using the herein mentioned cloning vectors and
transformation methods such as those which are published and cited
in: Plant Molecular Biology and Biotechnology (CRC Press, Boca
Raton, Fla.), chapter 6/7, pp. 71-119 (1993); F. F. White, Vectors
for Gene Transfer in Higher Plants; in: Transgenic Plants, vol. 1,
Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press,
1993, 15-38; B. Jenes et al., Techniques for Gene Transfer, in:
Transgenic Plants, vol. 1, Engineering and Utilization, Ed.: Kung
and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu. Rev.
Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225)) and further
cited below, the nucleic acids may be used for the recombinant
modification of a wide range of organisms, in particular
prokaryotic or eukaryotic microorganisms or plants, so that they
become a better and more efficient producer of the respective fine
chemical produced in the process according to the invention. This
improved production, or production efficiency, of the respective
fine chemical or products derived there from, such as modified
proteins, can be brought about by a direct effect of the
manipulation or by an indirect effect of this manipulation.
[0194] [0117.0.0.0] In one embodiment, the nucleic acid molecule
according to the invention originates from a plant, such as a plant
selected from the families Aceraceae, Anacardiaceae, Apiaceae,
Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Euphorbiaceae,
Fabaceae, Malvaceae, Nymphaeaceae, Papaveraceae, Rosaceae,
Salicaceae, Solanaceae, Arecaceae, Bromeliaceae, Cyperaceae,
Iridaceae, Liliaceae, Orchidaceae, Gentianaceae, Labiaceae,
Magnoliaceae, Ranunculaceae, Carifolaceae, Rubiaceae,
Scrophulariaceae, Caryophyllaceae, Ericaceae, Polygonaceae,
Violaceae, Juncaceae or Poaceae and preferably from a plant
selected from the group of the families Apiaceae, Asteraceae,
Brassicaceae, Cucurbitaceae, Fabaceae, Papaveraceae, Rosaceae,
Solanaceae, Liliaceae or Poaceae. Preferred are crop plants and in
particular plants mentioned herein above as host plants such as the
families and genera mentioned above for example preferred the
species Anacardium occidentale, Calendula officinalis, Carthamus
tinctorius, Cichorium intybus, Cynara scolymus, Helianthus annus,
Tagetes lucida, Tagetes erecta, Tagetes tenuifolia; Daucus carota;
Corylus avellana, Corylus columa, Borago officinalis; Brassica
napus, Brassica rapa ssp., Sinapis arvensis Brassica juncea,
Brassica juncea var. juncea, Brassica juncea var. crispifolia,
Brassica juncea var. foliosa, Brassica nigra, Brassica sinapioides,
Melanosinapis communis, Brassica oleracea, Arabidopsis thaliana,
Anana comosus, Ananas ananas, Bromelia comosa, Carica papaya,
Cannabis sative, Ipomoea batatus, Ipomoea pandurata, Convolvulus
batatas, Convolvulus tiliaceus, Ipomoea fastigiata, Ipomoea
tiliacea, Ipomoea triloba, Convolvulus panduratus, Beta vulgaris,
Beta vulgaris var. altissima, Beta vulgaris var. vulgaris, Beta
maritima, Beta vulgaris var. perennis, Beta vulgaris var.
conditiva, Beta vulgaris var. esculenta, Cucurbita maxima,
Cucurbita mixta, Cucurbita pepo, Cucurbita moschata, Olea europaea,
Manihot utilissima, Janipha manihotJatropha manihot., Manihot
aipil, Manihot dulcis, Manihot manihot, Manihot melanobasis,
Manihot esculenta, Ricinus communis, Pisum sativum, Pisum arvense,
Pisum humile, Medicago sativa, Medicago falcata, Medicago varia,
Glycine max Dolichos soja, Glycine gracilis, Glycine hispida,
Phaseolus max, Soja hispida, Soja max, Cocos nucifera, Pelargonium
grossularioides, Oleum cocoas, Laurus nobilis, Persea americana,
Arachis hypogaea, Linum usitatissimum, Linum humile, Linum
austriacum, Linum bienne, Linum angustifolium, Linum catharticum,
Linum flavum, Linum grandiflorum, Adenolinum grandiflorum, Linum
lewisii, Linum narbonense, Linum perenne, Linum perenne var.
lewisii, Linum pratense, Linum trigynum, Punica granatum, Gossypium
hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium
herbaceum, Gossypium thurberi, Musa nana, Musa acuminata, Musa
paradisiaca, Musa spp., Elaeis guineensis, Papaver orientale,
Papaver rhoeas, Papaver dubium, Sesamum indicum, Piper aduncum,
Piper amalago, Piper angustifolium, Piper auritum, Piper betel,
Piper cubeba, Piper longum, Piper nigrum, Piper retrofractum,
Artanthe adunca, Artanthe elongata, Peperomia elongata, Piper
elongatum, Steffensia elongata, Hordeum vulgare, Hordeum jubatum,
Hordeum murinum, Hordeum secalinum, Hordeum distichon Hordeum
aegiceras, Hordeum hexastichon., Hordeum hexastichum, Hordeum
irregulare, Hordeum sativum, Hordeum secalinum, Avena sativa, Avena
fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida,
Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghum
vulgare, Andropogon drummondii, Holcus bicolor, Holcus sorghum,
Sorghum aethiopicum, Sorghum arundinaceum, Sorghum caffrorum,
Sorghum cernuum, Sorghum dochna, Sorghum drummondii, Sorghum durra,
Sorghum guineense, Sorghum lanceolatum, Sorghum nervosum, Sorghum
saccharatum, Sorghum subglabrescens, Sorghum verticilliflorum,
Sorghum vulgare, Holcus halepensis, Sorghum miliaceum millet,
Panicum militaceum, Zea mays, Triticum aestivum, Triticum durum,
Triticum turgidum, Triticum hybernum, Triticum macha, Triticum
sativum or Triticum vulgare, Cofea spp., Coffea arabica, Coffea
canephora, Coffea liberica, Capsicum annuum, Capsicum annuum var.
glabriusculum, Capsicum frutescens, Capsicum annuum, Nicotiana
tabacum, Solanum tuberosum, Solanum melongena, Lycopersicon
esculentum, Lycopersicon lycopersicum., Lycopersicon pyriforme,
Solanum integrifolium, Solanum lycopersicum Theobroma cacao or
Camellia sinensis.
[0195] [0118.0.0.0] In one embodiment, the nucleic acid molecule
sequence originates advantageously from a microorganism as
mentioned above under host organism such as a fungus for example
the genera Aspergillus, Penicillium or Claviceps or from yeasts
such as the genera Pichia, Torulopsis, Hansenula,
Schizosaccharomyces, Candida, Rhodotorula or Saccharomyces, very
especially advantageously from the yeast of the family
Saccharomycetaceae, such as the advantageous genus Saccharomyces
and the very advantageous genus and species Saccharomyces
cerevisiae for the production of the respective fine chemical in
microorganims.
[0196] [0119.0.0.0] The skilled worker knows other suitable sources
for the production of respective fine chemicals, which present also
useful nucleic acid molecule sources. They include in general all
prokaryotic or eukaryotic cells, preferably unicellular
microorganisms, such as fungi like the genus Claviceps or
Aspergillus or gram-positive bacteria such as the genera Bacillus,
Corynebacterium, Micrococcus, Brevibacterium, Rhodococcus,
Nocardia, Caseobacter or Arthrobacter or gram-negative bacteria
such as the genera Escherichia, Flavobacterium or Salmonella, or
yeasts such as the genera Rhodotorula, Hansenula or Candida.
[0197] [0120.0.0.0] Production strains which are especially
advantageously selected in the process according to the invention
are microorganisms selected from the group of the families
Actinomycetaceae, Bacillaceae, Brevibacteriaceae,
Corynebacteriaceae, Enterobacteriacae, Gordoniaceae,
Micrococcaceae, Mycobacteriaceae, Nocardiaceae, Pseudomonaceae,
Rhizobiaceae, Streptomycetaceae, Chaetomiaceae, Choanephoraceae,
Cryptococcaceae, Cunninghamellaceae, Demetiaceae, Moniliaceae,
Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae,
Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae,
Sporobolomycetaceae, Tuberculariaceae, Adelotheciaceae,
Dinophyceae, Ditrichaceae and Prasinophyceaeor of the genera and
species consisting of Hansenula anomala, Candida utilis, Claviceps
purpurea, Bacillus circulans, Bacillus subtilis, Bacillus sp.,
Brevibacterium albidum, Brevibacterium album, Brevibacterium
cerinum, Brevibacterium flavum, Brevibacterium glutamigenes,
Brevibacterium iodinum, Brevibacterium ketoglutamicum,
Brevibacterium lactofermentum, Brevibacterium linens,
Brevibacterium roseum, Brevibacterium saccharolyticum,
Brevibacterium sp., Corynebacterium acetoacidophilum,
Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes,
Corynebacterium glutamicum (=Micrococcus glutamicum),
Corynebacterium melassecola, Corynebacterium sp. or Escherichia
coli, specifically Escherichia coli K12 and its described
strains.
[0198] [0121.0.0.0] However, it is also possible to use artificial
sequences, which differ in one or more bases from the nucleic acid
sequences found in organisms, or in one or more amino acid
molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 or the
functional homologues thereof as described herein, preferably
conferring above-mentioned activity, i.e. conferring a increase of
the respective fine chemical after increasing its activity.
[0199] [0122.0.0.0] In the process according to the invention
nucleic acid sequences can be used, which, if appropriate, contain
synthetic, non-natural or modified nucleotide bases, which can be
incorporated into DNA or RNA. Said synthetic, non-natural or
modified bases can for example increase the stability of the
nucleic acid molecule outside or inside a cell. The nucleic acid
molecules of the invention can contain the same modifications as
aforementioned.
[0200] [0123.0.0.0] As used in the present context the term
"nucleic acid molecule" may also encompass the untranslated
sequence located at the 3' and at the 5' end of the coding gene
region, for example at least 500, preferably 200, especially
preferably 100, nucleotides of the sequence upstream of the 5' end
of the coding region and at least 100, preferably 50, especially
preferably 20, nucleotides of the sequence downstream of the 3' end
of the coding gene region. It is often advantageous only to choose
the coding region for cloning and expression purposes.
[0201] [0124.0.0.0] Preferably, the nucleic acid molecule used in
the process according to the invention or the nucleic acid molecule
of the invention is an isolated nucleic acid molecule.
[0202] [0125.0.0.0] An "isolated" polynucleotide or nucleic acid
molecule is separated from other polynucleotides or nucleic acid
molecules, which are present in the natural source of the nucleic
acid molecule. An isolated nucleic acid molecule may be a
chromosomal fragment of several kb, or preferably, a molecule only
comprising the coding region of the gene. Accordingly, an isolated
nucleic acid molecule of the invention may comprise chromosomal
regions, which are adjacent 5' and 3' or further adjacent
chromosomal regions, but preferably comprises no such sequences
which naturally flank the nucleic acid molecule sequence in the
genomic or chromosomal context in the organism from which the
nucleic acid molecule originates (for example sequences which are
adjacent to the regions encoding the 5'- and 3'-UTRs of the nucleic
acid molecule). In various embodiments, the isolated nucleic acid
molecule used in the process according to the invention may, for
example comprise less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1
kb, 0.5 kb or 0.1 kb nucleotide sequences which naturally flank the
nucleic acid molecule in the genomic DNA of the cell from which the
nucleic acid molecule originates.
[0203] [0126.0.0.0] The nucleic acid molecules used in the process,
for example the polynucleotides of the invention or of a part
thereof can be isolated using molecular-biological standard
techniques and the sequence information provided herein. Also, for
example a homologous sequence or homologous, conserved sequence
regions at the DNA or amino acid level can be identified with the
aid of comparison algorithms. The former can be used as
hybridization probes under standard hybridization techniques (for
example those described in Sambrook et al., Molecular Cloning: A
Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) for
isolating further nucleic acid sequences useful in this
process.
[0204] [0127.0.0.0] A nucleic acid molecule encompassing a complete
sequence of the nucleic acid molecules used in the process, for
example the polynucleotide of the invention, or a part thereof may
additionally be isolated by polymerase chain reaction,
oligonucleotide primers based on this sequence or on parts thereof
being used. For example, a nucleic acid molecule comprising the
complete sequence or part thereof can be isolated by polymerase
chain reaction using oligonucleotide primers which have been
generated on the basis of this sequence for example, mRNA can be
isolated from cells (for example by means of the guanidinium
thiocyanate extraction method of Chirgwin et al. (1979)
Biochemistry 18:5294-5299) and cDNA can be generated by means of
reverse transcriptase (for example Moloney MLV reverse
transcriptase, available from Gibco/BRL, Bethesda, Md., or AMV
reverse transcriptase, obtainable from Seikagaku America, Inc., St.
Petersburg, Fla).
[0205] [0128.0.0.0] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, columns 7,
lines 1 to 5 and/or lines 334 to 338, by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table I, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338 or the sequences derived from
sequences as indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338.
[0206] [0129.0.0.0] Moreover, it is possible to identify conserved
regions from various organisms by carrying out protein sequence
alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention or the polypeptide used in the method of the invention,
from which conserved regions, and in turn, degenerate primers can
be derived. Conserved region for the polypeptide of the invention
or the polypeptide used in the method of the invention are
indicated in the alignments shown in the figures. Conserved regions
are those, which show a very little variation in the amino acid
sequence in one particular position of several homologs from
different origin. The consensus sequences indicated in Table IV,
columns 7, lines 1 to 5 and/or lines 334 to 338 are derived from
said alignments.
[0207] [0130.0.0.0] Degenerated primers can then be utilized by PCR
for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of the
respective fine chemical after increasing its expression or
activity or further functional homologs of the polypeptide of the
invention or the polypeptide used in the method of the invention
from other organisms.
[0208] [0131.0.0.0] These fragments can then be utilized as
hybridization probe for isolating the complete gene sequence. As an
alternative, the missing 5' and 3' sequences can be isolated by
means of RACE-PCR (rapid amplification of cDNA ends). A nucleic
acid molecule according to the invention can be amplified using
cDNA or, as an alternative, genomic DNA as template and suitable
oligonucleotide primers, following standard PCR amplification
techniques. The nucleic acid molecule amplified thus can be cloned
into a suitable vector and characterized by means of DNA sequence
analysis. Oligonucleotides, which correspond to one of the nucleic
acid molecules used in the process, can be generated by standard
synthesis methods, for example using an automatic DNA
synthesizer.
[0209] [0132.0.0.0] Nucleic acid molecules which are advantageously
for the process according to the invention can be isolated based on
their homology to the nucleic acid molecules disclosed herein using
the sequences or part thereof as hybridization probe and following
standard hybridization techniques under stringent hybridization
conditions. In this context, it is possible to use, for example,
isolated nucleic acid molecules of at least 15, 20, 25, 30, 35, 40,
50, 60 or more nucleotides, preferably of at least 15, 20 or 25
nucleotides in length which hybridize under stringent conditions
with the above-described nucleic acid molecules, in particular with
those which encompass a nucleotide sequence of the nucleic acid
molecule used in the process of the invention or encoding a protein
used in the invention or of the nucleic acid molecule of the
invention. Nucleic acid molecules with 30, 50, 100, 250 or more
nucleotides may also be used.
[0210] [0133.0.0.0] The term "homology" means that the respective
nucleic acid molecules or encoded proteins are functionally and/or
structurally equivalent. The nucleic acid molecules that are
homologous to the nucleic acid molecules described above and that
are derivatives of said nucleic acid molecules are, for example,
variations of said nucleic acid molecules which represent
modifications having the same biological function, in particular
encoding proteins with the same or substantially the same
biological function. They may be naturally occurring variations,
such as sequences from other plant varieties or species, or
mutations. These mutations may occur naturally or may be obtained
by mutagenesis techniques. The allelic variations may be naturally
occurring allelic variants as well as synthetically produced or
genetically engineered variants. Structurally equivalents can, for
example, be identified by testing the binding of said polypeptide
to antibodies or computer based predictions. Structurally
equivalent have the similar immunological characteristic, e.g.
comprise similar epitopes.
[0211] [0134.0.0.0] By "hybridizing" it is meant that such nucleic
acid molecules hybridize under conventional hybridization
conditions, preferably under stringent conditions such as described
by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989)) or in Current Protocols in Molecular Biology, John
Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
[0212] [0135.0.0.0] According to the invention, DNA as well as RNA
molecules of the nucleic acid of the invention can be used as
probes. Further, as template for the identification of functional
homologues Northern blot assays as well as Southern blot assays can
be performed. The Northern blot assay advantageously provides
further information about the expressed gene product: e.g.
expression pattern, occurrence of processing steps, like splicing
and capping, etc. The Southern blot assay provides additional
information about the chromosomal localization and organization of
the gene encoding the nucleic acid molecule of the invention.
[0213] [0136.0.0.0] A preferred, nonlimiting example of stringent
hydridization conditions are hybridizations in 6.times. sodium
chloride/sodium citrate (=SSC) at approximately 45.degree. C.,
followed by one or more wash steps in 0.2.times.SSC, 0.1% SDS at 50
to 65.degree. C., for example at 50.degree. C., 55.degree. C. or
60.degree. C. The skilled worker knows that these hybridization
conditions differ as a function of the type of the nucleic acid
and, for example when organic solvents are present, with regard to
the temperature and concentration of the buffer. The temperature
under "standard hybridization conditions" differs for example as a
function of the type of the nucleic acid between 42.degree. C. and
58.degree. C., preferably between 45.degree. C. and 50.degree. C.
in an aqueous buffer with a concentration of 0.1.times. 0.5.times.,
1.times., 2.times., 3.times., 4.times. or 5.times.SSC (pH 7.2). If
organic solvent(s) is/are present in the abovementioned buffer, for
example 50% formamide, the temperature under standard conditions is
approximately 40.degree. C., 42.degree. C. or 45.degree. C. The
hybridization conditions for DNA:DNA hybrids are preferably for
example 0.1.times.SSC and 20.degree. C., 25.degree. C., 30.degree.
C., 35.degree. C., 40.degree. C. or 45.degree. C., preferably
between 30.degree. C. and 45.degree. C. The hybridization
conditions for DNA:RNA hybrids are preferably for example
0.1.times.SSC and 30.degree. C., 35.degree. C., 40.degree. C.,
45.degree. C., 50.degree. C. or 55.degree. C., preferably between
45.degree. C. and 55.degree. C. The abovementioned hybridization
temperatures are determined for example for a nucleic acid
approximately 100 bp (=base pairs) in length and a G+C content of
50% in the absence of formamide. The skilled worker knows to
determine the hybridization conditions required with the aid of
textbooks, for example the ones mentioned above, or from the
following textbooks: Sambrook et al., "Molecular Cloning", Cold
Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985,
"Nucleic Acids Hybridization: A Practical Approach", IRL Press at
Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential
Molecular Biology: A Practical Approach", IRL Press at Oxford
University Press, Oxford.
[0214] [0137.0.0.0] A further example of one such stringent
hybridization condition is hybridization at 4.times.SSC at
65.degree. C., followed by a washing in 0.1.times.SSC at 65.degree.
C. for one hour. Alternatively, an exemplary stringent
hybridization condition is in 50 formamide, 4.times.SSC at
42.degree. C. Further, the conditions during the wash step can be
selected from the range of conditions delimited by low-stringency
conditions (approximately 2.times.SSC at 50.degree. C.) and
high-stringency conditions (approximately 0.2.times.SSC at
50.degree. C., preferably at 65.degree. C.) (20.times.SSC: 0.3M
sodium citrate, 3M NaCl, pH 7.0). In addition, the temperature
during the wash step can be raised from low-stringency conditions
at room temperature, approximately 22.degree. C., to
higher-stringency conditions at approximately 65.degree. C. Both of
the parameters salt concentration and temperature can be varied
simultaneously, or else one of the two parameters can be kept
constant while only the other is varied. Denaturants, for example
formamide or SDS, may also be employed during the hybridization. In
the presence of 50% formamide, hybridization is preferably effected
at 42.degree. C. Relevant factors like i) length of treatment, ii)
salt conditions, iii) detergent conditions, iv) competitor DNAs, v)
temperature and vi) probe selection can be combined case by case so
that not all possibilities can be mentioned herein.
[0215] Thus, in a preferred embodiment, Northern blots are
prehybridized with Rothi-Hybri-Quick buffer (Roth, Karlsruhe) at
68.degree. C. for 2 h. Hybridization with radioactive labelled
probe is done overnight at 68.degree. C. Subsequent washing steps
are performed at 68.degree. C. with 1.times.SSC.
[0216] For Southern blot assays the membrane is prehybridized with
Rothi-Hybri-Quick buffer (Roth, Karlsruhe) at 68.degree. C. for 2
h. The hybridization with radioactive labelled probe is conducted
over night at 68.degree. C. Subsequently the hybridization buffer
is discarded and the filter shortly washed using 2.times.SSC; 0.1%
SDS. After discarding the washing buffer new 2.times.SSC; 0.1% SDS
buffer is added and incubated at 68.degree. C. for 15 minutes. This
washing step is performed twice followed by an additional washing
step using 1.times.SSC; 0.1% SDS at 68.degree. C. for 10 min.
[0217] [0138.0.0.0] Some further examples of conditions for DNA
hybridization (Southern blot assays) and wash step are shown herein
below: [0218] (1) Hybridization conditions can be selected, for
example, from the following conditions: [0219] a) 4.times.SSC at
65.degree. C., [0220] b) 6.times.SSC at 45.degree. C., [0221] c)
6.times.SSC, 100 mg/ml denatured fragmented fish sperm DNA at
68.degree. C., [0222] d) 6.times.SSC, 0.5% SDS, 100 mg/ml denatured
salmon sperm DNA at 68.degree. C., [0223] e) 6.times.SSC, 0.5% SDS,
100 mg/ml denatured fragmented salmon sperm DNA, 50% formamide at
42.degree. C., [0224] f) 50% formamide, 4.times.SSC at 42.degree.
C., [0225] g) 50% (vol/vol) formamide, 0.1% bovine serum albumin,
0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate
buffer pH 6.5, 750 mM NaCl, 75 mM sodium citrate at 42.degree. C.,
[0226] h) 2.times. or 4.times.SSC at 50.degree. C. (low-stringency
condition), or [0227] i) 30 to 40% formamide, 2.times. or
4.times.SSC at 42.degree. C. (low-stringency condition). [0228] (2)
Wash steps can be selected, for example, from the following
conditions: [0229] a) 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS
at 50.degree. C. [0230] b) 0.1.times.SSC at 65.degree. C. [0231] c)
0.1.times.SSC, 0.5% SDS at 68.degree. C. [0232] d) 0.1.times.SSC,
0.5% SDS, 50% formamide at 42.degree. C. [0233] e) 0.2.times.SSC,
0.1% SDS at 42.degree. C. [0234] f) 2.times.SSC at 65.degree. C.
(low-stringency condition).
[0235] [0139.0.0.0] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical increase, derived from
other organisms, can be encoded by other DNA sequences which
hybridize to a sequences indicated in Table I, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338, preferably of Table I B,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 under relaxed
hybridization conditions and which code on expression for peptides
having the methionine increasing activity.
[0236] [0140.0.0.0] Further, some applications have to be performed
at low stringency hybridisation conditions, without any
consequences for the specificity of the hybridisation. For example,
a Southern blot analysis of total DNA could be probed with a
nucleic acid molecule of the present invention and washed at low
stringency (55.degree. C. in 2.times.SSPE0.1% SDS). The
hybridisation analysis could reveal a simple pattern of only genes
encoding polypeptides of the present invention or used in the
process of the invention, e.g. having herein-mentioned activity of
increasing the respective fine chemical. A further example of such
low-stringent hybridization conditions is 4.times.SSC at 50.degree.
C. or hybridization with 30 to 40% formamide at 42.degree. C. Such
molecules comprise those which are fragments, analogues or
derivatives of the polypeptide of the invention or used in the
process of the invention and differ, for example, by way of amino
acid and/or nucleotide deletion(s), insertion(s), substitution (s),
addition(s) and/or recombination (s) or any other modification(s)
known in the art either alone or in combination from the
above-described amino acid sequences or their underlying nucleotide
sequence(s). However, it is preferred to use high stringency
hybridisation conditions.
[0237] [0141.0.0.0] Hybridization should advantageously be carried
out with fragments of at least 5, 10, 15, 20, 25, 30, 35 or 40 bp,
advantageously at least 50, 60, 70 or 80 bp, preferably at least
90, 100 or 110 bp. Most preferably are fragments of at least 15,
20, 25 or 30 bp. Preferably are also hybridizations with at least
100 bp or 200, very especially preferably at least 400 bp in
length. In an especially preferred embodiment, the hybridization
should be carried out with the entire nucleic acid sequence with
conditions described above.
[0238] [0142.0.0.0] The terms "fragment", "fragment of a sequence"
or "part of a sequence" mean a truncated sequence of the original
sequence referred to. The truncated sequence (nucleic acid or
protein sequence) can vary widely in length; the minimum size being
a sequence of sufficient size to provide a sequence with at least a
comparable function and/or activity of the original sequence
referred to or hybridising with the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention or used in the process of the invention under stringent
conditions, while the maximum size is not critical. In some
applications, the maximum size usually is not substantially greater
than that required to provide the desired activity and/or
function(s) of the original sequence.
[0239] [0143.0.0.0] Typically, the truncated amino acid sequence
will range from about 5 to about 310 amino acids in length. More
typically, however, the sequence will be a maximum of about 250
amino acids in length, preferably a maximum of about 200 or 100
amino acids. It is usually desirable to select sequences of at
least about 10, 12 or 15 amino acids, up to a maximum of about 20
or 25 amino acids.
[0240] [0144.0.0.0] The term "epitope" relates to specific
immunoreactive sites within an antigen, also known as antigenic
determinates. These epitopes can be a linear array of monomers in a
polymeric composition--such as amino acids in a protein--or consist
of or comprise a more complex secondary or tertiary structure.
Those of skill will recognize that immunogens (i.e., substances
capable of eliciting an immune response) are antigens; however,
some antigen, such as haptens, are not immunogens but may be made
immunogenic by coupling to a carrier molecule. The term "antigen"
includes references to a substance to which an antibody can be
generated and/or to which the antibody is specifically
immunoreactive.
[0241] [0145.0.0.0] In one embodiment the present invention relates
to a epitope of the polypeptide of the present invention or used in
the process of the present invention and conferring above mentioned
activity, preferably conferring an increase in the respective fine
chemical.
[0242] [0146.0.0.0] The term "one or several amino acids" relates
to at least one amino acid but not more than that number of amino
acids, which would result in a homology of below 50% identity.
Preferably, the identity is more than 70% or 80%, more preferred
are 85%, 90%, 91%, 92%, 93%, 94% or 95%, even more preferred are
96%, 97%, 98%, or 99% identity.
[0243] [0147.0.0.0] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338, preferably
of Table I B, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338
is one which is sufficiently complementary to one of said
nucleotide sequences such that it can hybridize to one of said
nucleotide sequences thereby forming a stable duplex. Preferably,
the hybridisation is performed under stringent hybridization
conditions. However, a complement of one of the herein disclosed
sequences is preferably a sequence complement thereto according to
the base pairing of nucleic acid molecules well known to the
skilled person. For example, the bases A and G undergo base pairing
with the bases T and U or C, resp. and visa versa. Modifications of
the bases can influence the base-pairing partner.
[0244] [0148.0.0.0] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines 1
to 5 and/or lines 334 to 338, preferably of Table I B, columns 5 or
7, lines 1 to 5 and/or lines 334 to 338, or a functional portion
thereof and preferably has above mentioned activity, in particular
has the--fine-chemical-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homologs.
[0245] [0149.0.0.0] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridises, preferably
hybridises under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338, preferably of Table I B,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 or a portion
thereof and encodes a protein having above-mentioned activity and
as indicated in indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338, preferably of Table II B, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338, e.g. conferring an increase
of the respective fine chemical.
[0246] [0149.1.0.0] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338, preferably of
Table I B, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 has
further one or more of the activities annotated or known for the a
protein as indicated in Table II, column 3, lines 1 to 5 and/or
lines 334 to 338, preferably of Table II B, columns 3, lines 1 to 5
and/or lines 334 to 338.
[0247] [0150.0.0.0] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table I, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338,
preferably of Table I B, columns 5 or 7, lines 1 to 5 and/or lines
334 to 338, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of methionine if its activity
is increased. The nucleotide sequences determined from the cloning
of the present protein-according-to-the-invention-encoding gene
allows for the generation of probes and primers designed for use in
identifying and/or cloning its homologues in other cell types and
organisms. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, 15 preferably about 20 or 25, more
preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one of the sequences indicated in Table I, columns 5 or
7, lines 1 to 5 and/or lines 334 to 338, an anti-sense sequence of
one of the sequences indicated in Table I, columns 5 or 7, lines 1
to 5 and/or lines 334 to 338, or naturally occurring mutants
thereof. Primers based on a nucleotide sequence of the invention
can be used in PCR reactions to clone homologues of the polypeptide
of the invention or of the polypeptide used in the process of the
invention, e.g. as the primers described in the examples of the
present invention, e.g. as shown in the examples. A PCR with the
primer pairs indicated in Table III, column 7, lines 1 to 5 and/or
lines 334 to 338 will result in a fragment of a polynucleotide
sequence as indicated in Table I, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338. Preferred is Table II B, columns 7, lines
1 to 5 and/or lines 334 to 338.
[0248] [0151.0.0.0] Primer sets are interchangeable. The person
skilled in the art knows to combine said primers to result in the
desired product, e.g. in a full-length clone or a partial sequence.
Probes based on the sequences of the nucleic acid molecule of the
invention or used in the process of the present invention can be
used to detect transcripts or genomic sequences encoding the same
or homologous proteins. The probe can further comprise a label
group attached thereto, e.g. the label group can be a radioisotope,
a fluorescent compound, an enzyme, or an enzyme co-factor. Such
probes can be used as a part of a genomic marker test kit for
identifying cells which express an polypeptide of the invention or
used in the process of the present invention, such as by measuring
a level of an encoding nucleic acid molecule in a sample of cells,
e.g., detecting mRNA levels or determining, whether a genomic gene
comprising the sequence of the polynucleotide of the invention or
used in the processes of the present invention has been mutated or
deleted.
[0249] [0152.0.0.0] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 such that the protein or portion thereof
maintains the ability to participate in the respective fine
chemical production, in particular a methionine increasing activity
as mentioned above or as described in the examples in plants or
microorganisms is comprised.
[0250] [0153.0.0.0] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 1 to
5 and/or lines 334 to 338 such that the protein or portion thereof
is able to participate in the increase of the respective fine
chemical production. In one embodiment, a protein or portion
thereof as indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 has for example an activity of a
polypeptide indicated in Table II, column 3, lines 1 to 5 and/or
lines 334 to 338.
[0251] [0154.0.0.0] In one embodiment, the nucleic acid molecule of
the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 and has above-mentioned activity, e.g.
conferring preferably the increase of the respective fine
chemical.
[0252] [0155.0.0.0] Portions of proteins encoded by the nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention are preferably biologically active,
preferably having above-mentioned annotated activity, e.g.
conferring a increase the respective fine chemical after increase
of activity.
[0253] [0156.0.0.0] As mentioned herein, the term "biologically
active portion" is intended to include a portion, e.g., a
domain/motif, that confers increase of the respective fine chemical
or has an immunological activity such that it is binds to an
antibody binding specifically to the polypeptide of the present
invention or a polypeptide used in the process of the present
invention for producing the respective fine chemical;
[0254] [0157.0.0.0] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 1 to 5 and/or lines 334
to 338 (and portions thereof) due to degeneracy of the genetic code
and thus encode a polypeptide of the present invention, in
particular a polypeptide having above mentioned activity, e.g.
conferring an increase in the respective fine chemical in a
organism, e.g. as that polypeptides comprising the consensus
sequences as indicated in Table IV, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 or of the polypeptide as indicated in Table
II, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 or their
functional homologues. Advantageously, the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, a consensus sequences as indicated in Table IV,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 or of the
polypeptide as indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 or the functional homologues. In a still
further embodiment, the nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
encodes a full length protein which is substantially homologous to
an amino acid sequence comprising a consensus sequence as indicated
in Table IV, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338
or of a polypeptide as indicated in Table II, columns 5 or 7, lines
1 to 5 and/or lines 334 to 338 or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table I, columns 5 or 7, lines 1 to 5 and/or lines 334
to 338, preferably as indicated in Table I A, columns 5 or 7, lines
1 to 5 and/or lines 334 to 338. Preferably the nucleic acid
molecule of the invention is a functional homologue or identical to
a nucleic acid molecule indicated in Table I B, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338.
[0255] [0158.0.0.0] In addition, it will be appreciated by those
skilled in the art that DNA sequence polymorphisms that lead to
changes in the amino acid sequences may exist within a population.
Such genetic polymorphism in the gene encoding the polypeptide of
the invention or the polypeptide used in the method of the
invention or comprising the nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
may exist among individuals within a population due to natural
variation.
[0256] [0159.0.0.0] As used herein, the terms "gene" and
"recombinant gene" refer to nucleic acid molecules comprising an
open reading frame encoding the polypeptide of the invention or the
polypeptide used in the method of the invention or comprising the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention or encoding the polypeptide
used in the process of the present invention, preferably from a
crop plant or from a microorganism useful for the production of
respective fine chemicals, in particular for the production of the
respective fine chemical. Such natural variations can typically
result in 1-5% variance in the nucleotide sequence of the gene. Any
and all such nucleotide variations and resulting amino acid
polymorphisms in genes encoding a polypeptide of the invention or
the polypeptide used in the method of the invention or comprising a
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention that are the result of
natural variation and that do not alter the functional activity as
described are intended to be within the scope of the invention.
[0257] [0160.0.0.0] Nucleic acid molecules corresponding to natural
variants homologues of a nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention,
which can also be a cDNA, can be isolated based on their homology
to the nucleic acid molecules disclosed herein using the nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention, or a portion thereof, as a
hybridization probe according to standard hybridization techniques
under stringent hybridization conditions.
[0258] [0161.0.0.0] Accordingly, in another embodiment, a nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338. The nucleic acid molecule is preferably at
least 20, 30, 50, 100, 250 or more nucleotides in length.
[0259] [0162.0.0.0] The term "hybridizes under stringent
conditions" is defined above. In one embodiment, the term
"hybridizes under stringent conditions" is intended to describe
conditions for hybridization and washing under which nucleotide
sequences at least 30%, 40%, 50% or 65% identical to each other
typically remain hybridized to each other. Preferably, the
conditions are such that sequences at least about 70%, more
preferably at least about 75% or 80%, and even more preferably at
least about 85%, 90% or 95% or more identical to each other
typically remain hybridized to each other.
[0260] [0163.0.0.0] Preferably, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 1 to 5 and/or lines
334 to 338 corresponds to a naturally-occurring nucleic acid
molecule of the invention. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein). Preferably, the nucleic acid molecule encodes a natural
protein having above-mentioned activity, e.g. conferring the
respective fine chemical increase after increasing the expression
or activity thereof or the activity of an protein of the invention
or used in the process of the invention.
[0261] [0164.0.0.0] In addition to naturally-occurring variants of
the sequences of the polypeptide or nucleic acid molecule of the
invention as well as of the polypeptide or nucleic acid molecule
used in the process of the invention that may exist in the
population, the skilled artisan will further appreciate that
changes can be introduced by mutation into a nucleotide sequence of
the nucleic acid molecule encoding the polypeptide of the invention
or used in the process of the present invention, thereby leading to
changes in the amino acid sequence of the encoded said polypeptide,
without altering the functional ability of the polypeptide,
preferably not decreasing said activity.
[0262] [0165.0.0.0] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 1 to 5 and/or lines 334
to 338.
[0263] [0166.0.0.0] A "non-essential" amino acid residue is a
residue that can be altered from the wild-type sequence of one
without altering the activity of said polypeptide, whereas an
"essential" amino acid residue is required for an activity as
mentioned above, e.g. leading to an increase in the respective fine
chemical in an organism after an increase of activity of the
polypeptide. Other amino acid residues, however, (e.g., those that
are not conserved or only semi-conserved in the domain having said
activity) may not be essential for activity and thus are likely to
be amenable to alteration without altering said activity.
[0264] [0167.0.0.0] Further, a person skilled in the art knows that
the codon usage between organism can differ. Therefore, he may
adapt the codon usage in the nucleic acid molecule of the present
invention to the usage of the organism in which the polynucleotide
or polypeptide is expressed.
[0265] [0168.0.0.0] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338, preferably of Table II B,
column 7, lines 1 to 5 and/or lines 334 to 338 yet retain said
activity described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table II, columns 5 or 7, lines
1 to 5 and/or lines 334 to 338, preferably of Table II B, column 7,
lines 1 to 5 and/or lines 334 to 338 and is capable of
participation in the increase of production of the respective fine
chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table II,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338, preferably of
Table II B, column 7, lines 1 to 5 and/or lines 334 to 338, more
preferably at least about 70% identical to one of the sequences as
indicated in Table II, columns 5 or 7, lines 1 to 5 and/or lines
334 to 338, preferably of Table II B, column 7, lines 1 to 5 and/or
lines 334 to 338, even more preferably at least about 80%, 90%, or
95% homologous to a sequence as indicated in Table II, columns 5 or
7, lines 1 to 5 and/or lines 334 to 338, preferably of Table II B,
column 7, lines 1 to 5 and/or lines 334 to 338, and most preferably
at least about 96%, 97%, 98%, or 99% identical to the sequence as
indicated in Table II, columns 5 or 7, lines 1 to 5 and/or lines
334 to 338, preferably of Table II B, column 7, lines 1 to 5 and/or
lines 334 to 338.
[0266] [0169.0.0.0] To determine the percentage homology
(=identity) of two amino acid sequences or of two nucleic acid
molecules, the sequences are written one underneath the other for
an optimal comparison (for example gaps may be inserted into the
sequence of a protein or of a nucleic acid in order to generate an
optimal alignment with the other protein or the other nucleic
acid).
[0267] [0170.0.0.0] The amino acid residues or nucleic acid
molecules at the corresponding amino acid positions or nucleotide
positions are then compared. If a position in one sequence is
occupied by the same amino acid residue or the same nucleic acid
molecule as the corresponding position in the other sequence, the
molecules are homologous at this position (i.e. amino acid or
nucleic acid "homology" as used in the present context corresponds
to amino acid or nucleic acid "identity". The percentage homology
between the two sequences is a function of the number of identical
positions shared by the sequences (i.e. % homology=number of
identical positions/total number of positions.times.100). The terms
"homology" and "identity" are thus to be considered as
synonyms.
[0268] [0171.0.0.0] For the determination of the percentage
homology (=identity) of two or more amino acids or of two or more
nucleotide sequences several computer software programs have been
developed. The homology of two or more sequences can be calculated
with for example the software fasta, which presently has been used
in the version fasta 3 (W. R. Pearson and D. J. Lipman (1988),
Improved Tools for Biological Sequence Comparison. PNAS
85:2444-2448; W. R. Pearson (1990) Rapid and Sensitive Sequence
Comparison with FASTP and FASTA, Methods in Enzymology 183:63-98;
W. R. Pearson and D. J. Lipman (1988) Improved Tools for Biological
Sequence Comparison. PNAS 85:2444-2448; W. R. Pearson (1990); Rapid
and Sensitive Sequence Comparison with FASTP and FASTA Methods in
Enzymology 183:63-98). Another useful program for the calculation
of homologies of different sequences is the standard blast program,
which is included in the Biomax pedant software (Biomax, Munich,
Federal Republic of Germany). This leads unfortunately sometimes to
suboptimal results since blast does not always include complete
sequences of the subject and the query. Nevertheless as this
program is very efficient it can be used for the comparison of a
huge number of sequences. The following settings are typically used
for such a comparisons of sequences: -p Program Name [String]; -d
Database [String]; default=nr; -i Query File [File In];
default=stdin; -e Expectation value (E) [Real]; default=10.0; -m
alignment view options: 0=pairwise; 1=query-anchored showing
identities; 2=query-anchored no identities; 3=flat query-anchored,
show identities; 4=flat query-anchored, no identities;
5=query-anchored no identities and blunt ends; 6=flat
query-anchored, no identities and blunt ends; 7=XML Blast output;
8=tabular; 9 tabular with comment lines [Integer]; default=0; -o
BLAST report Output File [File Out] Optional; default=stdout; -F
Filter query sequence (DUST with blastn, SEG with others) [String];
default=T; -G Cost to open a gap (zero invokes default behavior)
[Integer]; default=0; -E Cost to extend a gap (zero invokes default
behavior) [Integer]; default=0; -X X dropoff value for gapped
alignment (in bits) (zero invokes default behavior); blastn 30,
megablast 20, tblastx 0, all others 15 [Integer]; default=0; -I
Show GI's in deflines [T/F]; default=F; -q Penalty for a nucleotide
mismatch (blastn only) [Integer]; default=-3; -r Reward for a
nucleotide match (blastn only) [Integer]; default=1; -v Number of
database sequences to show one-line descriptions for (V) [Integer];
default=500; -b Number of database sequence to show alignments for
(B) [Integer]; default=250; -f Threshold for extending hits,
default if zero; blastp 11, blastn 0, blastx 12, tblastn 13;
tblastx 13, megablast 0 [Integer]; default=0; -g Perfom gapped
alignment (not available with tblastx) [T/F]; default=T; -Q Query
Genetic code to use [Integer]; default=1; -D DB Genetic code (for
tblast[nx] only) [Integer]; default=1; -a Number of processors to
use [Integer]; default=1; -O Sec:Align file [File Out] Optional; -J
Believe the query defline [T/F]; default=F; -M Matrix [String];
default=BLOSUM62; -W Word size, default if zero (blastn 11,
megablast 28, all others 3) [Integer]; default=0; -z Effective
length of the database (use zero for the real size) [Real];
default=0; -K Number of best hits from a region to keep (off by
default, if used a value of 100 is recommended) [Integer];
default=0; -P 0 for multiple hit, 1 for single hit [Integer];
default=0; -Y Effective length of the search space (use zero for
the real size) [Real]; default=0; -S Query strands to search
against database (for blast[nx], and tblastx); 3 is both, 1 is top,
2 is bottom [Integer]; default=3; -T Produce HTML output [T/F];
default=F; -I Restrict search of database to list of GI's [String]
Optional; -U Use lower case filtering of FASTA sequence [T/F]
Optional; default=F; -y X dropoff value for ungapped extensions in
bits (0.0 invokes default behavior); blastn 20, megablast 10, all
others 7 [Real]; default=0.0; -Z X dropoff value for final gapped
alignment in bits (0.0 invokes default behavior); blastn/megablast
50, tblastx 0, all others 25 [Integer]; default=0; -R PSI-TBLASTN
checkpoint file [File In] Optional; -n MegaBlast search [T/F];
default=F; -L Location on query sequence [String] Optional; -A
Multiple Hits window size, default if zero (blastn/megablast 0, all
others 40 [Integer]; default=0; -w Frame shift penalty (OOF
algorithm for blastx) [Integer]; default=0; -t Length of the
largest intron allowed in tblastn for linking HSPs (0 disables
linking) [Integer]; default=0.
[0269] [0172.0.0.0] Results of high quality are reached by using
the algorithm of Needleman and Wunsch or Smith and Waterman.
Therefore programs based on said algorithms are preferred.
Advantageously the comparisons of sequences can be done with the
program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et
al., CABIOS, 5 1989: 151-153) or preferably with the programs Gap
and BestFit, which are respectively based on the algorithms of
Needleman and Wunsch [J. Mol. Biol. 48; 443-453 (1970)] and Smith
and Waterman [Adv. Appl. Math. 2; 482-489 (1981)]. Both programs
are part of the GCG software-package [Genetics Computer Group, 575
Science Drive, Madison, Wis., USA 53711 (1991); Altschul et al.
(1997) Nucleic Acids Res. 25:3389 et seq.]. Therefore preferably
the calculations to determine the percentages of sequence homology
are done with the program Gap over the whole range of the
sequences. The following standard adjustments for the comparison of
nucleic acid sequences were used: gap weight: 50, length weight: 3,
average match: 10.000, average mismatch: 0.000.
[0270] [0173.0.0.0] For example a sequence which has a 80% homology
with sequence SEQ ID NO: 1 at the nucleic acid level is understood
as meaning a sequence which, upon comparison with the sequence SEQ
ID NO: 1 by the above Gap program algorithm with the above
parameter set, has a 80% homology.
[0271] [0174.0.0.0] In the state of the art, homology between two
polypeptides is also understood as meaning the identity of the
amino acid sequence over in each case the entire sequence length
which is calculated by comparison with the aid of the program
algorithm GAP (Wisconsin Package Version 10.0, University of
Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting
the following parameters:
TABLE-US-00001 Gap weight: 8 Length weight: 2 Average match: 2,912
Average mismatch: -2,003
[0272] [0175.0.0.0] For example a sequence which has a 80% homology
with sequence SEQ ID NO: 2 at the protein level is understood as
meaning a sequence which, upon comparison with the sequence SEQ ID
NO: 2 by the above program algorithm with the above parameter set,
has a 80% homology.
[0273] [0176.0.0.0] Functional equivalents derived from one of the
polypeptides as indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 according to
the invention and are distinguished by essentially the same
properties as a polypeptide as indicated in Table II, columns 5 or
7, lines 1 to 5 and/or lines 334 to 338.
[0274] [0177.0.0.0] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338, preferably of Table I B, column 7, lines 1
to 5 and/or lines 334 to 338 according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of a polypeptide as indicated in Table II,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 according to
the invention and encode polypeptides having essentially the same
properties as a polypeptide as indicated in Table II, columns 5 or
7, lines 1 to 5 and/or lines 334 to 338, preferably of Table II B,
column 7, lines 1 to 5 and/or lines 334 to 338.
[0275] [0178.0.0.0] "Essentially the same properties" of a
functional equivalent is above all understood as meaning that the
functional equivalent has above mentioned activity, e.g. conferring
an increase in the respective fine chemical amount while increasing
the amount of protein, activity or function of said functional
equivalent in an organism, e.g. a microorganism, a plant or plant
or animal tissue, plant or animal cells or a part of the same.
[0276] [0179.0.0.0] A nucleic acid molecule encoding a homologous
to a protein sequence of as indicated in Table II, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338, preferably of Table II B,
column 7, lines 1 to 5 and/or lines 334 to 338 can be created by
introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of the nucleic acid molecule
of the present invention, in particular as indicated in Table I,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 such that one
or more amino acid substitutions, additions or deletions are
introduced into the encoded protein. Mutations can be introduced
into the encoding sequences for example into sequences as indicated
in Table I, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 by
standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[0277] [0180.0.0.0] Preferably, conservative amino acid
substitutions are made at one or more predicted non-essential amino
acid residues. A "conservative amino acid substitution" is one in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0278] [0181.0.0.0] Thus, a predicted nonessential amino acid
residue in a polypeptide of the invention or a polypeptide used in
the process of the invention is preferably replaced with another
amino acid residue from the same family. Alternatively, in another
embodiment, mutations can be introduced randomly along all or part
of a coding sequence of a nucleic acid molecule of the invention or
used in the process of the invention, such as by saturation
mutagenesis, and the resultant mutants can be screened for activity
described herein to identify mutants that retain or even have
increased above mentioned activity, e.g. conferring an increase in
content of the respective fine chemical.
[0279] [0182.0.0.0] Following mutagenesis of one of the sequences
shown herein, the encoded protein can be expressed recombinantly
and the activity of the protein can be determined using, for
example, assays described herein (see Examples).
[0280] [0183.0.0.0] The highest homology of the nucleic acid
molecule used in the process according to the invention was found
for the following database entries by Gap search.
[0281] [0184.0.0.0] Homologues of the nucleic acid sequences used,
with a sequence as indicated in Table I, columns 5 or 7, lines 1 to
5 and/or lines 334 to 338, preferably of Table I B, column 7, lines
1 to 5 and/or lines 334 to 338, or of the nucleic acid sequences
derived from a sequences as indicated in Table II, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338, preferably of Table II B,
column 7, lines 1 to 5 and/or lines 334 to 338, comprise also
allelic variants with at least approximately 30%, 35%, 40% or 45%
homology, by preference at least approximately 50%, 60% or 70%,
more preferably at least approximately 90%, 91%, 92%, 93%, 94% or
95% and even more preferably at least approximately 96%, 97%, 98%,
99% or more homology with one of the nucleotide sequences shown or
the abovementioned derived nucleic acid sequences or their
homologues, derivatives or analogues or parts of these. Allelic
variants encompass in particular functional variants which can be
obtained by deletion, insertion or substitution of nucleotides from
the sequences shown, preferably from a sequence as indicated in
Table I, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338, or
from the derived nucleic acid sequences, the intention being,
however, that the enzyme activity or the biological activity of the
resulting proteins synthesized is advantageously retained or
increased.
[0282] [0185.0.0.0] In one embodiment of the present invention, the
nucleic acid molecule of the invention or used in the process of
the invention comprises one or more sequences as indicated in Table
I, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338, preferably
of Table I B, column 7, lines 1 to 5 and/or lines 334 to 338. In
one embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotide sequences not
shown in any one of sequences as indicated in Table I, columns 5 or
7, lines 1 to 5 and/or lines 334 to 338, preferably of Table I B,
column 7, lines 1 to 5 and/or lines 334 to 338. In one embodiment,
the nucleic acid molecule comprises less than 500, 400, 300, 200,
100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a further
embodiment, the nucleic acid molecule comprises less than 30, 20 or
10 further nucleotides. In one embodiment, a nucleic acid molecule
used in the process of the invention is identical to a sequences as
indicated in Table I, columns 5 or 7, lines 1 to 5 and/or lines 334
to 338, preferably of Table I B, column 7, lines 1 to 5 and/or
lines 334 to 338.
[0283] [0186.0.0.0] Also preferred is that one or more nucleic acid
molecule(s) used in the process of the invention encode a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 1 to 5 and/or lines 334 to 338, preferably of Table
II B, column 7, lines 1 to 5 and/or lines 334 to 338. In one
embodiment, the nucleic acid molecule encodes less than 150, 130,
100, 80, 60, 50, 40 or 30 further amino acids. In a further
embodiment, the encoded polypeptide comprises less than 20, 15, 10,
9, 8, 7, 6 or 5 further amino acids. In one embodiment, the encoded
polypeptide used in the process of the invention is identical to
the sequences as indicated in Table II, columns 5 or 7, lines 1 to
5 and/or lines 334 to 338, preferably of Table II B, column 7,
lines 1 to 5 and/or lines 334 to 338.
[0284] [0187.0.0.0] In one embodiment, the nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table II, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338, preferably of Table II B,
column 7, lines 1 to 5 and/or lines 334 to 338 comprises less than
100 further nucleotides. In a further embodiment, said nucleic acid
molecule comprises less than 30 further nucleotides. In one
embodiment, the nucleic acid molecule used in the process is
identical to a coding sequence encoding a sequences as indicated in
Table II, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338,
preferably of Table II B, column 7, lines 1 to 5 and/or lines 334
to 338.
[0285] [0188.0.0.0] Polypeptides (=proteins), which still have the
essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338,
preferably compared to a sequence as indicated in Table II, column
3 and 5, lines 1 to 5 and/or lines 334 to 338, and expressed under
identical conditions.
[0286] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, columns 7, lines 1 to 5 and/or lines 334 to 338.
[0287] [0189.0.0.0] Homologues of a sequence as indicated in Table
I, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 or of a
derived sequence as indicated in Table II, columns 5 or 7, lines 1
to 5 and/or lines 334 to 338 also mean truncated sequences, cDNA,
single-stranded DNA or RNA of the coding and noncoding DNA
sequence. Homologues of said sequences are also understood as
meaning derivatives, which comprise noncoding regions such as, for
example, UTRs, terminators, enhancers or promoter variants. The
promoters upstream of the nucleotide sequences stated can be
modified by one or more nucleotide substitution(s), insertion(s)
and/or deletion(s) without, however, interfering with the
functionality or activity either of the promoters, the open reading
frame (=ORF) or with the 3'-regulatory region such as terminators
or other 3' regulatory regions, which are far away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[0288] [0190.0.0.0] In a further embodiment, the process according
to the present invention comprises the following steps: [0289] (a)
selecting an organism or a part thereof expressing the polypeptide
of this invention; [0290] (b) mutagenizing the selected organism or
the part thereof; [0291] (c) comparing the activity or the
expression level of said polypeptide in the mutagenized organism or
the part thereof with the activity or the expression of said
polypeptide in the selected organisms or the part thereof; [0292]
(d) selecting the mutagenized organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism (a) or the part
thereof; [0293] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [0294] (f) recovering, and
optionally isolating, the free or bound respective fine chemical
produced by the selected mutated organisms or parts thereof.
[0295] [0191.0.0.0] The organisms or part thereof produce according
to the herein mentioned process of the invention an increased level
of free and/or -bound respective fine chemical compared to said
control or selected organisms or parts thereof.
[0296] [0191.1.0.0] In one embodiment, the organisms or part
thereof produce according to the herein mentioned process of the
invention an increased level of protein-bound respective fine
chemical compared to said control or selected organisms or parts
thereof.
[0297] [0192.0.0.0] Advantageously the selected organisms are
mutagenized according to the invention. According to the invention
mutagenesis is any change of the genetic information in the genome
of an organism, that means any structural or compositional change
in the nucleic acid preferably DNA of an organism that is not
caused by normal segregation or genetic recombination processes.
Such mutations may occur spontaneously, or may be induced by
mutagens as described below. Such change can be induced either
randomly or selectively. In both cases the genetic information of
the organism is modified. In general this lead to the situation
that the activity of the gene product of the relevant genes inside
the cells or inside the organism is increased.
[0298] [0193.0.0.0] In case of the specific or so called site
directed mutagenesis a distinct gene is mutated and thereby its
activity and/or the activity or the encoded gene product is
repressed, reduced or increased, preferably increased. In the event
of a random mutagenesis one or more genes are mutated by chance and
their activities and/or the activities of their gene products are
repressed, reduced or increased, preferably increased.
[0299] [0194.0.0.0] For the purpose of a mutagenesis of a huge
population of organisms, such population can be transformed with a
DNA construct, which is useful for the activation of as much as
possible genes of an organism, preferably all genes. For example
the construct can contain a strong promoter or one or more
enhancers, which are capable of transcriptionally activate genes in
the vicinity of their integration side. With this method it is
possible to statistically mutagenize, e.g. activate nearly all
genes of an organism by the random integration of an activation
construct. Afterwards the skilled worker can identify those
mutagenized lines in which a gene of the invention has been
activated, which in turns leads to the desired increase in the
respective fine chemical production.
[0300] [0195.0.0.0] The genes of the invention can also be
activated by mutagenesis, either of regulatory or coding regions.
In the event of a random mutagenesis a huge number of organisms are
treated with a mutagenic agent. The amount of said agent and the
intensity of the treatment will be chosen in such a manner that
statistically nearly every gene is mutated once. The process for
the random mutagenesis as well as the respective agens is well
known by the skilled person. Such methods are disclosed for example
by A. M. van Harten [(1998), "Mutation breeding: theory and
practical applications", Cambridge University Press, Cambridge,
UK], E Friedberg, G Walker, W Siede [(1995), "DNA Repair and
Mutagenesis", Blackwell Publishing], or K. Sankaranarayanan, J. M.
Gentile, L. R. Ferguson [(2000) "Protocols in Mutagenesis",
Elsevier Health Sciences]. As the skilled worker knows the
spontaneous mutation rate in the cells of an organism is very low
and that a large number of chemical, physical or biological agents
are available for the mutagenesis of organisms. These agents are
named as mutagens or mutagenic agents. As mentioned before three
different kinds of mutagens (chemical, physical or biological
agents) are available.
[0301] [0196.0.0.0] There are different classes of chemical
mutagens, which can be separated by their mode of action. For
example base analogues such as 5-bromouracil, 2-amino purin. Other
chemical mutagens are interacting with the DNA such as sulphuric
acid, nitrous acid, hydroxylamine; or other alkylating agents such
as monofunctional agents like ethyl methanesulfonate,
dimethylsulfate, methyl methanesulfonate), bifunctional like
dichloroethyl sulphide, Mitomycin,
Nitrosoguanidine-dialkylnitrosamine, N-Nitrosoguanidin derivatives,
N-alkyl-N-nitro-N-nitroso-guanidine-), ntercalating dyes like
Acridine, ethidium bromide).
[0302] [0197.0.0.0] Physical mutagens are for example ionizing
irradiation (X ray), UV irradiation. Different forms of irradiation
are available and they are strong mutagens. Two main classes of
irradiation can be distinguished: a) non-ionizing irradiation such
as UV light or ionizing irradiation such as X ray. Biological
mutagens are for example transposable elements for example IS
elements such as IS100, transposons such as Tn5, Tn10, Tn916 or
Tn1000 or phages like Mu.sup.amplac, P1, T5, .lamda.plac etc.
Methods for introducing this phage DNA into the appropriate
microorganism are well known to the skilled worker (see
Microbiology, Third Edition, Eds. Davis, B. D., Dulbecco, R.,
Eisen, H. N. and Ginsberg, H. S., Harper International Edition,
1980). The common procedure of a transposon mutagenesis is the
insertion of a transposable element within a gene or nearby for
example in the promotor or terminator region and thereby leading to
a loss of the gene function. Procedures to localize the transposon
within the genome of the organisms are well known by a person
skilled in the art.
[0303] [0198.0.0.0] Preferably a chemical or biochemical procedure
is used for the mutagenesis of the organisms. A preferred chemical
method is the mutagenesis with
N-methyl-N-nitro-nitrosoguanidine.
[0304] [0199.0.0.0] Other biological method are disclosed by Spee
et al. (Nucleic Acids Research, Vol. 21, No. 3, 1993: 777-778).
Spee et al. teaches a PCR method using dITP for the random
mutagenesis. This method described by Spee et al. was further
improved by Rellos et al. (Protein Expr. Purif., 5, 1994: 270-277).
The use of an in vitro recombination technique for molecular
mutagenesis is described by Stemmer (Proc. Natl. Acad. Sci. USA,
Vol. 91, 1994: 10747-10751). Moore et al. (Nature Biotechnology
Vol. 14, 1996: 458-467) describe the combination of the PCR and
recombination methods for increasing the enzymatic activity of an
esterase toward a para-nitrobenzyl ester. Another route to the
mutagenesis of enzymes is described by Greener et al. in Methods in
Molecular Biology (Vol. 57, 1996: 375-385). Greener et al. use the
specific Escherichia coli strain XL1-Red to generate Escherichia
coli mutants which have increased antibiotic resistance.
[0305] [0200.0.0.0] In one embodiment, the protein according to the
invention or the nucleic acid molecule characterized herein
originates from a eukaryotic or prokaryotic organism such as a
non-human animal, a plant, a microorganism such as a fungi, a
yeast, an alga, a diatom or a bacterium. Nucleic acid molecules,
which advantageously can be used in the process of the invention
originate from yeasts, for example the family Saccharomycetaceae,
in particular the genus Saccharomyces, or yeast genera such as
Candida, Hansenula, Pichia, Yarrowia, Rhodotorula or
Schizosaccharomyces and the especially advantageous from the
species Saccharomyces cerevisiae.
[0306] [0201.0.0.0] In one embodiment, nucleic acid molecules,
which advantageously can be used in the process of the invention
originate from bacteria, for example from Proteobacteria, in
particular from Gammaproteobacteria, more preferred from
Enterobacteriales, e.g. from the family Enterobacteriaceae,
particularly from genera Escherichia, Salmonella, Klebsiella,
advantageously form the species Escherichia coli K12.
[0307] [0202.0.0.0] If, in the process according to the invention,
plants are selected as the donor organism, this plant may, in
principle, be in any phylogenetic relation of the recipient plant.
Donor and recipient plant may belong to the same family, genus,
species, variety or line, resulting in an increasing homology
between the nucleic acids to be integrated and corresponding parts
of the genome of the recipient plant. This also applies analogously
to microorganisms as donor and recipient organism. It might also be
advantageously to use nuclei acids molecules from very distinct
species, since these might exhibit reduced sensitivity against
endogenous regulatory mechanisms and such sequences might not be
recognized by endogenous silencing mechanisms.
[0308] [0203.0.0.0] Accordingly, one embodiment of the application
relates to the use of nucleic acid molecules in the process of the
invention from plants, e.g. crop plants, e.g. from: B. napus;
Glycine max; sunflower linseed or maize or their homologues.
[0309] [0204.0.0.0] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule which comprises a nucleic acid
molecule selected from the group consisting of: [0310] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338, preferably of Table II B, column 7, lines
1 to 5 and/or lines 334 to 338; or a fragment thereof conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof [0311] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338, preferably of Table I B, column 7, lines 1
to 5 and/or lines 334 to 338 or a fragment thereof conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [0312] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [0313]
d) nucleic acid molecule encoding a polypeptide whose sequence has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [0314] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under stringent hybridisation conditions and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [0315] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [0316] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to [0317] (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [0318]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
primers or primer pairs as indicated in Table III, column 7, lines
1 to 5 and/or lines 334 to 338 and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [0319] i) nucleic acid molecule encoding a polypeptide
which is isolated, e.g. from a expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (g), preferably to (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [0320] j) nucleic acid
molecule which encodes a polypeptide comprising a consensus
sequence as indicated in Table IV, columns 7, lines 1 to 5 and/or
lines 334 to 338 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [0321]
k) nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of a polypeptide as indicated in
Table II, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338,
preferably of Table II B, column 7, lines 1 to 5 and/or lines 334
to 338 and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; and [0322] l)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (h) or of a nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338, preferably of Table I B, column 7, lines 1
to 5 and/or lines 334 to 338 or a nucleic acid molecule encoding,
preferably at least the mature form of, the polypeptide as
indicated in Table II, columns 5 or 7, lines 1 to 5 and/or lines
334 to 338, preferably of Table II B, column 7, lines 1 to 5 and/or
lines 334 to 338, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
encompasses a sequence which is complementary thereto; whereby,
preferably, the nucleic acid molecule according to (a) to (l)
distinguishes over the sequence indicated in Table IA or I B,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338, by one or
more nucleotides. In one embodiment, the nucleic acid molecule does
not consist of the sequence shown and indicated in Table I A or I
B, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338: In one
embodiment, the nucleic acid molecule is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to a sequence indicated in Table I A
or I B, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338. In
another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II A or II B, columns
5 or 7, lines 1 to 5 and/or lines 334 to 338. In an other
embodiment, the nucleic acid molecule of the present invention is
at least 30%, 40%, 50%, or 60% identical and less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence indicated in
Table I A or I B, columns 5 or 7, lines 1 to 5 and/or lines 334 to
338. In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table II A or II B,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338. Accordingly,
in one embodiment, the nucleic acid molecule of the differs at
least in one or more residues from a nucleic acid molecule
indicated in Table I A or I B, columns 5 or 7, lines 1 to 5 and/or
lines 334 to 338. Accordingly, in one embodiment, the nucleic acid
molecule of the present invention encodes a polypeptide, which
differs at least in one or more amino acids from a polypeptide
indicated in Table II A or I B, columns 5 or 7, lines 1 to 5 and/or
lines 334 to 338. In another embodiment, a nucleic acid molecule
indicated in Table I A or I B, columns 5 or 7, lines 1 to 5 and/or
lines 334 to 338 does not encode a protein of a sequence indicated
in Table II A or II B, columns 5 or 7, lines 1 to 5 and/or lines
334 to 338. Accordingly, in one embodiment, the protein encoded by
a sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table II A or II B, columns 5
or 7, lines 1 to 5 and/or lines 334 to 338. In a further
embodiment, the protein of the present invention is at least 30%,
40%, 50%, or 60% identical to a protein sequence indicated in Table
II A or II B, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338
and less than 100%, preferably less than 99.999%, 99.99% or 99.9%,
more preferably less than 99%, 98%, 97%, 96% or 95% identical to a
sequence as indicated in Table I A or II B, columns 5 or 7, lines 1
to 5 and/or lines 334 to 338.
[0323] [0205.0.0.0] The nucleic acid sequences used in the process
are advantageously introduced in a nucleic acid construct,
preferably an expression cassette which makes possible the
expression of the nucleic acid molecules in an organism,
advantageously a plant or a microorganism.
[0324] [0206.0.0.0] Accordingly, the invention also relates to an
nucleic acid construct, preferably to an expression construct,
comprising the nucleic acid molecule of the present invention
functionally linked to one or more regulatory elements or
signals.
[0325] [0207.0.0.0] As described herein, the nucleic acid construct
can also comprise further genes, which are to be introduced into
the organisms or cells. It is possible and advantageous to
introduce into, and express in, the host organisms regulatory genes
such as genes for inductors, repressors or enzymes, which, owing to
their enzymatic activity, engage in the regulation of one or more
genes of a biosynthetic pathway. These genes can be of heterologous
or homologous origin. Moreover, further biosynthesis genes may
advantageously be present, or else these genes may be located on
one or more further nucleic acid constructs. Genes, which are
advantageously employed as biosynthesis genes are genes of the
amino acid metabolism, of glycolysis, of the tricarboxylic acid
metabolism or their combinations. As described herein, regulator
sequences or factors can have a positive effect on preferably the
gene expression of the genes introduced, thus increasing it. Thus,
an enhancement of the regulator elements may advantageously take
place at the transcriptional level by using strong transcription
signals such as promoters and/or enhancers. In addition, however,
an enhancement of translation is also possible, for example by
increasing mRNA stability or by inserting a translation enhancer
sequence.
[0326] [0208.0.0.0] In principle, the nucleic acid construct can
comprise the herein described regulator sequences and further
sequences relevant for the expression of the comprised genes. Thus,
the nucleic acid construct of the invention can be used as
expression cassette and thus can be used directly for introduction
into the plant, or else they may be introduced into a vector.
Accordingly in one embodiment the nucleic acid construct is an
expression cassette comprising a microorganism promoter or a
microorganism terminator or both. In another embodiment the
expression cassette encompasses a plant promoter or a plant
terminator or both.
[0327] [0209.0.0.0] Accordingly, in one embodiment, the process
according to the invention comprises the following steps: [0328]
(a) introducing of a nucleic acid construct comprising the nucleic
acid molecule of the invention or used in the process of the
invention or encoding the polypeptide of the present invention or
used in the process of the invention; or [0329] (b) introducing of
a nucleic acid molecule, including regulatory sequences or factors,
which expression increases the expression of the nucleic acid
molecule of the invention or used in the process of the invention
or encoding the polypeptide of the present invention or used in the
process of the invention; in a cell, or an organism or a part
thereof, preferably in a plant, plant cell or a microorganism, and
[0330] (c) expressing of the gene product encoded by the nucleic
acid construct or the nucleic acid molecule mentioned under (a) or
(b) in the cell or the organism.
[0331] [0210.0.0.0] After the introduction and expression of the
nucleic acid construct the transgenic organism or cell is
advantageously cultured and subsequently harvested. The transgenic
organism or cell may be a prokaryotic or eukaryotic organism such
as a microorganism, a non-human animal and plant for example a
plant or animal cell, a plant or animal tissue, preferably a crop
plant, or a part thereof.
[0332] [0211.0.0.0] To introduce a nucleic acid molecule into a
nucleic acid construct, e.g. as part of an expression cassette, the
codogenic gene segment is advantageously subjected to an
amplification and ligation reaction in the manner known by a
skilled person. It is preferred to follow a procedure similar to
the protocol for the Pfu DNA polymerase or a Pfu/Taq DNA polymerase
mixture. The primers are selected according to the sequence to be
amplified. The primers should expediently be chosen in such a way
that the amplificate comprise the codogenic sequence from the start
to the stop codon. After the amplification, the amplificate is
expediently analyzed. For example, the analysis may consider
quality and quantity and be carried out following separation by gel
electrophoresis. Thereafter, the amplificate can be purified
following a standard protocol (for example Qiagen). An aliquot of
the purified amplificate is then available for the subsequent
cloning step. Suitable cloning vectors are generally known to the
skilled worker.
[0333] [0212.0.0.0] They include, in particular, vectors which are
capable of replication in easy to handle cloning systems like as
bacterial yeast or insect cell based (e.g. baculovirus expression)
systems, that is to say especially vectors which ensure efficient
cloning in E. coli, and which make possible the stable
transformation of plants. Vectors, which must be mentioned in
particular are various binary and cointegrated vector systems which
are suitable for the T-DNA-mediated transformation. Such vector
systems are generally characterized in that they contain at least
the vir genes, which are required for the Agrobacterium-mediated
transformation, and the T-DNA border sequences.
[0334] [0213.0.0.0] In general, vector systems preferably also
comprise further cis-regulatory regions such as promoters and
terminators and/or selection markers by means of which suitably
transformed organisms can be identified. While vir genes and T-DNA
sequences are located on the same vector in the case of
cointegrated vector systems, binary systems are based on at least
two vectors, one of which bears vir genes, but no T-DNA, while a
second one bears T-DNA, but no vir gene. Owing to this fact, the
last-mentioned vectors are relatively small, easy to manipulate and
capable of replication in E. coli and in Agrobacterium. These
binary vectors include vectors from the series pBIB-HYG, pPZP,
pBecks, pGreen. Those which are preferably used in accordance with
the invention are Bin19, pBI101, pBinAR, pGPTV and pCAMBIA. An
overview of binary vectors and their use is given by Hellens et al,
Trends in Plant Science (2000) 5, 446-451.
[0335] [0214.0.0.0] For a vector preparation, vectors may first be
linearized using restriction endonuclease(s) and then be modified
enzymatically in a suitable manner. Thereafter, the vector is
purified, and an aliquot is employed in the cloning step. In the
cloning step, the enzyme-cleaved and, if required, purified
amplificate is cloned together with similarly prepared vector
fragments, using ligase. In this context, a specific nucleic acid
construct, or vector or plasmid construct, may have one or else
more codogenic gene segments. The codogenic gene segments in these
constructs are preferably linked operably to regulatory sequences.
The regulatory sequences include, in particular, plant sequences
like the above-described promoters and terminators. The constructs
can advantageously be propagated stably in microorganisms, in
particular Escherichia coli and/or Agrobacterium tumefaciens, under
selective conditions and enable the transfer of heterologous DNA
into plants or other microorganisms. In accordance with a
particular embodiment, the constructs are based on binary vectors
(overview of a binary vector: Hellens et al., 2000). As a rule,
they contain prokaryotic regulatory sequences, such as replication
origin and selection markers, for the multiplication in
microorganisms such as Escherichia coli and Agrobacterium
tumefaciens. Vectors can further contain agrobacterial T-DNA
sequences for the transfer of DNA into plant genomes or other
eukaryotic regulatory sequences for transfer into other eukaryotic
cells, e.g. Saccharomyces sp. or other prokaryotic regulatory
sequences for the transfer into other prokaryotic cells, e.g.
Corynebacterium sp. or Bacillus sp. For the transformation of
plants, the right border sequence, which comprises approximately 25
base pairs, of the total agrobacterial T-DNA sequence is
advantageously included. Usually, the plant transformation vector
constructs according to the invention contain T-DNA sequences both
from the right and from the left border region, which contain
expedient recognition sites for site-specific acting enzymes which,
in turn, are encoded by some of the vir genes.
[0336] [0215.0.0.0] Suitable host organisms are known to the
skilled worker. Advantageous organisms are described further above
in the present application. They include in particular eukaryotes
or eubacteria, e.g. prokaryotes or archae bacteria. Advantageously
host organisms are microorganisms selected from the group
consisting of Actinomycetaceae, Bacillaceae, Brevibacteriaceae,
Corynebacteriaceae, Enterobacteriacae, Gordoniaceae,
Micrococcaceae, Mycobacteriaceae, Nocardiaceae, Pseudomonaceae,
Rhizobiaceae, Streptomycetaceae, Chaetomiaceae, Choanephoraceae,
Cryptococcaceae, Cunninghamellaceae, Demetiaceae, Moniliaceae,
Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae,
Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae,
Sporobolomycetaceae, Tuberculariaceae, Adelotheciaceae,
Dinophyceae, Ditrichaceae and Prasinophyceae. Preferably are
unicellular, microorganisms, e.g. fungi, bacteria or protoza, such
as fungi like the genus Claviceps or Aspergillus or gram-positive
bacteria such as the genera Bacillus, Corynebacterium, Micrococcus,
Brevibacterium, Rhodococcus, Nocardia, Caseobacter or Arthrobacter
or gram-negative bacteria such as the genera Escherichia,
Flavobacterium or Salmonella, or yeasts such as the genera
Rhodotorula, Hansenula, Pichia, Yerrowia, Saccharomyces,
Schizosaccharomyces or Candida.
[0337] [0216.0.0.0] Host organisms which are especially
advantageously selected in the process according to the invention
are microorganisms selected from the group of the genera and
species consisting of Hansenula anomala, Candida utilis, Claviceps
purpurea, Bacillus circulans, Bacillus subtilis, Bacillus sp.,
Brevibacterium albidum, Brevibacterium album, Brevibacterium
cerinum, Brevibacterium flavum, Brevibacterium glutamigenes,
Brevibacterium iodinum, Brevibacterium ketoglutamicum,
Brevibacterium lactofermentum, Brevibacterium linens,
Brevibacterium roseum, Brevibacterium saccharolyticum,
Brevibacterium sp., Corynebacterium acetoacidophilum,
Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes,
Corynebacterium glutamicum (=Micrococcus glutamicum),
Corynebacterium melassecola, Corynebacterium sp. or Escherichia
coli, specifically Escherichia coli K12 and its described
strains.
[0338] [0217.0.0.0] Advantageously preferred in accordance with the
invention are host organisms of the genus Agrobacterium tumefaciens
or plants. Preferred plants are selected from among the families
Aceraceae, Anacardiaceae, Apiaceae, Asteraceae, Apiaceae,
Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Cactaceae,
Caricaceae, Caryophyllaceae, Cannabaceae, Convolvulaceae,
Chenopodiaceae, Elaeagnaceae, Geraniaceae, Gramineae, Juglandaceae,
Lauraceae, Leguminosae, Linaceae, Cucurbitaceae, Cyperaceae,
Euphorbiaceae, Fabaceae, Malvaceae, Nymphaeaceae, Papaveraceae,
Rosaceae, Salicaceae, Solanaceae, Arecaceae, Iridaceae, Liliaceae,
Orchidaceae, Gentianaceae, Labiaceae, Magnoliaceae, Ranunculaceae,
Carifolaceae, Rubiaceae, Scrophulariaceae, Ericaceae, Polygonaceae,
Violaceae, Juncaceae, Poaceae, perennial grass, fodder crops,
vegetables and ornamentals.
[0339] [0218.0.0.0] Especially preferred are plants selected from
the groups of the families Apiaceae, Asteraceae, Brassicaceae,
Cucurbitaceae, Fabaceae, Papaveraceae, Rosaceae, Solanaceae,
Liliaceae or Poaceae. Especially advantageous are, in particular,
crop plants. Accordingly, an advantageous plant preferably belongs
to the group of the genus peanut, oilseed rape, canola, sunflower,
safflower, olive, sesame, hazelnut, almond, avocado, bay,
pumpkin/squash, linseed, soya, pistachio, borage, maize, wheat,
rye, oats, sorghum and millet, triticale, rice, barley, cassava,
potato, sugarbeet, fodder beet, egg plant, and perennial grasses
and forage plants, oil palm, vegetables (brassicas, root
vegetables, tuber vegetables, pod vegetables, fruiting vegetables,
onion vegetables, leafy vegetables and stem vegetables), buckwheat,
Jerusalem artichoke, broad bean, vetches, lentil, alfalfa, dwarf
bean, lupin, clover and lucerne.
[0340] [0219.0.0.0] In order to introduce, into a plant, the
nucleic acid molecule of the invention or used in the process
according to the invention, it has proved advantageous first to
transfer them into an intermediate host, for example a bacterium or
a eukaryotic unicellular cell. The transformation into E. coli,
which can be carried out in a manner known per se, for example by
means of heat shock or electroporation, has proved itself expedient
in this context. Thus, the transformed E. coli colonies can be
analysed for their cloning efficiency. This can be carried out with
the aid of a PCR. Here, not only the identity, but also the
integrity, of the plasmid construct can be verified with the aid of
a defined colony number by subjecting an aliquot of the colonies to
said PCR. As a rule, universal primers which are derived from
vector sequences are used for this purpose, it being possible, for
example, for a forward primer to be arranged upstream of the start
ATG and a reverse primer to be arranged downstream of the stop
codon of the codogenic gene segment. The amplificates are separated
by electrophoresis and assessed with regard to quantity and
quality.
[0341] [0220.0.0.0] The nucleic acid constructs, which are
optionally verified, are subsequently used for the transformation
of the plants or other hosts, e.g. other eukaryotic cells or other
prokaryotic cells. To this end, it may first be necessary to obtain
the constructs from the intermediate host. For example, the
constructs may be obtained as plasmids from bacterial hosts by a
method similar to conventional plasmid isolation.
[0342] [0221.0.0.0] The nucleic acid molecule of the invention or
used in the process according to the invention can also be
introduced into modified viral vectors like baculovirus vectors for
expression in insect cells or plant viral vectors like tobacco
mosaic virus or potato virus X-based vectors. Approaches leading to
the expression of proteins from the modified viral genome including
the the nucleic acid molecule of the invention or used in the
process according to the invention involve for example the
inoculation of tobacco plants with infectious RNA transcribed in
vitro from a cDNA copy of the recombinant viral genome. Another
approach utilizes the transfection of whole plants from wounds
inoculated with Agrobacterium tumefaciens containing cDNA copies of
recombinant plus-sense RNA viruses. Different vectors and virus are
known to the skilled worker for expression in different target eg.
production plants.
[0343] [0222.0.0.0] A large number of methods for the
transformation of plants are known. Since, in accordance with the
invention, a stable integration of heterologous DNA into the genome
of plants is advantageous, the T-DNA-mediated transformation has
proved expedient in particular. For this purpose, it is first
necessary to transform suitable vehicles, in particular
agrobacteria, with a codogenic gene segment or the corresponding
plasmid construct comprising the nucleic acid molecule of the
invention. This can be carried out in a manner known per se. For
example, said nucleic acid construct of the invention, or said
expression construct or said plasmid construct, which has been
generated in accordance with what has been detailed above, can be
transformed into competent agrobacteria by means of electroporation
or heat shock. In principle, one must differentiate between the
formation of cointegrated vectors on the one hand and the
transformation with binary vectors on the other hand. In the case
of the first alternative, the constructs, which comprise the
codogenic gene segment or the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention have no T-DNA sequences, but the formation of the
cointegrated vectors or constructs takes place in the agrobacteria
by homologous recombination of the construct with T-DNA. The T-DNA
is present in the agrobacteria in the form of Ti or Ri plasmids in
which exogenous DNA has expediently replaced the oncogenes. If
binary vectors are used, they can be transferred to agrobacteria
either by bacterial conjugation or by direct transfer. These
agrobacteria expediently already comprise the vector bearing the
vir genes (currently referred to as helper Ti(Ri) plasmid).
[0344] [0223.0.0.0] One or more markers may expediently also be
used together with the nucleic acid construct, or the vector of the
invention and, if plants or plant cells shall be transformed
together with the T-DNA, with the aid of which the isolation or
selection of transformed organisms, such as agrobacteria or
transformed plant cells, is possible. These marker genes enable the
identification of a successful transfer of the nucleic acid
molecules according to the invention via a series of different
principles, for example via visual identification with the aid of
fluorescence, luminescence or in the wavelength range of light
which is discernible for the human eye, by a resistance to
herbicides or antibiotics, via what are known as nutritive markers
(auxotrophism markers) or antinutritive markers, via enzyme assays
or via phytohormones. Examples of such markers which may be
mentioned are GFP (=green fluorescent protein); the
luciferin/luceferase system, the .beta.-galactosidase with its
colored substrates, for example X-Gal, the herbicide resistances
to, for example, imidazolinone, glyphosate, phosphinothricin or
sulfonylurea, the antibiotic resistances to, for example,
bleomycin, hygromycin, streptomycin, kanamycin, tetracyclin,
chloramphenicol, ampicillin, gentamycin, geneticin (G418),
spectinomycin or blasticidin, to mention only a few, nutritive
markers such as the utilization of mannose or xylose, or
antinutritive markers such as the resistance to 2-deoxyglucose.
This list is a small number of possible markers. The skilled worker
is very familiar with such markers. Different markers are
preferred, depending on the organism and the selection method.
[0345] [0224.0.0.0] As a rule, it is desired that the plant nucleic
acid constructs are flanked by T-DNA at one or both sides of the
codogenic gene segment. This is particularly useful when bacteria
of the species Agrobacterium tumefaciens or Agrobacterium
rhizogenes are used for the transformation. A method, which is
preferred in accordance with the invention, is the transformation
with the aid of Agrobacterium tumefaciens. However, biolistic
methods may also be used advantageously for introducing the
sequences in the process according to the invention, and the
introduction by means of PEG is also possible. The transformed
agrobacteria can be grown in the manner known per se and are thus
available for the expedient transformation of the plants. The
plants or plant parts to be transformed are grown or provided in
the customary manner. The transformed agrobacteria are subsequently
allowed to act on the plants or plant parts until a sufficient
transformation rate is reached. Allowing the agrobacteria to act on
the plants or plant parts can take different forms. For example, a
culture of morphogenic plant cells or tissue may be used. After the
T-DNA transfer, the bacteria are, as a rule, eliminated by
antibiotics, and the regeneration of plant tissue is induced. This
is done in particular using suitable plant hormones in order to
initially induce callus formation and then to promote shoot
development.
[0346] [0225.0.0.0] The transfer of foreign genes into the genome
of a plant is called transformation. In doing this the methods
described for the transformation and regeneration of plants from
plant tissues or plant cells are utilized for transient or stable
transformation. An advantageous transformation method is the
transformation in planta. To this end, it is possible, for example,
to allow the agrobacteria to act on plant seeds or to inoculate the
plant meristem with agrobacteria. It has proved particularly
expedient in accordance with the invention to allow a suspension of
transformed agrobacteria to act on the intact plant or at least the
flower primordia. The plant is subsequently grown on until the
seeds of the treated plant are obtained (Clough and Bent, Plant J.
(1998) 16, 735-743). To select transformed plants, the plant
material obtained in the transformation is, as a rule, subjected to
selective conditions so that transformed plants can be
distinguished from untransformed plants. For example, the seeds
obtained in the above-described manner can be planted and, after an
initial growing period, subjected to a suitable selection by
spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into
plants. Further advantageous transformation methods, in particular
for plants, are known to the skilled worker and are described
hereinbelow.
[0347] [0226.0.0.0] Further advantageous and suitable methods are
protoplast transformation by poly(ethylene glycol)-induced DNA
uptake, the "biolistic" method using the gene cannon--referred to
as the particle bombardment method, electroporation, the incubation
of dry embryos in DNA solution, microinjection and gene transfer
mediated by Agrobacterium. Said methods are described by way of
example in B. Jenes et al., Techniques for Gene Transfer, in:
Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D.
Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu.
Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The
nucleic acids or the construct to be expressed is preferably cloned
into a vector, which is suitable for transforming Agrobacterium
tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12
(1984) 8711). Agrobacteria transformed by such a vector can then be
used in known manner for the transformation of plants, in
particular of crop plants such as by way of example tobacco plants,
for example by bathing bruised leaves or chopped leaves in an
agrobacterial solution and then culturing them in suitable media.
The transformation of plants by means of Agrobacterium tumefaciens
is described, for example, by Hofgen and Willmitzer in Nucl. Acid
Res. (1988) 16, 9877 or is known inter alia from F. F. White,
Vectors for Gene Transfer in Higher Plants; in Transgenic Plants,
Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu,
Academic Press, 1993, pp. 15-38.
[0348] [0227.0.0.0] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[0349] In addition to a sequence indicated in Table I, columns 5 or
7, lines 1 to 5 and/or lines 334 to 338 or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the amino acid biosynthetic pathway such as for
L-lysine, L-threonine and/or L-methionine is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine a sequence as indicated in Table I,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 with genes
which generally support or enhances to growth or yield of the
target organisms, for example genes which lead to faster growth
rate of microorganisms or genes which produces stress-, pathogen,
or herbicide resistant plants.
[0350] [0228.0.0.0] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the amino acid
metabolism, in particular in amino acid synthesis.
[0351] [0229.0.0.0] A further advantageous nucleic acid sequence
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes is the
sequence of the ATP/ADP translocator as described in WO 01/20009.
This ATP/ADP translocator leads to an increased synthesis of the
essential amino acids lysine and/or methionine. Furthermore, an
advantageous nucleic acid sequence coexpressed can be threonine
adlolase and/or lysine decarboxylase as described in the state of
the art.
[0352] [0230.0.0.0] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously at least one of the aforementioned
genes or one of the aforementioned nucleic acids is mutated so that
the activity of the corresponding proteins is influenced by
metabolites to a smaller extent compared with the unmutated
proteins, or not at all, and that in particular the production
according to the invention of the respective fine chemical is not
impaired, or so that their specific enzymatic activity is
increased. Less influence means in this connection that the
regulation of the enzymic activity is less by at least 10%,
advantageously at least 20, 30 or 40%, particularly advantageously
by at least 50, 60, 70, 80 or 90%, compared with the starting
organism, and thus the activity of the enzyme is increased by these
figures mentioned compared with the starting organism. An increase
in the enzymatic activity means an enzymatic activity which is
increased by at least 10%, advantageously at least 20, 30, 40 or
50%, particularly advantageously by at least 60, 70, 80, 90, 100,
200, 300, 500 or 1000%, compared with the starting organism. This
leads to an increased productivity of the desired respective fine
chemical or of the desired respective fine chemicals.
[0353] [0231.0.0.0] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a methionine degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[0354] [0232.0.0.0] In another embodiment of the process of the
invention, the organisms used in the process are those in which
simultaneously at least one of the aforementioned nucleic acids or
of the aforementioned genes is mutated in such a way that the
enzymatic activity of the corresponding protein is partially
reduced or completely blocked. A reduction in the enzymatic
activity means an enzymatic activity, which is reduced by at least
10%, advantageously at least 20, 30 or 40%, particularly
advantageously by at least 50, 60 or 70%, preferably more, compared
with the starting organism.
[0355] [0233.0.0.0] If it is intended to transform the host cell,
in particular the plant cell, with several constructs or vectors,
the marker of a preceding transformation must be removed or a
further marker employed in a following transformation. The markers
can be removed from the host cell, in particular the plant cell, as
described hereinbelow via methods with which the skilled worker is
familiar. In particular plants without a marker, in particular
without resistance to antibiotics, are an especially preferred
embodiment of the present invention.
[0356] [0234.0.0.0] In the process according to the invention, the
nucleic acid sequences used in the process according to the
invention are advantageously linked operably to one or more
regulatory signals in order to increase gene expression. These
regulatory sequences are intended to enable the specific expression
of the genes and the expression of protein. Depending on the host
organism for example plant or microorganism, this may mean, for
example, that the gene is expressed and/or overexpressed after
induction only, or that it is expressed and/or overexpressed
constitutively. These regulatory sequences are, for example,
sequences to which the inductors or repressors bind and which thus
regulate the expression of the nucleic acid. In addition to these
novel regulatory sequences, or instead of these sequences, the
natural regulation of these sequences may still be present before
the actual structural genes and, if appropriate, may have been
genetically modified so that the natural regulation has been
switched off and gene expression has been increased. However, the
nucleic acid construct of the invention suitable as expression
cassette (=expression construct=gene construct) can also be simpler
in construction, that is to say no additional regulatory signals
have been inserted before the nucleic acid sequence or its
derivatives, and the natural promoter together with its regulation
has not been removed. Instead, the natural regulatory sequence has
been mutated in such a way that regulation no longer takes place
and/or gene expression is increased. These modified promoters can
also be introduced on their own before the natural gene in the form
of part sequences (=promoter with parts of the nucleic acid
sequences according to the invention) in order to increase the
activity. Moreover, the gene construct can advantageously also
comprise one or more of what are known as enhancer sequences in
operable linkage with the promoter, and these enable an increased
expression of the nucleic acid sequence. Also, it is possible to
insert additional advantageous sequences at the 3' end of the DNA
sequences, such as, for example, further regulatory elements or
terminators.
[0357] [0235.0.0.0] The nucleic acid molecules, which encode
proteins according to the invention and nucleic acid molecules,
which encode other polypeptides may be present in one nucleic acid
construct or vector or in several ones. Advantageously, only one
copy of the nucleic acid molecule of the invention or the nucleic
acid molecule used in the method of the invention or its encoding
genes is present in the nucleic acid construct or vector. Several
vectors or nucleic acid construct or vector can be expressed
together in the host organism. The nucleic acid molecule or the
nucleic acid construct or vectoraccording to the invention can be
inserted in a vector and be present in the cell in a free form. If
a stable transformation is preferred, a vector is used, which is
stably duplicated over several generations or which is else be
inserted into the genome. In the case of plants, integration into
the plastid genome or, in particular, into the nuclear genome may
have taken place. For the insertion of more than one gene in the
host genome the genes to be expressed are present together in one
gene construct, for example in above-described vectors bearing a
plurality of genes.
[0358] [0236.0.0.0] As a rule, regulatory sequences for the
expression rate of a gene are located upstream (5'), within, and/or
downstream (3') relative to to the coding sequence of the nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention or another codogenic gene segment. They
control in particular transcription and/or translation and/or the
transcript stability. The expression level is dependent on the
conjunction of further cellular regulatory systems, such as the
protein biosynthesis and degradation systems of the cell.
[0359] [0237.0.0.0] Regulatory sequences include transcription and
translation regulating sequences or signals, e.g. sequences located
upstream (5'), which concern in particular the regulation of
transcription or translation initiation, such as promoters or start
codons, and sequences located downstream (3'), which concern in
particular the regulation of transcription or translation
termination and transcript stability, such as polyadenylation
signals or stop codons. Regulatory sequences can also be present in
transcribed coding regions as well in transcribed non-coding
regions, e.g. in introns, as for example splicing sites. Promoters
for the regulation of expression of the nucleic acid molecule
according to the invention in a cell and which can be employed are,
in principle, all those which are capable of stimulating the
transcription of genes in the organisms in question, such as
microorganisms or plants. Suitable promoters, which are functional
in these organisms are generally known. They may take the form of
constitutive or inducible promoters. Suitable promoters can enable
the development- and/or tissue-specific expression in multi-celled
eukaryotes; thus, leaf-, root-, flower-, seed-, stomata-, tuber- or
fruit-specific promoters may advantageously be used in plants.
[0360] [0238.0.0.0] The regulatory sequences or factors can, as
described above, have a positive effect on, the expression of the
genes introduced, thus increasing their expression. Thus, an
enhancement of the expression can advantageously take place at the
transcriptional level by using strong transcription signals such as
strong promoters and/or strong enhancers. In addition, enhancement
of expression on the translational level is also possible, for
example by introducing translation enhancer sequences, e.g., the
.OMEGA. enhancer e.g. improving the ribosomal binding to the
transcript, or by increasing the stability of the mRNA, e.g. by
replacing the 3'UTR coding region by a region encoding a 3'UTR
known as conferring an high stability of the transcript or by
stabilization of the transcript through the elimination of
transcript instability, so that the mRNA molecule is translated
more often than the wild type. For example in plants AU-rich
elements (AREs) and DST (downstream) elements destabilized
transcripts. Mutagenesis studies have demonstrated that residues
within two of the conserved domains, the ATAGAT and the GTA
regions, are necessary for instability function. Therefore removal
or mutation of such elements would obviously lead to more stable
transcripts, higher transcript rates and higher protein activity.
Translation enhancers are also the "overdrive sequence", which
comprises the tobacco mosaic virus 5'-untranslated leader sequence
and which increases the protein/RNA ratio (Gallie et al., 1987,
Nucl. Acids Research 15:8693-8711)
[0361] Enhancers are generally defined as cis active elements,
which can stimulate gene transcription independent of position and
orientation. Different enhancers have been identified in plants,
which can either stimulate transcription constitutively or tissue
or stimuli specific. Well known examples for constitutive enhancers
are the enhancer from the 35S promoter (Odell et al., 1985, Nature
313:810-812) or the ocs enhancer (Fromm et al., 1989, Plant Cell 1:
977:984) Another examples are the G-Box motif tetramer which
confers high-level constitutive expression in dicot and monocot
plants (Ishige et al., 1999, Plant Journal, 18, 443-448) or the
petE, a A/T-rich sequence which act as quantitative enhancers of
gene expression in transgenic tobacco and potato plants (Sandhu et
al., 1998; Plant Mol Biol. 37(5):885-96). Beside that, a large
variety of cis-active elements have been described which contribute
to specific expression pattern, like organ specific expression or
induced expression in response to biotic or abiotic stress.
Examples are elements which provide pathogen or wound-induced
expression (Rushton, 2002, Plant Cell, 14, 749-762) or guard
cell-specific expression (Plesch, 2001, Plant Journal 28,
455-464).
[0362] [0239.0.0.0] Advantageous regulatory sequences for the
expression of the nucleic acid molecule according to the invention
in microorganisms are present for example in promoters such as the
cos, tac, rha, trp, tet, trp-tet, lpp, lac, lpp-lac, lacl.sup.q-,
T7, T5, T3, gal, trc, ara, SP6, .lamda.-P.sub.R or .lamda.-P.sub.L
promoter, which are advantageously used in Gram-negative bacteria.
Further advantageous regulatory sequences are present for example
in the Gram-positive promoters amy, dnaK, xylS and SPO2, in the
yeast or fungal promoters ADC1, MF.alpha., AC, P-60, UASH, MCB,
PHO, CYC1, GAPDH, TEF, rp28, ADH. Promoters, which are particularly
advantageous, are constitutive, tissue or compartment specific and
inducible promoters. In general, "promoter" is understood as
meaning, in the present context, a regulatory sequence in a nucleic
acid molecule, which mediates the expression of a coding sequence
segment of a nucleic acid molecule. In general, the promoter is
located upstream to the coding sequence segment. Some elements, for
example expression-enhancing elements such as enhancer may,
however, also be located downstream or even in the transcribed
region.
[0363] [0240.0.0.0] In principle, it is possible to use natural
promoters together with their regulatory sequences, such as those
mentioned above, for the novel process. It is also possible
advantageously to use synthetic promoters, either additionally or
alone, in particular when they mediate seed-specific expression
such as described in, for example, WO 99/16890.
[0364] [0241.0.0.0] The expression of the nucleic acid molecules
used in the process may be desired alone or in combination with
other genes or nucleic acids. Multiple nucleic acid molecules
conferring the expression of advantageous genes can be introduced
via the simultaneous transformation of several individual suitable
nucleic acid constructs, i.e. expression constructs, or,
preferably, by combining several expression cassettes on one
construct. It is also possible to transform several vectors with in
each case several expression cassettes stepwise into the recipient
organisms.
[0365] [0242.0.0.0] As described above the transcription of the
genes introduced should advantageously be terminated by suitable
terminators at the 3' end of the biosynthesis genes introduced
(behind the stop codon). A terminator, which may be used for this
purpose is, for example, the OCS1 terminator, the nos3 terminator
or the 35S terminator. As is the case with the promoters, different
terminator sequences should be used for each gene. Terminators,
which are useful in microorganism are for example the fimA
terminator, txn terminator or trp terminator. Such terminators can
be rho-dependent or rho-independent.
[0366] [0243.0.0.0] Different plant promoters such as, for example,
the USP, the LegB4-, the DC3 promoter or the ubiquitin promoter
from parsley or other herein mentioned promoter and different
terminators may advantageously be used in the nucleic acid
construct.
[0367] [0244.0.0.0] In order to ensure the stable integration, into
the transgenic plant, of nucleic acid molecules used in the process
according to the invention in combination with further biosynthesis
genes over a plurality of generations, each of the coding regions
used in the process should be expressed under the control of its
own, preferably unique, promoter since repeating sequence motifs
may lead to recombination events or to silencing or, in plants, to
instability of the T-DNA.
[0368] [0245.0.0.0] The nucleic acid construct is advantageously
constructed in such a way that a promoter is followed by a suitable
cleavage site for insertion of the nucleic acid to be expressed,
advantageously in a polylinker, followed, if appropriate, by a
terminator located behind the polylinker. If appropriate, this
order is repeated several times so that several genes are combined
in one construct and thus can be introduced into the transgenic
plant in order to be expressed. The sequence is advantageously
repeated up to three times. For the expression, the nucleic acid
sequences are inserted via the suitable cleavage site, for example
in the polylinker behind the promoter. It is advantageous for each
nucleic acid sequence to have its own promoter and, if appropriate,
its own terminator, as mentioned above. However, it is also
possible to insert several nucleic acid sequences behind a promoter
and, if appropriate, before a terminator if a polycistronic
transcription is possible in the host or target cells. In this
context, the insertion site, or the sequence of the nucleic acid
molecules inserted, in the nucleic acid construct is not decisive,
that is to say a nucleic acid molecule can be inserted in the first
or last position in the cassette without this having a substantial
effect on the expression. However, it is also possible to use only
one promoter type in the construct. However, this may lead to
undesired recombination events or silencing effects, as said.
[0369] [0246.0.0.0] Accordingly, in a preferred embodiment, the
nucleic acid construct according to the invention confers
expression of the nucleic acid molecule of the invention or the
nucleic acid molecule used in the method of the invention, and,
optionally further genes, in a plant and comprises one or more
plant regulatory elements. Said nucleic acid construct according to
the invention advantageously encompasses a plant promoter or a
plant terminator or a plant promoter and a plant terminator.
[0370] [0247.0.0.0] A "plant" promoter comprises regulatory
elements, which mediate the expression of a coding sequence segment
in plant cells. Accordingly, a plant promoter need not be of plant
origin, but may originate from viruses or microorganisms, in
particular for example from viruses which attack plant cells.
[0371] [0248.0.0.0] The plant promoter can also originates from a
plant cell, e.g. from the plant, which is transformed with the
nucleic acid construct or vector as described herein.
[0372] This also applies to other "plant" regulatory signals, for
example in "plant" terminators.
[0373] [0249.0.0.0] A nucleic acid construct suitable for plant
expression preferably comprises regulatory elements which are
capable of controlling the expression of genes in plant cells and
which are operably linked so that each sequence can fulfill its
function. Accordingly, the nucleic acid construct can also comprise
transcription terminators. Examples for transcriptional termination
arepolyadenylation signals. Preferred polyadenylation signals are
those which originate from Agrobacterium tumefaciens T-DNA, such as
the gene 3 of the Ti plasmid pTiACH5, which is known as octopine
synthase (Gielen et al., EMBO J. 3 (1984) 835 et seq.) or
functional equivalents thereof, but all the other terminators which
are functionally active in plants are also suitable.
[0374] [0250.0.0.0] The nucleic acid construct suitable for plant
expression preferably also comprises other operably linked
regulatory elements such as translation enhancers, for example the
overdrive sequence, which comprises the tobacco mosaic virus
5'-untranslated leader sequence, which increases the protein/RNA
ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711).
[0375] [0251.0.0.0] Other preferred sequences for use in operable
linkage in gene expression constructs are targeting sequences,
which are required for targeting the gene product into specific
cell compartments (for a review, see Kermode, Crit. Rev. Plant Sci.
15, 4 (1996) 285-423 and references cited therein), for example
into the vacuole, the nucleus, all types of plastids, such as
amyloplasts, chloroplasts, chromoplasts, the extracellular space,
the mitochondria, the endoplasmic reticulum, elaioplasts,
peroxisomes, glycosomes, and other compartments of cells or
extracellular. Sequences, which must be mentioned in this context
are, in particular, the signal-peptide- or transit-peptide-encoding
sequences which are known per se. For example,
plastid-transit-peptide-encoding sequences enable the targeting of
the expression product into the plastids of a plant cellTargeting
sequences are also known for eukaryotic and to a lower extent for
prokaryotic organisms and can advantageously be operable linked
with the nucleic acid molecule of the present invention to achieve
an expression in one of said compartments or extracellular.
[0376] [0252.0.0.0] For expression in plants, the nucleic acid
molecule must, as described above, be linked operably to or
comprise a suitable promoter which expresses the gene at the right
point in time and in a cell- or tissue-specific manner. Usable
promoters are constitutive promoters (Benfey et al., EMBO J. 8
(1989) 2195-2202), such as those which originate from plant
viruses, such as 35S CAMV (Franck et al., Cell 21 (1980) 285-294),
19S CaMV (see also U.S. Pat. No. 5,352,605 and WO 84/02913), 34S
FMV (Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443), the
parsley ubiquitin promoter, or plant promoters such as the Rubisco
small subunit promoter described in U.S. Pat. No. 4,962,028 or the
plant promoters PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)],
SSU, PGEL1, OCS [Leisner (1988) Proc Natl Acad Sci USA
85(5):2553-2557], lib4, usp, mas [Comai (1990) Plant Mol Biol 15
(3):373-381], STLS1, ScBV (Schenk (1999) Plant Mol Biol
39(6):1221-1230), B33, SAD1 or SAD2 (flax promoters, Jain et al.,
Crop Science, 39 (6), 1999: 1696-1701) or nos [Shaw et al. (1984)
Nucleic Acids Res. 12(20):7831-7846]. Stable, constitutive
expression of the proteins according to the invention a plant can
be advantageous. However, inducible expression of the polypeptide
of the invention or the polypeptide used in the method of the
invention is advantageous, if a late expression before the harvest
is of advantage, as metabolic manipulation may lead to a plant
growth retardation.
[0377] [0253.0.0.0] The expression of plant genes can also be
facilitated as described above via a chemical inducible promoter
(for a review, see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol.
Biol., 48:89-108). Chemically inducible promoters are particularly
suitable when it is desired to express the gene in a time-specific
manner. Examples of such promoters are a salicylic acid inducible
promoter (WO 95/19443), and abscisic acid-inducible promoter (EP
335 528), a tetracyclin-inducible promoter (Gatz et al. (1992)
Plant J. 2, 397-404), a cyclohexanol- or ethanol-inducible promoter
(WO 93/21334) or others as described herein.
[0378] [0254.0.0.0] Other suitable promoters are those which react
to biotic or abiotic stress conditions, for example the
pathogen-induced PRP1 gene promoter (Ward et al., Plant. Mol. Biol.
22 (1993) 361-366), the tomato heat-inducible hsp80 promoter (U.S.
Pat. No. 5,187,267), the potato chill-inducible alpha-amylase
promoter (WO 96/12814) or the wound-inducible pinII promoter
(EP-A-0 375 091) or others as described herein.
[0379] [0255.0.0.0] Preferred promoters are in particular those
which bring about gene expression in tissues and organs in which
the biosynthesis of amino acids takes place, in seed cells, such as
endosperm cells and cells of the developing embryo. Suitable
promoters are the oilseed rape napin gene promoter (U.S. Pat. No.
5,608,152), the Vicia faba USP promoter (Baeumlein et al., Mol Gen
Genet, 1991, 225 (3):459-67), the Arabidopsis oleosin promoter (WO
98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No.
5,504,200), the Brassica Bce4 promoter (WO 91/13980), the bean arcs
promoter, the carrot DcG3 promoter, or the Legumin B4 promoter
(LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2):233-9), and
promoters which bring about the seed-specific expression in
monocotyledonous plants such as maize, barley, wheat, rye, rice and
the like. Advantageous seed-specific promoters are the sucrose
binding protein promoter (WO 00/26388), the phaseolin promoter and
the napin promoter. Suitable promoters which must be considered are
the barley Ipt2 or Ipt1 gene promoter (WO 95/15389 and WO
95/23230), and the promoters described in WO 99/16890 (promoters
from the barley hordein gene, the rice glutelin gene, the rice
oryzin gene, the rice prolamin gene, the wheat gliadin gene, the
wheat glutelin gene, the maize zein gene, the oat glutelin gene,
the sorghum kasirin gene and the rye secalin gene). Further
suitable promoters are Amy32b, Amy 6-6 and Aleurain [U.S. Pat. No.
5,677,474], Bce4 (oilseed rape) [U.S. Pat. No. 5,530,149], glycinin
(soya) [EP 571 741], phosphoenolpyruvate carboxylase (soya) [JP
06/62870], ADR12-2 (soya) [WO 98/08962], isocitrate lyase (oilseed
rape) [U.S. Pat. No. 5,689,040] or .alpha.-amylase (barley) [EP 781
849]. Other promoters which are available for the expression of
genes in plants are leaf-specific promoters such as those described
in DE-A 19644478 or light-regulated promoters such as, for example,
the pea petE promoter.
[0380] [0256.0.0.0] Further suitable plant promoters are the
cytosolic FBPase promoter or the potato ST-LSI promoter (Stockhaus
et al., EMBO J. 8, 1989, 2445), the Glycine max
phosphoribosylpyrophosphate amidotransferase promoter (GenBank
Accession No. U87999) or the node-specific promoter described in
EP-A-0 249 676.
[0381] [0257.0.0.0] Other promoters, which are particularly
suitable, are those which bring about plastid-specific expression.
Suitable promoters such as the viral RNA polymerase promoter are
described in WO 95/16783 and WO 97/06250, and the Arabidopsis clpP
promoter, which is described in WO 99/46394.
[0382] [0258.0.0.0] Other promoters, which are used for the strong
expression of heterologous sequences in as many tissues as
possible, in particular also in leaves, are, in addition to several
of the abovementioned viral and bacterial promoters, preferably,
plant promoters of actin or ubiquitin genes such as, for example,
the rice actin1 promoter. Further examples of constitutive plant
promoters are the sugarbeet V-ATPase promoters (WO 01/14572).
Examples of synthetic constitutive promoters are the Super promoter
(WO 95/14098) and promoters derived from G-boxes (WO 94/12015). If
appropriate, chemical inducible promoters may furthermore also be
used, compare EP-A 388186, EP-A 335528, WO 97/06268.
[0383] [0259.0.0.0] As already mentioned herein, further regulatory
sequences, which may be expedient, if appropriate, also include
sequences, which target the transport and/or the localization of
the expression products. Sequences, which must be mentioned in this
context are, in particular, the signal-peptide- or
transit-peptide-encoding sequences which are known per se. For
example, plastid-transit-peptide-encoding sequences enable the
targeting of the expression product into the plastids of a plant
cell.
[0384] [0260.0.0.0] Preferred recipient plants are, as described
above, in particular those plants, which can be transformed in a
suitable manner. These include monocotyledonous and dicotyledonous
plants. Plants which must be mentioned in particular are
agriculturally useful plants such as cereals and grasses, for
example Triticum spp., Zea mays, Hordeum vulgare, oats, Secale
cereale, Oryza sativa, Pennisetum glaucum, Sorghum bicolor,
Triticale, Agrostis spp., Cenchrus ciliaris, Dactylis glomerata,
Festuca arundinacea, Lolium spp., Medicago spp. and Saccharum spp.,
legumes and oil crops, for example Brassica juncea, Brassica napus,
Glycine max, Arachis hypogaea, Gossypium hirsutum, Cicer arietinum,
Helianthus annuus, Lens culinaris, Linum usitatissimum, Sinapis
alba, Trifolium repens and Vicia narbonensis, vegetables and
fruits, for example bananas, grapes, Lycopersicon esculentum,
asparagus, cabbage, watermelons, kiwi fruit, Solanum tuberosum,
Beta vulgaris, cassava and chicory, trees, for example Coffea
species, Citrus spp., Eucalyptus spp., Picea spp., Pinus spp. and
Populus spp., medicinal plants and trees, and flowers.
[0385] [0261.0.0.0] One embodiment of the present invention also
relates to a method for generating a vector, which comprises the
insertion, into a vector, of the nucleic acid molecule
characterized herein, the nucleic acid molecule according to the
invention or the expression cassette according to the invention.
The vector can, for example, be introduced in to a cell, e.g. a
microorganism or a plant cell, as described herein for the nucleic
acid construct, or below under transformation or transfection or
shown in the examples. A transient or stable transformation of the
host or target cell is possible, however, a stable transformation
is preferred. The vector according to the invention is preferably a
vector, which is suitable for expressing the polypeptide according
to the invention in a plant. The method can thus also encompass one
or more steps for integrating regulatory signals into the vector,
in particular signals, which mediate the expression in
microorganisms or plants.
[0386] [0262.0.0.0] Accordingly, the present invention also relates
to a vector comprising the nucleic acid molecule characterized
herein as part of a nucleic acid construct suitable for plant
expression or the nucleic acid molecule according to the
invention.
[0387] [0263.0.0.0] The advantageous vectors of the
inventioncomprise the nucleic acid molecules which encode proteins
according to the invention, nucleic acid molecules which are used
in the process, or nucleic acid construct suitable for plant
expression comprising the nucleic acid molecules used, either alone
or in combination with further genes such as the biosynthesis or
regulatory genes of the respective fine chemical metabolism e.g.
with the genes mentioned herein above. In accordance with the
invention, the term "vector" refers to a nucleic acid molecule,
which is capable of transporting another nucleic acid to which it
is linked. One type of vector is a "plasmid", which means a
circular double-stranded DNA loop into which additional DNA
segments can be ligated. A further type of vector is a viral
vector, it being possible to ligate additional nucleic acids
segments into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they have been
introduced (for example bacterial vectors with bacterial
replication origin). Other preferred vectors are advantageously
completely or partly integrated into the genome of a host cell when
they are introduced into the host cell and thus replicate together
with the host genome. Moreover, certain vectors are capable of
controlling the expression of genes with which they are in operable
linkage. In the present context, these vectors are referred to as
"expression vectors". As mentioned above, they are capable of
autonomous replication or may be integrated partly or completely
into the host genome. Expression vectors, which are suitable for
DNA recombination techniques usually take the form of plasmids. In
the present description, "plasmid" and "vector" can be used
interchangeably since the plasmid is the most frequently used form
of a vector. However, the invention is also intended to encompass
these other forms of expression vectors, such as viral vectors,
which exert similar functions. The term vector is furthermore also
to encompass other vectors which are known to the skilled worker,
such as phages, viruses such as SV40, CMV, TMV, transposons, IS
elements, phasmids, phagemids, cosmids, and linear or circular
DNA.
[0388] [0264.0.0.0] The recombinant expression vectors which are
advantageously used in the process comprise the nucleic acid
molecules according to the invention or the nucleic acid construct
according to the invention in a form which is suitable for
expressing, in a host cell, the nucleic acid molecules according to
the invention or described herein. Accordingly, the the recombinant
expression vectors comprise one or more regulatory signals selected
on the basis of the host cells to be used for the expression, in
operable linkage with the nucleic acid sequence to be
expressed.
[0389] [0265.0.0.0] In a recombinant expression vector, "operable
linkage" means that the nucleic acid molecule of interest is linked
to the regulatory signals in such a way that expression of the
nucleic acid molecule is possible: they are linked to one another
in such a way that the two sequences fulfill the predicted function
assigned to the sequence (for example in an in-vitro
transcription/translation system, or in a host cell if the vector
is introduced into the host cell).
[0390] [0266.0.0.0] The term "regulatory sequence" is intended to
comprise promoters, enhancers and other expression control elements
(for example polyadenylation signalsThese regulatory sequences are
described, for example, in Goeddel: Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990), or see: Gruber and Crosby, in: Methods in Plant Molecular
Biology and Biotechnolgy, CRC Press, Boca Raton, Fla., Ed.: Glick
and Thompson, chapter 7, 89-108, including the references cited
therein. Regulatory sequences encompass those, which control the
constitutive expression of a nucleotide sequence in many types of
host cells and those which control the direct expression of the
nucleotide sequence in specific host cells only, and under specific
conditions. The skilled worker knows that the design of the
expression vector may depend on factors such as the selection of
the host cell to be transformed, the extent to which the desired
protein is expressed, and the like. A preferred selection of
regulatory sequences is described above, for example promoters,
terminators, enhancers and the like. The term regulatory sequence
is to be considered as being encompassed by the term regulatory
signal. Several advantageous regulatory sequences, in particular
promoters and terminators are described above. In general, the
regulatory sequences described as advantageous for nucleic acid
construct suitable for expression are also applicable for
vectors.
[0391] [0267.0.0.0] The recombinant expression vectors used can be
designed specifically for the expression, in prokaryotic and/or
eukaryotic cells, of nucleic acid molecules used in the process.
This is advantageous since intermediate steps of the vector
construction are frequently carried out in microorganisms for the
sake of simplicity. For example, the genes according to the
invention and other genes can be expressed in bacterial cells,
insect cells (using baculovirus expression vectors), yeast cells
and other fungal cells [Romanos (1992), Yeast 8:423-488; van den
Hondel, (1991), in: More Gene Manipulations in Fungi, J. W. Bennet
& L. L. Lasure, Ed., pp. 396-428: Academic Press: San Diego;
and van den Hondel, C. A. M. J. J. (1991), in: Applied Molecular
Genetics of Fungi, Peberdy, J. F., et al., Ed., pp. 1-28, Cambridge
University Press: Cambridge], algae [Falciatore et al., 1999,
Marine Biotechnology. 1, 3:239-251] using vectors and following a
transformation method as described in WO 98/01572, and preferably
in cells of multi-celled plants [see Schmidt, R. and Willmitzer, L.
(1988) Plant Cell Rep.:583-586; Plant Molecular Biology and
Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, pp. 71-119
(1993); F. F. White, in: Transgenic Plants, Bd. 1, Engineering and
Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-43;
Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991),
205-225 (and references cited therein)]. Suitable host cells are
furthermore discussed in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). As an alternative, the sequence of the recombinant
expression vector can be transcribed and translated in vitro, for
example using T7 promotor-regulatory sequences and T7
polymerase.
[0392] [0268.0.0.0] Proteins can be expressed in prokaryotes using
vectors comprising constitutive or inducible promoters, which
control the expression of fusion proteins or nonfusion proteins.
Typical fusion expression vectors are, inter alia, pGEX (Pharmacia
Biotech Inc; Smith, D. B., and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, M A) and pRIT5
(Pharmacia, Piscataway, N.J.), in which glutathione-S-transferase
(GST), maltose-E-binding protein or protein A is fused with the
recombinant target protein. Examples of suitable inducible
nonfusion E. coli expression vectors are, inter alia, pTrc (Amann
et al. (1988) Gene 69:301-315) and pET 11d [Studier et al., Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990) 60-89]. The target gene expression of the
pTrc vector is based on the transcription of a hybrid trp-lac
fusion promoter by the host RNA polymerase. The target gene
expression from the pET 11d vector is based on the transcription of
a T7-gn10-lac fusion promoter, which is mediated by a coexpressed
viral RNA polymerase (T7 gn1). This viral polymerase is provided by
the host strains BL21 (DE3) or HMS174 (DE3) by a resident
A-prophage which harbors a T7 gn1 gene under the transcriptional
control of the lacUV 5 promoter.
[0393] [0269.0.0.0] Other vectors which are suitable in prokaryotic
organisms are known to the skilled worker; these vectors are for
example in E. coli pLG338, pACYC184, the pBR series, such as
pBR322, the pUC series such as pUC18 or pUC19, the M113mp series,
pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200, pUR290,
pIN-III.sup.113-B1, .lamda.gt11 or pBdCl, in Streptomyces pIJ101,
pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in
Corynebacterium pSA77 or pAJ667.
[0394] [0270.0.0.0] In a further embodiment, the expression vector
is a yeast expression vector. Examples of vectors for expression in
the yeasts S. cerevisiae encompass pYeDesaturasec1 (Baldari et al.
(1987) Embo J. 6:229-234), pMF.alpha.(Kurjan and Herskowitz (1982)
Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123)
and pYES2 (Invitrogen Corporation, San Diego, Calif.). Vectors and
methods for the construction of vectors which are suitable for use
in other fungi, such as the filamentous fungi, encompass those
which are described in detail in: van den Hondel, C. A. M. J. J.
[(1991), J. F. Peberdy, Ed., pp. 1-28, Cambridge University Press:
Cambridge; or in: More Gene Manipulations in Fungi; J. W. Bennet
& L. L. Lasure, Ed., pp. 396-428: Academic Press: San Diego].
Examples of other suitable yeast vectors are 2 .mu.M, pAG-1, YEp6,
YEp13 or pEMBLYe23.
[0395] [0271.0.0.0] Further vectors, which may be mentioned by way
of example, are pALS1, pIL2 or pBB116 in fungi or pLGV23, pGHlac+,
pBIN19, pAK2004 or pDH51 in plants.
[0396] [0272.0.0.0] As an alternative, the nucleic acid sequences
can be expressed in insect cells using baculovirus expression
vectors. Baculovirus vectors, which are available for expressing
proteins in cultured insect cells (for example Sf9 cells) encompass
the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)
and the pVL series (Lucklow and Summers (1989) Virology
170:31-39).
[0397] [0273.0.0.0] The abovementioned vectors are only a small
overview of potentially suitable vectors. Further plasmids are
known to the skilled worker and are described, for example, in:
Cloning Vectors (Ed. Pouwels, P. H., et al., Elsevier,
Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Further
suitable expression systems for prokaryotic and eukaryotic cells,
see the chapters 16 and 17 by Sambrook, J., Fritsch, E. F., and
Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Edition,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989.
[0398] [0274.0.0.0] Accordingly, one embodiment of the invention
relates to a vector where the nucleic acid molecule according to
the invention is linked operably to regulatory sequences which
permit the expression in a prokaryotic or eukaryotic or in a
prokaryotic and eukaryotic host.
[0399] [0275.0.0.0] Accordingly, one embodiment of the invention
relates to a host cell, which has been transformed stably or
transiently with the vector according to the invention or the
nucleic acid molecule according to the invention or the nucleic
acid construct according to the invention.
[0400] [0276.0.0.0] Depending on the host organism, the organisms
used in the process according to the invention are cultured or
grown in a manner with which the skilled worker is familiar. As a
rule, microorganisms are grown in a liquid medium comprising a
carbon source, usually in the form of sugars, a nitrogen source,
usually in the form of organic nitrogen sources such as yeast
extract or salts such as ammonium sulfate, trace elements such as
iron salts, manganese salts, magnesium salts, and, if appropriate,
vitamins, at temperatures between 0.degree. C. and 100.degree. C.,
preferably between 10.degree. C. and 60.degree. C., while passing
in oxygen. In the event the microorganism is anaerobe, no oxygen is
blown through the culture medium. The pH value of the liquid
nutrient medium may be kept constant, that is to say regulated
during the culturing phase, or not. The organisms may be cultured
batchwise, semibatchwise or continuously. Nutrients may be provided
at the beginning of the fermentation or fed in semicontinuously or
continuously.
[0401] [0277.0.0.0] The amino acids produced can be isolated from
the organism by methods with which the skilled worker is familiar.
For example via extraction, salt precipitation and/or ion-exchange
chromatography. To this end, the organisms may be disrupted
beforehand. The process according to the invention can be conducted
batchwise, semibatchwise or continuously. A summary of known
culture and isolation techniques can be found in the textbook by
Chmiel [Bioproze.beta.technik 1, Einfuhrung in die
Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)],
Demain et al. (Industrial Microbiology and Biotechnology, second
edition, ASM Press, Washington, D.C., 1999, ISBN 1-55581-128-0] or
in the textbook by Storhas (Bioreaktoren and periphere
Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
[0402] [0278.0.0.0] In one embodiment, the present invention
relates to a polypeptide encoded by the nucleic acid molecule
according to the present invention, preferably conferring an
increase in the respective fine chemical content in an organism or
cell after increasing the expression or activity.
[0403] [0279.0.0.0] The present invention also relates to a process
for the production of a polypeptide according to the present
invention, the polypeptide being expressed in a host cell according
to the invention, preferably in a microorganism or a transgenic
plant cell.
[0404] [0280.0.0.0] In one embodiment, the nucleic acid molecule
used in the process for the production of the polypeptide is
derived from a microorganism, preferably from a prokaryotic or
protozoic cell with an eukaryotic organism as host cell. E.g., in
one embodiment the polypeptide is produced in a plant cell or plant
with a nucleic acid molecule derived from a prokaryote or a fungus
or an alga or an other microorganism but not from plant.
[0405] [0281.0.0.0] The skilled worker knows that protein and DNA
expressed in different organisms differ in many respects and
properties, e.g. DNA modulation and imprinting, such as methylation
or post-translational modification, as for example glucosylation,
phosphorylation, acetylation, myristoylation, ADP-ribosylation,
farnesylation, carboxylation, sulfation, ubiquination, etc. though
having the same coding sequence. Preferably, the cellular
expression control of the corresponding protein differs accordingly
in the control mechanisms controlling the activity and expression
of an endogenous protein or another eukaryotic protein. One major
difference between proteins expressed in prokaryotic or eukaryotic
organisms is the amount and pattern of glycosylation. For example
in E. coli there are no glycosylated proteins. Proteins expressed
in yeasts have high mannose content in the glycosylated proteins,
whereas in plants the glycosylation pattern is complex.
[0406] [0282.0.0.0] The polypeptide of the present invention is
preferably produced by recombinant DNA techniques. For example, a
nucleic acid molecule encoding the protein is cloned into a vector
(as described above), the vector is introduced into a host cell (as
described above) and said polypeptide is expressed in the host
cell. Said polypeptide can then be isolated from the cells by an
appropriate purification scheme using standard protein purification
techniques. Alternative to recombinant expression, the polypeptide
or peptide of the present invention can be synthesized chemically
using standard peptide synthesis techniques.
[0407] [0283.0.0.0] Moreover, a native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an antibody against a protein as indicated in
Table II, column 3, lines 1 to 5 and/or lines 334 to 338. E.g. an
antibody against a polypeptide as indicated in Table II, columns 5
or 7, lines 1 to 5 and/or lines 334 to 338, or an antigenic part
thereof which can be produced by standard techniques utilizing
polypeptides comprising or consisting of above mentioned sequences,
e.g. the polypeptid of the present invention or fragment thereof.
Preferred are monoclonal antibodies specifically binding to
polypeptide as indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338.
[0408] [0284.0.0.0] In one embodiment, the present invention
relates to a polypeptide having the amino acid sequence encoded by
a nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention or obtainable by a
process of the invention. Said polypeptide confers preferably the
aforementioned activity, in particular, the polypeptide confers the
increase of the respective fine chemical in a cell or an organism
or a part thereof after increasing the cellular activity, e.g. by
increasing the expression or the specific activity of the
polypeptide.
[0409] [0285.0.0.0] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 or as
encoded by a nucleic acid molecule as indicated in Table I, columns
5 or 7, lines 1 to 5 and/or lines 334 to 338 or functional
homologues thereof.
[0410] [0286.0.0.0] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 1 to 5 and/or lines 334 to 338 and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table IV, column 7, lines 1 to 5 and/or lines 334 to 338, whereby
20 or less, preferably 15 or 10, preferably 9, 8, 7, or 6, more
preferred 5 or 4, even more preferred 3, even more preferred 2,
even more preferred 1, most preferred 0 of the amino acids
positions indicated can be replaced by any amino acid or, in an
further embodiment, can be replaced and/or absent. In one
embodiment, the present invention relates to the method of the
present invention comprising a polypeptide or to a polypeptide
comprising more than one consensus sequences (of an individual
line) as indicated in Table IV, column 7, lines 1 to 5 and/or lines
334 to 338.
[0411] [0287.0.0.0] In one embodiment not more than 15%, preferably
10%, even more preferred 5%, 4%, 3%, or 2%, most preferred 1% or 0%
of the amino acid position indicated by a letter are/is replaced
another amino acid or, in an other embodiment, are/is absent and/or
replaced. In another embodiment the stretches of non-conserved
amino acids, indicated by (X), [whereas n indicates the number of
X], vary in their length by 20%, preferably by 15 or 10%, even more
preferred by 5%, 4%, 3%, 2% or most preferred by only 1%.
[0412] [0288.0.0.0] In one embodiment 20 or less, preferably 15 or
10, preferably 9, 8, 7, or 6, more preferred 5 or 4, even more
preferred 3, even more preferred 2, even more preferred 1, most
preferred 0 amino acids are inserted into the consensus sequence
or, in an other embodiment, are absent and/or replaced.
[0413] [0289.0.0.0] The consensus sequence shown herein was derived
from a multiple alignment of the sequences as listed in table II.
The consensus sequences of specified domains were derived from a
multiple alignment of all sequences. The letters represent the one
letter amino acid code and indicate that the amino acids are
conserved in all aligned proteins. The letter X stands for amino
acids, which are not conserved in all sequences.
[0414] In one example, in the cases where only a small selected
subset of amino acids are possible at a certain position these
amino acids are given in brackets. The number of given X indicates
the distances between conserved amino acid residues, e.g.
YX(21-23)F means that conserved tyrosine and phenylalanine residues
are separated from each other by minimum 21 and maximum 23 amino
acid residues in all investigated sequences.
[0415] [0290.0.0.0] The alignment was performed with the Software
AlignX (Sep. 25, 2002) a component of Vector NTI Suite 8.0,
InforMax.TM., Invitrogen.TM. life science software, U.S. Main
Office, 7305 Executive Way, Frederick, Md. 21704, USA with the
following settings: For pairwise alignments: gap opening penalty:
10.0; gap extension penalty 0.1. For multiple alignments: Gap
opening penalty: 10.0; Gap extension penalty: 0.1; Gap separation
penalty range: 8; Residue substitution matrix: blosum62;
Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0.5;
Consensus calculation options: Residue fraction for consensus: 0.9.
Presettings were selected to allow also for the alignment of
conserved amino acids.
[0416] [0291.0.0.0] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of
plant or microorganism specific consensus sequences.
[0417] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table II A or IIB,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 by one or more
amino acids. In one embodiment, polypeptide distinguishes form a
sequence as indicated in Table II A or IIB, columns 5 or 7, lines 1
to 5 and/or lines 334 to 338 by more than 1, 2, 3, 4, 5, 6, 7, 8 or
9 amino acids, preferably by more than 10, 15, 20, 25 or 30 amino
acids, even more preferred are more than 40, 50, or 60 amino acids
and, preferably, the sequence of the polypeptide of the invention
distinguishes from a sequence as indicated in Table II A or II B,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 by not more
than 80% or 70% of the amino acids, preferably not more than 60% or
50%, more preferred not more than 40% or 30%, even more preferred
not more than 20% or 10%. In an other embodiment, said polypeptide
of the invention does not consist of a sequence as indicated in
Table II A or II B, columns 5 or 7, lines 1 to 5 and/or lines 334
to 338.
[0418] [0292.0.0.0] In one embodiment, the polypeptide of the
invention comprises any one of the sequences not known to the
public before. In one embodiment, the polypeptide of the invention
originates from a non-plant cell, in particular from a
microorganism, and was expressed in a plant cell. In one
embodiment, the present invention relates to a polypeptide encoded
by the nucleic acid molecule of the invention or used in the
process of the invention for which an activity has not been
described yet.
[0419] [0293.0.0.0] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the process
of the invention.
[0420] In one embodiment, the polypeptide of the invention has a
sequence which distinguishes from a sequence as indicated in Table
II A or II B, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338
by one or more amino acids. In an other embodiment, said
polypeptide of the invention does not consist of the sequence as
indicated in Table II A or II B, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338. In a further embodiment, said polypeptide
of the present invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical. In one embodiment, said polypeptide does not
consist of the sequence encoded by a nucleic acid molecules as
indicated in Table I A or IB, columns 5 or 7, lines 1 to 5 and/or
lines 334 to 338.
[0421] [0294.0.0.0] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 1 to 5 and/or lines 334 to
338, which distinguishes over a sequence as indicated in Table II A
or II B, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 by
one or more amino acids, preferably by more than 5, 6, 7, 8 or 9
amino acids, preferably by more than 10, 15, 20, 25 or 30 amino
acids, even more preferred are more than 40, 50, or 60 amino acids
but even more preferred by less than 70% of the amino acids, more
preferred by less than 50%, even more preferred my less than 30% or
25%, more preferred are 20% or 15%, even more preferred are less
than 10%.
[0422] [0295.0.0.0] The terms "protein" and "polypeptide" used in
this application are interchangeable. "Polypeptide" refers to a
polymer of amino acids (amino acid sequence) and does not refer to
a specific length of the molecule. Thus peptides and oligopeptides
are included within the definition of polypeptide. This term does
also refer to or include post-translational modifications of the
polypeptide, for example, glycosylations, acetylations,
phosphorylations and the like. Included within the definition are,
for example, polypeptides containing one or more analogs of an
amino acid (including, for example, unnatural amino acids, etc.),
polypeptides with substituted linkages, as well as other
modifications known in the art, both naturally occurring and
non-naturally occurring.
[0423] [0296.0.0.0] Preferably, the polypeptide is isolated. An
"isolated" or "purified" protein or nucleic acid molecule or
biologically active portion thereof is substantially free of
cellular material when produced by recombinant DNA techniques or
chemical precursors or other chemicals when chemically
synthesized.
[0424] [0297.0.0.0] The language "substantially free of cellular
material" includes preparations of the polypeptide of the invention
in which the protein is separated from cellular components of the
cells in which it is naturally or recombinantly produced. In one
embodiment, the language "substantially free of cellular material"
includes preparations having less than about 30% (by dry weight) of
"contaminating protein", more preferably less than about 20% of
"contaminating protein", still more preferably less than about 10%
of "contaminating protein", and most preferably less than about 5%
"contaminating protein". The term "Contaminating protein" relates
to polypeptides, which are not polypeptides of the present
invention. When the polypeptide of the present invention or
biologically active portion thereof is recombinantly produced, it
is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less
than about 10%, and most preferably less than about 5% of the
volume of the protein preparation. The language "substantially free
of chemical precursors or other chemicals" includes preparations in
which the polypeptide of the present invention is separated from
chemical precursors or other chemicals, which are involved in the
synthesis of the protein. The language "substantially free of
chemical precursors or other chemicals" includes preparations
having less than about 30% (by dry weight) of chemical precursors
or non-polypeptide of the invention-chemicals, more preferably less
than about 20% chemical precursors or non-polypeptide of the
invention-chemicals, still more preferably less than about 10%
chemical precursors or non-polypeptide of the invention-chemicals,
and most preferably less than about 5% chemical precursors or
non-polypeptide of the invention-chemicals. In preferred
embodiments, isolated proteins or biologically active portions
thereof lack contaminating proteins from the same organism from
which the polypeptide of the present invention is derived.
Typically, such proteins are produced by recombinant
techniques.
[0425] [0297.10] Non-polypeptide of the invention-chemicals are
e.g. polypeptides having not the activity and/or amino acid
sequence of a polypeptide indicated in Table II, columns 3, 5 or 7,
lines 1 to 5 and/or lines 334 to 338.
[0426] [0298.0.0.0] A polypeptide of the invention can participate
in the process of the present invention. The polypeptide or a
portion thereof comprises preferably an amino acid sequence which
is sufficiently homologous to an amino acid sequence as indicated
in Table II, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338
such that the protein or portion thereof maintains the ability to
confer the activity of the present invention. The portion of the
protein is preferably a biologically active portion as described
herein. Preferably, the polypeptide used in the process of the
invention has an amino acid sequence identical to a sequence as
indicated in Table II, columns 5 or 7, lines 1 to 5 and/or lines
334 to 338.
[0427] [0299.0.0.0] Further, the polypeptide can have an amino acid
sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
1 to 5 and/or lines 334 to 338. The preferred polypeptide of the
present invention preferably possesses at least one of the
activities according to the invention and described herein. A
preferred polypeptide of the present invention includes an amino
acid sequence encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions, to a nucleotide
sequence as indicated in Table I, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 or which is homologous thereto, as defined
above.
[0428] [0300.0.0.0] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 in amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of a sequence as indicated in Table
II A or IIB, columns 5 or 7, lines 1 to 5 and/or lines 334 to
338.
[0429] [0301.0.0.0] For the comparison of amino acid sequences the
same algorithms as described above or nucleic acid sequences can be
used. Results of high quality are reached by using the algorithm of
Needleman and Wunsch or Smith and Waterman. Therefore programs
based on said algorithms are preferred. Advantageously the
comparisons of sequences can be done with the program PileUp (J.
Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989:
151-153) or preferably with the programs Gap and BestFit, which are
respectively based on the algorithms of Needleman and Wunsch [J.
Mol. Biol. 48; 443-453 (1970)] and Smith and Waterman [Adv. Appl.
Math. 2; 482-489 (1981)]. Both programs are part of the GCG
software-package [Genetics Computer Group, 575 Science Drive,
Madison, Wis., USA 53711 (1991); Altschul et al. (1997) Nucleic
Acids Res. 25:3389 et seq.]. Therefore preferably the calculations
to determine the percentages of sequence homology are done with the
program Gap over the whole range of the sequences. The following
standard adjustments for the comparison of amino acid sequences
were used: gap weight: 8, length weight: 2, average match: 2.912,
average mismatch: -2.003.
[0430] [0302.0.0.0] Biologically active portions of an polypeptide
of the present invention include peptides comprising amino acid
sequences derived from the amino acid sequence of the polypeptide
of the present invention or used in the process of the present
invention, e.g., an amino acid sequence as indicated in Table II,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 or the amino
acid sequence of a protein homologous thereto, which include fewer
amino acids than a full length polypeptide of the present invention
or used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[0431] [0303.0.0.0] Typically, biologically (or immunologically)
active portions i.e. peptides, e.g., peptides which are, for
example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more
amino acids in length comprise a domain or motif with at least one
activity or epitope of a polypeptide of the present invention or
used in the process of the present invention. Moreover, other
biologically active portions, in which other regions of the
polypeptide are deleted, can be prepared by recombinant techniques
and evaluated for one or more of the activities described
herein.
[0432] [0304.0.0.0] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 1 to 5 and/or lines 334 to 338 but having
differences in the sequence from said wild-type protein. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity in relation to the wild type protein.
[0433] [0305.0.0.0] Any mutagenesis strategies for the polypeptide
of the present invention or the polypeptide used in the process of
the present invention to result in increasing said activity are not
meant to be limiting; variations on these strategies will be
readily apparent to one skilled in the art. Using such strategies,
and incorporating the mechanisms disclosed herein, the nucleic acid
molecule and polypeptide of the invention or the polypeptide used
in the method of the invention may be utilized to generate plants
or parts thereof, expressing one or more wildtype protein(s) or one
or more mutated protein encoding nucleic acid molecule(s) or
polypeptide molecule(s) of the invention such that the yield,
production, and/or efficiency of production of a desired compound
is improved.
[0434] [0306.0.0.0] This desired compound may be any natural
product of plants, which includes the final products of
biosynthesis pathways and intermediates of naturally-occurring
metabolic pathways, as well as molecules which do not naturally
occur in the metabolism of said cells, but which are produced by a
said cells of the invention. Preferably, the compound is a
composition comprising the respective fine chemical or a recovered
respective fine chemical, in particular, the fine chemical, free or
in protein-bound form.
[0435] [0306.1.0.0] Preferably, the compound is a composition
comprising the methionine or a recovered methionine, in particular,
the fine chemical, free or in protein-bound form.
[0436] [0307.0.0.0] The invention also provides chimeric or fusion
proteins.
[0437] [0308.0.0.0] As used herein, an "chimeric protein" or
"fusion protein" comprises an polypeptide operatively linked to a
polypeptide which does not confer above-mentioned activity, in
particular, which does not confer an increase of content of the
respective fine chemical in a cell or an organism or a part
thereof, if its activity is increased.
[0438] [0309.0.0.0] In one embodiment, an reference to a "protein
(=polypeptide) of the invention" or as indicated in Table II,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 refers to a
polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas a "non-polypeptide of the invention" or "other
polypeptide" not being indicated in Table II, columns 5 or 7, lines
1 to 5 and/or lines 334 to 338 refers to a polypeptide having an
amino acid sequence corresponding to a protein which is not
substantially homologous a polypeptide of the invention, preferably
which is not substantially homologous to a polypeptide as indicated
in Table II, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338,
e.g., a protein which does not confer the activity described herein
or annotated or known for as indicated in Table II, column 3, lines
1 to 5 and/or lines 334 to 338, and which is derived from the same
or a different organism. In one embodiment, a "non-polypeptide of
the invention" or "other polypeptide" not being indicated in Table
II, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 does not
confer an increase of the respective fine chemical in an organism
or part thereof.
[0439] [0310.0.0.0] Within the fusion protein, the term
"operatively linked" is intended to indicate that the polypeptide
of the invention or a polypeptide used in the process of the
invention and the "other polypeptide" or a part thereof are fused
to each other so that both sequences fulfil the proposed function
addicted to the sequence used. The "other polypeptide" can be fused
to the N-terminus or C-terminus of the polypeptide of the invention
or used in the process of the invention. For example, in one
embodiment the fusion protein is a GST-LMRP fusion protein in which
the sequences of the polypeptide of the invention or the
polypeptide used in the process of the invention are fused to the
C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant polypeptides of the
invention or a polypeptide useful in the process of the
invention.
[0440] [0311.0.0.0] In another embodiment, the fusion protein is a
polypeptide of the invention or a polypeptide used in the process
of the invention containing a heterologous signal sequence at its
N-terminus. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of a polypeptide of the invention or a
polypeptide used in the process of the invention can be increased
through use of a heterologous signal sequence. As already mentioned
above, targeting sequences, are required for targeting the gene
product into specific cell compartment (for a review, see Kermode,
Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 and references cited
therein), for example into the vacuole, the nucleus, all types of
plastids, such as amyloplasts, chloroplasts, chromoplasts, the
extracellular space, the mitochondria, the endoplasmic reticulum,
elaioplasts, peroxisomes, glycosomes, and other compartments of
cells or extracellular. Sequences, which must be mentioned in this
context are, in particular, the signal-peptide- or
transit-peptide-encoding sequences which are known per se. For
example, plastid-transit-peptide-encoding sequences enable the
targeting of the expression product into the plastids of a plant
cell. Targeting sequences are also known for eukaryotic and to a
lower extent for prokaryotic organisms and can advantageously be
operable linked with the nucleic acid molecule of the present
invention to achieve an expression in one of said compartments or
extracellular.
[0441] [0312.0.0.0] Preferably, a chimeric or fusion protein of the
invention is produced by standard recombinant DNA techniques. For
example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. The fusion gene can be synthesized
by conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of gene fragments can be carried
out using anchor primers, which give rise to complementary
overhangs between two consecutive gene fragments which can
subsequently be annealed and reamplified to generate a chimeric
gene sequence (see, for example, Current Protocols in Molecular
Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
Moreover, many expression vectors are commercially available that
already encode a fusion moiety (e.g., a GST polypeptide). The
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention can be cloned into such an
expression vector such that the fusion moiety is linked in-frame to
the encoded protein.
[0442] [0313.0.0.0] Furthermore, folding simulations and computer
redesign of structural motifs of the protein of the invention can
be performed using appropriate computer programs (Olszewski,
Proteins 25 (1996), 286-299; Hoffman, Comput. Appl. Biosci. 11
(1995), 675-679). Computer modelling of protein folding can be used
for the conformational and energetic analysis of detailed peptide
and protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012;
Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45). The appropriate
programs can be used for the identification of interactive sites
the polypeptide of the invention or polypeptides used in the
process of the invention and its substrates or binding factors or
other interacting proteins by computer assistant searches for
complementary peptide sequences (Fassina, Immunomethods (1994),
114-120). Further appropriate computer systems for the design of
protein and peptides are described in the prior art, for example in
Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y.
Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986),
5987-5991. The results obtained from the above-described computer
analysis can be used for, e.g., the preparation of peptidomimetics
of the protein of the invention or fragments thereof. Such
pseudopeptide analogues of the, natural amino acid sequence of the
protein may very efficiently mimic the parent protein (Benkirane,
J. Biol. Chem. 271 (1996), 33218-33224). For example, incorporation
of easily available achiral Q-amino acid residues into a protein of
the invention or a fragment thereof results in the substitution of
amide bonds by polymethylene units of an aliphatic chain, thereby
providing a convenient strategy for constructing a peptidomimetic
(Banerjee, Biopolymers 39 (1996), 769-777).
[0443] [0314.0.0.0] Superactive peptidomimetic analogues of small
peptide hormones in other systems are described in the prior art
(Zhang, Biochem. Biophys. Res. Commun. 224 (1996), 327-331).
Appropriate peptidomimetics of the protein of the present invention
can also be identified by the synthesis of peptidomimetic
combinatorial libraries through successive amide alkylation and
testing the resulting compounds, e.g., for their binding and
immunological properties. Methods for the generation and use of
peptidomimetic combinatorial libraries are described in the prior
art, for example in Ostresh, Methods in Enzymology 267 (1996),
220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.
[0444] [0315.0.0.0] Furthermore, a three-dimensional and/or
crystallographic structure of the protein of the invention can be
used for the design of peptidomimetic inhibitors of the biological
activity of the protein of the invention (Rose, Biochemistry 35
(1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996),
1545-1558).
[0445] [0316.0.0.0] Furthermore, a three-dimensional and/or
crystallographic structure of the protein of the invention and the
identification of interactive sites the polypeptide of the
invention or the polypeptide used in the method of the invention
and its substrates or binding factors can be used for the
identification or design of mutants with modulated binding or turn
over activities. For example, the active centre of the polypeptide
of the present invention can be modelled and amino acid residues
participating in the catalytic reaction can be modulated to
increase or decrease the binding of the substrate to activate or
improve the polypeptide. The identification of the active centre
and the amino acids involved in the catalytic reaction facilitates
the screening for mutants having an increased activity.
[0446] [0317.0.0.0] The sequences shown in column 5 of the Tables I
to IV herein have also been described under their Gene/ORF Locus
Name as described in the Table I, II, III or IV, column 3.
[0447] [0318.0.0.0] In an especially preferred embodiment, the
polypeptide according to the invention furthermore also does not
have the sequences of those proteins which are encoded by the
sequences shown in the known listed Gene/ORF Locus Names or as
described in the Tables, column 3.
[0448] [0319.0.0.0] One embodiment of the invention also relates to
an antibody, which binds specifically to the polypeptide according
to the invention or parts, i.e. specific fragments or epitopes of
such a protein.
[0449] [0320.0.0.0] The antibodies of the invention can be used to
identify and isolate the polypeptide according to the invention and
encoding genes in any organism, preferably plants, prepared in
plants described herein. These antibodies can be monoclonal
antibodies, polyclonal antibodies or synthetic antibodies as well
as fragments of antibodies, such as Fab, Fv or scFv fragments etc.
Monoclonal antibodies can be prepared, for example, by the
techniques as originally described in KOhler and Milstein, Nature
256 (1975), 495, and Galfr6, Meth. Enzymol. 73 (1981), 3, which
comprise the fusion of mouse myeloma cells to spleen cells derived
from immunized mammals.
[0450] [0321.0.0.0] Furthermore, antibodies or fragments thereof to
the aforementioned peptides can be obtained by using methods, which
are described, e.g., in Harlow and Lane "Antibodies, A Laboratory
Manual", CSH Press, Cold Spring Harbor, 1988. These antibodies can
be used, for example, for the immunoprecipitation and
immunolocalization of proteins according to the invention as well
as for the monitoring of the synthesis of such proteins, for
example, in recombinant organisms, and for the identification of
compounds interacting with the protein according to the invention.
For example, surface plasmon resonance as employed in the BIAcore
system can be used to increase the efficiency of phage antibodies
selections, yielding a high increment of affinity from a single
library of phage antibodies, which bind to an epitope of the
protein of the invention (Schier, Human Antibodies Hybridomas 7
(1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). In
many cases, the binding phenomena of antibodies to antigens are
equivalent to other ligand/anti-ligand binding.
[0451] [0322.0.0.0] In one embodiment, the present invention
relates to an antisense nucleic acid molecule comprising the
complementary sequence of the nucleic acid molecule of the present
invention.
[0452] [0323.0.0.0] Methods to modify the expression levels and/or
the activity are known to persons skilled in the art and include
for instance overexpression, co-suppression, the use of ribozymes,
sense and anti-sense strategies or other gene silencing approaches
like RNA interference (RNAi) or promoter methylation. "Sense
strand" refers to the strand of a double-stranded DNA molecule that
is homologous to an mRNA transcript thereof. The "anti-sense
strand" contains an inverted sequence, which is complementary to
that of the "sense strand".
[0453] In addition the expression levels and/or the activity can be
modified by the introduction of mutations in the regulatory or
coding regions of the nucleic acids of the invention. Furthermore
antibodies can be expressed which specifically binds to a
polypeptide of interest and thereby blocks it activity. The
protein-binding factors can, for example, also be aptamers [Famulok
M and Mayer G (1999) Curr. Top Microbiol. Immunol. 243: 123-36] or
antibodies or antibody fragments or single-chain antibodies.
Obtaining these factors has been described, and the skilled worker
is familiar therewith. For example, a cytoplasmic scFv antibody has
been employed for modulating activity of the phytochrome A protein
in genetically modified tobacco plants [Owen M et al. (1992)
Biotechnology (NY) 10(7): 790-794; Franken E et al. (1997) Curr.
Opin. Biotechnol. 8(4): 411-416; Whitelam (1996) Trend Plant Sci.
1: 286-272].
[0454] [0324.0.0.0] An "antisense" nucleic acid molecule comprises
a nucleotide sequence, which is complementary to a "sense" nucleic
acid molecule encoding a protein, e.g., complementary to the coding
strand of a double-stranded cDNA molecule or complementary to an
encoding mRNA sequence. Accordingly, an antisense nucleic acid
molecule can bond via hydrogen bonds to a sense nucleic acid
molecule. The antisense nucleic acid molecule can be complementary
to an entire coding strand of a nucleic acid molecule conferring
the expression of the polypeptide of the invention or used in the
process of the present invention, as the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention coding strand, or to only a portion thereof.
Accordingly, an antisense nucleic acid molecule can be antisense to
a "coding region" of the coding strand of a nucleotide sequence of
a nucleic acid molecule of the present invention. The term "coding
region" refers to the region of the nucleotide sequence comprising
codons, which are translated into amino acid residues. Further, the
antisense nucleic acid molecule is antisense to a "noncoding
region" of the coding strand of a nucleotide sequence encoding the
polypeptide of the invention or a polypeptide used in the process
of the invention. The term "noncoding region" refers to 5' and 3'
sequences which flank the coding region that are not translated
into a polypeptide, i.e., also referred to as 5' and 3'
untranslated regions (5'-UTR or 3'-UTR).
[0455] [0325.0.0.0] Given the coding strand sequences encoding the
polypeptide of the present invention antisense nucleic acid
molecules of the invention can be designed according to the rules
of Watson and Crick base pairing.
[0456] [0326.0.0.0] The antisense nucleic acid molecule can be
complementary to the entire coding region of the mRNA encoding the
nucleic acid molecule to the invention or used in the process of
the present invention, but can also be an oligonucleotide which is
antisense to only a portion of the coding or noncoding region of
said mRNA. For example, the antisense oligonucleotide can be
complementary to the region surrounding the translation start site
of said mRNA. An antisense oligonucleotide can be, for example,
about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or 200 nucleotides
in length. An antisense nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention can
be constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid molecule (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense
and sense nucleic acids, e.g., phosphorothioate derivatives and
acridine substituted nucleotides can be used. Examples of modified
nucleotides which can be used to generate the antisense nucleic
acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid molecule has been subcloned in an antisense
orientation (i.e., RNA transcribed from the inserted nucleic acid
molecule will be of an antisense orientation to a target nucleic
acid molecule of interest, described further in the following
subsection).
[0457] [0327.0.0.0] The antisense nucleic acid molecules of the
invention are typically administered to a cell or generated in situ
such that they hybridize with or bind to cellular mRNA and/or
genomic DNA encoding a polypeptide of the invention or the
polypeptide used in the method of the invention having
aforementioned the respective fine chemical increasing activity to
thereby inhibit expression of the protein, e.g., by inhibiting
transcription and/or translation.
[0458] [0328.0.0.0] The hybridization can be by conventional
nucleotide complementarity to form a stable duplex, or, for
example, in the case of an antisense nucleic acid molecule which
binds to DNA duplexes, through specific interactions in the major
groove of the double helix. The antisense nucleic acid molecule can
also be delivered to cells using the vectors described herein. To
achieve sufficient intracellular concentrations of the antisense
molecules, vector in which the antisense nucleic acid molecule is
placed under the control of a strong prokaryotic, viral, or
eukaryotic including plant promoters are preferred.
[0459] [0329.0.0.0] In a further embodiment, the antisense nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention can be an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methyl-ribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0460] [0330.0.0.0] Further the antisense nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention can be also a ribozyme. Ribozymes are catalytic RNA
molecules with ribonuclease activity, which are capable of cleaving
a single-stranded nucleic acid, such as an mRNA, to which they have
a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can
be used to catalytically cleave mRNA transcripts encoding the
polypeptide of the invention or the polypeptide used in the method
of the invention to thereby inhibit translation of said mRNA. A
ribozyme having specificity for a nucleic acid molecule encoding
the polypeptide of the invention or used in the process of the
invention can be designed based upon the nucleotide sequence of the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention or coding a protein used in the
process of the invention or on the basis of a heterologous sequence
to be isolated according to methods taught in this invention. For
example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved in an
encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071 and
Cech et al. U.S. Pat. No. 5,116,742. Alternatively, mRNA encoding
the polypeptide of the invention or a polypeptide used in the
process of the invention can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science
261:1411-1418.
[0461] [0331.0.0.0] The antisense molecule of the present invention
comprises also a nucleic acid molecule comprising a nucleotide
sequences complementary to the regulatory region of an nucleotide
sequence encoding the natural occurring polypeptide of the
invention or the polypeptide used in the method of the invention,
e.g. the polypeptide sequences shown in the sequence listing, or
identified according to the methods described herein, e.g., its
promoter and/or enhancers, e.g. to form triple helical structures
that prevent transcription of the gene in target cells. See
generally, Helene, C. (1991) Anticancer Drug Des. 6(6): 569-84;
Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher,
L. J. (1992) Bioassays 14(12): 807-15.
[0462] [0332.0.0.0] Furthermore the present invention relates to a
double stranded RNA molecule capable for the reduction or
inhibition of the activity of the gene product of a gene encoding
the polypeptide of the invention, a polypeptide used in the process
of the invention, the nucleic acid molecule of the invention or a
nucleic acid molecule used in the process of the invention
encoding.
[0463] [0333.0.0.0] The method of regulating genes by means of
double-stranded RNA ("double-stranded RNA interference"; dsRNAi)
has been described extensively for animal, yeast, fungi and plant
organisms such as Neurospora, zebrafish, Drosophila, mice,
planaria, humans, Trypanosoma, petunia or Arabidopsis (for example
Matzke M A et al. (2000) Plant Mol. Biol. 43: 401-415; Fire A. et
al. (1998) Nature 391: 806-811; WO 99/32619; WO 99/53050; WO
00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364). In
addition RNAi is also documented as an advantageously tool for the
repression of genes in bacteria such as E. coli for example by
Tchurikov et al. [J. Biol. Chem., 2000, 275 (34): 26523-26529].
Fire et al. named the phenomenon RNAi for "RNA interference". The
techniques and methods described in the above references are
expressly referred to. Efficient gene suppression can also be
observed in the case of transient expression or following transient
transformation, for example as the consequence of a biolistic
transformation (Schweizer P et al. (2000) Plant J 2000 24:
895-903). dsRNAi methods are based on the phenomenon that the
simultaneous introduction of complementary strand and counterstrand
of a gene transcript brings about highly effective suppression of
the expression of the gene in question. The resulting phenotype is
very similar to that of an analogous knock-out mutant (Waterhouse P
M et al. (1998) Proc. Natl. Acad. Sci. USA 95: 13959-64).
[0464] [0334.0.0.0] Tuschl et al. [Gens Dev., 1999, 13 (24):
3191-3197] was able to show that the efficiency of the RNAi method
is a function of the length of the duplex, the length of the 3'-end
overhangs, and the sequence in these overhangs. Based on the work
of Tuschl et al. the following guidelines can be given to the
skilled worker: To achieve good results the 5' and 3' untranslated
regions of the used nucleic acid sequence and regions close to the
start codon should be avoided as this regions are richer in
regulatory protein binding sites and interactions between RNAi
sequences and such regulatory proteins might lead to undesired
interactions. Preferably a region of the used mRNA is selected,
which is 50 to 100 nt (=nucleotides or bases) downstream of the AUG
start codon. Only dsRNA (=double-stranded RNA) sequences from exons
are useful for the method, as sequences from introns have no
effect. The G/C content in this region should be greater than 30%
and less than 70% ideally around 50%. A possible secondary
structure of the target mRNA is less important for the effect of
the RNAi method.
[0465] [0335.0.0.0] The dsRNAi method has proved to be particularly
effective and advantageous for reducing the expression of a nucleic
acid sequences as indicated in Table I, columns 5 or 7, lines 1 to
5 and/or lines 334 to 338 and/or homologs thereof. As described
inter alia in WO 99/32619, dsRNAi approaches are clearly superior
to traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived there from),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table I,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 and/or
homologs thereof. In a double-stranded RNA molecule for reducing
the expression of an protein encoded by a nucleic acid sequence of
one of the sequences as indicated in Table I, columns 5 or 7, lines
1 to 5 and/or lines 334 to 338 and/or homologs thereof, one of the
two RNA strands is essentially identical to at least part of a
nucleic acid sequence, and the respective other RNA strand is
essentially identical to at least part of the complementary strand
of a nucleic acid sequence.
[0466] [0336.0.0.0] The term "essentially identical" refers to the
fact that the dsRNA sequence may also include insertions, deletions
and individual point mutations in comparison to the target sequence
while still bringing about an effective reduction in expression.
Preferably, the homology as defined above amounts to at least 30%,
preferably at least 40%, 50%, 60%, 70% or 80%, very especially
preferably at least 90%, most preferably 100%, between the "sense"
strand of an inhibitory dsRNA and a part-segment of a nucleic acid
sequence of the invention (or between the "antisense" strand and
the complementary strand of a nucleic acid sequence, respectively).
The part-segment amounts to at least 10 bases, preferably at least
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 bases,
especially preferably at least 40, 50, 60, 70, 80 or 90 bases, very
especially preferably at least 100, 200, 300 or 400 bases, most
preferably at least 500, 600, 700, 800, 900 or more bases or at
least 1000 or 2000 bases or more in length. In another preferred
embodiment of the invention the part-segment amounts to 17, 18, 19,
20, 21, 22, 23, 24, 25, 26 or 27 bases, preferably to 20, 21, 22,
23, 24 or 25 bases. These short sequences are preferred in animals
and plants. The longer sequences preferably between 200 and 800
bases are preferred in non-mammalian animals, preferably in
invertebrates, in yeast, fungi or bacteria, but they are also
useable in plants. Long double-stranded RNAs are processed in the
organisms into many siRNAs (=small/short interfering RNAs) for
example by the protein Dicer, which is a ds-specific Rnase III
enzyme. As an alternative, an "essentially identical" dsRNA may
also be defined as a nucleic acid sequence, which is capable of
hybridizing with part of a gene transcript (for example in 400 mM
NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50.degree. C. or 70.degree.
C. for 12 to 16 h).
[0467] [0337.0.0.0] The dsRNA may consist of one or more strands of
polymerized ribonucleotides. Modification of both the
sugar-phosphate backbone and of the nucleosides may furthermore be
present. For example, the phosphodiester bonds of the natural RNA
can be modified in such a way that they encompass at least one
nitrogen or sulfur heteroatom. Bases may undergo modification in
such a way that the activity of, for example, adenosine deaminase
is restricted. These and other modifications are described herein
below in the methods for stabilizing antisense RNA.
[0468] [0338.0.0.0] The dsRNA can be prepared enzymatically; it may
also be synthesized chemically, either in full or in part.
[0469] [0339.0.0.0] The double-stranded structure can be formed
starting from a single, self-complementary strand or starting from
two complementary strands. In a single, self-complementary strand,
"sense" and "antisense" sequence can be linked by a linking
sequence ("linker") and form for example a hairpin structure.
Preferably, the linking sequence may take the form of an intron,
which is spliced out following dsRNA synthesis. The nucleic acid
sequence encoding a dsRNA may contain further elements such as, for
example, transcription termination signals or polyadenylation
signals. If the two strands of the dsRNA are to be combined in a
cell or an organism advantageously in a plant, this can be brought
about in a variety of ways.
[0470] [0340.0.0.0] Formation of the RNA duplex can be initiated
either outside the cell or within the cell. As shown in WO
99/53050, the dsRNA may also encompass a hairpin structure, by
linking the "sense" and "antisense" strands by a "linker" (for
example an intron). The self-complementary dsRNA structures are
preferred since they merely require the expression of a construct
and always encompass the complementary strands in an equimolar
ratio.
[0471] [0341.0.0.0] The expression cassettes encoding the
"antisense" or the "sense" strand of the dsRNA or the
self-complementary strand of the dsRNA are preferably inserted into
a vector and stably inserted into the genome of a plant, using the
methods described herein below (for example using selection
markers), in order to ensure permanent expression of the dsRNA.
[0472] [0342.0.0.0] The dsRNA can be introduced using an amount
which makes possible at least one copy per cell. A larger amount
(for example at least 5, 10, 100, 500 or 1 000 copies per cell) may
bring about more efficient reduction.
[0473] [0343.0.0.0] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 or its homolog is not necessarily required
in order to bring about effective reduction in the expression. The
advantage is, accordingly, that the method is tolerant with regard
to sequence deviations as may be present as a consequence of
genetic mutations, polymorphisms or evolutionary divergences. Thus,
for example, using the dsRNA, which has been generated starting
from a sequence as indicated in Table I, columns 5 or 7, lines 1 to
5 and/or lines 334 to 338 or homologs thereof of the one organism,
may be used to suppress the corresponding expression in another
organism.
[0474] [0344.0.0.0] Due to the high degree of sequence homology
between sequences from various organisms (e.g. plants), allows the
conclusion that these proteins may be conserved to a high degree
within, for example other, plants, it is optionally possible that
the expression of a dsRNA derived from one of the disclosed
sequences as shown herein or homologs thereof should also have has
an advantageous effect in other plant species. Preferably the
consensus sequences shown herein can be used for the construction
of useful dsRNA molecules.
[0475] [0345.0.0.0] The dsRNA can be synthesized either in vivo or
in vitro. To this end, a DNA sequence encoding a dsRNA can be
introduced into an expression cassette under the control of at
least one genetic control element (such as, for example, promoter,
enhancer, silencer, splice donor or splice acceptor or
polyadenylation signal). Suitable advantageous constructs are
described herein below. Polyadenylation is not required, nor do
elements for initiating translation have to be present.
[0476] [0346.0.0.0] A dsRNA can be synthesized chemically or
enzymatically. Cellular RNA polymerases or bacteriophage RNA
polymerases (such as, for example T3, T7 or SP6 RNA polymerase) can
be used for this purpose. Suitable methods for the in-vitro
expression of RNA are described (WO 97/32016; U.S. Pat. No.
5,593,874; U.S. Pat. No. 5,698,425, U.S. Pat. No. 5,712,135, U.S.
Pat. No. 5,789,214, U.S. Pat. No. 5,804,693). Prior to introduction
into a cell, tissue or organism, a dsRNA which has been synthesized
in vitro either chemically or enzymatically can be isolated to a
higher or lesser degree from the reaction mixture, for example by
extraction, precipitation, electrophoresis, chromatography or
combinations of these methods. The dsRNA can be introduced directly
into the cell or else be applied extracellularly (for example into
the interstitial space).
[0477] [0347.0.0.0] Advantageously the RNAi method leads to only a
partial loss of gene function and therefore enables the skilled
worker to study a gene dose effect in the desired organism and to
fine tune the process of the invention. Furthermore it enables a
person skilled in the art to study multiple functions of a
gene.
[0478] [0348.0.0.0] Stable transformation of the plant with an
expression construct, which brings about the expression of the
dsRNA is preferred, however. Suitable methods are described herein
below.
[0479] [0349.0.0.0] A further embodiment of the invention also
relates to a method for the generation of a transgenic host or host
cell, e.g. a eukaryotic or prokaryotic cell, preferably a
transgenic microorganism, a transgenic plant cell or a transgenic
plant tissue or a transgenic plant, which comprises introducing,
into the plant, the plant cell or the plant tissue, the nucleic
acid construct according to the invention, the vector according to
the invention, or the nucleic acid molecule according to the
invention.
[0480] [0350.0.0.0] A further embodiment of the invention also
relates to a method for the transient generation of a host or host
cell, eukaryotic or prokaryotic cell, preferably a transgenic
microorganism, a transgenic plant cell or a transgenic plant tissue
or a transgenic plant, which comprises introducing, into the plant,
the plant cell or the plant tissue, the nucleic acid construct
according to the invention, the vector according to the invention,
the nucleic acid molecule characterized herein as being contained
in the nucleic acid construct of the invention or the nucleic acid
molecule according to the invention, whereby the introduced nucleic
acid molecules, nucleic acid construct and/or vector is not
integrated into the genome of the host or host cell. Therefore the
transformants are not stable during the propagation of the host in
respect of the introduced nucleic acid molecules, nucleic acid
construct and/or vector.
[0481] [0351.0.0.0] In the process according to the invention,
transgenic organisms are also to be understood as meaning--if they
take the form of plants--plant cells, plant tissues, plant organs
such as root, shoot, stem, seed, flower, tuber or leaf, or intact
plants which are grown for the production of the respective fine
chemical.
[0482] [0352.0.0.0] Growing is to be understood as meaning for
example culturing the transgenic plant cells, plant tissue or plant
organs on or in a nutrient medium or the intact plant on or in a
substrate, for example in hydroponic culture, potting compost or on
a field soil.
[0483] [0353.0.0.0] In a further advantageous embodiment of the
process, the nucleic acid molecules can be expressed in
single-celled plant cells (such as algae), see Falciatore et al.,
1999, Marine Biotechnology 1 (3): 239-251 and references cited
therein, and plant cells from higher plants (for example
spermatophytes such as crops). Examples of plant expression vectors
encompass those which are described in detail herein or in: Becker,
D. [(1992) Plant Mol. Biol. 20:1195-1197] and Bevan, M. W. [(1984),
Nucl. Acids Res. 12:8711-8721; Vectors for Gene Transfer in Higher
Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization,
Ed.: Kung and R. Wu, Academic Press, 1993, pp. 15-38]. An overview
of binary vectors and their use is also found in Hellens, R.
[(2000), Trends in Plant Science, Vol. 5 No. 10, 446-451.
[0484] [0354.0.0.0] Vector DNA can be introduced into prokaryotic
or eukaryotic cells via conventional transformation or transfection
techniques. The terms "transformation" and "transfection" include
conjugation and transduction and, as used in the present context,
are intended to encompass a multiplicity of prior-art methods for
introducing foreign nucleic acid molecules (for example DNA) into a
host cell, including calcium phosphate coprecipitation or calcium
chloride coprecipitation, DEAE-dextran-mediated transfection,
PEG-mediated transfection, lipofection, natural competence,
chemically mediated transfer, electroporation or particle
bombardment. Suitable methods for the transformation or
transfection of host cells, including plant cells, can be found in
Sambrook et al. (Molecular Cloning: A Laboratory Manual., 2nd Ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989) and in other laboratory handbooks
such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium
protocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J.
[0485] [0355.0.0.0] The above-described methods for the
transformation and regeneration of plants from plant tissues or
plant cells are exploited for transient or stable transformation of
plants. Suitable methods are the transformation of protoplasts by
polyethylene-glycol-induced DNA uptake, the biolistic method with
the gene gun--known as the particle bombardment method--,
electroporation, the incubation of dry embryos in DNA-containing
solution, microinjection and the Agrobacterium-mediated gene
transfer. The abovementioned methods are described for example in
B. Jenes, Techniques for Gene Transfer, in: Transgenic Plants, Vol.
1, Engineering and Utilization, edited by S. D. Kung and R. Wu,
Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant
Physiol. Plant Molec. Biol. 42 (1991) 205-225. The construct to be
expressed is preferably cloned into a vector, which is suitable for
transforming Agrobacterium tumefaciens, for example pBin19 (Bevan,
Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed with
such a vector can then be used in the known manner for the
transformation of plants, in particular crop plants, such as, for
example, tobacco plants, for example by bathing scarified leaves or
leaf segments in an agrobacterial solution and subsequently
culturing them in suitable media. The transformation of plants with
Agrobacterium tumefaciens is described for example by Hofgen and
Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or known from, inter
alia, F. F. White, Vectors for Gene Transfer in Higher Plants; in
Transgenic Plants, Vol. 1, Engineering and Utilization, edited by
S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
[0486] [0356.0.0.0] To select for the successful transfer of the
nucleic acid molecule, vector or nucleic acid construct of the
invention according to the invention into a host organism, it is
advantageous to use marker genes as have already been described
above in detail. It is known of the stable or transient integration
of nucleic acids into plant cells that only a minority of the cells
takes up the foreign DNA and, if desired, integrates it into its
genome, depending on the expression vector used and the
transfection technique used. To identify and select these
integrants, a gene encoding for a selectable marker (as described
above, for example resistance to antibiotics) is usually introduced
into the host cells together with the gene of interest. Preferred
selectable markers in plants comprise those, which confer
resistance to an herbicide such as glyphosate or gluphosinate.
Other suitable markers are, for example, markers, which encode
genes involved in biosynthetic pathways of, for example, sugars or
amino acids, such as .beta.-galactosidase, ura3 or ilv2. Markers,
which encode genes such as luciferase, gfp or other fluorescence
genes, are likewise suitable. These markers and the aforementioned
markers can be used in mutants in whom these genes are not
functional since, for example, they have been deleted by
conventional methods. Furthermore, nucleic acid molecules, which
encode a selectable marker, can be introduced into a host cell on
the same vector as those, which encode the polypeptides of the
invention or used in the process or else in a separate vector.
Cells which have been transfected stably with the nucleic acid
introduced can be identified for example by selection (for example,
cells which have integrated the selectable marker survive whereas
the other cells die).
[0487] [0357.0.0.0] Since the marker genes, as a rule specifically
the gene for resistance to antibiotics and herbicides, are no
longer required or are undesired in the transgenic host cell once
the nucleic acids have been introduced successfully, the process
according to the invention for introducing the nucleic acids
advantageously employs techniques which enable the removal, or
excision, of these marker genes. One such a method is what is known
as cotransformation. The cotransformation method employs two
vectors simultaneously for the transformation, one vector bearing
the nucleic acid according to the invention and a second bearing
the marker gene(s). A large proportion of transformants receives
or, in the case of plants, comprises (up to 40% of the
transformants and above), both vectors. In case of transformation
with Agrobacteria, the transformants usually receive only a part of
the vector, the sequence flanked by the T-DNA, which usually
represents the expression cassette. The marker genes can
subsequently be removed from the transformed plant by performing
crosses. In another method, marker genes integrated into a
transposon are used for the transformation together with desired
nucleic acid (known as the Ac/Ds technology). The transformants can
be crossed with a transposase resource or the transformants are
transformed with a nucleic acid construct conferring expression of
a transposase, transiently or stable. In some cases (approx. 10%),
the transposon jumps out of the genome of the host cell once
transformation has taken place successfully and is lost. In a
further number of cases, the transposon jumps to a different
location. In these cases, the marker gene must be eliminated by
performing crosses. In microbiology, techniques were developed
which make possible, or facilitate, the detection of such events. A
further advantageous method relies on what are known as
recombination systems, whose advantage is that elimination by
crossing can be dispensed with. The best-known system of this type
is what is known as the Cre/lox system. Cre1 is a recombinase,
which removes the sequences located between the loxP sequences. If
the marker gene is integrated between the loxP sequences, it is
removed, once transformation has taken place successfully, by
expression of the recombinase. Further recombination systems are
the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., J. Biol.
Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol.,
149, 2000: 553-566). A site-specific integration into the plant
genome of the nucleic acid sequences according to the invention is
possible. Naturally, these methods can also be applied to
microorganisms such as yeast, fungi or bacteria.
[0488] [0358.0.0.0] Agrobacteria transformed with an expression
vector according to the invention may also be used in the manner
known per se for the transformation of plants such as experimental
plants like Arabidopsis or crop plants, such as, for example,
cereals, maize, oats, rye, barley, wheat, soya, rice, cotton,
sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato,
carrot, bell peppers, oilseed rape, tapioca, cassava, arrow root,
tagetes, alfalfa, lettuce and the various tree, nut, and grapevine
species, in particular oil-containing crop plants such as soya,
peanut, castor-oil plant, sunflower, maize, cotton, flax, oilseed
rape, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa
beans, for example by bathing scarified leaves or leaf segments in
an agrobacterial solution and subsequently growing them in suitable
media.
[0489] [0359.0.0.0] In addition to the transformation of somatic
cells, which then has to be regenerated into intact plants, it is
also possible to transform the cells of plant meristems and in
particular those cells which develop into gametes. In this case,
the transformed gametes follow the natural plant development,
giving rise to transgenic plants. Thus, for example, seeds of
Arabidopsis are treated with agrobacteria and seeds are obtained
from the developing plants of which a certain proportion is
transformed and thus transgenic (Feldman, K A and Marks M D (1987).
Mol Gen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua
and J Shell, eds, Methods in Arabidopsis Research. Word Scientific,
Singapore, pp. 274-289). Alternative methods are based on the
repeated removal of the influorescences and incubation of the
excision site in the center of the rosette with transformed
agrobacteria, whereby transformed seeds can likewise be obtained at
a later point in time (Chang (1994). Plant J. 5: 551-558; Katavic
(1994). Mol Gen Genet, 245: 363-370). However, an especially
effective method is the vacuum infiltration method with its
modifications such as the "floral dip" method. In the case of
vacuum infiltration of Arabidopsis, intact plants under reduced
pressure are treated with an agrobacterial suspension (Bechthold, N
(1993). C R Acad Sci Paris Life Sci, 316: 1194-1199), while in the
case of the "floral dip" method the developing floral tissue is
incubated briefly with a surfactant-treated agrobacterial
suspension (Clough, S J and Bent, A F (1998). The Plant J. 16,
735-743). A certain proportion of transgenic seeds are harvested in
both cases, and these seeds can be distinguished from nontransgenic
seeds by growing under the above-described selective conditions. In
addition the stable transformation of plastids is of advantages
because plastids are inherited maternally is most crops reducing or
eliminating the risk of transgene flow through pollen. The
transformation of the chloroplast genome is generally achieved by a
process, which has been schematically displayed in Klaus et al.,
2004 (Nature Biotechnology 22(2), 225-229). Briefly the sequences
to be transformed are cloned together with a selectable marker gene
between flanking sequences homologous to the chloroplast genome.
These homologous flanking sequences direct site specific
integration into the plastome. Plastidal transformation has been
described for many different plant species and an overview can be
taken from Bock (2001) Transgenic plastids in basic research and
plant biotechnology. J Mol Biol. 2001 September 21; 312 (3): 425-38
or Maliga, P (2003) Progress towards commercialization of plastid
transformation technology. Trends Biotechnol. 21, 20-28. Further
biotechnological progress has recently been reported in form of
marker free plastid transformants, which can be produced by a
transient cointegrated maker gene (Klaus et al., 2004, Nature
Biotechnology 22 (2), 225-229).
[0490] [0360.0.0.0] The genetically modified plant cells can be
regenerated via all methods with which the skilled worker is
familiar. Suitable methods can be found in the abovementioned
publications by S. D. Kung and R. Wu, Potrykus or Hofgen and
Willmitzer.
[0491] [0361.0.0.0] Accordingly, the present invention thus also
relates to a plant cell comprising the nucleic acid construct
according to the invention, the nucleic acid molecule according to
the invention or the vector according to the invention.
[0492] [0362.0.0.0] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, the nucleic acid construct of
the invention, the antisense molecule of the invention, the vector
of the invention or a nucleic acid molecule encoding the
polypeptide of the invention or the polypeptide used in the method
of the invention, e.g. the polypeptide as indicated in Table II,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338. Due to the
above mentioned activity the respective fine chemical content in a
cell or an organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or the polypeptide used in the method of the invention or
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention is increased, e.g. due
to an increased expression or specific activity of the subject
matters of the invention in a cell or an organism or a part
thereof. In one embodiment, transgenic for a polypeptide having an
activity of a polypeptide as indicated in Table II, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338 means herein that due to
modulation or manipulation of the genome, an activity as annotated
for a polypeptide as indicated in Table II, column 3, lines 1 to 5
and/or lines 334 to 338, e.g. having a sequence as indicated in
Table II, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338, is
increased in a cell or an organism or a part thereof. Examples are
described above in context with the process of the invention
[0493] [0363.0.0.0] "Transgenic", for example regarding a nucleic
acid molecule, an nucleic acid construct or a vector comprising
said nucleic acid molecule or an organism transformed with said
nucleic acid molecule, nucleic acid construct or vector, refers to
all those subjects originating by recombinant methods in which
either [0494] a) the nucleic acid sequence, or [0495] b) a genetic
control sequence linked operably to the nucleic acid sequence, for
example a promoter, or [0496] c) (a) and (b) are not located in
their natural genetic environment or have been modified by
recombinant methods, an example of a modification being a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide residues. Natural genetic environment refers to the
natural chromosomal locus in the organism of origin, or to the
presence in a genomic library. In the case of a genomic library,
the natural genetic environment of the nucleic acid sequence is
preferably retained, at least in part. The environment flanks the
nucleic acid sequence at least at one side and has a sequence of at
least 50 bp, preferably at least 500 bp, especially preferably at
least 1000 bp, very especially preferably at least 5000 bp, in
length.
[0497] [0364.0.0.0] A naturally occurring expression cassette--for
example the naturally occurring combination of a promoter of a
polypeptide of the invention with the corresponding
protein-encoding sequence--becomes a transgenic expression cassette
when it is modified by non-natural, synthetic "artificial" methods
such as, for example, mutagenization. Such methods have been
described (U.S. Pat. No. 5,565,350; WO 00/15815; also see
above).
[0498] [0365.0.0.0] Further, the plant cell, plant tissue or plant
can also be transformed such that further enzymes and proteins are
(over)expressed which expression supports an increase of the
respective fine chemical.
[0499] [0366.0.0.0] However, transgenic also means that the nucleic
acids according to the invention are located at their natural
position in the genome of an organism, but that the sequence has
been modified in comparison with the natural sequence and/or that
the regulatory sequences of the natural sequences have been
modified. Preferably, transgenic/recombinant is to be understood as
meaning the transcription of the nucleic acids used in the process
according to the invention occurs at a non-natural position in the
genome, that is to say the expression of the nucleic acids is
homologous or, preferably, heterologous. This expression can be
transiently or of a sequence integrated stably into the genome.
[0500] [0367.0.0.0] The term "transgenic plants" used in accordance
with the invention also refers to the progeny of a transgenic
plant, for example the T.sub.1, T.sub.2, T.sub.3 and subsequent
plant generations or the BC.sub.1, BC.sub.2, BC.sub.3 and
subsequent plant generations. Thus, the transgenic plants according
to the invention can be raised and selfed or crossed with other
individuals in order to obtain further transgenic plants according
to the invention. Transgenic plants may also be obtained by
propagating transgenic plant cells vegetatively. The present
invention also relates to transgenic plant material, which can be
derived from a transgenic plant population according to the
invention. Such material includes plant cells and certain tissues,
organs and parts of plants in all their manifestations, such as
seeds, leaves, anthers, fibers, tubers, roots, root hairs, stems,
embryo, calli, cotelydons, petioles, harvested material, plant
tissue, reproductive tissue and cell cultures, which are derived
from the actual transgenic plant and/or can be used for bringing
about the transgenic plant.
[0501] [0368.0.0.0] Any transformed plant obtained according to the
invention can be used in a conventional breeding scheme or in in
vitro plant propagation to produce more transformed plants with the
same characteristics and/or can be used to introduce the same
characteristic in other varieties of the same or related species.
Such plants are also part of the invention. Seeds obtained from the
transformed plants genetically also contain the same characteristic
and are part of the invention. As mentioned before, the present
invention is in principle applicable to any plant and crop that can
be transformed with any of the transformation method known to those
skilled in the art.
[0502] [0369.0.0.0] In an especially preferred embodiment, the
organism, the host cell, plant cell, plant, microorganism or plant
tissue according to the invention is transgenic.
[0503] [0370.0.0.0] Accordingly, the invention therefore relates to
transgenic organisms transformed with at least one nucleic acid
molecule, nucleic acid construct or vector according to the
invention, and to cells, cell cultures, tissues, parts--such as,
for example, in the case of plant organisms, plant tissue, for
example leaves, roots and the like--or propagation material derived
from such organisms, or intact plants. The terms "recombinant
(host)", and "transgenic (host)" are used interchangeably in this
context. Naturally, these terms refer not only to the host organism
or target cell in question, but also to the progeny, or potential
progeny, of these organisms or cells. Since certain modifications
may occur in subsequent generations owing to mutation or
environmental effects, such progeny is not necessarily identical
with the parental cell, but still comes within the scope of the
term as used herein.
[0504] [0371.0.0.0] Suitable organisms for the process according to
the invention or as hosts are all these eukaryotic or prokaryotic
organisms, which are capable of synthesizing the respective fine
chemical. The organisms used as hosts are microorganisms, such as
bacteria, fungi, yeasts or algae, non-human animals, or plants,
such as dictotyledonous or monocotyledonous plants.
[0505] [0372.0.0.0] In principle all plants can be used as host
organism, especially the plants mentioned above as source organism.
Preferred transgenic plants are, for example, selected from the
families Aceraceae, Anacardiaceae, Apiaceae, Asteraceae,
Brassicaceae, Cactaceae, Cucurbitaceae, Euphorbiaceae, Fabaceae,
Malvaceae, Nymphaeaceae, Papaveraceae, Rosaceae, Salicaceae,
Solanaceae, Arecaceae, Bromeliaceae, Cyperaceae, Iridaceae,
Liliaceae, Orchidaceae, Gentianaceae, Labiaceae, Magnoliaceae,
Ranunculaceae, Carifolaceae, Rubiaceae, Scrophulariaceae,
Caryophyllaceae, Ericaceae, Polygonaceae, Violaceae, Juncaceae or
Poaceae and preferably from a plant selected from the group of the
families Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae,
Fabaceae, Papaveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae.
Preferred are crop plants such as plants advantageously selected
from the group of the genus peanut, oilseed rape, canola,
sunflower, safflower, olive, sesame, hazelnut, almond, avocado,
bay, pumpkin/squash, linseed, soya, pistachio, borage, maize,
wheat, rye, oats, sorghum and millet, triticale, rice, barley,
cassava, potato, sugarbeet, egg plant, alfalfa, and perennial
grasses and forage plants, oil palm, vegetables (brassicas, root
vegetables, tuber vegetables, pod vegetables, fruiting vegetables,
onion vegetables, leafy vegetables and stem vegetables), buckwheat,
Jerusalem artichoke, broad bean, vetches, lentil, dwarf bean,
lupin, clover and Lucerne for mentioning only some of them.
[0506] [0373.0.0.0] Preferred plant cells, plant organs, plant
tissues or parts of plants originate from the under source organism
mentioned plant families, preferably from the abovementioned plant
genus, more preferred from abovementioned plants species.
[0507] [0374.0.0.0] Transgenic plants comprising the amino acids
synthesized in the process according to the invention can be
marketed directly without isolation of the compounds synthesized.
In the process according to the invention, plants are understood as
meaning all plant parts, plant organs such as leaf, stalk, root,
tubers or seeds or propagation material or harvested material or
the intact plant. In this context, the seed encompasses all parts
of the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue. The amino acids produced in the
process according to the invention may, however, also be isolated
from the plant in the form of their free amino acids or bound in
proteins. Amino acids produced by this process can be harvested by
harvesting the organisms either from the culture in which they grow
or from the field. This can be done via expressing, grinding and/or
extraction, salt precipitation and/or ion-exchange chromatography
of the plant parts, preferably the plant seeds, plant fruits, plant
tubers and the like.
[0508] [0375.0.0.0] In a further embodiment, the present invention
relates to a process for the generation of a microorganism,
comprising the introduction, into the microorganism or parts
thereof, of the nucleic acid construct of the invention, or the
vector of the invention or the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention.
[0509] [0376.0.0.0] In another embodiment, the present invention
relates also to a transgenic microorganism comprising the nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention, the nucleic acid construct of the
invention or the vector as of the invention. Appropriate
microorganisms have been described herein before under source
organism, preferred are in particular aforementioned strains
suitable for the production of fine chemicals.
[0510] [0377.0.0.0] Accordingly, the present invention relates also
to a process according to the present invention whereby the
produced amino acid composition or the produced respective fine
chemical is isolated.
[0511] [0378.0.0.0] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the fine
chemicals produced in the process can be isolated. The resulting
fine chemicals can, if appropriate, subsequently be further
purified, if desired mixed with other active ingredients such as
vitamins, amino acids, carbohydrates, antibiotics and the like,
and, if appropriate, formulated.
[0512] [0379.0.0.0] In one embodiment, the fatty acid is the fine
chemical.
[0513] [0380.0.0.0] The amino acids obtained in the process are
suitable as starting material for the synthesis of further products
of value. For example, they can be used in combination with each
other or alone for the production of pharmaceuticals, foodstuffs,
animal feeds or cosmetics. Accordingly, the present invention
relates a method for the production of a pharmaceuticals, food
stuff, animal feeds, nutrients or cosmetics comprising the steps of
the process according to the invention, including the isolation of
the amino acid composition produced or the fine chemical produced
if desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the amino acids produced in the
process or of the transgenic organisms in animal feeds, foodstuffs,
medicines, food supplements, cosmetics or pharmaceuticals.
[0514] [0381.0.0.0] In principle all microorganisms can be used as
host organism especially the ones mentioned under source organism
above. It is advantageous to use in the process of the invention
transgenic microorganisms such as fungi such as the genus Claviceps
or Aspergillus or Gram-positive bacteria such as the genera
Bacillus, Corynebacterium, Micrococcus, Brevibacterium,
Rhodococcus, Nocardia, Caseobacter or Arthrobacter or Gram-negative
bacteria such as the genera Escherichia, Flavobacterium or
Salmonella or yeasts such as the genera Rhodotorula, Hansenula or
Candida. Particularly advantageous organisms are selected from the
group of genera Corynebacterium, Brevibacterium, Escherichia,
Bacillus, Rhodotorula, Hansenula, Candida, Claviceps or
Flavobacterium. It is very particularly advantageous to use in the
process of the invention microorganisms selected from the group of
genera and species consisting of Hansenula anomala, Candida utilis,
Claviceps purpurea, Bacillus circulans, Bacillus subtilis, Bacillus
sp., Brevibacterium albidum, Brevibacterium album, Brevibacterium
cerinum, Brevibacterium flavum, Brevibacterium glutamigenes,
Brevibacterium iodinum, Brevibacterium ketoglutamicum,
Brevibacterium lactofermentum, Brevibacterium linens,
Brevibacterium roseum, Brevibacterium saccharolyticum,
Brevibacterium sp., Corynebacterium acetoacidophilum,
Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes,
Corynebacterium glutamicum (=Micrococcus glutamicum),
Corynebacterium melassecola, Corynebacterium sp. or Escherichia
coli, specifically Escherichia coli K12 and its described
strains.
[0515] [0382.0.0.0] The process of the invention is, when the host
organisms are microorganisms, advantageously carried out at a
temperature between 0.degree. C. and 95.degree. C., preferably
between 10.degree. C. and 85.degree. C., particularly preferably
between 15.degree. C. and 75.degree. C., very particularly
preferably between 15.degree. C. and 45.degree. C. The pH is
advantageously kept at between pH 4 and 12, preferably between pH 6
and 9, particularly preferably between pH 7 and 8, during this. The
process of the invention can be operated batchwise, semibatchwise
or continuously. A summary of known cultivation methods is to be
found in the textbook by Chmiel (Bioproze.beta.technik 1.
Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag,
Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and
periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden,
1994)). The culture medium to be used must meet the requirements of
the respective strains in a suitable manner. Descriptions of
culture media for various microorganisms are present in the
handbook "Manual of Methods for General Bacteriology" of the
American Society for Bacteriology (Washington D. C., USA, 1981).
These media, which can be employed according to the invention
include, as described above, usually one or more carbon sources,
nitrogen sources, inorganic salts, vitamins and/or trace elements.
Preferred carbon sources are sugars such as mono-, di- or
polysaccharides. Examples of very good carbon sources are glucose,
fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,
maltose, sucrose, raffinose, starch or cellulose. Sugars can also
be added to the media via complex compounds such as molasses, or
other byproducts of sugar refining. It may also be advantageous to
add mixtures of various carbon sources. Other possible carbon
sources are oils and fats such as, for example, soybean oil,
sunflower oil, peanut oil and/or coconut fat, fatty acids such as,
for example, palmitic acid, stearic acid and/or linoleic acid,
alcohols and/or polyalcohols such as, for example, glycerol,
methanol and/or ethanol and/or organic acids such as, for example,
acetic acid and/or lactic acid. Nitrogen sources are usually
organic or inorganic nitrogen compounds or materials, which contain
these compounds. Examples of nitrogen sources include ammonia in
liquid or gaseous form or ammonium salts such as ammonium sulfate,
ammonium chloride, ammonium phosphate, ammonium carbonate or
ammonium nitrate, nitrates, urea, amino acids or complex nitrogen
sources such as corn steep liquor, soybean meal, soybean protein,
yeast extract, meat extract and others. The nitrogen sources may be
used singly or as a mixture. Inorganic salt compounds, which may be
present in the media include the chloride, phosphorus or sulfate
salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium,
manganese, zinc, copper and iron.
[0516] [0383.0.0.0] For preparing sulfur-containing fine chemicals,
in particular the respective fine chemical, e.g. amino acids
containing sulfur it is possible to use as sulfur source inorganic
sulfur-containing compounds such as, for example, sulfates,
sulfites, dithionites, tetrathionates, thiosulfates, sulfides or
else organic sulfur compounds such as mercaptans and thiols.
[0517] [0384.0.0.0] It is possible to use as phosphorus source
phosphoric acid, potassium dihydrogenphosphate or dipotassium
hydrogenphosphate or the corresponding sodium-containing salts.
Chelating agents can be added to the medium in order to keep the
metal ions in solution. Particularly suitable chelating agents
include dihydroxyphenols such as catechol or protocatechuate, or
organic acids such as citric acid. The fermentation media employed
according to the invention for cultivating microorganisms normally
also contain other growth factors such as vitamins or growth
promoters, which include, for example, biotin, riboflavin,
thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine.
Growth factors and salts are often derived from complex media
components such as yeast extract, molasses, corn steep liquor and
the like. Suitable precursors can moreover be added to the culture
medium. The exact composition of the media compounds depends
greatly on the particular experiment and is chosen individually for
each specific case. Information about media optimization is
obtainable from the textbook "Applied Microbiol. Physiology, A
Practical Approach" (editors P. M. Rhodes, P. F. Stanbury, IRL
Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also
be purchased from commercial suppliers such as Standard 1 (Merck)
or BHI (Brain heart infusion, DIEGO) and the like. All media
components are sterilized either by heat (1.5 bar and 121.degree.
C. for 20 min) or by sterilizing filtration. The components can be
sterilized either together or, if necessary, separately. All media
components can be present at the start of the cultivation or
optionally be added continuously or batchwise. The temperature of
the culture is normally between 15.degree. C. and 45.degree. C.,
preferably at 25.degree. C. to 40.degree. C., and can be kept
constant or changed during the experiment. The pH of the medium
should be in the range from 5 to 8.5, preferably around 7. The pH
for the cultivation can be controlled during the cultivation by
adding basic compounds such as sodium hydroxide, potassium
hydroxide, ammonia or aqueous ammonia or acidic compounds such as
phosphoric acid or sulfuric acid. Foaming can be controlled by
employing antifoams such as, for example, fatty acid polyglycol
esters. The stability of plasmids can be maintained by adding to
the medium suitable substances having a selective effect, for
example antibiotics. Aerobic conditions are maintained by
introducing oxygen or oxygen-containing gas mixtures such as, for
example, ambient air into the culture. The temperature of the
culture is normally from 20.degree. C. to 45.degree. C. and
preferably from 25.degree. C. to 40.degree. C. The culture is
continued until formation of the desired product is at a maximum.
This aim is normally achieved within 10 hours to 160 hours.
[0518] [0385.0.0.0] The fermentation broths obtained in this way,
containing in particular L-methionine, L-threonine and/or L-lysine,
normally have a dry matter content of from 7.5 to 25% by weight.
Sugar-limited fermentation is additionally advantageous, at least
at the end, but especially over at least 30% of the fermentation
time. This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 3 g/l during
this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed entirely or
partly by separation methods, such as, for example, centrifugation,
filtration, decantation or a combination of these methods, from the
fermentation broth or left completely in it. The fermentation broth
can then be thickened or concentrated by known methods, such as,
for example, with the aid of a rotary evaporator, thin-film
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. This concentrated fermentation broth can then be
worked up by freeze-drying, spray drying, spray granulation or by
other processes.
[0519] [0386.0.0.0] However, it is also possible to purify the
amino acid produced further. For this purpose, the
product-containing composition is subjected to a chromatography on
a suitable resin, in which case the desired product or the
impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use. The purified product can be
concentrated by filtration or ultrafiltration and stored at a
temperature at which the stability of the product is a maximum.
[0520] [0387.0.0.0] The identity and purity of the isolated
compound(s) can be determined by prior art techniques. These
include high performance liquid chromatography (HPLC),
spectroscopic methods, mass spectrometry (MS), staining methods,
thin-layer chromatography, NIRS, enzyme assay or microbiological
assays. These analytical methods are summarized in: Patek et al.
(1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al.
(1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540,
pp. 540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, Vol. 17.
[0521] [0388.0.0.0] In yet another aspect, the invention also
relates to harvestable parts and to propagation material of the
transgenic plants according to the invention which either contain
transgenic plant cells expressing a nucleic acid molecule according
to the invention or which contains cells which show an increased
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. an increased
expression level or higher activity of the described protein.
[0522] [0389.0.0.0] Harvestable parts can be in principle any
useful parts of a plant, for example, flowers, pollen, seedlings,
tubers, leaves, stems, fruit, seeds, roots etc. Propagation
material includes, for example, seeds, fruits, cuttings, seedlings,
tubers, rootstocks etc. Preferred are seeds, fruits, seedlings or
tubers as harvestable or propagation material.
[0523] [0390.0.0.0] The invention furthermore relates to the use of
the transgenic organisms according to the invention and of the
cells, cell cultures, parts--such as, for example, roots, leaves
and the like as mentioned above in the case of transgenic plant
organisms--derived from them, and to transgenic propagation
material such as seeds or fruits and the like as mentioned above,
for the production of foodstuffs or feeding stuffs, pharmaceuticals
or fine chemicals.
[0524] [0391.0.0.0] Accordingly in another embodiment, the present
invention relates to the use of the nucleic acid molecule, the
organism, e.g. the microorganism, the plant, plant cell or plant
tissue, the vector, or the polypeptide of the present invention for
making fatty acids, carotenoids, isoprenoids, vitamins, lipids, wax
esters, (poly)saccharides and/or polyhydroxyalkanoates, and/or its
metabolism products, in particular, steroid hormones, cholesterol,
prostaglandin, triacylglycerols, bile acids and/or ketone bodies
producing cells, tissues and/or plants. There are a number of
mechanisms by which the yield, production, and/or efficiency of
production of fatty acids, carotenoids, isoprenoids, vitamins, wax
esters, lipids, (poly)saccharides and/or polyhydroxyalkanoates,
and/or its metabolism products, in particular, steroid hormones,
cholesterol, triacylglycerols, prostaglandin, bile acids and/or
ketone bodies or further of above defined fine chemicals
incorporating such an altered protein can be affected. In the case
of plants, by e.g. increasing the expression of acetyl-CoA which is
the basis for many products, e.g., fatty acids, carotenoids,
isoprenoids, vitamines, lipids, (poly)saccharides, wax esters,
and/or polyhydroxyalkanoates, and/or its metabolism products, in
particular, prostaglandin, steroid hormones, cholesterol,
triacylglycerols, bile acids and/or ketone bodies in a cell, it may
be possible to increase the amount of the produced said compounds
thus permitting greater ease of harvesting and purification or in
case of plants more efficient partitioning. Further, one or more of
said metabolism products, increased amounts of the cofactors,
precursor molecules, and intermediate compounds for the appropriate
biosynthetic pathways maybe required. Therefore, by increasing the
number and/or activity of transporter proteins involved in the
import of nutrients, such as carbon sources (i.e., sugars),
nitrogen sources (i.e., amino acids, ammonium salts), phosphate,
and sulfur, it may be possible to improve the production of acetyl
CoA and its metabolism products as mentioned above, due to the
removal of any nutrient supply limitations on the biosynthetic
process. In particular, it may be possible to increase the yield,
production, and/or efficiency of production of said compounds, e.g.
fatty acids, carotenoids, isoprenoids, vitamins, was esters,
lipids, (poly)saccharides, and/or polyhydroxyalkanoates, and/or its
metabolism products, in particular, steroid hormones, cholesterol,
prostaglandin, triacylglycerols, bile acids and/or ketone bodies
molecules etc. in plants.
[0525] [0392.0.0.0] Furthermore preferred is a method for the
recombinant production of pharmaceuticals or fine chemicals in host
organisms, wherein a host organism is transformed with one of the
above-described nucleic acid constructs comprising one or more
structural genes which encode the desired fine chemical or catalyze
the biosynthesis of the desired fine chemical, the transformed host
organism is cultured, and the desired fine chemical is isolated
from the culture medium. This method can be applied widely to fine
chemicals such as enzymes, vitamins, amino acids, sugars, fatty
acids, and natural and synthetic flavourings, aroma substances and
colorants or compositions comprising these. Especially preferred is
the additional production of further amino acids, tocopherols and
tocotrienols and carotenoids or compositions comprising said
compounds. The transformed host organisms are cultured and the
products are recovered from the host organisms or the culture
medium by methods known to the skilled worker or the organism
itself servers as food or feed supplement. The production of
pharmaceuticals such as, for example, antibodies or vaccines, is
described by Hood E E, Jilka J M. Curr Opin Biotechnol. 1999
August; 10(4):382-6; Ma J K, Vine N D. Curr Top Microbiol Immunol.
1999; 236:275-92.
[0526] [0393.0.0.0] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [0527] (a) contacting,
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [0528] (b) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to a nucleic acid
molecule sequence as indicated in Table I, columns 5 or 7, lines 1
to 5 and/or lines 334 to 338, preferably in Table I B, columns 5 or
7, lines 1 to 5 and/or lines 334 to 338 and, optionally, isolating
the full length cDNA clone or complete genomic clone; [0529] (c)
introducing the candidate nucleic acid molecules in host cells,
preferably in a plant cell or a microorganism, appropriate for
producing the respective fine chemical; [0530] (d) expressing the
identified nucleic acid molecules in the host cells; [0531] (e)
assaying the respective fine chemical level in the host cells; and
[0532] (f) identifying the nucleic acid molecule and its gene
product which expression confers an increase in the respective fine
chemical level in the host cell after expression compared to the
wild type.
[0533] [0394.0.0.0] Relaxed hybridisation conditions are: After
standard hybridisation procedures washing steps can be performed at
low to medium stringency conditions usually with washing conditions
of 40.degree.-55.degree. C. and salt conditions between 2.times.SSC
and 0.2.times.SSC with 0.1% SDS in comparison to stringent washing
conditions as e.g. 60.degree.-68.degree. C. with 0.1% SDS. Further
examples can be found in the references listed above for the
stringent hybridization conditions. Usually washing steps are
repeated with increasing stringency and length until a useful
signal to noise ratio is detected and depend on many factors as the
target, e.g. its purity, GC-content, size etc, the probe, e.g. its
length, is it a RNA or a DNA probe, salt conditions, washing or
hybridisation temperature, washing or hybridisation time etc.
[0534] [0395.0.0.0] In an other embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [0535] (a) identifying
nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the respective fine chemical after expression, which are at least
20%, preferably 25%, more preferably 30%, even more preferred are
35%. 40% or 50%, even more preferred are 60%, 70% or 80%, most
preferred are 90% or 95% or more homology to the nucleic acid
molecule of the present invention, for example via homology search
in a data bank; [0536] (b) introducing the candidate nucleic acid
molecules in host cells, preferably in a plant cells or
microorganisms, appropriate for producing the respective fine
chemical; [0537] (c) expressing the identified nucleic acid
molecules in the host cells; [0538] (d) assaying the the respective
fine chemical level in the host cells; and [0539] (e) identifying
the nucleic acid molecule and its gene product which expression
confers an increase in the the respective fine chemical level in
the host cell after expression compared to the wild type. [0540]
Eventually gene products conferring the increase in the respective
fine chemical production can also be identify according to a
identical or similar 3D structure in step (a) and by the above
described method.
[0541] [0396.0.0.0] The nucleic acid molecules identified can then
be used for the production of the respective fine chemical in the
same way as the nucleic acid molecule of the present invention.
Accordingly, in one embodiment, the present invention relates to a
process for the production of the respective fine chemical,
comprising (a) identifying a nucleic acid molecule according to
aforementioned steps (a) to (f) or (a) to (e) and recovering the
free or bound fine chemical from a organism having an increased
cellular activity of a polypeptide encoded by the isolated nucleic
acid molecule compared to a wild type.
[0542] [0397.0.0.0] Furthermore, in one embodiment, the present
invention relates to a method for the identification of a compound
stimulating production of the respective fine chemical to said
plant comprising: [0543] a) contacting cells which express the
polypeptide of the present invention or its mRNA with a candidate
compound under cell cultivation conditions; [0544] b) assaying an
increase in expression of said polypeptide or said mRNA; [0545] c)
comparing the expression level to a standard response made in the
absence of said candidate compound; whereby, an increased
expression over the standard indicates that the compound is
stimulating production of the respective fine chemical.
[0546] [0398.0.0.0] Furthermore, in one embodiment, the present
invention relates to a method for the screening for agonists or an
antagonist of the activity of the polypeptide of the present
invention or used in the process of the present invention, e.g. a
polypeptide conferring an increase of the respective fine chemical
in an organism or a part thereof after increasing the activity in
an organism or a part thereof, comprising: [0547] (a) contacting
cells, tissues, plants or microorganisms which express the
polypeptide according to the invention with a candidate compound or
a sample comprising a plurality of compounds under conditions which
permit the expression the polypeptide of the present invention or
used in the process of the present invention; [0548] (b) assaying
the respective fine chemical level or the polypeptide expression
level in the cell, tissue, plant or microorganism or the media the
cell, tissue, plant or microorganisms is cultured or maintained in;
and [0549] (c) identifying a agonist or antagonist by comparing the
measured the respective fine chemical level or polypeptide of the
invention or used in the invention expression level with a standard
the respective fine chemical or polypeptide expression level
measured in the absence of said candidate compound or a sample
comprising said plurality of compounds, whereby an increased level
over the standard indicates that the compound or the sample
comprising said plurality of compounds is an agonist and a
decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an
antagonist.
[0550] [0399.0.0.0] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increased the respective fine chemical production in a
plant or microorganism, comprising the steps: [0551] (a) culturing
a cell or tissue or microorganism or maintaining a plant expressing
the polypeptide according to the invention or a nucleic acid
molecule encoding said polypeptide and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and the
polypeptide of the present invention or used in the process of the
invention; and [0552] (b) identifying if the compound is an
effective agonist by detecting the presence or absence or increase
of a signal produced by said readout system. The screen for a gene
product or an agonist conferring an increase in the respective fine
chemical production can be performed by growth of an organism for
example a microorganism in the presence of growth reducing amounts
of an inhibitor of the synthesis of the respective fine chemical.
Better growth, e.g. higher dividing rate or high dry mass in
comparison to the control under such conditions would identify a
gene or gene product or an agonist conferring an increase in fine
chemical production.
[0553] [0399.1.0.0] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 or a homolog
thereof, e.g. comparing the phenotype of nearly identical organisms
with low and high activity of a protein as indicated in Table II,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 after
incubation with the drug.
[0554] [0400.0.0.0] Said compound may be chemically synthesized or
microbiologically produced and/or comprised in, for example,
samples, e.g., cell extracts from, e.g., plants, animals or
microorganisms, e.g. pathogens. Furthermore, said compound(s) may
be known in the art but hitherto not known to be capable of
suppressing or activating the polypeptide of the present invention.
The reaction mixture may be a cell free extract or may comprise a
cell or tissue culture. Suitable set ups for the method of the
invention are known to the person skilled in the art and are, for
example, generally described in Alberts et al., Molecular Biology
of the Cell, third edition (1994), in particular Chapter 17. The
compounds may be, e.g., added to the reaction mixture, culture
medium, injected into the cell or sprayed onto the plant.
[0555] [0401.0.0.0] If a sample containing a compound is identified
in the method of the invention, then it is either possible to
isolate the compound from the original sample identified as
containing the compound capable of activating or increasing the
content of the respective fine chemical in an organism or part
thereof, or one can further subdivide the original sample, for
example, if it consists of a plurality of different compounds, so
as to reduce the number of different substances per sample and
repeat the method with the subdivisions of the original sample.
Depending on the complexity of the samples, the steps described
above can be performed several times, preferably until the sample
identified according to the method of the invention only comprises
a limited number of or only one substance(s). Preferably said
sample comprises substances of similar chemical and/or physical
properties, and most preferably said substances are identical.
Preferably, the compound identified according to the above
described method or its derivative is further formulated in a form
suitable for the application in plant breeding or plant cell and
tissue culture.
[0556] [0402.0.0.0] The compounds which can be tested and
identified according to a method of the invention may be expression
libraries, e.g., cDNA expression libraries, peptides, proteins,
nucleic acids, antibodies, small organic compounds, hormones,
peptidomimetics, PNAs or the like (Milner, Nature Medicine 1
(1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79
(1994), 193-198 and references cited supra). Said compounds can
also be functional derivatives or analogues of known inhibitors or
activators. Methods for the preparation of chemical derivatives and
analogues are well known to those skilled in the art and are
described in, for example, Beilstein, Handbook of Organic
Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New
York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York,
USA. Furthermore, said derivatives and analogues can be tested for
their effects according to methods known in the art. Furthermore,
peptidomimetics and/or computer aided design of appropriate
derivatives and analogues can be used, for example, according to
the methods described above. The cell or tissue that may be
employed in the method of the invention preferably is a host cell,
plant cell or plant tissue of the invention described in the
embodiments hereinbefore.
[0557] [0403.0.0.0] Thus, in a further embodiment the invention
relates to a compound obtained or identified according to the
method for identifying an agonist of the invention said compound
being an agonist of the polypeptide of the present invention or
used in the process of the present invention.
[0558] [0404.0.0.0] Accordingly, in one embodiment, the present
invention further relates to a compound identified by the method
for identifying a compound of the present invention.
[0559] [0405.0.0.0] Said compound is, for example, a homologous of
the polypeptide of the present invention. Homologues of the
polypeptid of the present invention can be generated by
mutagenesis, e.g., discrete point mutation or truncation of the
polypeptide of the present invention. As used herein, the term
"homologue" refers to a variant form of the protein, which acts as
an agonist of the activity of the polypeptide of the present
invention. An agonist of said protein can retain substantially the
same, or a subset, of the biological activities of the polypeptide
of the present invention. In particular, said agonist confers the
increase of the expression level of the polypeptide of the present
invention and/or the expression of said agonist in an organisms or
part thereof confers the increase of free and/or bound the
respective fine chemical in the organism or part thereof.
[0560] [0406.0.0.0] In one embodiment, the invention relates to an
antibody specifically recognizing the compound or agonist of the
present invention.
[0561] [0407.0.0.0] The invention also relates to a diagnostic
composition comprising at least one of the aforementioned nucleic
acid molecules, vectors, proteins, antibodies or compounds of the
invention and optionally suitable means for detection.
[0562] [0408.0.0.0] The diagnostic composition of the present
invention is suitable for the isolation of mRNA from a cell and
contacting the mRNA so obtained with a probe comprising a nucleic
acid probe as described above under hybridizing conditions,
detecting the presence of mRNA hybridized to the probe, and thereby
detecting the expression of the protein in the cell. Further
methods of detecting the presence of a protein according to the
present invention comprise immunotechniques well known in the art,
for example enzyme linked immunosorbent assay. Furthermore, it is
possible to use the nucleic acid molecules according to the
invention as molecular markers or primer in plant breeding.
Suitable means for detection are well known to a person skilled in
the arm, e.g. buffers and solutions for hydridization assays, e.g.
the aforementioned solutions and buffers, further and means for
Southern-, Western-, Northern--etc.--blots, as e.g. described in
Sambrook et al. are known.
[0563] [0409.0.0.0] In another embodiment, the present invention
relates to a kit comprising the nucleic acid molecule, the vector,
the host cell, the polypeptide, the antisense nucleic acid, the
antibody, plant cell, the plant or plant tissue, the harvestable
part, the propagation material and/or the compound or agonist or
antagonists identified according to the method of the
invention.
[0564] [0410.0.0.0] The compounds of the kit of the present
invention may be packaged in containers such as vials, optionally
with/in buffers and/or solution. If appropriate, one or more of
said components might be packaged in one and the same container.
Additionally or alternatively, one or more of said components might
be adsorbed to a solid support as, e.g. a nitrocellulose filter, a
glass plate, a chip, or a nylon membrane or to the well of a micro
titerplate. The kit can be used for any of the herein described
methods and embodiments, e.g. for the production of the host cells,
transgenic plants, pharmaceutical compositions, detection of
homologous sequences, identification of antagonists or agonists, as
food or feed or as a supplement thereof, as supplement for the
treating of plants, etc.
[0565] [0411.0.0.0] Further, the kit can comprise instructions for
the use of the kit for any of said embodiments, in particular for
the use for producing organisms or part thereof having an increased
free or bound the respective fine chemical content.
[0566] [0412.0.0.0] In one embodiment said kit comprises further a
nucleic acid molecule encoding one or more of the aforementioned
protein, and/or an antibody, a vector, a host cell, an antisense
nucleic acid, a plant cell or plant tissue or a plant.
[0567] [0413.0.0.0] In a further embodiment, the present invention
relates to a method for the production of a agricultural
composition providing the nucleic acid molecule, the vector or the
polypeptide of the invention or the polypeptide used in the method
of the invention or comprising the steps of the method according to
the invention for the identification of said compound, agonist or
antagonist; and formulating the nucleic acid molecule, the vector
or the polypeptide of the invention or the polypeptide used in the
method of the invention or the agonist, or compound identified
according to the methods or processes of the present invention or
with use of the subject matters of the present invention in a form
applicable as plant agricultural composition.
[0568] [0414.0.0.0] In another embodiment, the present invention
relates to a method for the production of a "the respective fine
chemical"-production supporting plant culture composition
comprising the steps of the method for of the present invention;
and formulating the compound identified in a form acceptable as
agricultural composition.
[0569] [0415.0.0.0] Under "acceptable as agricultural composition"
is understood, that such a composition is in agreement with the
laws regulating the content of fungicides, plant nutrients,
herbicides, etc. Preferably such a composition is without any harm
for the protected plants and the animals (humans included) fed
therewith.
[0570] [0416.0.0.0] The present invention also pertains to several
embodiments relating to further uses and methods. The nucleic acid
molecule, polypeptide, protein homologues, fusion proteins,
primers, vectors, host cells, described herein can be used in one
or more of the following methods: identification of plants useful
for the respective fine chemical production as mentioned and
related organisms; mapping of genomes; identification and
localization of sequences of interest; evolutionary studies;
determination of regions required for function; modulation of an
activity.
[0571] [0417.0.0.0] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention, the
vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the amino acid production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect plants against herbicides, which
block the amino acid, in particular the respective fine chemical,
synthesis in said plant. Inhibitors may inhibit one or more of the
steps for the synthesis of methionine. The first committed step for
the synthesis of Lys, Met and Thr is the first step, in which
aspartate is phosphorylated to aspartyl-b-phosphate, catalyzed by
aspartokinase: E. coli has 3 isozymes of aspartokinase that respond
differently to each of the 3 amino acids, with regard to enzyme
inhibition and feedback inhibition. The biosynthesis of lysine,
methionine and threonine are not, then, controlled as a group. The
pathway from aspartate to lysine has 10 steps. The pathway from
aspartate to threonine has 5 steps. The pathway from aspartate to
methionine has 7 steps. Regulation of the three pathways also
occurs at the two branch points: [0572] b-Aspartate-semialdehyde
(homoserine and lysine) [0573] Homoserine (threonine and
methionine)
[0574] The regulation results from feedback inhibition by the amino
acid products of the branches, indicated in the brackets above. One
important step in the synthesis of this group of 3 amino acids is
the step in which homocysteine is converted to methionine,
catalyzed by the enzyme methionine synthase:
##STR00001##
[0575] In this reaction, homocysteine is methylated to methionine,
and the Cl donor is N5-methyl-THF. Thus, inhibition of one or more
of the methionine synthesis enzymes, including also the provision
of donor molecules, can inhibit the synthesis of methionine.
[0576] Examples of herbicides blocking the amino acid synthesis in
plants are for example sulfonylurea and imidazolinone herbicides,
which catalyze the first step in branched-chain amino acid
biosynthesis. Inhibitors of the methionine synthesis may for
example described in Danishpajooh 10, 2001 Nitric oxide inhibits
methionine synthase activity in vivo and disrupts carbon flow
through the folate pathway. J. Biol. Chem. 276: 27296-27303; Datko
A H, 1982 Methionine biosynthesis in Lemna-inhibitor studies. Plant
Physiol. 69: 1070-1076; Lavrador K, 1998 A new series of cyclic
amino acids as inhibitors of S-adenosyl L-methionine synthetase.
Bioorg. Med. Chem. Lett. 8: 1629-1634; Thompson G A, 1982
Methionine synthesis in Lemna-inhibition of cystathionine
gamma-synthase by propargylglycine. Plant Physiol. 70: 1347-1352.
In some organisms the methionine synthesis is inhibited by ethanol,
lead, mercury, aluminium, thimerosal, cupper, N20, as e.g.
discussed in M. Waly, H. Oleteanu et al., 2004, Molecular
Psychiatry, 1-13.
[0577] Interestingly, Arabidopsis seed germination was strongly
delayed in the presence of DL-propargylglycine, a specific
inhibitor of methionine synthesis. Furthermore, this compound
totally inhibited seedling growth. These phenotypic effects were
largely alleviated upon methionine supplementation in the
germination medium. The results indicated that methionine synthase
and S-adenosylmethionine synthetase are fundamental components
controlling metabolism in the transition from a quiescent to a
highly active state during seed germination. Moreover, the observed
temporal patterns of accumulation of these proteins are consistent
with an essential role of endogenous ethylene in Arabidopsis only
after radicle protrusion; s. Gallarado, K., 2002, Importance of
methionine biosynthesis for Arabidopsis seed germination and
seedling growth, Physiolgia Plantarum, 116(2), pp 238-247.
Accordingly, the overexpression of a polypeptide of the present
invention in a plant may protect the plant against a herbicide
inhibiting methionine synthesis.
[0578] [0418.0.0.0] Accordingly, the nucleic acid molecules of the
present invention have a variety of uses. First, they may be used
to identify an organism or a close relative thereof. Also, they may
be used to identify the presence thereof or a relative thereof in a
mixed population of microorganisms or plants. By probing the
extracted genomic DNA of a culture of a unique or mixed population
of plants under stringent conditions with a probe spanning a region
of the gene of the present invention which is unique to this, one
can ascertain whether the present invention has been used or
whether it or a close relative is present.
[0579] [0419.0.0.0] Further, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention may be sufficiently homologous to the sequences of
related species such that these nucleic acid molecules may serve as
markers for the construction of a genomic map in related
organism.
[0580] [0420.0.0.0] Accordingly, the present invention relates to a
method for breeding plants for the production of the respective
fine chemical, comprising [0581] (a) providing a first plant
variety produced according to the process of the invention
preferably (over)expressing the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention; [0582] (b) crossing the first plant variety with a
second plant variety; and [0583] (c) selecting the offspring plants
which overproduce the respective fine chemical by means of analysis
the distribution of a molecular marker in the offspring
representing the first plant variety and its capability to
(over)produce the respective fine chemical.
[0584] Details about the use of molecular markers in breeding can
be found in Kumar et al., 1999 (Biotech Adv., 17:143-182) and
Peleman and van der Voort 2003 (Trends Plant Sci. 2003 July;
8(7):330-334)
[0585] The molecular marker can e.g. relate to the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention and/or its expression level. Accordingly,
the molecular marker can be a probe or a PCR primer set useful for
identification of the genomic existence or genomic localisation of
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention, e.g. in a Southern
blot analysis or a PCR or its expression level, i.g. in a Northern
Blot analysis or a quantitative PCR.
[0586] Accordingly, in one embodiment, the present invention
relates to the use of the nucleic acid molecule of the present
invention or encoding the polypeptide of the present invention as
molecular marker for breeding, especially for breeding for a high
or low respective fine chemical production.
[0587] [0421.0.0.0] The nucleic acid molecules of the invention are
also useful for evolutionary and protein structural studies. By
comparing the sequences of the invention or used in the process of
the invention to those encoding similar enzymes from other
organisms, the evolutionary relatedness of the organisms can be
assessed. Similarly, such a comparison permits an assessment of
which regions of the sequence are conserved and which are not,
which may aid in determining those regions of the protein which are
essential for the functioning of the enzyme. This type of
determination is of value for protein engineering studies and may
give an indication of what the protein can tolerate in terms of
mutagenesis without losing function.
[0588] [0422.0.0.0] Accordingly, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention can be used for the identification of other nucleic acids
conferring an increase of the respective fine chemical after
expression.
[0589] [0423.0.0.0] Further, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention or a fragment of a gene conferring the expression of the
polypeptide of the invention or the polypeptide used in the method
of the invention, preferably comprising the nucleic acid molecule
of the invention, can be used for marker assisted breeding or
association mapping of the respective fine chemical derived
traits
[0590] [0424.0.0.0] Accordingly, the nucleic acid of the invention,
the polypeptide of the invention or the polypeptide used in the
method of the invention, the nucleic acid construct of the
invention, the organisms, the host cell, the microorganisms, the
plant, plant tissue, plant cell, or the part thereof of the
invention, the vector of the invention, the agonist identified with
the method of the invention, the nucleic acid molecule identified
with the method of the present invention, can be used for the
production of the respective fine chemical or of the fine chemical
and one or more other amino acids, in particular Threoinine,
Alanine, Glutamin, Glutamic acid, Valine, Asparagine,
Phenylalanine, Leucine, Proline, Tryptophan Tyrosine, Valine,
Isoleucine and Arginine. Accordingly, the nucleic acid of the
invention, or the nucleic acid molecule identified with the method
of the present invention or the complement sequences thereof, the
polypeptide of the invention or the polypeptide used in the method
of the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the respective fine chemical in a organism or part
thereof, e.g. in a cell.
[0591] [0425.0.0.0] Further, the nucleic acid of the invention, the
polypeptide of the invention or the polypeptide used in the method
of the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist or the agonist identified
with the method of the invention, the antibody of the present
invention, the antisense molecule of the present invention or the
nucleic acid molecule identified with the method of the present
invention, can be used for the preparation of an agricultural
composition.
[0592] [0426.0.0.0] Furthermore, the nucleic acid of the invention,
the polypeptide of the invention or the polypeptide used in the
method of the invention, the nucleic acid construct of the
invention, the organisms, the host cell, the microorganisms, the
plant, plant tissue, plant cell, or the part thereof of the
invention, the vector of the invention, antagonist or the agonist
identified with the method of the invention, the antibody of the
present invention, the antisense molecule of the present invention
or the nucleic acid molecule identified with the method of the
present invention, can be used for the identification and
production of compounds capable of conferring a modulation of the
respective fine chemical levels in an organism or parts thereof,
preferably to identify and produce compounds conferring an increase
of the respective fine chemical levels in an organism or parts
thereof, if said identified compound is applied to the organism or
part thereof, i.e. as part of its food, or in the growing or
culture media.
[0593] [0427.0.0.0] These and other embodiments are disclosed and
encompassed by the description and examples of the present
invention. Further literature concerning any one of the methods,
uses and compounds to be employed in accordance with the present
invention may be retrieved from public libraries, using for example
electronic devices. For example the public database "Medline" may
be utilized which is available on the Internet, for example under
hftp://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases
and addresses, such as hftp://www.ncbi.nlm.nih.gov/,
hftp://www.infobiogen.fr/,
hftp://www.fmi.ch/biology/research-tools.html,
hftp://www.tigr.org/, are known to the person skilled in the art
and can also be obtained using, e.g., hftp://www.lycos.com. An
overview of patent information in biotechnology and a survey of
relevant sources of patent information useful for retrospective
searching and for current awareness is given in Berks, TIBTECH 12
(1994), 352-364.
[0594] [0428.0.0.0] Table 1 gives an overview about the sequences
disclosed in the present invention.
TABLE-US-00002 1) Increase of the metabolites: Max: maximal x-fold
(normalised to wild type)- Min: minimal x-fold (normalised to wild
type) 2) Decrease of the metabolites: Max: maximal x-fold
(normalised to wild type) (minimal decrease) Min: minimal x-fold
(normalised to wild type) (maximal decrease)
[0595] [0429.0.0.0] The present invention is illustrated by the
examples, which follow. The present examples illustrate the basic
invention without being intended as limiting the subject of the
invention. The content of all of the references, patent
applications, patents and published patent applications cited in
the present patent application is herewith incorporated by
reference.
[0430.0.0.0] EXAMPLES
[0431.0.0.0] Example 1
Cloning into in Escherichia coli
[0596] [0432.0.0.0] A DNA polynucleotide with a sequence as
indicated in Table I, column 5 and encoding a polypeptide as listed
in Table 1 below, was cloned into the plasmids pBR322 (Sutcliffe,
J. G. (1979) Proc. Natl Acad. Sci. USA, 75: 3737-3741); pACYC177
(Change & Cohen (1978) J. Bacteriol. 134: 1141-1156); plasmids
of the pBS series (pBSSK+, pBSSK- and others; Stratagene, LaJolla,
USA) or cosmids such as SuperCosi (Stratagene, LaJolla, USA) or
Lorist6 (Gibson, T. J. Rosenthal, A., and Waterson, R. H. (1987)
Gene 53: 283-286) for expression in E. coli using known,
well-established procedures (see, for example, Sambrook, J. et al.
(1989) "Molecular Cloning: A Laboratory Manual". Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons).
[0433.0.0.0] Example 2
DNA Sequencing and Computerized Functional Analysis
[0597] [0434.0.0.0] The DNA was sequenced by standard procedures,
in particular the chain determination method, using ABI377
sequencers (see, for example, Fleischman, R. D. et al. (1995)
"Whole-genome Random Sequencing and Assembly of Haemophilus
Influenzae Rd., Science 269; 496-512)".
[0435.0.0.0] Example 3
In-Vivo and In-Vitro Mutagenesis
[0598] [0436.0.0.0] An in vivo mutagenesis of Corynebacterium
glutamicum for the production of the respective fine chemical can
be carried out by passing a plasmid DNA (or another vector DNA)
through E. coli and other microorganisms (for example Bacillus spp.
or yeasts such as Saccharomyces cerevisiae), which are not capable
of maintaining the integrity of its genetic information. Usual
mutator strains have mutations in the genes for the DNA repair
system [for example mutHLS, mutD, mutT and the like; for
comparison, see Rupp, W. D. (1996) DNA repair mechanisms in
Escherichia coli and Salmonella, pp. 2277-2294, ASM: Washington].
The skilled worker knows these strains. The use of these strains is
illustrated for example in Greener, A. and Callahan, M. (1994)
Strategies 7; 32-34.
[0599] [0436.1.0.0] In-vitro mutation methods such as increasing
the spontaneous mutation rates by chemical or physical treatment
are well known to the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagenesis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[0600] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[0601] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.0.0] Example 4
DNA Transfer Between Escherichia coli and Corynebacterium
glutamicum
[0602] [0438.0.0.0] Several Corynebacterium and Brevibacterium
species comprise endogenous plasmids (such as, for example, pHM1519
or pBL1) which replicate autonomously (for a review, see, for
example, Martin, J. F. et al. (1987) Biotechnology 5: 137-146).
Shuttle vectors for Escherichia coli and Corynebacterium glutamicum
can be constructed easily using standard vectors for E. coli
(Sambrook, J. et al., (1989), "Molecular Cloning: A Laboratory
Manual", Cold Spring Harbor Laboratory Press or Ausubel, F. M. et
al. (1994) "Current Protocols in Molecular Biology", John Wiley
& Sons), which have a replication origin for, and suitable
marker from, Corynebacterium glutamicum added. Such replication
origins are preferably taken from endogenous plasmids, which have
been isolated from Corynebacterium and Brevibacterium species.
Genes, which are used in particular as transformation markers for
these species are genes for kanamycin resistance (such as those
which originate from the Tn5 or Tn-903 transposon) or for
chloramphenicol resistance (Winnacker, E. L. (1987) "From Genes to
Clones--Introduction to Gene Technology, VCH, Weinheim). There are
many examples in the literature of the preparation of a large
multiplicity of shuttle vectors which are replicated in E. coli and
C. glutamicum and which can be used for various purposes including
the overexpression of genes (see, for example, Yoshihama, M. et al.
(1985) J. Bacteriol. 162: 591-597, Martin, J. F. et al., (1987)
Biotechnology, 5: 137-146 and Eikmanns, B. J. et al. (1992) Gene
102: 93-98). Suitable vectors, which replicate in coryneform
bacteria are, for example, pZ1 (Menke) et al., Appl. Environ.
Microbiol., 64, 1989: 549-554) pEkEx1 (Eikmanns et al., Gene 102,
1991: 93-98) or pHS2-1 (Sonnen et al, Gene 107, 1991: 69-74). These
vectors are based on the cryptic plasmids pHM1519, pBL1 or pGA1.
Other plasmid vectors such as, for example, those based on pCG4
(U.S. Pat. No. 4,489,160), pNG2 (Serwold-Davis et al., FEMS
Microbiol. Lett., 66, 1990: 119-124) or pAG1 (U.S. Pat. No.
5,158,891) can be used in the same manner.
[0603] [0439.0.0.0] Using standard methods, it is possible to clone
a gene of interest into one of the above-described shuttle vectors
and to introduce such hybrid vectors into Corynebacterium
glutamicum strains. The transformation of C. glutamicum can be
achieved by protoplast transformation (Kastsumata, R. et al.,
(1984) J. Bacteriol. 159, 306-311), electroporation (Liebl, E. et
al., (1989) FEMS Microbiol. Letters, 53: 399-303) and in those
cases where specific vectors are used also by conjugation (such as,
for example, described in Schafer, A., et al. (1990) J. Bacteriol.
172: 1663-1666). Likewise, it is possible to transfer the shuttle
vectors for C. glutamicum to E. coli by preparing plasmid DNA from
C. glutamicum (using standard methods known in the art) and
transforming it into E. coli. This transformation step can be
carried out using standard methods, but preferably using an
Mcr-deficient E. coli strain, such as NM522 (Gough & Murray
(1983) J. Mol. Biol. 166: 1-19).
[0604] [0440.0.0.0] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the coryneform
bacteria, standard techniques known to the skilled worker also
exist for this purpose. Examples, which are used for this purpose
are plasmid vectors as they have been described by Remscheid et al.
(Appl. Environ. Microbiol., 60, 1994: 126-132) for the duplication
and amplification of the hom-thrB operon. In this method, the
complete gene is cloned into a plasmid vector which is capable of
replication in a host such as E. coli, but not in C. glutamicum.
Suitable vectors are, for example, pSUP301 (Simon et al.,
Bio/Technology 1, 1983: 784-791), pKlBmob or pKl 9mob (Schafer et
al., Gene 145, 1994: 69-73), pGEM-T (Promega Corp., Madison, Wis.,
USA), pCR2.1-TOPO (Schuman, J. Biol. Chem., 269, 1994: 32678-32684,
U.S. Pat. No. 5,487,993), pCR.RTM.Blunt (Invitrogen, Groningen, the
Netherlands) or pEM1 (Schrumpf et al., J. Bacteriol., 173, 1991:
4510-4516).
[0441.0.0.0] Example 5
Determining the Expression of the Mutant/Transgenic Protein
[0605] [0442.0.0.0] The observations of the activity of a mutated,
or transgenic, protein in a transformed host cell are based on the
fact that the protein is expressed in a similar manner and in a
similar quantity as the wild-type protein. A suitable method for
determining the transcription quantity of the mutant, or
transgenic, gene (a sign for the amount of mRNA which is available
for the translation of the gene product) is to carry out a Northern
blot (see, for example, Ausubel et al., (1988) Current Protocols in
Molecular Biology, Wiley: New York), where a primer which is
designed in such a way that it binds to the gene of interest is
provided with a detectable marker (usually a radioactive or
chemiluminescent marker) so that, when the total RNA of a culture
of the organism is extracted, separated on a gel, applied to a
stable matrix and incubated with this probe, the binding and
quantity of the binding of the probe indicates the presence and
also the amount of mRNA for this gene. Another method is a
quantitative PCR. This information detects the extent to which the
gene has been transcribed. Total cell RNA can be isolated from
Corynebacterium glutamicum or other microorganisms by a variety of
methods, which are known in the art, e.g. as described in Bormann,
E. R. et al., (1992) Mol. Microbiol. 6: 317-326.
[0606] [0443.0.0.0] Standard techniques, such as Western blot, may
be employed to determine the presence or relative amount of protein
translated from this mRNA (see, for example, Ausubel et al. (1988)
"Current Protocols in Molecular Biology", Wiley, New York). In this
method, total cell proteins are extracted, separated by gel
electrophoresis, transferred to a matrix such as nitrocellulose and
incubated with a probe, such as an antibody, which binds
specifically to the desired protein. This probe is usually provided
directly or indirectly with a chemiluminescent or colorimetric
marker, which can be detected readily. The presence and the
observed amount of marker indicates the presence and the amount of
the sought mutant protein in the cell. However, other methods are
also known.
[0444.0.0.0] Example 6
Growth of Genetically Modified Corynebacterium Glutamicum: Media
and Culture Conditions
[0607] [0444.0.0.0] Genetically modified Corynebacteria are grown
in synthetic or natural growth media. A number of different growth
media for Corynebacteria are known and widely available (Lieb et
al. (1989) Appl. Microbiol. Biotechnol. 32: 205-210; von der Osten
et al. (1998) Biotechnology Letters 11: 11-16; Patent DE 4 120 867;
Liebl (1992) "The Genus Corynebacterium", in: The Procaryotes, Vol.
II, Balows, A., et al., Ed. Springer-Verlag).
[0608] [0445.0.0.0] Said media, which can be used according to the
invention usually consist of one or more carbon sources, nitrogen
sources, inorganic salts, vitamins and trace elements. Preferred
carbon sources are sugars such as mono-, di- or polysaccharides.
Examples of very good carbon sources are glucose, fructose,
mannose, galactose, ribose, sorbose, ribulose, lactose, maltose,
sucrose, raffinose, starch or cellulose. Sugars may also be added
to the media via complex compounds such as molasses or other
by-products of sugar refining. It may also be advantageous to add
mixtures of various carbon sources. Other possible carbon sources
are alcohols and/or organic acids such as methanol, ethanol, acetic
acid or lactic acid. Nitrogen sources are usually organic or
inorganic nitrogen compounds or materials containing said
compounds. Examples of nitrogen sources include ammonia gas,
aqueous ammonia solutions or ammonium salts such as NH.sub.4Cl, or
(NH.sub.4).sub.2SO.sub.4, NH.sub.4OH, nitrates, urea, amino acids
or complex nitrogen sources such as cornsteep liquor, soybean
flour, soybean protein, yeast extract, meat extract and others.
Mixtures of the above nitrogen sources may be used
advantageously.
[0609] [0446.0.0.0] Inorganic salt compounds, which may be included
in the media comprise the chloride, phosphorus or sulfate salts of
calcium, magnesium, sodium, cobalt, molybdenum, potassium,
manganese, zinc, copper and iron. Chelating agents may be added to
the medium in order to keep the metal ions in solution.
Particularly suitable chelating agents include dihydroxyphenols
such as catechol or protocatechulate or organic acids such as
citric acid. The media usually also contain other growth factors
such as vitamins or growth promoters, which include, for example,
biotin, riboflavin, thiamine, folic acid, nicotinic acid,
panthothenate and pyridoxine.
[0610] [0447.0.0.0] Growth factors and salts are frequently derived
from complex media components such as yeast extract, molasses,
cornsteep liquor and the like. The exact composition of the
compounds used in the media depends heavily on the particular
experiment and is decided upon individually for each specific case.
Information on the optimization of media can be found in the
textbook "Applied Microbiol. Physiology, A Practical Approach" (Ed.
P. M. Rhodes, P. F. Stanbury, IRL Press (1997) S. 53-73, ISBN 0 19
963577 3). Growth media can also be obtained from commercial
suppliers, for example Standard 1 (Merck) or BHI (Brain heart
infusion, DIEGO) and the like.
[0611] [0448.0.0.0] All media components are sterilized, either by
heat (20 min at 1.5 bar and 121.degree. C.) or by filter
sterilization. The components may be sterilized either together or,
if required, separately. All media components may be present at the
start of the cultivation or added continuously or batchwise, as
desired.
[0612] [0449.0.0.0] The culture conditions are defined separately
for each experiment. The temperature is normally between 15.degree.
C. and 45.degree. C. and may be kept constant or may be altered
during the experiment. The pH of the medium should be in the range
from 5 to 8.5, preferably around 7.0, and can be maintained by
adding buffers to the media. An example of a buffer for this
purpose is a potassium phosphate buffer. Synthetic buffers such as
MOPS, HEPES, ACES and the like may be used as an alternative or
simultaneously. The culture pH value may also be kept constant
during the culture period by addition of, for example, NaOH or
NH.sub.4OH. If complex media components such as yeast extract are
used, additional buffers are required less since many complex
compounds have a high buffer capacity. When using a fermenter for
the culture of microorganisms, the pH value can also be regulated
using gaseous ammonia.
[0613] [0450.0.0.0] The incubation period is generally in a range
of from several hours to several days. This time period is selected
in such a way that the maximum amount of product accumulates in the
fermentation broth. The growth experiments, which are disclosed can
be carried out in a multiplicity of containers such as microtiter
plates, glass tubes, glass flasks or glass or metal fermenters of
various sizes. To screen a large number of clones, the
microorganisms should be grown in microtiter plates, glass tubes or
shake flasks, either using simple flasks or baffle flasks. 100 ml
shake flasks filled with 10% (based on the volume) of the growth
medium required are preferably used. The flasks should be shaken on
an orbital shaker (amplitude 25 mm) at a rate ranging from 100 to
300 rpm. Evaporation losses can be reduced by maintaining a humid
atmosphere; as an alternative, a mathematical correction should be
carried out for the evaporation losses.
[0614] [0451.0.0.0] If genetically modified clones are examined, an
unmodified control clone, or a control clone, which contains the
basic plasmid without insertion, should also be included in the
tests. If a transgenic sequence is expressed, a control clone
should advantageously again be included in these tests. The medium
is advantageously inoculated to an OD600 of 0.5 to 1.5 using cells
which have been grown on agar plates, such as CM plates (10 g/l
glucose, 2.5 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast
extract, 5 g/l meat extract, 22 g/l agar, pH value 6.8 established
with 2M NaOH), which have been incubated at 30.degree. C. The media
are inoculated for example by introducing of a preculture of seed
organisms.
[0615] [0451.1.0.0] For example, the media are inoculated by
introducing of a saline solution of C. glutamicum cells from CM
plates or by addition of a liquid preculture of this bacterium.
[0452.0.0.0] Example 7
In-Vitro Analysis of the Function of the Proteins Encoded by the
Transformed Sequences
[0616] [0453.0.0.0] The determination of the activities and kinetic
parameters of enzymes is well known in the art. Experiments for
determining the activity of a specific modified enzyme must be
adapted to the specific activity of the wild-enzyme type, which is
well within the capabilities of the skilled worker. Overviews of
enzymes in general and specific details regarding the structure,
kinetics, principles, methods, applications and examples for the
determination of many enzyme activities can be found for example in
the following literature: Dixon, M., and Webb, E. C: (1979)
Enzymes, Longmans, London; Fersht (1985) Enzyme Structure and
Mechanism, Freeman, New York; Walsh (1979) Enzymatic Reaction
Mechanisms. Freeman, San Francisco; Price, N. C., Stevens, L.
(1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford;
Boyer, P. D: Ed. (1983) The Enzymes, 3rd Ed. Academic Press, New
York; Bisswanger, H. (1994) Enzymkinetik, 2nd Ed. VCH, Weinheim
(ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J., Gra.beta.l, M.
Ed. (1983-1986) Methods of Enzymatic Analysis, 3rd Ed. Vol. I-XII,
Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial
Chemistry (1987) Vol. A9, "Enzymes", VCH, Weinheim, pp.
352-363.
[0454.0.0.0] Example 8
Analysis of the Effect of the Nucleic Acid Molecule on the
Production of the Amino Acids
[0617] [0455.0.0.0] The effect of the genetic modification in C.
glutamicum on the production of an amino acid can be determined by
growing the modified microorganisms under suitable conditions (such
as those described above) and analyzing the medium and/or the
cellular components for the increased production of the amino acid.
Such analytical techniques are well known to the skilled worker and
encompass spectroscopy, thin-layer chromatography, various types of
staining methods, enzymatic and microbiological methods and
analytical chromatography such as high-performance liquid
chromatography (see, for example, Ullman, Encyclopedia of
Industrial Chemistry, Vol. A2, pp. 89-90 and pp. 443-613, VCH:
Weinheim (1985); Fallon, A., et al., (1987) "Applications of HPLC
in Biochemistry" in: Laboratory Techniques in Biochemistry and
Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol.
3, Chapter III: "Product recovery and purification", pp. 469-714,
VCH: Weinheim; Belter, P. A. et al. (1988) Bioseparations:
downstream processing for Biotechnology, John Wiley and Sons;
Kennedy, J. F. and Cabral, J. M. S. (1992) Recovery processes for
biological Materials, John Wiley and Sons; Shaeiwitz, J. A. and
Henry, J. D. (1988) Biochemical Separations, in Ullmann's
Encyclopedia of Industrial Chemistry, Vol. B3; chapter 11, pp.
1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation and
purification techniques in biotechnology, Noyes Publications).
[0618] [0456.0.0.0] In addition to the determination of the
fermentation end product, other components of the metabolic
pathways which are used for the production of the desired compound,
such as intermediates and by-products, may also be analyzed in
order to determine the total productivity of the organism, the
yield and/or production efficiency of the compound. The analytical
methods encompass determining the amounts of nutrients in the
medium (for example sugars, hydrocarbons, nitrogen sources,
phosphate and other ions), determining biomass composition and
growth, analyzing the production of ordinary metabolites from
biosynthetic pathways and measuring gases generated during the
fermentation. Standard methods for these are described in Applied
Microbial Physiology; A Practical Approach, P. M. Rhodes and P. F.
Stanbury, Ed. IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN:
0199635773) and the references cited therein.
[0457.0.0.0] Example 9
Purification of the Amino Acid
[0619] [0458.0.0.0] The amino acid can be recovered from cells or
from the supernatant of the above-described culture by a variety of
methods known in the art. For example, the culture supernatant is
recovered first. To this end, the cells are harvested from the
culture by slow centrifugation. Cells can generally be disrupted or
lysed by standard techniques such as mechanical force or
sonication. The cell debris is removed by centrifugation and the
supernatant fraction, if appropriate together with the culture
supernatant, is used for the further purification of the amino
acid. However, it is also possible to process the supernatant alone
if the amino acid is present in the supernatant in sufficiently
high a concentration. In this case, the amino acid, or the amino
acid mixture, can be purified further for example via extraction
and/or salt precipitation or via ion-exchange chromatography.
[0620] [0459.0.0.0] If required and desired, further chromatography
steps with a suitable resin may follow, the amino acid, but not
many contaminants in the sample, being retained on the
chromatography resin or the contaminants, but not the sample with
the product (amino acid), being retained on the resin. If
necessary, these chromatography steps may be repeated, using
identical or other chromatography resins. The skilled worker is
familiar with the selection of suitable chromatography resin and
the most effective use for a particular molecule to be purified.
The purified product can be concentrated by filtration or
ultrafiltration and stored at a temperature at which maximum
product stability is ensured. Many purification methods, which are
not limited to the above purification method are known in the art.
They are described, for example, in Bailey, J. E. & Ollis, D.
F. Biochemical Engineering Fundamentals, McGraw-Hill: New York
(1986).
[0621] [0460.0.0.0] Identity and purity of the amino acid isolated
can be determined by standard techniques of the art. They encompass
high-performance liquid chromatography (HPLC), spectroscopic
methods, mass spectrometry (MS), staining methods, thin-layer
chromatography, NIRS, enzyme assay or microbiological assays. These
analytical methods are compiled in: Patek et al. (1994) Appl.
Environ. Microbiol. 60: 133-140; Malakhova et al. (1996)
Biotekhnologiya 11:27-32; and Schmidt et al. (1998) Bioprocess
Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry
(1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, Vol. 17.
[0461.0.0.0] Example 10
Cloning SEQ ID NO: 1 for the Expression in Plants
[0622] [0462.0.0.0] Unless otherwise specified, standard methods as
described in Sambrook et al., Molecular Cloning: A laboratory
manual, Cold Spring Harbor 1989, Cold Spring Harbor Laboratory
Press are used.
[0623] [0463.0.0.0] SEQ ID NO: 1 is amplified by PCR as described
in the protocol of the Pfu Turbo or DNA Herculase polymerase
(Stratagene).
[0624] [0464.0.0.0] The composition for the protocol of the Pfu
Turbo DNA polymerase was as follows: 1.times.PCR buffer
(Stratagene), 0.2 mM of each dNTP, 100 ng genomic DNA of
Saccharomyces cerevisiae (strain S288C; Research Genetics, Inc.,
now Invitrogen) or Escherichia coli (strain MG1655; E. coli Genetic
Stock Center), 50 .mu.mol forward primer, 50 .mu.mol reverse
primer, 2.5 u Pfu Turbo DNA polymerase. The amplification cycles
were as follows:
[0625] [0465.0.0.0] 1 cycle of 3 minutes at 94-95.degree. C.,
followed by 25-36 cycles of in each case 1 minute at 95.degree. C.
or 30 seconds at 94.degree. C., 45 seconds at 50.degree. C., 30
seconds at 50.degree. C. or 30 seconds at 55.degree. C. and 210-480
seconds at 72.degree. C., followed by 1 cycle of 8 minutes at
72.degree. C., then 4.degree. C. The composition for the protocol
of the Herculase polymerase was as follows: 1.times.PCR buffer
(Stratagene), 0.2 mM of each dNTP, 100 ng genomic DNA of
Saccharomyces cerevisiae (strain S288C; Research Genetics, Inc.,
now Invitrogen) or Escherichia coli (strain MG1655; E. coli Genetic
Stock Center), 50 .mu.mol forward primer, 50 .mu.mol reverse
primer, 2.5 u Herculase polymerase. The amplification cycles were
as follows:
[0626] [0466.0.0.0] 1 cycle of 2-3 minutes at 94.degree. C.,
followed by 25-30 cycles of in each case 30 seconds at 94.degree.
C., 30 seconds at 55-60.degree. C. and 5-10 minutes at 72.degree.
C., followed by 1 cycle of 10 minutes at 72.degree. C., then
4.degree. C.
[0627] [0467.0.0.0] The following primer sequences were selected
for the gene SEQ ID NO: 1:
TABLE-US-00003 i) forward primer (SEQ ID NO: 3)
ATGGAACAGAACAGGTTCAAGAAAG ii) reverse primer (SEQ ID NO: 4)
TTACAGTTTTTGTTTAGTCGTTTTAAC
[0628] [0468.0.0.0] Thereafter, the amplificate was purified over
QIAquick columns following the standard protocol (Qiagen).
[0629] [0469.0.0.0] For the cloning of PCR-products, produced by
Pfu Turbo DNA polymerase, the vector DNA (30 ng) was restricted
with Smal following the standard protocol (MBI Fermentas) and
stopped by addition of high-salt buffer. The restricted vector
fragments were purified via Nucleobond columns using the standard
protocol (Macherey-Nagel). Thereafter, the linearized vector was
dephosphorylated following the standard protocol (MBI
Fermentas).
[0630] [0470.0.0.0] The PCR-products, produced by Pfu Turbo DNA
polymerase, were directly cloned into the processed binary vector.
The PCR-products, produced by Pfu Turbo DNA polymerase, were
phosphorylated using a T4 DNA polymerase using a standard protocol
(e.g. MBI Fermentas) and cloned into the processed binary
vector.
[0631] [0471.0.0.0] The DNA termini of the PCR-products, produced
by Herculase DNA polymerase, were blunted in a second synthesis
reaction using Pfu Turbo DNA polymerase. The composition for the
protocol of the blunting the DNA-termini was as follows: 0.2 mM
blunting dTTP and 1.25 u Pfu Turbo DNA polymerase. The reaction was
incubated at 72.degree. C. for 30 minutes. Then the PCR-products
were cloned into the processed vector as well. The DNA termini of
the PCR-products, produced by Herculase DNA polymerase, were
blunted in a second synthesis reaction using Pfu Turbo DNA
polymerase. The composition for the protocol of the blunting the
DNA-termini was as follows: 0.2 mM blunting dTTP and 1.25 u Pfu
Turbo DNA polymerase. The reaction was incubated at 72.degree. C.
for 30 minutes. Then the PCR-products were phosphorylated using a
T4 DNA polymerase using a standard protocol (e.g. MBI Fermentas)
and cloned into the processed vector as well.
[0632] [0472.0.0.0] A binary vector comprising a selection cassette
(promoter, selection marker, terminator) and an expression cassette
with promoter, cloning cassette and terminator sequence between the
T-DNA border sequences was used. In addition to those within the
cloning cassette, the binary vector has no Smal cleavage site.
Binary vectors which can be used are known to the skilled worker;
an overview of binary vectors and their use can be found in
Hellens, R., Mullineaux, P. and Klee H., [(2000) "A guide to
Agrobacterium binary vectors", Trends in Plant Science, Vol. 5 No.
10, 446-451. Depending on the vector used, cloning may
advantageously also be carried out via other restriction enzymes.
Suitable advantageous cleavage sites can be added to the ORF by
using suitable primers for the PCR amplification.
[0633] [0473.0.0.0] Approximately 30 ng of prepared vector and a
defined amount of prepared amplificate were mixed and ligated by
addition of ligase.
[0634] [0474.0.0.0] The ligated vectors were transformed in the
same reaction vessel by addition of competent E. coli cells (strain
DH5alpha) and incubation for 20 minutes at 1.degree. C. followed by
a heat shock for 90 seconds at 42.degree. C. and cooling to
4.degree. C. Then, complete medium (SOC) was added and the mixture
was incubated for 45 minutes at 37.degree. C. The entire mixture
was subsequently plated onto an agar plate with antibiotics
(selected as a function of the binary vector used) and incubated
overnight at 37.degree. C.
[0635] [0475.0.0.0] The outcome of the cloning step was verified by
amplification with the aid of primers which bind upstream and
downstream of the integration site, thus allowing the amplification
of the insertion. In addition combinations of the above mentioned
gene specific primers and upstream and downstream primers were used
in PCR reactions to identify clones with the correct insert
orientation. The amplifications were carried as described in the
protocol of Taq DNA polymerase (Gibco-BRL).
[0636] [0476.0.0.0] The amplification cycles were as follows: 1
cycle of 5 minutes at 94.degree. C., followed by 35 cycles of in
each case 15 seconds at 94.degree. C., 15 seconds at 50-66.degree.
C. and 5 minutes at 72.degree. C., followed by 1 cycle of 10
minutes at 72.degree. C., then 4.degree. C.
[0637] [0477.0.0.0] Several colonies were checked, but only one
colony for which a PCR product of the expected size was detected
was used in the following steps.
[0638] [0478.0.0.0] A portion of this positive colony was
transferred into a reaction vessel filled with complete medium (LB)
and incubated overnight at 37.degree. C. The LB medium contained an
antibiotic chosen to suit the binary vector (see above) used and
the resistance gene present therein in order to select the
clone.
[0639] [0479.0.0.0] The plasmid preparation was carried out as
specified in the Qiaprep standard protocol (Qiagen).
[0480.0.0.0] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 1
[0640] [0481.0.0.0] 1 ng of the plasmid DNA isolated was
transformed by electroporation into competent cells of
Agrobacterium tumefaciens, of strain GV 3101 pMP90 (Koncz and
Schell, Mol. Gen. Gent. 204, 383-396, 1986). The choice of the
agrobacterial strain depends on the choice of the binary vector. An
overview of possible strains and their properties is found in
Hellens, R., Mullineaux, P. and Klee H., (2000) "A guide to
Agrobacterium binary vectors, Trends in Plant Science, Vol. 5 No.
10, 446-451. Thereafter, complete medium (YEP) was added and the
mixture was transferred into a fresh reaction vessel for 3 hours at
28.degree. C. Thereafter, all of the reaction mixture was plated
onto YEP agar plates supplemented with the respective antibiotics,
for example rifampicin and gentamycin for GV3101 pMP90, and a
further antibiotic for the selection onto the binary vector, was
plated, and incubated for 48 hours at 28.degree. C.
[0641] [0482.0.0.0] The agrobacteria generated in Example 10, which
contains the plasmid construct were then used for the
transformation of plants.
[0642] [0483.0.0.0] A colony was picked from the agar plate with
the aid of a pipette tip and taken up in 3 ml of liquid TB medium,
which also contained suitable antibiotics, depending on the
agrobacterial strain and the binary plasmid. The preculture was
grown for 48 hours at 28.degree. C. and 120 rpm.
[0643] [0484.0.0.0] 400 ml of LB medium containing the same
antibiotics as above were used for the main culture. The preculture
was transferred into the main culture. It was grown for 18 hours at
28.degree. C. and 120 rpm. After centrifugation at 4 000 rpm, the
pellet was resuspended in infiltration medium (MS medium, 10%
sucrose).
[0644] [0485.0.0.0] In order to grow the plants for the
transformation, dishes (Piki Saat 80, green, provided with a screen
bottom, 30.times. 20.times. 4.5 cm, from Wiesauplast,
Kunststofftechnik, Germany) were half-filled with a GS 90 substrate
(standard soil, Werkverband E.V., Germany). The dishes were watered
overnight with 0.05% Proplant solution (Chimac-Apriphar, Belgium).
Arabidopsis thaliana C24 seeds (Nottingham Arabidopsis Stock
Centre, UK; NASC Stock N906) were scattered over the dish,
approximately 1 000 seeds per dish. The dishes were covered with a
hood and placed in the stratification facility (8 h, 110
.mu.mol/m.sup.2/s.sup.-1, 22.degree. C.; 16 h, dark, 6.degree. C.).
After 5 days, the dishes were placed into the short-day controlled
environment chamber (8 h 130 .mu.mol/m.sup.2/s.sup.-1, 22.degree.
C.; 16 h, dark 20.degree. C.), where they remained for
approximately 10 days until the first true leaves had formed.
[0645] [0486.0.0.0] The seedlings were transferred into pots
containing the same substrate (Teku pots, 7 cm, LC series,
manufactured by Poppelmann GmbH & Co, Germany). Five plants
were pricked out into each pot. The pots were then returned into
the short-day controlled environment chamber for the plant to
continue growing.
[0646] [0487.0.0.0] After 10 days, the plants were transferred into
the greenhouse cabinet (supplementary illumination, 16 h, 340
.mu.E, 22.degree. C.; 8 h, dark, 20.degree. C.), where they were
allowed to grow for further 17 days.
[0647] [0488.0.0.0] For the transformation, 6-week-old Arabidopsis
plants which had just started flowering were immersed for 10
seconds into the above-described agrobacterial suspension which had
previously been treated with 10 .mu.l Silwett L77 (Crompton S. A.,
Osi Specialties, Switzerland). The method in question is described
in Clough and Bent, 1998 (Clough, J C and Bent, A F. 1998 Floral
dip: a simplified method for Agrobacterium-mediated transformation
of Arabidopsis thaliana, Plant J. 16:735-743.
[0648] [0489.0.0.0] The plants were subsequently placed for 18
hours into a humid chamber. Thereafter, the pots were returned to
the greenhouse for the plants to continue growing. The plants
remained in the greenhouse for another 10 weeks until the seeds
were ready for harvesting.
[0649] [0490.0.0.0] Depending on the resistance marker used for the
selection of the transformed plants the harvested seeds were
planted in the greenhouse and subjected to a spray selection or
else first sterilized and then grown on agar plates supplemented
with the respective selection agent. In case of
BASTA.RTM.-resistance, plantlets were sprayed four times at an
interval of 2 to 3 days with 0.02% BASTA.RTM. and transformed
plants were allowed to set seeds. The seeds of the transgenic A.
thaliana plants were stored in the freezer (at -20.degree. C.).
[0491.0.0.0] Example 12
Plant Culture for Bioanalytical Analyses
[0650] [0492.0.0.0] For the bioanalytical analyses of the
transgenic plants, the latter were grown uniformly a specific
culture facility. To this end the GS-90 substrate as the compost
mixture was introduced into the potting machine (Laible System
GmbH, Singen, Germany) and filled into the pots. Thereafter, 35
pots were combined in one dish and treated with Previcur. For the
treatment, 25 ml of Previcur were taken up in 10 l of tap water.
This amount was sufficient for the treatment of approximately 200
pots. The pots were placed into the Previcur solution and
additionally irrigated overhead with tap water without Previcur.
They were used within four days.
[0651] [0493.0.0.0] For the sowing, the seeds, which had been
stored in the refrigerator (at -20.degree. C.), were removed from
the Eppendorf tubes with the aid of a toothpick and transferred
into the pots with the compost. In total, approximately 5 to 12
seeds were distributed in the middle of the pot.
[0652] [0494.0.0.0] After the seeds had been sown, the dishes with
the pots were covered with matching plastic hood and placed into
the stratification chamber for 4 days in the dark at 4.degree. C.
The humidity was approximately 90%. After the stratification, the
test plants were grown for 22 to 23 days at a 16-h-light, 8-h-dark
rhythm at 20.degree. C., an atmospheric humidity of 60% and a
CO.sub.2 concentration of approximately 400 ppm. The light sources
used were Powerstar HQI-T 250 W/D Daylight lamps from Osram, which
generate a light resembling the solar color spectrum with a light
intensity of approximately 220 .mu.E/m2/s-1.
[0653] [0495.0.0.0] When the plants were 8, 9 and 10 days old, they
were subjected to selection for the resistance marker Approximately
1400 pots with transgenic plants were treated with 1 l 0.015%
vol/vol of Basta.RTM. (Glufosinate-ammonium) solution in water
(Aventis Cropsience, Germany). After a further 3 to 4 days, the
transgenic, resistant seedlings (plantlets in the 4-leaf stage)
could be distinguished clearly from the untransformed plantlets.
The nontransgenic seedlings were bleached or dead. The transgenic
resistance plants were thinned when they had reached the age of 14
days. The plants, which had grown best in the center of the pot
were considered the target plants. All the remaining plants were
removed carefully with the aid of metal tweezers and discarded.
[0654] [0496.0.0.0] During their growth, the plants received
overhead irrigation with distilled water (onto the compost) and
bottom irrigation into the placement grooves. Once the grown plants
had reached the age of 23 days, they were harvested.
[0497.0.0.0] Example 13
Metabolic Analysis of Transformed Plants
[0655] [0498.0.0.0] The modifications identified in accordance with
the invention, in the content of above-described metabolites, were
identified by the following procedure.
[0656] a) Sampling and Storage of the Samples
[0657] [0499.0.0.0] Sampling was performed directly in the
controlled-environment chamber. The plants were cut using small
laboratory scissors, rapidly weighed on laboratory scales,
transferred into a pre-cooled extraction sleeve and placed into an
aluminum rack cooled by liquid nitrogen. If required, the
extraction sleeves can be stored in the freezer at -80.degree. C.
The time elapsing between cutting the plant to freezing it in
liquid nitrogen amounted to not more than 10 to 20 seconds.
[0658] b) Lyophilization
[0659] [0500.0.0.0] During the experiment, care was taken that the
plants either remained in the deep-frozen state (temperatures
<-40.degree. C.) or were freed from water by lyophilization
until the first contact with solvents.
[0660] [0501.0.0.0] The aluminum rack with the plant samples in the
extraction sleeves was placed into the pre-cooled (-40.degree. C.)
lyophilization facility. The initial temperature during the main
drying phase was -35.degree. C. and the pressure was 0.120 mbar.
During the drying phase, the parameters were altered following a
pressure and temperature program. The final temperature after 12
hours was +30.degree. C. and the final pressure was 0.001 to 0.004
mbar. After the vacuum pump and the refrigerating machine had been
switched off, the system was flushed with air (dried via a drying
tube) or argon.
[0661] c) Extraction
[0662] [0502.0.0.0] Immediately after the lyophilization apparatus
had been flushed, the extraction sleeves with the lyophilized plant
material were transferred into the 5 ml extraction cartridges of
the ASE device (Accelerated Solvent Extractor ASE 200 with Solvent
Controller and AutoASE software (DIONEX)).
[0663] [0503.0.0.0] The 24 sample positions of an ASE device
(Accelerated Solvent Extractor ASE 200 with Solvent Controller and
AutoASE software (DIONEX)) were filled with plant samples,
including some samples for testing quality control.
[0664] [0504.0.0.0] The polar substances were extracted with
approximately 10 ml of methanol/water (80/20, v/v) at T=70.degree.
C. and p=140 bar, 5 minutes heating-up phase, 1 minute static
extraction. The more lipophilic substances were extracted with
approximately 10 ml of methanol/dichloromethane (40/60, v/v) at
T=70.degree. C. and p=140 bar, 5 minute heating-up phase, 1 minute
static extraction. The two solvent mixtures were extracted into the
same glass tubes (centrifuge tubes, 50 ml, equipped with screw cap
and pierceable septum for the ASE (DIONEX)).
[0665] [0505.0.0.0] The solution was treated with internal
standards: ribitol, L-glycine-2,2-d.sub.2,
L-alanine-2,3,3,3-d.sub.4, methionine-methyl-d.sub.3, and
.alpha.-methylglucopyranoside and methyl nonadecanoate, methyl
undecanoate, methyl tridecanoate, methyl pentadecanoate, methyl
nonacosanoate.
[0666] [0506.0.0.0] The total extract was treated with 8 ml of
water. The solid residue of the plant sample and the extraction
sleeve were discarded.
[0667] [0507.0.0.0] The extract was shaken and then centrifuged for
5 to 10 minutes at least at 1 400 g in order to accelerate phase
separation. 1 ml of the supernatant methanol/water phase ("polar
phase", colorless) was removed for the further GC analysis, and 1
ml was removed for the LC analysis. The remainder of the
methanol/water phase was discarded. 0.5 ml of the organic phase
("lipid phase", dark green) was removed for the further GC analysis
and 0.5 ml was removed for the LC analysis. All the portions
removed were evaporated to dryness using the IR Dancer infrared
vacuum evaporator (Hettich). The maximum temperature during the
evaporation process did not exceed 40.degree. C. Pressure in the
apparatus was not less than 10 mbar.
[0668] d) Processing the Lipid Phase for the LC/MS or LC/MS/MS
Analysis
[0669] [0508.0.0.0] The lipid extract, which had been evaporated to
dryness was taken up in mobile phase. The HPLC was run with
gradient elution.
[0670] [0509.0.0.0] The polar extract, which had been evaporated to
dryness was taken up in mobile phase. The HPLC was run with
gradient elution.
[0671] e) Derivatization of the Lipid Phase for the GC/MS
Analysis
[0672] [0510.0.0.0] For the transmethanolysis, a mixture of 140
.mu.l of chloroform, 37 .mu.l of hydrochloric acid (37% by weight
HCl in water), 320 .mu.l of methanol and 20 .mu.l of toluene was
added to the evaporated extract. The vessel was sealed tightly and
heated for 2 hours at 100.degree. C., with shaking. The solution
was subsequently evaporated to dryness. The residue was dried
completely.
[0673] [0511.0.0.0] The methoximation of the carbonyl groups was
carried out by reaction with methoxyamine hydrochloride (5 mg/ml in
pyridine, 100 .mu.l for 1.5 hours at 60.degree. C.) in a tightly
sealed vessel. 20 .mu.l of a solution of odd-numbered,
straight-chain fatty acids (solution of each 0.3 mg/mL of fatty
acids from 7 to 25 carbon atoms and each 0.6 mg/mL of fatty acids
with 27, 29 and 31 carbon atoms in 3/7 (v/v) pyridine/toluene) were
added as time standards. Finally, the derivatization with 100 .mu.l
of N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was
carried out for 30 minutes at 60.degree. C., again in the tightly
sealed vessel. The final volume before injection into the GC was
220 .mu.l.
[0674] f) Derivatization of the Polar Phase for the GC/MS
Analysis
[0675] [0512.0.0.0] The methoximation of the carbonyl groups was
carried out by reaction with methoxyamine hydrochloride (5 mg/ml in
pyridine, 50 .mu.l for 1.5 hours at 60.degree. C.) in a tightly
sealed vessel. 10 .mu.l of a solution of odd-numbered,
straight-chain fatty acids (solution of each 0.3 mg/mL of fatty
acids from 7 to 25 carbon atoms and each 0.6 mg/mL of fatty acids
with 27, 29 and 31 carbon atoms in 3/7 (v/v) pyridine/toluene) were
added as time standards. Finally, the derivatization with 50 .mu.l
of N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was
carried out for 30 minutes at 60.degree. C., again in the tightly
sealed vessel. The final volume before injection into the GC was
110 .mu.l.
[0676] g) Analysis of the Various Plant Samples
[0677] [0513.0.0.0] The samples were measured in individual series
of 20 plant samples each (also referred to as sequences), each
sequence containing at least 5 wild-type plants as controls. The
peak area of each analyte was divided by the peak area of the
respective internal standard. The data were standardized for the
fresh weight established for the plant. The values calculated thus
were related to the wild-type control group by being divided by the
mean of the corresponding data of the wild-type control group of
the same sequence. The values obtained were referred to as
ratio_by_WT, they are comparable between sequences and indicate how
much the analyte concentration in the mutant differs in relation to
the wild-type control. Appropriate controls were done before to
proof that the vector and transformation procedure itself has no
significant influence on the metabolic composition of the plants.
Therefore the described changes in comparison with wildtypes were
caused by the introduced genes.
[0678] [0514.0.0.0] As an alternative, the amino acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55). [0679] The results of the different plant analyses
can be seen from the table 1 which follows:
TABLE-US-00004 [0679] ORF ANNOTATION Metabolite Min Max Method
YBL015W acetyl-CoA hydrolase Methionine 1.42 2.16 LC YER173W
checkpoint protein, Methionine 1.35 1.60 GC YLR375W involved in
pre-tRNA Methionine 1.27 2.93 LC + GC splicing and in uptake of
branched-chain amino acids YOR084W putative peroxisomal Methionine
3.18 3.18 GC lipase b1829 heat shock protein with Methionine 1.29
3.73 GC protease activity b4232 fructose-1,6- Methionine 1.20 1.21
LC bisphosphatase b0464 transcriptional repressor Methionine 1.35
4.66 GC for multidrug efflux pump (TetR/AcrR family) b1343
ATP-dependent RNA Methionine 1.38 1.51 GC helicase, stimulated by
23S rRNA b2414 subunit of cysteine Methionine 1.37 1.75 LC synthase
A and O- acetylserine sulfhydrolase A, PLP-dependent enzyme b2762
3'-phosphoadenosine 5'- Methionine 1.43 1.69 LC + GC phosphosulfate
(PAPS) reductase
[0680] [0515.0.0.0] Column 3 shows the metabolite/respective fine
chemical analyzed. Columns 4 and 5 shows the ratio of the analyzed
metabolite/respective fine chemical between the transgenic plants
and the wild type; Increase of the metabolites: Max: maximal x-fold
(normalised to wild type)-Min: minimal x-fold (normalised to wild
type). Decrease of the metabolites: Max: maximal x-fold (normalised
to wild type) (minimal decrease), Min: minimal x-fold (normalised
to wild type) (maximal decrease). Column 6 indicates the analytical
method.
[0681] [0516.0.0.0] When the analyses were repeated independently,
all results proved to be significant.
[0517.0.0.0] Example 14a
Engineering Ryegrass Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, e.g. Derived from Saccharomyces
cerevisiae, E. Coli or Plants or an Other Organism
[0682] [0518.0.0.0] Seeds of several different ryegrass varieties
can be used as explant sources for transformation, including the
commercial variety Gunne available from Svalof Weibull seed company
or the variety Affinity. Seeds are surface-sterilized sequentially
with 1% Tween-20 for 1 minute, 100% bleach for 60 minutes, 3 rinses
with 5 minutes each with de-ionized and distilled H2O, and then
germinated for 3-4 days on moist, sterile filter paper in the dark.
Seedlings are further sterilized for 1 minute with 1% Tween-20, 5
minutes with 75% bleach, and rinsed 3 times with ddH2O, 5 min
each.
[0683] [0519.0.0.0] Surface-sterilized seeds are placed on the
callus induction medium containing Murashige and Skoog basal salts
and vitamins, 20 g/l sucrose, 150 mg/l asparagine, 500 mg/l casein
hydrolysate, 3 g/l Phytagel, 10 mg/l BAP, and 5 mg/l dicamba.
Plates are incubated in the dark at 25.degree. C. for 4 weeks for
seed germination and embryogenic callus induction.
[0684] [0520.0.0.0] After 4 weeks on the callus induction medium,
the shoots and roots of the seedlings are trimmed away, the callus
is transferred to fresh media, is maintained in culture for another
4 weeks, and is then transferred to MS0 medium in light for 2
weeks. Several pieces of callus (11-17 weeks old) are either
strained through a 10 mesh sieve and put onto callus induction
medium, or are cultured in 100 ml of liquid ryegrass callus
induction media (same medium as for callus induction with agar) in
a 250 ml flask. The flask is wrapped in foil and shaken at 175 rpm
in the dark at 23.degree. C. for 1 week. Sieving the liquid culture
with a 40-mesh sieve is collected the cells. The fraction collected
on the sieve is plated and is cultured on solid ryegrass callus
induction medium for 1 week in the dark at 25.degree. C. The callus
is then transferred to and is cultured on MS medium containing 1%
sucrose for 2 weeks.
[0685] [0521.0.0.0] Transformation can be accomplished with either
Agrobacterium or with particle bombardment methods. An expression
vector is created containing a constitutive plant promoter and the
cDNA of the gene in a pUC vector. The plasmid DNA is prepared from
E. coli cells using with Qiagen kit according to manufacturer's
instruction. Approximately 2 g of embryogenic callus is spread in
the center of a sterile filter paper in a Petri dish. An aliquot of
liquid MS0 with 10 g/l sucrose is added to the filter paper. Gold
particles (1.0 .mu.m in size) are coated with plasmid DNA according
to method of Sanford et al., 1993 and are delivered to the
embryogenic callus with the following parameters: 500 .mu.g
particles and 2 .mu.g DNA per shot, 1300 psi and a target distance
of 8.5 cm from stopping plate to plate of callus and 1 shot per
plate of callus.
[0686] [0522.0.0.0] After the bombardment, calli are transferred
back to the fresh callus development medium and maintained in the
dark at room temperature for a 1-week period. The callus is then
transferred to growth conditions in the light at 25.degree. C. to
initiate embryo differentiation with the appropriate selection
agent, e.g. 250 nM Arsenal, 5 mg/l PPT or 50 mg/L Kanamycin. Shoots
resistant to the selection agent are appearing and once rooted are
transferred to soil.
[0687] [0523.0.0.0] Samples of the primary transgenic plants (TO)
are analyzed by PCR to confirm the presence of T-DNA. These results
are confirmed by Southern hybridization in which DNA is
electrophoresed on a 1% agarose gel and transferred to a positively
charged nylon membrane (Roche Diagnostics). The PCR DIG Probe
Synthesis Kit (Roche Diagnostics) is used to prepare a
digoxigenin-labelled probe by PCR, and used as recommended by the
manufacturer.
[0688] [0524.0.0.0] Transgenic T0 ryegrass plants are propagated
vegetatively by excising tillers. The transplanted tillers are
maintained in the greenhouse for 2 months until well established.
The shoots are defoliated and allowed to grow for 2 weeks.
[0525.0.0.0] Example 14b
Engineering Soybean Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, e.g. Derived from Saccharomyces
cerevisiae, E. Coli or Plants or Another Organism
[0689] [0526.0.0.0] Soybean can be transformed according to the
following modification of the method described in the Texas A&M
patent U.S. Pat. No. 5,164,310. Several commercial soybean
varieties are amenable to transformation by this method. The
cultivar Jack (available from the Illinois Seed Foundation) is
commonly used for transformation. Seeds are sterilized by immersion
in 70% (v/v) ethanol for 6 min and in 25 commercial bleach (NaOCI)
supplemented with 0.1% (v/v) Tween for 20 min, followed by rinsing
4 times with sterile double distilled water. Removing the radicle,
hypocotyl and one cotyledon from each seedling propagates seven-day
seedlings. Then, the epicotyl with one cotyledon is transferred to
fresh germination media in petri dishes and incubated at 25.degree.
C. under a 16-hr photoperiod (approx. 100 .mu.E-m-2s-1) for three
weeks. Axillary nodes (approx. 4 mm in length) are cut from 3-4
week-old plants. Axillary nodes are excised and incubated in
Agrobacterium LBA4404 culture.
[0690] [0527.0.0.0] Many different binary vector systems have been
described for plant transformation (e.g. An, G. in Agrobacterium
Protocols. Methods in Molecular Biology vol 44, pp 47-62, Gartland
K M A and M R Davey eds. Humana Press, Totowa, N.J.). Many are
based on the vector pBIN19 described by Bevan (Nucleic Acid
Research. 1984. 12:8711-8721) that includes a plant gene expression
cassette flanked by the left and right border sequences from the Ti
plasmid of Agrobacterium tumefaciens. A plant gene expression
cassette consists of at least two genes--a selection marker gene
and a plant promoter regulating the transcription of the cDNA or
genomic DNA of the trait gene. Various selection marker genes can
be used as described above, including the Arabidopsis gene encoding
a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S. Pat. Nos.
5,767,3666 and 6,225,105). Similarly, various promoters can be used
to regulate the trait gene to provide constitutive, developmental,
tissue or environmental regulation of gene transcription as
described above. In this example, the 34S promoter (GenBank
Accession numbers M59930 and X16673) is used to provide
constitutive expression of the trait gene.
[0691] [0528.0.0.0] After the co-cultivation treatment, the
explants are washed and transferred to selection media supplemented
with 500 mg/L timentin. Shoots are excised and placed on a shoot
elongation medium. Shoots longer than 1 cm are placed on rooting
medium for two to four weeks prior to transplanting to soil.
[0692] [0529.0.0.0] The primary transgenic plants (TO) are analyzed
by PCR to confirm the presence of T-DNA. These results are
confirmed by Southern hybridization in which DNA is electrophoresed
on a 1% agarose gel and transferred to a positively charged nylon
membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit
(Roche Diagnostics) is used to prepare a digoxigenin-labelled probe
by PCR, and is used as recommended by the manufacturer.
[0530.0.0.0] Example 14c
Engineering Corn Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, e.g. Derived from Saccharomyces
cerevisiae, E. Coli or Plants or Another Organism
[0693] [0530.1.0.0] Amplification of for example SEQ ID NO: 1 was
achieved as described in example 10 except that the upstream primer
SEQ ID NO:3 and the reverse primer SEQ ID NO: 4 contained the
following 5''extensions:
TABLE-US-00005 SEQ ID NO: 68243 i) forward primer:
5'-GGGTCGCTCCTACGCG-3' SEQ ID NO: 68246 ii) reverse primer
5'-CTCGGGCTCGGCGTCC-3'
[0694] [0531.0.0.0] Vector Construction
[0695] The maize transformation vector for constitutive expression
was constructed as follows. As base vectors, the vectors EG073qcz
(SEQ ID NO 68240) and EG065qcz (SEQ ID NO: 68241) were chosen. The
MCS from EG065qcz was deleted by digestion of the vector with
Asp718 and PstI, followed by blunting of the vector using T4 DNA
polymerase. The blunted vector was religated. The vector generated
was called EG065-MCS. The LIC cassette was cloned in the vector
EG065-MCS by hybridizing the following oligos, generating a DNA
fragment with ends able to ligate into a Smal and SacI digested
vector. This fragment was ligated into the vector EG065-MCS that
had been digested with Smal and SacI. The generated vector was
called EG065-LIC. The complete expression cassette comprising ScBV
(Schenk (1999) Plant Mol BioI 39(6):1221-1230) promoter, LIC
cassette and terminator was cut out of EG065-LIC with AscI and PacI
and ligated into the vector EG073qcz that had previously been
digested with AscI and PacI. The resulting binary vector for corn
transformation was called pMME0607 (SEQ ID NO: 68242).
TABLE-US-00006 Oligo POCCLicMluISacIIfw: (SEQ ID NO: 68244)
gggtcgctcctacgcgtcaatgatccgcggacgccgagcccgagct Oligo
POCCLicMluISacIrev: (SEQ ID NO: 68245)
cgggctcggcgtccgcggatcattgacgcgtaggagcgaccc
[0696] For cloning of a polynucleotide of the invention, for
example the ORF of SEQ ID NO: 1, from S. cerevisiae the vector DNA
was treated with the restriction enzyme Mlul and SaII. The reaction
was stopped by inactivation at 70.degree. C. for 20 minutes and
purified over QIAquick columns following the standard protocol
(Qiagen).
[0697] Then the PCR-product representing the amplified ORF and the
vector DNA were treated with T4 DNA polymerase according to the
standard protocol (MBI Fermentas) to produce single stranded
overhangs with the parameters 1 unit T4 DNA polymerase at
37.degree. C. for 2-10 minutes for the vector and 1 u T4 DNA
polymerase at 15.degree. C. for 10-60 minutes for the PCR product
representing SEQ ID NO: 1.
[0698] The reaction was stopped by addition of high-salt buffer and
purified over QIAquick columns following the standard protocol
(Qiagen).
[0699] Approximately 30 ng of prepared vector and a defined amount
of prepared amplificate were mixed and hybridized at 65.degree. C.
for 15 minutes followed by 37.degree. C. 0.1.degree. C./1 seconds,
followed by 37.degree. C. 10 minutes, followed by 0.1.degree. C./1
seconds, then 4.degree. C.
[0700] The ligated constructs were transformed in the same reaction
vessel by addition of competent E. coli cells (strain DH5alpha) and
incubation for 20 minutes at 1.degree. C. followed by a heat shock
for 90 seconds at 42.degree. C. and cooling to 4.degree. C. Then,
complete medium (SOC) was added and the mixture was incubated for
45 minutes at 37.degree. C. The entire mixture was subsequently
plated onto an agar plate with 0.05 mg/ml kanamycine and incubated
overnight at 37.degree. C.
[0701] The outcome of the cloning step was verified by
amplification with the aid of primers which bind upstream and
downstream of the integration site, thus allowing the amplification
of the insertion. The amplifications were carried as described in
the protocol of Taq DNA polymerase (Gibco-BRL).
[0702] The amplification cycles were as follows: 1 cycle of 5
minutes at 94.degree. C., followed by 35 cycles of in each case 15
seconds at 94.degree. C., 15 seconds at 50-66.degree. C. and 5
minutes at 72.degree. C., followed by 1 cycle of 10 minutes at
72.degree. C., then 4.degree. C.
[0703] Several colonies were checked, but only one colony for which
a PCR product of the expected size was detected was used in the
following steps.
[0704] A portion of this positive colony was transferred into a
reaction vessel filled with complete medium (LB) supplemented with
kanamycin 0 and incubated overnight at 37.degree. C.
[0705] The plasmid preparation was carried out as specified in the
Qiaprep standard protocol (Qiagen).
[0530.3.0.0] Example 14 c.a.
Corn Transformation
[0706] The preparation of the immature embryos and Agrobacterium
were basically as stated in U.S. Pat. No. 5,591,616. In brief, the
Agrobacterium strain LBA4404 transformed with the plasmid by a
standard method, such as the triple cross method or the
electroporation, was grown on LB plates for 2 days prior to
cocultivation. A loop of cells was resuspended in liquid infection
media at an O.D. of approximately 1.0. Immature Embryos of about
1.5 mm in size were incubated in the soln of agrobacterium for
around 30 minutes. Excised embryos were removed from liquid and
then co-cultivated in the dark at 22.degree. C. with Agrobacterium
tumefaciens on solid MS-based callus induction medium containing 2
mg/l 2,4-D, 10 um AgNO3, and 200 um Acetosyringone. After several
days of co-cultivation, embryos were transferred to MS-based media
containing 2 mg/l 2,4, 10 um AgNO3 and 200 mg/l Timentin the dark
at 27.degree. C. for 1 week. Embryos were transferred to MS-based
selection media containing imidazoline herbicide (500 nM Pursuit)
as a selection agent in the dark for 3 weeks. After 3 weeks
putative transgenic events were transferred to an MS-based media
containing 2 mg/L Kinetin 500 nM Pursuit, 200 mg/l Timentin and
incubated under cool white fluorescent light (100 uE/m2/s-1 with
photoperiod of 16 hrs) at 25.degree. C. for 2-3 weeks, or until
shoots develop. The shoots were transferred to MS-based rooting
medium and incubated under light at 25.degree. C. for 2 weeks. The
rooted shoots were transplanted to 4 inch pots containing
artificial soil mix. Metro-Mix.RTM. 360 in and grown in an
environmental chamber for 1-2 weeks. The environmental chamber
maintained 16-h-light, 8-h-dark cycles at 27.degree. C. day and
22.degree. C. respectively. Light was supplied by a mixture of
incandescent and cool white fluorescent bulbs with an intensity of
.about.400 uE/m2/s-1. After plants were grown to 4-6 leaf stage
they were moved to 14 inch pots containing Metro-Mix.RTM. 360.
Supplemental metal-halide lamps were used to maintain >800
uE/m2/s-1 with a 16-h-light, 8-h-dark cycles at 28.degree. C. day
and 22.degree. C. Transplantation occurs weekly on Tuesday. Peters
20-20-20 plus micronutrients (200 ppm) is used to fertilize plants
2.times. weekly on Monday and Thursday after sampling of TO's is
performed. T1 seeds were produced from plants that exhibit
tolerance to the imidazolinone herbicides and which are PCR
positive for the transgenes. T0 plants with single locus insertions
of the T-DNA (self-pollinated) produced T1 generation that
segregated for the transgene in a 3:1 ratio. Progeny containing
copies of the transgene were tolerant of imidazolinone herbicides
and could be detected by PCR analysis.
[0530.4.0.0] Example 14 c.b.
Growth of T0 Corn Plants for Metabolic Analysis
[0707] Plants were grown under the following standardized
conditions to properly stage them for TO sampling. T0 plantlets
were transferred to 14'' pots in the greenhouse after they grow to
4-6 leaf stage (1-3 weeks). pBSMM232 containing plants were
produced carried along with each experiment to serve as controls
for TO samples. Plantlets were moved to 14'' pots on Tuesday of
each week. Plants were grown for 9 days until the 7-13 leaf stage
is reached. On Thursday between 10 am and 2 .mu.m leaf sampling was
performed on the 3rd youngest (1.sup.st fully elongated). Within 30
seconds 250-500 mg of leaf material (without midrib), were removed
weighed and placed into pre-extracted glass thimbles in liquid
nitrogen. A second sample (opposite side of the midrib) from each
plant was sampled as described above for qPCR analysis.
[0530.5.0.0] Example 14 c.c.
Growth of T1 Corn Plant for Metabolic Analysis
[0708] For the bioanalytical analyses of the transgenic plants, the
latter were grown uniformly in a specific culture facility. To this
end the GS-90 substrate as the compost mixture was introduced into
the potting machine (Laible System GmbH, Singen, Germany) and
filled into the pots. Thereafter, 26 pots were combined in one dish
and treated with Previcur. For the treatment, 25 ml of Previcur
were taken up in 10 l of tap water. This amount was sufficient for
the treatment of approximately 150 pots. The pots were placed into
the Previcur solution and additionally irrigated overhead with tap
water without Previcur. They were used within four days.
[0709] For the sowing, the seeds, which had been stored at room
temperature were removed from the paper-bag and transferred into
the pots with the soil. In total, approximately 1 to 3 seeds were
distributed in the middle of the pot.
[0710] After the seeds had been sown, the dishes with the pots were
covered with matching plastic hood and placed into growth chambers
for 2 days. After this time the plastic hood was removed and plants
were placed on the growth table and cultivated for 22 to 24 days
under following growth conditions: 16-h-light, 8-h-dark rhythm at
20.degree. C., an atmospheric humidity of 60% and a CO.sub.2
concentration of approximately 400 ppm. The light sources used were
Powerstar HQI-T 250 W/D Daylight lamps from Osram, which generate a
light resembling the solar color spectrum with a light intensity of
approximately 220 pE/m2/s-1.
[0711] When the plants were 7 days old, they were subjected to
select transgenic plants. For this purposes pieces of plant leaves
were sampled and a PCR reaction with the respective primers for the
transgene were performed. Plants exhibiting the transgene were used
for the metabolic analysis. The nontransgenic seedlings were
removed. The transgenic plants were thinned when they had reached
the age of 18 days. The transgenic plants, which had grown best in
the center of the pot were considered the target plants. All the
remaining plants were removed carefully with the aid of metal
tweezers and discarded.
[0712] During their growth, the plants received overhead irrigation
with distilled water (onto the compost) and bottom irrigation into
the placement grooves. Once the grown plants had reached the age of
24 days, they were harvested.
[0530.6.0.0] Example 14 c.d.
Metabolic Analysis of Maize Leaves
[0713] The modifications identified in accordance with the
invention, in the content of above-described metabolites, were
identified by the following procedure.
[0714] a) Sampling and Storage of the Samples
[0715] Sampling was performed in corridor next to the green house.
The leaves were incised twice using small laboratory scissors and
this part of the leave was removed manually from the middle rib.
The sample was rapidly weighed on laboratory scales, transferred
into a pre-cooled extraction sleeve and placed into kryo-box cooled
by liquid nitrogen. The time elapsing between cutting the leave to
freezing it in liquid nitrogen amounted to not more than 30
seconds. The boxes were stored in a freezer at -80.degree. C., an
shipped on dry ice.
[0716] b) Lyophilization
[0717] During the experiment, care was taken that the plants either
remained in the deep-frozen state (temperatures <-40.degree. C.)
or were freed from water by lyophilization until the first contact
with solvents. Before entering the analytical process the
extraction sleeves with the samples were transferred to a
pre-cooled aluminium rack.
[0718] The aluminum rack with the plant samples in the extraction
sleeves was placed into the pre-cooled (-40.degree. C.)
lyophilization facility. The initial temperature during the main
drying phase was -35.degree. C. and the pressure was 0.120 mbar.
During the drying phase, the parameters were altered following a
pressure and temperature program. The final temperature after 12
hours was +30.degree. C. and the final pressure was 0.001 to 0.004
mbar. After the vacuum pump and the refrigerating machine had been
switched off, the system was flushed with air (dried via a drying
tube) or argon.
[0719] c) Extraction
[0720] Immediately after the lyophilization apparatus had been
flushed, the extraction sleeves with the lyophilized plant material
were transferred into the 5 ml extraction cartridges of the ASE
device (Accelerated Solvent Extractor ASE 200 with Solvent
Controller and AutoASE software (DIONEX)).
[0721] Immediately after the lyophilization apparatus had been
flushed, the extraction sleeves with the lyophilized plant material
were transferred into the 5 ml extraction cartridges of the ASE
device (Accelerated Solvent Extractor ASE 200 with Solvent
Controller and AutoASE software (DIONEX)).
[0722] The 24 sample positions of an ASE device (Accelerated
Solvent Extractor ASE 200 with Solvent Controller and AutoASE
software (DIONEX)) were filled with plant samples, including some
samples for testing quality control.
[0723] The polar substances were extracted with approximately 10 ml
of methanol/water (80/20, v/v) at T=70.degree. C. and p=140 bar, 5
minutes heating-up phase, 1 minute static extraction. The more
lipophilic substances were extracted with approximately 10 ml of
methanol/dichloromethane (40/60, v/v) at T=70.degree. C. and p=140
bar, 5 minute heating-up phase, 1 minute static extraction. The two
solvent mixtures were extracted into the same glass tubes
(centrifuge tubes, 50 ml, equipped with screw cap and pierceable
septum for the ASE (DIONEX)).
[0724] The solution was treated with internal standards: ribitol,
L-glycine-2,2-d.sub.2, L-alanine-2,3,3,3-d.sub.4,
methionine-methyl-d.sub.3, and .alpha.-methylglucopyranoside and
methyl nonadecanoate, methyl undecanoate, methyl tridecanoate,
methyl pentadecanoate, methyl nonacosanoate.
[0725] The total extract was treated with 8 ml of water. The solid
residue of the plant sample and the extraction sleeve were
discarded.
[0726] The extract was shaken and then centrifuged for 5 to 10
minutes at least at 1 400 g in order to accelerate phase
separation. 0.5 ml of the supernatant methanol/water phase ("polar
phase", colorless) was removed for the further GC analysis, and 0.5
ml was removed for the LC analysis. The remainder of the
methanol/water phase of all samples was used for additional quality
controls. 0.5 ml of the organic phase ("lipid phase", dark green)
was removed for the further GC analysis and 0.5 ml was removed for
the LC analysis. All the portions removed were evaporated to
dryness using the IR Dancer infrared vacuum evaporator (Hettich).
The maximum temperature during the evaporation process did not
exceed 40.degree. C. Pressure in the apparatus was not less than 10
mbar.
[0727] d) Processing the Lipid Phase for the LC/MS or LC/MS/MS
Analysis
[0728] The lipid extract, which had been evaporated to dryness was
taken up in mobile phase. The HPLC was run with gradient
elution.
[0729] The polar extract, which had been evaporated to dryness was
taken up in mobile phase. The HPLC was run with gradient
elution.
[0730] e) Derivatization of the Lipid Phase for the GC/MS
Analysis
[0731] For the transmethanolysis, a mixture of 140 .mu.l of
chloroform, 37 .mu.l of hydrochloric acid (37% by weight HCl in
water), 320 .mu.l of methanol and 20 .mu.l of toluene was added to
the evaporated extract. The vessel was sealed tightly and heated
for 2 hours at 100.degree. C., with shaking. The solution was
subsequently evaporated to dryness. The residue was dried
completely.
[0732] The methoximation of the carbonyl groups was carried out by
reaction with methoxyamine hydrochloride (20 mg/ml in pyridine, 100
.mu.l for 1.5 hours at 60.degree. C.) in a tightly sealed vessel.
20 .mu.l of a solution of odd-numbered, straight-chain fatty acids
(solution of each 0.3 mg/mL of fatty acids from 7 to 25 carbon
atoms and each 0.6 mg/mL of fatty acids with 27, 29 and 31 carbon
atoms in 3/7 (v/v) pyridine/toluene) were added as time standards.
Finally, the derivatization with 100 .mu.l of
N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was
carried out for 30 minutes at 60.degree. C., again in the tightly
sealed vessel. The final volume before injection into the GC was
220 .mu.l.
[0733] f) Derivatization of the Polar Phase for the GC/MS
Analysis
[0734] The methoximation of the carbonyl groups was carried out by
reaction with methoxyamine hydrochloride (20 mg/ml in pyridine, 50
.mu.l for 1.5 hours at 60.degree. C.) in a tightly sealed vessel.
10 .mu.l of a solution of odd-numbered, straight-chain fatty acids
(solution of each 0.3 mg/mL of fatty acids from 7 to 25 carbon
atoms and each 0.6 mg/mL of fatty acids with 27, 29 and 31 carbon
atoms in 3/7 (v/v) pyridine/toluene) were added as time standards.
Finally, the derivatization with 50 .mu.l of
N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was
carried out for 30 minutes at 60.degree. C., again in the tightly
sealed vessel. The final volume before injection into the GC was
110 .mu.l.
[0735] g) Analysis of the Various Plant Samples
[0736] The samples were measured in individual series of 20 plant
(leaf) samples each (also referred to as sequences), each sequence
containing at least 5 samples from individual control plants
containing GUS. The peak area of each analyte was divided by the
peak area of the respective internal standard. The data were
standardized for the fresh weight established for the respective
harvested sample. The values calculated were then related to the
GUS-containing control group by being divided by the mean of the
corresponding data of the control group of the same sequence. The
values obtained were referred to as ratio_by_WT, they are
comparable between sequences and indicate how much the analyte
concentration in the mutant differs in relation to the control. The
GUS-containing plants were chosen in order to assure that the
vector and transformation procedure itself has no significant
influence on the metabolic composition of the plants. Therefore the
described changes in comparison with the controls were caused by
the introduced genes.
[0737] [0531.0.0.0] Transformation of maize (Zea Mays L.) can also
be performed with a modification of the method described by Ishida
et al. (1996. Nature Biotech 14745-50). Transformation is
genotype-dependent in corn and only specific genotypes are amenable
to transformation and regeneration. The inbred line A188
(University of Minnesota) or hybrids with A188 as a parent are good
sources of donor material for transformation (Fromm et al. 1990
Biotech 8:833-839), but other genotypes can be used successfully as
well. Ears are harvested from corn plants at approximately 11 days
after pollination (DAP) when the length of immature embryos is
about 1 to 1.2 mm. Immature embryos are co-cultivated with
Agrobacterium tumefaciens that carry "super binary" vectors and
transgenic plants are recovered through organogenesis. The super
binary vector system of Japan Tobacco is described in WO patents
WO94/00977 and WO95/06722. Vectors can be constructed as described.
Various selection marker genes can be used including the maize gene
encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S.
Pat. No. 6,025,541). Similarly, various promoters can be used to
regulate the trait gene to provide constitutive, developmental,
tissue or environmental regulation of gene transcription. In this
example, the 34S promoter (GenBank Accession numbers M59930 and
X16673 can be used to provide constitutive expression of the trait
gene.
[0738] [0532.0.0.0] Excised embryos can be grown on callus
induction medium, then maize regeneration medium, containing
imidazolinone as a selection agent. The Petri plates can be
incubated in the light at 25.degree. C. for 2-3 weeks, or until
shoots develop. The green shoots can be transferred from each
embryo to maize rooting medium and incubated at 25.degree. C. for
2-3 weeks, until roots develop. The rooted shoots can be
transplanted to soil in the greenhouse. T1 seeds can be produced
from plants that exhibit tolerance to the imidazolinone herbicides
and which can be PCR positive for the transgenes.
[0739] [0533.0.0.0] The T1 generation of single locus insertions of
the T-DNA can segregate for the transgene in a 3:1 ratio. Those
progeny containing one or two copies of the transgene can be
tolerant of the imidazolinone herbicide. Homozygous T2 plants can
exhibited similar phenotypes as the T1 plants. Hybrid plants (F1
progeny) of homozygous transgenic plants and non-transgenic plants
can also exhibit increased similar phenotypes.
[0534.0.0.0] Example 14d
Engineering Wheat Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, e.g. Derived from Saccharomyces
cerevisiae, E. Coli or Plants or Another Organism
[0740] [0535.0.0.0] Transformation of wheat can be performed with
the method described by Ishida et al. (1996 Nature Biotech.
14745-50). The cultivar Bobwhite (available from CYMMIT, Mexico)
can commonly be used in transformation. Immature embryos can be
co-cultivated with Agrobacterium tumefaciens that carry "super
binary" vectors, and transgenic plants are recovered through
organogenesis. The super binary vector system of Japan Tobacco is
described in WO patents WO94/00977 and WO95/06722. Vectors can be
constructed as described. Various selection marker genes can be
used including the maize gene encoding a mutated acetohydroxy acid
synthase (AHAS) enzyme (U.S. Pat. No. 6,025,541). Similarly,
various promoters can be used to regulate the trait gene to provide
constitutive, developmental, tissue or environmental regulation of
gene transcription. The 34S promoter (GenBank Accession numbers
M59930 and X16673) can be used to provide constitutive expression
of the trait gene.
[0741] [0536.0.0.0] After incubation with Agrobacterium, the
embryos can be grown on callus induction medium, then regeneration
medium, containing imidazolinone as a selection agent. The Petri
plates can be incubated in the light at 25.degree. C. for 2-3
weeks, or until shoots develop. The green shoots can be transferred
from each embryo to rooting medium and incubated at 25.degree. C.
for 2-3 weeks, until roots develop. The rooted shoots can be
transplanted to soil in the greenhouse. T1 seeds can be produced
from plants that exhibit tolerance to the imidazolinone herbicides
and which are PCR positive for the transgenes.
[0742] [0537.0.0.0] The T1 generation of single locus insertions of
the T-DNA can segregate for the transgene in a 3:1 ratio. Those
progeny containing one or two copies of the transgene can be
tolerant of the imidazolinone herbicide. Homozygous T2 plants
exhibited similar phenotypes.
[0538.0.0.0] Example 14e
Engineering Rapeseed/Canola Plants by Over-Expressing the
Polynucleotide Characterized in the Invention, e.g. Derived from
Saccharomyces cerevisiae, E. Coli or Plants or Another Organism
[0743] [0539.0.0.0] Cotyledonary petioles and hypocotyls of 5-6
day-old young seedlings can be used as explants for tissue culture
and transformed according to Babic et al. (1998, Plant Cell Rep 17:
183-188). The commercial cultivar Westar (Agriculture Canada) can
be the standard variety used for transformation, but other
varieties can be used.
[0744] [0540.0.0.0] Agrobacterium tumefaciens LBA4404 containing a
binary vector can be used for canola transformation. Many different
binary vector systems have been described for plant transformation
(e.g. An, G. in Agrobacterium Protocols. Methods in Molecular
Biology vol 44, pp 47-62, Gartland K M A and M R Davey eds. Humana
Press, Totowa, N.J.). Many are based on the vector pBIN19 described
by Bevan (Nucleic Acid Research. 1984. 12:8711-8721) that includes
a plant gene expression cassette flanked by the left and right
border sequences from the Ti plasmid of Agrobacterium tumefaciens.
A plant gene expression cassette can consist of at least two
genes--a selection marker gene and a plant promoter regulating the
transcription of the cDNA or genomic DNA of the trait gene. Various
selection marker genes can be used including the Arabidopsis gene
encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S.
Pat. Nos. 5,767,3666 and 6,225,105). Similarly, various promoters
can be used to regulate the trait gene to provide constitutive,
developmental, tissue or environmental regulation of gene
transcription. The 34S promoter (GenBank Accession numbers M59930
and X16673) can be used to provide constitutive expression of the
trait gene.
[0745] [0541.0.0.0] Canola seeds can be surface-sterilized in 70%
ethanol for 2 min., and then in 30% Clorox with a drop of Tween-20
for 10 min, followed by three rinses with sterilized distilled
water. Seeds can be then germinated in vitro 5 days on half
strength MS medium without hormones, 1% sucrose, 0.7% Phytagar at
23.degree. C., 16 hr. light. The cotyledon petiole explants with
the cotyledon attached can be excised from the in vitro seedlings,
and can be inoculated with Agrobacterium by dipping the cut end of
the petiole explant into the bacterial suspension. The explants can
be then cultured for 2 days on MSBAP-3 medium containing 3 mg/l
BAP, 3% sucrose, 0.7% Phytagar at 23.degree. C., 16 hr light. After
two days of co-cultivation with Agrobacterium, the petiole explants
can be transferred to MSBAP-3 medium containing 3 mg/l BAP,
cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and
can then be cultured on MSBAP-3 medium with cefotaxime,
carbenicillin, or timentin and selection agent until shoot
regeneration. When the shoots are 5-10 mm in length, they can be
cut and transferred to shoot elongation medium (MSBAP-0.5,
containing 0.5 mg/l BAP). Shoots of about 2 cm in length can be
transferred to the rooting medium (MS0) for root induction.
[0746] [0542.0.0.0] Samples of the primary transgenic plants (TO)
can be analyzed by PCR to confirm the presence of T-DNA. These
results can be confirmed by Southern hybridization in which DNA is
electrophoresed on a 1% agarose gel and are transferred to a
positively charged nylon membrane (Roche Diagnostics). The PCR DIG
Probe Synthesis Kit (Roche Diagnostics) can be used to prepare a
digoxigenin-labelled probe by PCR, and used as recommended by the
manufacturer.
[0543.0.0.0] Example 14f
Engineering Alfalfa Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, e.g. Derived from Saccharomyces
cerevisiae or E. coli or Plants or Another Organism
[0747] [0544.0.0.0] A regenerating clone of alfalfa (Medicago
sativa) can be transformed using the method of (McKersie et al.,
1999 Plant Physiol 119: 839-847). Regeneration and transformation
of alfalfa can be genotype dependent and therefore a regenerating
plant is required. Methods to obtain regenerating plants have been
described. For example, these can be selected from the cultivar
Rangelander (Agriculture Canada) or any other commercial alfalfa
variety as described by Brown D C W and A Atanassov (1985. Plant
Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3
variety (University of Wisconsin) can be selected for use in tissue
culture (Walker et al., 1978 Am J Bot 65:654-659).
[0748] [0545.0.0.0] Petiole explants can be cocultivated with an
overnight culture of Agrobacterium tumefaciens C58C1 pMP90
(McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404
containing a binary vector. Many different binary vector systems
have been described for plant transformation (e.g. An, G. in
Agrobacterium Protocols. Methods in Molecular Biology vol 44, pp
47-62, Gartland K M A and M R Davey eds. Humana Press, Totowa,
N.J.). Many are based on the vector pBIN19 described by Bevan
(Nucleic Acid Research. 1984. 12:8711-8721) that includes a plant
gene expression cassette flanked by the left and right border
sequences from the Ti plasmid of Agrobacterium tumefaciens. A plant
gene expression cassette can consist of at least two genes--a
selection marker gene and a plant promoter regulating the
transcription of the cDNA or genomic DNA of the trait gene. Various
selection marker genes can be used including the Arabidopsis gene
encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S.
Pat. Nos. 5,767,3666 and 6,225,105). Similarly, various promoters
can be used to regulate the trait gene that provides constitutive,
developmental, tissue or environmental regulation of gene
transcription. The 34S promoter (GenBank Accession numbers M59930
and X16673) can be used to provide constitutive expression of the
trait gene.
[0749] [0546.0.0.0] The explants can be cocultivated for 3 d in the
dark on SH induction medium containing 288 mg/L Pro, 53 mg/L
thioproline, 4.35 g/L K2SO4, and 100 .mu.m acetosyringinone. The
explants can be washed in half-strength Murashige-Skoog medium
(Murashige and Skoog, 1962) and plated on the same SH induction
medium without acetosyringinone but with a suitable selection agent
and suitable antibiotic to inhibit Agrobacterium growth. After
several weeks, somatic embryos can be transferred to BOi2Y
development medium containing no growth regulators, no antibiotics,
and 50 g/L sucrose. Somatic embryos are subsequently germinated on
half-strength Murashige-Skoog medium. Rooted seedlings can be
transplanted into pots and grown in a greenhouse.
[0750] [0547.0.0.0] The T0 transgenic plants are propagated by node
cuttings and rooted in Turface growth medium. The plants are
defoliated and grown to a height of about 10 cm (approximately 2
weeks after defoliation).
[0548.0.0.0] Example 14 g
Engineering Alfalfa Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, Derived e.g. from Saccharomyces
cerevisiae, E. Coli or Plants or Another Organism
[0751] [0549.0.0.0] A regenerating clone of alfalfa (Medicago
sativa) can be transformed using the method of (McKersie et al.,
1999 Plant Physiol 119: 839-847). Regeneration and transformation
of alfalfa can be genotype dependent and therefore a regenerating
plant is required. Methods to obtain regenerating plants have been
described. For example, these can be selected from the cultivar
Rangelander (Agriculture Canada) or any other commercial alfalfa
variety as described by Brown D C W and A Atanassov (1985. Plant
Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3
variety (University of Wisconsin) has been selected for use in
tissue culture (Walker et al., 1978 Am J Bot 65:654-659).
[0752] [0550.0.0.0] Petiole explants can be cocultivated with an
overnight culture of Agrobacterium tumefaciens C58C1 pMP90
(McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404
containing a binary vector. Many different binary vector systems
have been described for plant transformation (e.g. An, G. in
Agrobacterium Protocols. Methods in Molecular Biology vol 44, pp
47-62, Gartland K M A and M R Davey eds. Humana Press, Totowa,
N.J.). Many are based on the vector pBIN19 described by Bevan
(Nucleic Acid Research. 1984. 12:8711-8721) that includes a plant
gene expression cassette flanked by the left and right border
sequences from the Ti plasmid of Agrobacterium tumefaciens. A plant
gene expression cassette consists of at least two genes--a
selection marker gene and a plant promoter regulating the
transcription of the cDNA or genomic DNA of the trait gene. Various
selection marker genes can be used including the Arabidopsis gene
encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S.
Pat. Nos. 5,767,3666 and 6,225,105). Similarly, various promoters
can be used to regulate the trait gene that provides constitutive,
developmental, tissue or environmental regulation of gene
transcription. In this example, the 34S promoter (GenBank Accession
numbers M59930 and X16673) can be used to provide constitutive
expression of the trait gene.
[0753] [0551.0.0.0] The explants are cocultivated for 3 d in the
dark on SH induction medium containing 288 mg/L Pro, 53 mg/L
thioproline, 4.35 g/L K2SO4, and 100 .mu.m acetosyringinone. The
explants are washed in half-strength Murashige-Skoog medium
(Murashige and Skoog, 1962) and plated on the same SH induction
medium without acetosyringinone but with a suitable selection agent
and suitable antibiotic to inhibit Agrobacterium growth. After
several weeks, somatic embryos are transferred to BOi2Y development
medium containing no growth regulators, no antibiotics, and 50 g/L
sucrose. Somatic embryos are subsequently germinated on
half-strength Murashige-Skoog medium. Rooted seedlings are
transplanted into pots and grown in a greenhouse.
[0754] [0552.0.0.0] The T0 transgenic plants are propagated by node
cuttings and rooted in Turface growth medium. The plants are
defoliated and grown to a height of about 10 cm (approximately 2
weeks after defoliation).
[0552.1.0.0] Example 15
Metabolite Profiling Info from Zea mays
[0755] Zea mays plants were engineered, grown and analyzed as
described in Example 14c.
[0756] The results of the different Zea mays plants analysed can be
seen from Table 2 which follows:
TABLE-US-00007 TABLE 2 ORF_NAME Metabolite Min Max b2414 Methionine
1.36 2.61
[0757] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in methionine in
genetically modified corn plants expressing the E. coli nucleic
acid sequence b2414.
[0758] In one embodiment, in case the activity of the E. coli
protein b2414 or its homologs, e.g. "the activity of a subunit of
cysteine synthase A and O-acetylserine sulfhydrolase
[0759] A, PLP-dependent enzyme", is increased in corn plants,
preferably, an increase of the fine chemical methionine between 36%
and 161% is conferred.
[0552.2.0.0] Example 16
Preparation of Homologous Sequences from Plants
[0760] Different plants can be grown under standard or varying
conditions in the greenhouse. RNA can be extracted following the
protocol of Jones, Dunsmuir and Bedbrook (1985) EMBO J. 4:
2411-2418. Approx. 1 gram of tissue material from various organs is
ground in liquid nitrogen. The powder is transferred to a 13 ml
Falcon tube containing 4.5 ml NTES buffer (100 mM NaCl, 10 mM
Tris/HCl pH 7.5, 1 mM EDTA, 1% SDS; in RNase-free water) and 3 ml
phenol/chloroform/isoamylalcohol (25/24/1), immediately mixed and
stored on ice. The mixture is spun for 10 minutes at 7000 rpm using
a centrifuge (Sorval; SM24 or SS34 rotor). The supernatant is
transferred to a new tube, 1/10th volume of 3 M NaAcetate (pH 5.2;
in RNase-free water) and 1 volume of isopropanol is added, mixed at
stored for 1 hour or overnight at -20.degree. C. The mixture is
spun for 10 minutes at 7000 rpm. The supernatant is discarded and
the pellet washed with 70% ethanol (v/v). The mixture is spun for 5
minutes at 7000 rpm, the supernatant is discarded and the pellet is
air-dried. 1 ml RNase-free water is added and allow the DNA/RNA
pellet to dissolve on ice at 4 C. The nucleic acid solution is
transferred to a 2 ml Eppendorf tube and 1 ml of 4 M LiAcetate is
added. After mixing the solution is kept for at least 3 hours, or
overnight, at 4 C. The mixture is spun for 10 minutes at 14000 rpm,
the supernatant discarded, the pellet washed with 70% Ethanol,
air-dried and dissolved in 200 .mu.l of RNase-free water.
[0761] Total RNA can be used to construct a cDNA-library according
to the manufacturer's protocol (for example using the ZAP-cDNA
synthesis and cloning kit of Stratagene, La Jolla, USA). Basically,
messenger RNA (mRNA) is primed in the first strand synthesis with a
oligo(dT) linker-primer and is reverse-transcribed using reverse
transcriptase. After second strand cDNA synthesis, the
double-stranded cDNA is ligated into the Uni-ZAP XR vector. The
Uni-ZAP XR vector allows in vivo excision of the pBluescript
phagemid. The polylinker of the pBluescript phagemid has 21 unique
cloning sites flanked by T3 and T7 promoters and a choice of 6
different primer sites for DNA sequencing. Systematic single run
sequencing of the expected 5 prime end of the clones can allow
preliminary annotation of the sequences for example with the help
of the pedant pro Software package (Biomax, Munchen). Clones for
the nucleic acids of the invention or used in the process according
to the invention can be identified based on homology search with
standard algorithms like blastp or gap. Identified putative full
length clones with identity or high homology can be subjected to
further sequencing in order to obtain the complete sequence.
[0762] Additional new homologous sequences can be identified in a
similar manner by preparing respective cDNA libraries from various
plant sources as described above. Libraries can then be screened
with available sequences of the invention under low stringency
conditions for example as described in Sambrook et al., Molecular
Cloning: A laboratory manual, Cold Spring Harbor 1989, Cold Spring
Harbor Laboratory Press. Purified positive clones can be subjected
to the in vivo excision and complete sequencing. A pairwise
sequence alignment of the original and the new sequence using the
blastp or gap program allows the identification of orthologs,
meaning homologous sequences from different organisms, which should
have a sequence identity of at least 30%. Furthermore the
conservation of functionally important amino acid residues or
domains, which can be identified by the alignment of several
already available paralogs, can identify a new sequence as an new
orthologs.
[0763] Alternatively libraries can be subjected to mass sequencing
and obtained sequences can be stored in a sequence database, which
then can be screened for putative orthologs by different search
algorithms, for example the tbastn algorithm to search the obtained
nucleic acid sequences with a amino acid sequence of the invention.
Clones with the highest sequence identity are used for a complete
sequence determination and orthologs can be identified as described
above. [0764] 1. A process for the production of methionine, which
comprises [0765] (a) increasing or generating the activity of a
protein as indicated in Table II, columns 5 or 7, lines 1 to 5
and/or lines 334 to 338 or a functional equivalent thereof in a
non-human organism, or in one or more parts thereof; and [0766] (b)
growing the organism under conditions which permit the production
of methionine in said organism. [0767] 2. A process for the
production of methionine, comprising the increasing or generating
in an organism or a part thereof the expression of at least one
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: [0768] a) nucleic acid molecule
encoding of a polypeptide as indicated in Table II, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338 or a fragment thereof, which
confers an increase in the amount of methionine in an organism or a
part thereof; [0769] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 1 to 5 and/or lines 334 to 338; [0770] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of methionine in an organism or a part thereof; [0771] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of methionine in an organism or a part
thereof; [0772] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of methionine
in an organism or a part thereof; [0773] f) nucleic acid molecule
which encompasses a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers or primer pairs as indicated in Table
III, columns 7, lines 1 to 5 and/or lines 334 to 338 and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [0774] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
methionine in an organism or a part thereof; [0775] h) nucleic acid
molecule encoding a polypeptide comprising a consensus sequence as
indicated in Table IV, columns 7, lines 1 to 5 and/or lines 334 to
338 and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; and [0776] i) nucleic
acid molecule which is obtainable by screening a suitable nucleic
acid library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof. [0777] or comprising a sequence which is complementary
thereto. [0778] 3. The process of claim 1 or 2, comprising
recovering of the free or bound methionine. [0779] 4. The process
of any one of claims 1 to 3, comprising the following steps: [0780]
(a) selecting an organism or a part thereof expressing a
polypeptide encoded by the nucleic acid molecule characterized in
claim 2; [0781] (b) mutagenizing the selected organism or the part
thereof; [0782] (c) comparing the activity or the expression level
of said polypeptide in the mutagenized organism or the part thereof
with the activity or the expression of said polypeptide of the
selected organisms or the part thereof; [0783] (d) selecting the
mutated organisms or parts thereof, which comprise an increased
activity or expression level of said polypeptide compared to the
selected organism or the part thereof; [0784] (e) optionally,
growing and cultivating the organisms or the parts thereof; and
[0785] (f) recovering, and optionally isolating, the free or bound
methionine produced by the selected mutated organisms or parts
thereof. [0786] 5. The process of any one of claims 1 to 4, wherein
the activity of said protein or the expression of said nucleic acid
molecule is increased or generated transiently or stably. [0787] 6.
An isolated nucleic acid molecule comprising a nucleic acid
molecule selected from the group consisting of: [0788] a) nucleic
acid molecule encoding of a polypeptide as indicated in Table II,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 or a fragment
thereof, which confers an increase in the amount of methionine in
an organism or a part thereof; [0789] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 1 to 5 and/or lines 334 to 338; [0790] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of methionine in an organism
or a part thereof; [0791] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of methionine
in an organism or a part thereof; [0792] e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of methionine in an organism or a part thereof; [0793]
f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table III, columns 7, lines 1 to 5 and/or lines 334 to
338 and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [0794] g) nucleic acid
molecule encoding a polypeptide which is isolated with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (f) and conferring an increase in
the amount of methionine in an organism or a part thereof; [0795]
h) nucleic acid molecule encoding a polypeptide comprising a
consensus sequence as indicated in Table IV, columns 7, lines 1 to
5 and/or lines 334 to 338 and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
and [0796] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof. [0797] whereby the nucleic acid molecule distinguishes
over the sequence as indicated in Table I A, columns 5 or 7, lines
1 to 5 and/or lines 334 to 338 by one or more nucleotides. [0798]
7. A nucleic acid construct which confers the expression of the
nucleic acid molecule of claim 6, comprising one or more regulatory
elements. [0799] 8. A vector comprising the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7.
[0800] 9. The vector as claimed in claim 8, wherein the nucleic
acid molecule is in operable linkage with regulatory sequences for
the expression in a prokaryotic or eukaryotic, or in a prokaryotic
and eukaryotic, host. [0801] 10. A host cell, which has been
transformed stably or transiently with the vector as claimed in
claim 8 or 9 or the nucleic acid molecule as claimed in claim 6 or
the nucleic acid construct of claim 7 or produced as described in
claim any one of claims 2 to 5. [0802] 11. The host cell of claim
10, which is a transgenic host cell. [0803] 12. The host cell of
claim 10 or 11, which is a plant cell, an animal cell, a
microorganism, or a yeast cell, a fungus cell, a prokaryotic cell,
an eukaryotic cell or an archaebacterium. [0804] 13. A process for
producing a polypeptide, wherein the polypeptide is expressed in a
host cell as claimed in any one of claims 10 to 12. [0805] 14. A
polypeptide produced by the process as claimed in claim 13 or
encoded by the nucleic acid molecule as claimed in claim 6 whereby
the polypeptide distinguishes over a sequence as indicated in Table
II A, columns 5 or 7, lines 1 to 5 and/or lines 334 to 338 by one
or more amino acids. [0806] 15. An antibody, which binds
specifically to the polypeptide as claimed in claim 14. [0807] 16.
A plant tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [0808] 17. A method for screening for agonists
and antagonists of the activity of a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of methionine in an organism or a part thereof comprising:
[0809] (a) contacting cells, tissues, plants or microorganisms
which express the a polypeptide encoded by the nucleic acid
molecule of claim 5 conferring an increase in the amount of
methionine in an organism or a part thereof with a candidate
compound or a sample comprising a plurality of compounds under
conditions which permit the expression the polypeptide; [0810] (b)
assaying the methionine level or the polypeptide expression level
in the cell, tissue, plant or microorganism or the media the cell,
tissue, plant or microorganisms is cultured or maintained in; and
[0811] (c) identifying a agonist or antagonist by comparing the
measured methionine level or polypeptide expression level with a
standard methionine or polypeptide expression level measured in the
absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. [0812] 18. A process for
the identification of a compound conferring increased methionine
production in a plant or microorganism, comprising the steps:
[0813] (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of methionine in an organism or a part thereof and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
dais readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
methionine in an organism or a part thereof; [0814] (b) identifying
if the compound is an effective agonist by detecting the presence
or absence or increase of a signal produced by said readout system.
[0815] 19. A method for the identification of a gene product
conferring an increase in methionine production in a cell,
comprising the following steps: [0816] (a) contacting the nucleic
acid molecules of a sample, which can contain a candidate gene
encoding a gene product conferring an increase in methionine after
expression with the nucleic acid molecule of claim 6; [0817] (b)
identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; [0818] (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing methionine;
[0819] (d) expressing the identified nucleic acid molecules in the
host cells; [0820] (e) assaying the methionine level in the host
cells; and [0821] (f) identifying nucleic acid molecule and its
gene product which expression confers an increase in the methionine
level in the host cell in the host cell after expression compared
to the wild type. [0822] 20. A method for the identification of a
gene product conferring an increase in methionine production in a
cell, comprising the following steps: [0823] (a) identifying in a
data bank nucleic acid molecules of an organism; which can contain
a candidate gene encoding a gene product conferring an increase in
the methionine amount or level in an organism or a part thereof
after expression, and which are at least 20% homolog to the nucleic
acid molecule of claim 6; [0824] (b) introducing the candidate
nucleic acid molecules in host cells appropriate for producing
methionine; [0825] (c) expressing the identified nucleic acid
molecules in the host cells; [0826] (d) assaying the methionine
level in the host cells; and [0827] (e) identifying nucleic acid
molecule and its gene product which expression confers an increase
in the methionine level in the host cell after expression compared
to the wild type. [0828] 21. A method for the production of an
agricultural composition comprising the steps of the method of any
one of claims 17 to 20 and formulating the compound identified in
any one of claims 17 to 20 in a form acceptable for an application
in agriculture. [0829] 22. A composition comprising the nucleic
acid molecule of claim 6, the polypeptide of claim 14, the nucleic
acid construct of claim 7, the vector of any one of claim 8 or 9,
an antagonist or agonist identified according to claim 17, the
compound of claim 18, the gene product of claim 19 or 20, the
antibody of claim 15, and optionally an agricultural acceptable
carrier. [0830] 23. Use of the nucleic acid molecule as claimed in
claim 6 for the identification of a nucleic acid molecule
conferring an increase of methionine after expression. [0831] 24.
Use of the polypeptide of claim 14 or the nucleic acid construct
claim 7 or the gene product identified according to the method of
claim 19 or 20 for identifying compounds capable of conferring a
modulation of methionine levels in an organism.
[0832] 25. Food or feed composition comprising the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the harvested material of
claim 16, the host cell of claim 10 to 12 or the gene product
identified according to the method of claim 19 or 20.
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[0833] [0000.0.0.1] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and corresponding embodiments as described herein
as follows.
[0834] [0001.0.0.1] to [0007.0.0.1]: see [0001.0.0.0] to
[0007.0.0.0]
[0835] [0007.1.0.1] Following the approach of deregulating specific
enzymes in the amino acid biosynthetic pathway an increase of the
levels of free threonine is disclosed in U.S. Pat. No. 5,942,660
which is achieved by overexpression of either a wild-type or
deregulated aspartate kinase, homoserine dehydrogenase or threonine
synthase.
[0836] [0008.0.0.1] see [0008.0.0.0]
[0837] [0009.0.1.1] As described above, the essential amino acids
are necessary for humans and many mammals, for example for
livestock. Threonine is an important constituent in many body
proteins and is necessary for the formation of tooth enamel
protein, collagen and elastin, which both needed for healthy skin
and wound healing. It is a precursor to the amino acids glycine and
serine. It acts as a lipotropic in controlling fat build-up in the
liver. Threonine is an immune stimulant because it promotes thymus
growth and activity. It is a component of digestive enzymes and
immune secretions from the gut, particularly mucins. It has been
used as a supplement to help alleviate anxiety and some cases of
depression. In animal production, as an important essential amino
acid, threonine is normally the second limiting amino acid for pigs
and the third limiting amino acid for chicken (Gallus gallus f.
domestica, e.g. laying hen or broiler).
[0838] [0010.0.0.1] see [0010.0.0.0]
[0839] [0011.0.0.1] see [0011.0.0.0]
[0840] [0012.0.1.1] It is an object of the present invention to
develop an inexpensive process for the synthesis of threonine,
preferably L-threonine. Threonine is together with lysine and
methionine (depending on the organism) one of the amino acids which
are most frequently limiting.
[0841] [0013.0.0.1] see [0013.0.0.0]
[0842] [0014.0.1.1] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is threonine, preferably
L-threonine. Accordingly, in the present invention, the term "the
fine chemical" as used herein relates to "threonine". Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising threonine.
[0843] [0015.0.1.1] In one embodiment, the term "the fine chemical"
means threonine, preferably L-threonine. Throughout the
specification the term "the fine chemical" means threonine,
preferably L-threonine, its salts, ester or amids in free form or
bound to proteins. In a preferred embodiment, the term "the fine
chemical" means threonine, preferably L-threonine, in free form or
its salts or bound to proteins.
[0844] [0016.0.1.1] Accordingly, the present invention relates to a
process comprising [0845] (a) increasing or generating the activity
of one or more
[0846] YFL050C, YKR057W, YIL150C, YNL046W, YNL120C, b0186, b0730,
b1829, b2170, b0019, b0464, b1360, b1738, b1830, b1896, b2270,
b2414, b2552, b2664, b3074, b3160, b3231, b3462, b3791, b3966,
b4004, YOR245C--protein(s) in a non-human organism in one or more
parts thereof and [0847] (b) growing the organism under conditions
which permit the production of the fine chemical, thus, threonine
or fine chemicals comprising threonine, in said organism.
[0848] Accordingly, the present invention relates to a process for
the production of a fine chemical comprising [0849] (a) increasing
or generating the activity of one or more proteins having the
activity of a protein indicated in Table IIA or IIB, column 3,
lines 6 to 15, 339 to 355 or having the sequence of a polypeptide
encoded by a nucleic acid molecule indicated in Table IA or IB,
column 5 or 7, lines 6 to 15, 339 to 355, in a non-human organism
in one or more parts thereof and [0850] (b) growing the organism
under conditions which permit the production of the fine chemical,
in particular threonine.
[0851] [0017.0.0.1] see [0017.0.0.0]
[0852] [0018.0.0.1] see [0018.0.0.0]
[0853] [0019.0.1.1] Advantageously the process for the production
of the fine chemical leads to an enhanced production of the fine
chemical. The terms "enhanced" or "increase" mean at least a 10%,
20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or
100%, more preferably 150%, 200%, 300%, 400% or 500% higher
production of the fine chemical in comparison to the reference as
defined below, e.g. that means in comparison to an organism without
the aforementioned modification of the activity of a protein having
the activity of a protein indicated in Table IIA or IIB, column 3,
lines 6 to 15, 339 to 355 or encoded by nucleic acid molecule
indicated in Table IIA or IIB, columns 5 or 7, lines 6 to 15, 339
to 355.
[0854] [0020.0.1.1] Surprisingly it was found, that the transgenic
expression of at least one of the Saccaromyces cerevisiae
protein(s) indicated in Table IIA or IIB, Column 3, lines 6 to 10
and line 355 and/or at least one of the Escherichia coli K12
proteins indicated in Table IIA or IIB, Column 3, line 11-15, 339
to 354 in Arabidopsis thaliana conferred an increase in the
threonine (or fine chemical) content of the transformed plants.
[0855] [0021.0.0.1] see [0021.0.0.0]
[0856] [0022.0.1.1] The sequence of YFL050C from Saccharomyces
cerevisiae has been published in Murakami et al., Nat. Genet. 10
(3), 261-268, 1995 and Goffeau et al., Science 274 (5287), 546-547,
1996, and its activity is defined as a di- trivalent inorganic
cation transporter. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product defined
as di- trivalent inorganic cation transporter from Saccaromyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the fine chemical, meaning threonine, in particular for
increasing the amount of threonine, preferably L-threonine in free
or bound form in an organism or a part thereof, as mentioned.
[0857] The sequence of YKR057W from Saccharomyces cerevisiae has
been published in Dujon et al., Nature 369 (6479), 371-378, 1994
and Goffeau et al., Science 274 (5287), 546-547, 1996 and its
activity is being defined as an ribosomal protein, similar to S21
ribosomal proteins, involved in ribosome biogenesis and
translation. Accordingly, in one embodiment, the process of the
present invention comprises the use of a ribosomal protein, similar
to S21 ribosomal proteins, involved in ribosome biogenesis and
translation from Saccaromyces cerevisiae or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
threonine, in particular for increasing the amount of threonine,
preferably L-threonine in free or bound form in an organism or a
part thereof, as mentioned.
[0858] The sequence of YIL150C from Saccharomyces cerevisiae has
been published in Goffeau et al., Science 274 (5287), 546-547, 1996
and Churcher et al., Nature 387 (6632 Suppl), 84-87, 1997 and its
activity is being defined as a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a chromatin binding protein, required for S-phase (DNA
synthesis) initiation or completion, from Saccaromyces cerevisiae
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of threonine, in particular for increasing
the amount of threonine, preferably L-threonine in free or bound
form in an organism or a part thereof, as mentioned.
[0859] The sequence of YNL046W from Saccharomyces cerevisiae has
been published in Goffeau et al., Science 274 (5287), 546-547, 1996
and Philippsen et al., Nature 387 (6632 Suppl), 93-98, 1997 and its
activity is being defined as a probable membrane protein of the
endoplasmatic reticulum. Accordingly, in one embodiment, the
process of the present invention comprises the use of a YNL046W, as
a probable membrane protein of the endoplasmatic reticulum, from
Saccaromyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of threonine, in
particular for increasing the amount of threonine, preferably
threonine in free or bound form in an organism or a part thereof,
as mentioned.
[0860] The sequence of YNL120C from Saccharomyces cerevisiae has
been published in de Antoni et al, Yeast 13:261-266, 1997, and its
cellular activity has not been characterized yet. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a YNL120C activity from Saccaromyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of threonine, in particular for increasing the
amount of threonine, preferably threonine in free or bound form in
an organism or a part thereof, as mentioned.
[0861] The sequence of b0186 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a lysine decarboxylase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a lysine decarboxylase from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of threonine, in particular for increasing
the amount of threonine, preferably threonine in free or bound form
in an organism or a part thereof, as mentioned. In one embodiment,
in the process of the present invention the activity of a lysine
decarboxylase is increased or generated, e.g. from E. coli or a
homolog thereof.
[0862] The sequence of b0730 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as transcriptional regulator of
succinylCoA synthetase operon and fatty acyl response regulator.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a transcriptional regulator of
succinylCoA synthetase operon or a fatty acid response regulator
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of threonine, in
particular for increasing the amount of threonine, preferably
L-threonine in free or bound form in an organism or a part thereof,
as mentioned.
[0863] The sequence of b1829 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a heat shock protein.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a "heat shock protein" from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of threonine, in particular for increasing
the amount of threonine in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a htpX heat shock protein is
increased or generated, e.g. from E. coli or a homolog thereof. The
htpX heat shock protein is also annotated as having a protease
activity. Accordingly, in one embodiment, in the process of the
present invention the activity of a protease, preferably of a heat
shock protease, more preferred of a htpX protease or its homolog is
increased for the production of the fine chemical, meaning of
threonine, in particular for increasing the amount of threonine in
free or bound form in an organism or a part thereof, as
mentioned.
[0864] The sequence of b2170 from Escherichia coli K12 has been
published in Blattner et al, Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a sugar efflux transporter.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a sugar efflux transporter B from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of threonine, in particular for
increasing the amount of threonine, preferably L-threonine in free
or bound form in an organism or a part thereof, as mentioned.
[0865] The sequence of b0019 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as protein for the transport; the
transport of small molecules, preferably cations. In a more
preferred embodiment the protein has the activity of a Na+/H+
antiporter, responsive to stress, especially to high salinity and
pH. Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein for the transport;
preferably a stress responsive Na+/H+ antiporter from E. coli or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of threonine, in particular for increasing the
amount of threonine, preferably L-threonine in free or bound form
in an organism or a part thereof, as mentioned.
[0866] The sequence of b0464 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a transcriptional repressor
for multidrug efflux pump (TetR/AcrR family). Accordingly, in one
embodiment, the process of the present invention comprises the use
of a transcriptional repressor for multidrug efflux pump (TetR/AcrR
family) from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of threonine, in
particular for increasing the amount of threonine, preferably
threonine in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a transcriptional repressor for multidrug
efflux pump (TetR/AcrR family) is increased or generated, e.g. from
E. coli or a homolog thereof.
[0867] The sequence of b1360 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative DNA replication
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of a putative DNA replication protein
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of threonine, in
particular for increasing the amount of threonine, preferably
threonine in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a putative DNA replication protein is
increased or generated, e.g. from E. coli or a homolog thereof.
[0868] The sequence of b1738 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a PEP-dependent
phosphotransferase. Accordingly, in one embodiment, the process of
the present invention comprises the use of a PEP-dependent
phosphotransferase from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
threonine, in particular for increasing the amount of threonine,
preferably threonine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a PEP-dependent
phosphotransferase is increased or generated, e.g. from E. coli or
a homolog thereof.
[0869] The sequence of b1830 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a carboxy-terminal protease
for penicillin-binding protein 4. Accordingly, in one embodiment,
the process of the present invention comprises the use of a
carboxy-terminal protease for penicillin-binding protein 4 from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of threonine, in particular for
increasing the amount of threonine, preferably threonine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a carboxy-terminal protease for penicillin-binding protein 4 is
increased or generated, e.g. from E. coli or a homolog thereof.
[0870] The sequence of b1896 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a trehalose-6-phosphate
synthase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a trehalose-6-phosphate
synthase from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of threonine, in
particular for increasing the amount of threonine, preferably
threonine in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a trehalose-6-phosphate synthase is
increased or generated, e.g. from E. coli or a homolog thereof.
[0871] The sequence of b2414 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a subunit of cysteine synthase
A and O-acetylserine sulfhydrolase A, PLP-dependent enzyme.
[0872] Accordingly, in one embodiment, the process of the present
invention comprises the use of a subunit of cysteine synthase A and
O-acetylserine sulfhydrolase A, PLP-dependent enzyme from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of threonine, in particular for increasing
the amount of threonine, preferably threonine in free or bound form
in an organism or a part thereof, as mentioned. In one embodiment,
in the process of the present invention the activity of a subunit
of cysteine synthase A and O-acetylserine sulfhydrolase A,
PLP-dependent enzyme is increased or generated, e.g. from E. coli
or a homolog thereof.
[0873] The sequence of b2552 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a dihydropteridine reductase
(nitric oxide dioxygenase). Accordingly, in one embodiment, the
process of the present invention comprises the use of a
dihydropteridine reductase (nitric oxide dioxygenase) from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of threonine, in particular for increasing
the amount of threonine, preferably threonine in free or bound form
in an organism or a part thereof, as mentioned. In one embodiment,
in the process of the present invention the activity of a
dihydropteridine reductase (nitric oxide dioxygenase) is increased
or generated, e.g. from E. coli or a homolog thereof.
[0874] The sequence of b4004 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a transcriptional regulatory
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of a transcriptional regulatory protein
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of threonine, in
particular for increasing the amount of threonine, preferably
threonine in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a transcriptional regulatory protein is
increased or generated, e.g. from E. coli or a homolog thereof.
[0875] The sequence of b2664 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative transcriptional
repressor with DNA-binding Winged helix domain (GntR familiy).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a putative transcriptional repressor
with DNA-binding Winged helix domain
[0876] (GntR familiy) from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
threonine, in particular for increasing the amount of threonine,
preferably threonine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a putative transcriptional
repressor with DNA-binding Winged helix domain (GntR familiy) is
increased or generated, e.g. from E. coli or a homolog thereof.
[0877] The sequence of b3074 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative tRNA synthetase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a putative tRNA synthetase from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of threonine, in particular for
increasing the amount of threonine, preferably threonine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a putative tRNA synthetase is increased or generated, e.g. from E.
coli or a homolog thereof.
[0878] The sequence of b2270 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has not been characterized yet. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a protein b2270 from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
threonine, in particular for increasing the amount of threonine,
preferably threonine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of the protein encoded by b2270 is
increased or generated, e.g. from E. coli or a homolog thereof.
[0879] The sequence of b3160 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative monooxygenase with
luciferase-like ATPase activity. Accordingly, in one embodiment,
the process of the present invention comprises the use of a
putative monooxygenase with luciferase-like ATPase activity from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of threonine, in particular for
increasing the amount of threonine, preferably threonine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a putative monooxygenase with luciferase-like ATPase activity is
increased or generated, e.g. from E. coli or a homolog thereof.
[0880] The sequence of b3231 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a 50S ribosomal subunit
protein L13. Accordingly, in one embodiment, the process of the
present invention comprises the use of a 50S ribosomal subunit
protein L13 from E. coli or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of threonine, in
particular for increasing the amount of threonine, preferably
threonine in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a 50S ribosomal subunit protein L13 is
increased or generated, e.g. from E. coli or a homolog thereof.
[0881] The sequence of b3462 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as an integral membrane cell
division protein. Accordingly, in one embodiment, the process of
the present invention comprises the use of a integral membrane cell
division protein from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of threonine, in
particular for increasing the amount of threonine, preferably
threonine in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a integral membrane cell division protein
is increased or generated, e.g. from E. coli or a homolog
thereof.
[0882] The sequence of b3791 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a transaminase involved in
lipopolysaccharide biosynthesis. Accordingly, in one embodiment,
the process of the present invention comprises the use of a
transaminase involved in lipopolysaccharide biosynthesis from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of threonine, in particular for
increasing the amount of threonine, preferably threonine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a transaminase involved in lipopolysaccharide biosynthesis is
increased or generated, e.g. from E. coli or a homolog thereof.
[0883] The sequence of b3966 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as an outer membrane porin.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a outer membrane porin from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of threonine, in particular for increasing
the amount of threonine, preferably threonine in free or bound form
in an organism or a part thereof, as mentioned. In one embodiment,
in the process of the present invention the activity of a outer
membrane porin is increased or generated, e.g. from E. coli or a
homolog thereof.
[0884] The sequence of YOR245C from Saccharomyces cerevisiae has
been published in Dujon, B. et al., Nature 387 (6632 Suppl), 98-102
(1997) and its activity is defined as a acyl-CoA:diacylglycerol
acyltransferase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product defined as a
acyl-CoA:diacylglycerol acyltransferase from Saccaromyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the fine chemical, meaning threonine, in particular for
increasing the amount of threonine, preferably L-threonine in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a acyl-CoA:diacylglycerol acyltransferase is increased
or generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[0885] [0023.0.1.1] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the fine chemical amount or content. Further, in the
present invention, the term "homologue" relates to the sequence of
an organism having the highest sequence homology to the herein
mentioned or listed sequences of all expressed sequences of said
organism. However, the person skilled in the art knows, that,
preferably, the homologue has said the--fine-chemical-increasing
activity and, if known, the same biological function or activity in
the organism as at least one of the protein(s) indicated in Table
IIA or IIB, Column 3, lines 6 to 15, 339 to 355, e.g. having the
sequence of a polypeptide encoded by a nucleic acid molecule
comprising the sequence indicated in in Table IA or IB, Column 5 or
7, lines 6 to 15, 339 to 355. In one embodiment, the homolog of any
one of the polypeptides indicated in Table IIA or IIB, lines 6 to
10, 339 to 355 is a homolog having the same or a similar activity,
in particular an increase of activity confers an increase in the
content of the fine chemical in the organsims and being derived
from an eukaryot. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, lines 11 to 15, 339 to 355
is a homolog having the same or a similar activity, in particular
an increase of activity confers an increase in the content of the
fine chemical in the organisms or part thereof, and being derived
from bacteria. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, lines 6 to 10, 339 to 355
is a homolog having the same or a similar activity, in particular
an increase of activity confers an increase in the content of the
fine chemical in an organisms or part thereof, and being derived
from Fungi. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, lines 11 to 15, 339 to 355
is a homolog having the same or a similar activity, in particular
an increase of activity confers an increase in the content of the
fine chemical in the organisms or part thereof and being derived
from
[0886] Proteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, lines 6 to 10,
339 to 355 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof and
being derived from Ascomyceta. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, lines 11 to
15, 339 to 355 is a homolog having the same or a similar activity,
in particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or part thereof, and
being derived from Gammaproteobacteria. In one embodiment, the
homolog of a polypeptide polypeptide indicated in Table IIA or IIB,
column 3, lines 6 to 10, 339 to 355 is a homolog having the same or
a similar activity, in particular an increase of activity confers
an increase in the content of the fine chemical in the organisms or
part thereof, and being derived from Saccharomycotina. In one
embodiment, the homolog of a polypeptide indicated in Table IIA or
IIB, column 3, lines 11 to 15, 339 to 355 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, lines 6 to 15, 339 to 355
is a homolog having the same or a similar activity, in particular
an increase of activity confers an increase in the content of the
fine chemical in the organisms or a part thereof, and being derived
from Saccharomycetes. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, lines 11 to
15, 339 to 354 is a homolog having the same or a similar activity,
in particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or part thereof, and
being derived from Enterobacteriaceae. In one embodiment, the
homolog of a polypeptide indicated in Table IIA or IIB, column 3,
lines 6 to 10, 355 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms, and being
derived from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, lines 11 to
15, 339 to 354 is a homolog having the same or a similar activity,
in particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof,
and being derived from Escherichia. In one embodiment, the homolog
of a polypeptide indicated in Table IIA or IIB, column 3, lines 6
to 10, 355 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof,
and being derived from Saccharomycetaceae. In one embodiment, the
homolog of a polypeptide indicated in Table IIA or IIB, column 3,
line 6 to 10, 355 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or a part
thereof, and being derived from Saccharomycetes.
[0887] [0023.1.0.1] Homologs of the polypeptides polypeptide
indicated in Table IIA or IIB, column 3, lines 6 to 15, 339 to 355
may be the polypetides encoded by the nucleic acid molecules
polypeptide indicated in Table IA or IB, column 7, lines 6 to 10,
339 to 355 or may be the polypeptides indicated in Table IIA or
IIB, column 7, lines 6 to 10, 339 to355. Homologs of the
polypeptides polypeptide indicated in Table IIA or IIB, column 3,
lines 6 to 15, 339 to 355 may be the polypetides encoded by the
nucleic acid molecules polypeptide indicated in Table IA or IB,
column 7, lines 6 to 10, 339 to 355 or may be the polypeptides
indicated in Table IIA or IIB, column 7, lines 11-15, 339 to
355.
[0888] [0024.0.0.1]: see [0024.0.0.0]
[0889] [0025.0.1.1] In accordance with the invention, a protein or
polypeptide has the "activity of an protein of the invention", e.g.
the activity of a protein indicated in Table IIA or IIB, column 3,
lines 6 to 15, 339 to 355 if its de novo activity, or its increased
expression directly or indirectly leads to an increased threonine
level in the organism or a part thereof, preferably in a cell of
said organism Throughout the specification the activity or
preferably the biological activity of such a protein or polypeptide
or an nucleic acid molecule or sequence encoding such protein or
polypeptide is identical or similar if it still has the biological
or enzymatic activity of any one of the proteins indicated in Table
IIA or IIB, column 3, lines 6 to 15, 339 to 355, i.e. or which has
at least 10% of the original enzymatic activity, preferably 20%,
particularly preferably 30%, most particularly preferably 40% in
comparison to an any one of the proteins indicated in Table IIA or
IIB, column 3, lines 6 to 10, 339 to 355 and/or any one of the
proteins indicated in Table IIA or IIB, column 3, lines 11 to 15,
339 to 354.
[0890] [0025.1.0.1] In one embodiment, the polypeptide of the
invention confers said activity, e.g. the increase of the fine
chemical in an organism or a part thereof, if it is derived from an
organism, which is evolutionary distant to the organism in which it
is expressed. For example origin and expressing organism are
derived from different families, orders, classes or phylums.
[0891] [0026.0.0.1] to [0033.0.0.1]: see [0026.0.0.0] to
[0033.0.0.0]
[0892] [0034.0.1.1] Preferably, the reference, control or wild type
differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention, e.g., it differs by or in the
expression level or activity of an protein having the activity of
protein as indicated in Table IIA or IIB, column 3, lines 6 to 15,
339 to 355 or being encoded by a nucleic acid molecule indicated in
Table IA or IB, column 5, lines 6 to 15, 339 to 355 or its
homologs, e.g. as indicated in Table IA or IB, column 7, lines 6 to
15, 339 to 355, its biochemical or genetical causes and therefore
shows the increased amount of the fine chemical.
[0893] [0035.0.0.1] to [0044.0.0.1]: see [0035.0.0.0] to
[0044.0.0.0]
[0894] [0045.0.1.1] In one embodiment, in case the activity of the
Saccharomyces cerevisiae protein YFL050C or di- trivalent inorganic
cation transporter or its homologs, e.g. as indicated in Table IA
or IB, columns 5 or 7, line 6, is increased, preferably, an
increase of the fine chemical threonine between 19% and 56% is
conferred.
[0895] In case the activity of the Saccharomyces cerevisiae protein
YKR057W or a ribosomal protein, similar to S21 ribosomal proteins,
involved in ribosome biogenesis and translation or its homolog e.g.
as indicated in Table IA or IB, columns 5 or 7, line 7, is
increased, preferably, in one embodiment the increase of the fine
chemical threonine between 34% and 142% is conferred.
[0896] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YIL150C or a "protein required for S-phase (DNA
synthesis) initiation or completion" or a chromatin binding
protein, required for S-phase (DNA synthesis) initiation or
completion or its homologs, e.g. a cell division cycle protein e.g.
as indicated in Table IA or IB, columns 5 or 7, line 8, is
increased, preferably, in one embodiment the increase of the fine
chemical threonine between 25% and 319% is conferred.
[0897] In case the activity of the Saccharomyces cerevisiae protein
YNL046W or its homologs, e.g. a probable membrane protein of the
endoplasmatic reticulum e.g. as indicated in Table IA or IB,
columns 5 or 7, line 9 is increased, preferably, in one embodiment
an increase of the fine chemical threonine between 18% and 53% is
conferred.
[0898] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YNL120C or its homologs, e.g. as indicated in
Table IA or IB, Columns 5 or 7, line 10, is increased, preferably,
the increase of the fine chemical threonine of 44% is
conferred.
[0899] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0186 or a lysine decarboxylases or its homologs,
e.g. as indicated in Table IA or IB, columns 5 or 7, line 11, is
increased, preferably, the increase of the fine chemical threonine
between 49% and 228% is conferred.
[0900] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0730 or a protein with the activity defined as
transcriptional regulator of succinylCoA synthetase operon or its
homologs, e.g. as indicated in Table IA or IB, columns 5 or 7, line
12, is increased, preferably, in one embodiment an increase of the
fine chemical threonine between 53% and 177% is conferred.
[0901] In case the activity of the Escherichia coli K12 protein
b1829 or its homologs is increased, e.g. the activity of a protease
is increased, preferably, the activity of a heat shock protein is
increased, more preferred the activity of a htpX protein or its
homolog e.g. as indicated in Table IA or IB, columns 5 or 7, line
13, is increased preferably, in one embodiment the increase of the
fine chemical threonine between 17% and 114% is conferred.
[0902] In case the activity of the Escherichia coli K12 protein
b2170 or a sugar efflux transporter or its homologs e.g. as
indicated in Table IA or IB, columns 5 or 7, line 14, is increased,
preferably, in one embodiment the increase of the fine chemical
threonine between 35% and 79% is conferred.
[0903] In case the activity of the Escherichia coli K12 protein
b0019 or a protein for the transport of cations or its homologs,
e.g. a Na.sup.+/H.sup.+ antiporter, e.g. as indicated in Table IA
or IB, columns 5 or 7, line 15, is increased, preferably, in one
embodiment the increase of the fine chemical threonine between 24%
and 44% is conferred.
[0904] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0464 or a protein with the activity defined as
transcriptional repressor for multidrug efflux pump (TetR/AcrR
family) or its homologs, e.g. as indicated in Table IA or IB,
columns 5 or 7, line 339, is increased, preferably, in one
embodiment an increase of the fine chemical threonine between 23%
and 43% is conferred.
[0905] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1360 or a protein with the activity defined as
putative DNA replication protein or its homologs, e.g. as indicated
in Table IA or IB, columns 5 or 7, line 340, is increased,
preferably, in one embodiment an increase of the fine chemical
threonine between 16% and 38% is conferred.
[0906] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1738 or a protein with the activity defined as
PEP-dependent phosphotransferase or its homologs, e.g. as indicated
in Table IA or IB, columns 5 or 7, line 341, is increased,
preferably, in one embodiment an increase of the fine chemical
threonine between 27% and 361% is conferred.
[0907] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1830 or a protein with the activity defined as
carboxy-terminal protease for penicillin-binding protein 4 or its
homologs, e.g. as indicated in Table IA or IB, columns 5 or 7, line
342, is increased, preferably, in one embodiment an increase of the
fine chemical threonine between 24% and 43% is conferred.
[0908] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or a protein with the activity defined as
trehalose-6-phosphate synthase or its homologs, e.g. e.g. as
indicated in Table IA or IB, columns 5 or 7, line 343, is
increased, preferably, in one embodiment an increase of the fine
chemical threonine between 46% and 108% is conferred.
[0909] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2414 or a protein with the activity defined as
subunit of cysteine synthase A and O-acetylserine sulfhydrolase A,
PLP-dependent enzyme or its homologs, e.g. as indicated in Table IA
or IB, columns 5 or 7, line 345, is increased, preferably, in one
embodiment an increase of the fine chemical threonine between24%
and 46% is conferred.
[0910] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2552 or a protein with the activity defined as
dihydropteridine reductase (nitric oxide dioxygenase) or its
homologs, e.g. as indicated in Table IA or IB, columns 5 or 7, line
346, is increased, preferably, in one embodiment an increase of the
fine chemical threonine between 17% and 37% is conferred.
[0911] In one embodiment, in case the activity of the Escherichia
coli K12 protein b4004 or a protein with the activity defined as
transcriptional regulatory protein or its homologs, e.g. as
indicated in Table IA or IB, columns 5 or 7, line 354, is
increased, preferably, in one embodiment an increase of the fine
chemical threonine between 17% and 37% is conferred.
[0912] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2664 or a protein with the activity defined as
putative transcriptional repressor with DNA-binding Winged helix
domain (GntR familiy) or its homologs, e.g. as indicated in Table
IA or IB, columns 5 or 7, line 347, is increased, preferably, in
one embodiment an increase of the fine chemical threonine
between29% and 284% is conferred.
[0913] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3074 or a protein with the activity defined as
putative tRNA synthetase or its homologs, e.g. as indicated in
Table IA or IB, columns 5 or 7, line 348, is increased, preferably,
in one embodiment an increase of the fine chemical threonine
between 31% and 59% is conferred.
[0914] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2270 or its homologs, e.g. as indicated in Table
IA or IB, columns 5 or 7, line 344, is increased, preferably, in
one embodiment an increase of the fine chemical threonine between
31% and 59% is conferred.
[0915] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3160 or a protein with the activity defined as
putative monooxygenase with luciferase-like ATPase activity or its
homologs, e.g. as indicated in Table IA or IB, columns 5 or 7, line
349, is increased, preferably, in one embodiment an increase of the
fine chemical threonine between 25% and 56% is conferred.
[0916] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3231 or a protein with the activity defined as
50S ribosomal subunit protein L13 or its homologs, e.g. as
indicated in Table IA or IB, columns 5 or 7, line 350, is
increased, preferably, in one embodiment an increase of the fine
chemical threonine between 17% and 32% is conferred.
[0917] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3462 or a protein with the activity defined as
integral membrane cell division protein or its homologs, e.g. as
indicated in Table IA or IB, columns 5 or 7, line 351, is
increased, preferably, in one embodiment an increase of the fine
chemical threonine between 18% and 51% is conferred.
[0918] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3791 or a protein with the activity defined as
transaminase involved in lipopolysaccharide biosynthesis or its
homologs, e.g. as indicated in Table IA or IB, columns 5 or 7, line
352, is increased, preferably, in one embodiment an increase of the
fine chemical threonine between 38% and 44% is conferred.
[0919] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3966 or a protein with the activity defined as
outer membrane porin or its homologs, e.g. as indicated in Table IA
or IB, columns 5 or 7, line 353, is increased, preferably, in one
embodiment an increase of the fine chemical threonine between 19%
and 47% is conferred.
[0920] In case the activity of the Saccharomyces cerevisiae protein
YOR245C or a protein with the activity defined as
acyl-CoA:diacylglycerol acyltransferase or its homologs, e.g. as
indicated in Table IA or IB, columns 5 or 7, line 355, is
increased, preferably, in one embodiment an increase of the fine
chemical threonine between 18% and 81% is conferred.
[0921] [0046.0.1.1] In one embodiment, in case the activity of the
Saccaromyces cerevisiae protein YFL050C or its homologs, e.g. di-
trivalent inorganic cation transporter, is increased, preferably,
an increase of the fine chemical threonine and of alanine is
conferred.
[0922] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YKR057W or its homologs, e.g. an ribosomal
protein, similar to S21 ribosomal proteins, involved in ribosome
biogenesis and translation is increased, preferably, an increase of
the fine chemical threonine and of arginine, is conferred.
[0923] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YIL150C or its homologs, e.g. "a chromatin
binding protein, required for S-phase (DNA synthesis) initiation or
completion" or its homologs, is increased, preferably, an increase
of the fine chemical threonine and of fumaric acid is
conferred.
[0924] In case the activity of the Escherichia coli K12 protein
b0186 or its homologs, e.g. a lysine decarboxylases or its
homologs, is increased preferably, an increase of the fine chemical
threonine and of methionine is conferred.
[0925] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0730 or its homologs, e.g. a protein with the
activity defined as transcriptional regulator of succinylCoA
synthetase operon and fatty acyl response regulator or its homologs
is increased preferably an increase of the fine chemical threonine
and of beta-carotene is conferred.
[0926] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1829 or its homologs is increased, e.g. the
activity of a protease is increased, preferably, the activity of a
heat shock protein is increased, more preferred the activity of a
htpX protein or its homolog is increased preferably in an increase
of the fine chemical threonine and of C18:0 is conferred.
[0927] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2170 or its homologs is increased, e.g. the
activity of a sugar efflux transporter B is increased, preferably
an increase of the fine chemical threonine and of isopentenyl
pyrophosphate is conferred.
[0928] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0019 or its homologs, e.g. a protein for the
transport of cations or its homologs, e.g. a Na+/H+ antiporter, is
increased, preferably an increase of the fine chemical threonine
and of .beta.-sitosterol is conferred.
[0929] [0047.0.0.1] see [0047.0.0.0]
[0930] [0048.0.0.1] see [0048.0.0.0]
[0931] [0049.0.1.1] A protein having an activity conferring an
increase in the amount or level of the fine chemical preferably has
the structure of the polypeptide described herein, in particular of
a polypeptides comprising a consensus sequence as indicated in
Table IV, column 7, line 6 to 15,339 to 355 or of a polypeptide as
indicated in Table IIA or IIB, columns 5 or 7, line 6 to 15,339 to
355 or the functional homologs thereof as described herein, or is
encoded by the nucleic acid molecule characterized herein or the
nucleic acid molecule according to the invention, for example by a
nucleic acid molecule as indicated in Table IA or IB, columns 5 or
7, line 6 to 15,339 to 355 or its herein described functional
homologs and has the herein mentioned activity.
[0932] [0050.0.1.1] For the purposes of the present invention, the
term "threonine" and "L-threonine" also encompass the corresponding
salts, such as, for example, threonine hydrochloride or threonine
sulfate. Preferably the term threonine is intended to encompass the
term L-threonine.
[0933] [0051.0.0.1] see [0051.0.0.0]
[0934] [0052.0.0.1] see [0052.0.0.0]
[0935] [0053.0.1.1] In one embodiment, the process of the present
invention comprises one or more of the following steps [0936] (a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention, e.g. of a polypeptide having an
activity of a protein as indicated in Table IIA or IIB, column 3,
line 6 to 15,339 to 355 or its homologs activity, e.g. as indicated
in Table IIA or IIB, columns 5 or 7, line 6 to 15,339 to 355,
having herein-mentioned the fine chemical-increasing activity;
[0937] (b) stabilizing a mRNA conferring the increased expression
of a protein encoded by the nucleic acid molecule of the invention,
e.g. of a polypeptide having an activity of a protein as indicated
in Table IIA or IIB, column 3, line 6 to 15,339 to 355 or its
homologs activity, e.g. as indicated in Table IIA or IIB, columns 5
or 7, line 6 to 15,339 to 355 or of a mRNA encoding the polypeptide
of the present invention having herein-mentioned threonine
increasing activity; [0938] (c) increasing the specific activity of
a protein conferring the increasd expression of a protein encoded
by the nucleic acid molecule of the invention or of the polypeptide
of the present invention having herein-mentioned threonine
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table IIA or IIB, column 3, line 6 to 15,
339 to 355 or its homologs activity, e.g. as indicated in Table IIA
or IIB, columns 5 or 7, line 6 to 15, 339 to 355, or decreasing the
inhibiitory regulation of the polypeptide of the invention; [0939]
(d) generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned threonine increasing
activity, e.g. of a polypeptide having an activity of a protein as
indicated in Table IIA or IIB, column 3, line 6 to 15, 339 to 355
or its homologs activity, e.g. as indicated in Table IIA or IIB,
columns 5 or 7, line 6 to 15, 339 to 355; [0940] (e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned threonine increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
IIA or IIB, column 3, line 6 to 15, 339 to 355 or its homologs
activity, e.g. as indicated in Table IIA or IIB, columns 5 or 7,
line 6 to 15, 339 to 355 by adding one or more exogenous inducing
factors to the organisms or parts thereof; [0941] (f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned threonine increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
IIA or IIB, column 3, line 6 to 15, 339 to 355 or its homologs
activity, e.g. as indicated in Table IIA or IIB, columns 5 or 7,
line 6 to 15, 339 to 355; [0942] (g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned threonine increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
IIA or IIB, column 3, line 6 to 15, 339 to 355 or its homologs
activity, e.g. as indicated in Table IIA or IIB, columns 5 or 7,
line 6 to 15, 339 to 355; [0943] (h) Increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having an activity of a protein as indicated in Table
IIA or IIB, column 3, line 6 to 15, 339 to 355 or its homologs
activity, e.g. as indicated in Table IIA or IIB, columns 5 or 7,
line 6 to 15, 339 to 355 by adding positive expression or removing
negative expression elements; e.g. homologous recombination can be
used to either introduce positive regulatory elements like for
plants the 35S enhancer into the promoter or to remove repressor
elements form regulatory regions. Further gene conversion methods
can be used to disrupt repressor elements or to enhance to activity
of positive elements. Positive elements can be randomly introduced
in plants by T-DNA or transposon mutagenesis and lines can be
identified in which the positive elements have be integrated near
to a gene of the invention, the expression of which is thereby
enhanced; [0944] (i) Modulating growth conditions of an organism in
such a manner, that the expression or activity of the gene encoding
the protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown under a higher
temperature regime leading to an enhanced expression of heat shock
proteins, e.g. the heat shock protein of the invention, which can
lead an enhanced the fine chemical production; and/or [0945] (j)
selecting of organisms with expecially high activity of the
proteins of the invention from natural or from mutagenized
resources and breeding them into the target organisms, eg the elite
crops.
[0946] [0054.0.1.1] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of threonine after
increasing the expression or activity of the encoded polypeptide or
having the activity of a polypeptide having an activity of a
protein as indicated in Table II, column 5, line 6 to 15, 339 to
355 or its homologs activity, e.g. as indicated in Table IIA or
IIB, column 7, line 6 to 15, 339 to 355.
[0947] [0055.0.0.1] to [0064.0.0.1] see [0055.0.0.0] to
[0064.0.0.0]
[0948] [0065.0.1.1] The activation of an endogenous polypeptide
having above-mentioned activity, of the polypeptide of the
invention, e.g. conferring the increase of the fine chemical after
increase of expression or activity can also be increased by
introducing a synthetic transcription factor, which binds close to
the coding region of an endogenous polypeptide of the invention- or
its endogenous homolog-encoding gene and activates its
transcription. A chimeric zinc finger protein can be construed,
which comprises a specific DNA-binding domain and an activation
domain as e.g. the VP16 domain of Herpes Simplex virus. The
specific binding domain can bind to the regulatory region of the
endogenous protein-coding region. The expression of the chimeric
transcription factor in a organism, in particular in a plant, leads
to a specific expression of an endogenous polypeptid of the
invention, in particular a plant homolog thereof, see e.g. in
WO01/52620, Oriz, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, 13290
or Guan, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, 13296.
[0949] [0066.0.0.1] to [0069.0.0.1]: see [0066.0.0.0] to
[0069.0.0.0]
[0950] [0070.0.1.1] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, into an
organism alone or in combination with other genes, it is possible
not only to increase the biosynthetic flux towards the end product,
but also to increase, modify or create de novo an advantageous,
preferably novel metabolites composition in the organism, e.g. an
advantageous amino acid composition comprising a higher content of
(from a viewpoint of nutrional physiology limited) fine chemicals,
in particular amino acids, likewise the fine chemical.
[0951] [0071.0.0.1] see [0071.0.0.0]
[0952] [0072.0.1.1] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous hydroxy containing compounds. Examples of such
compounds are, in addition to threonine, serine, homoserine,
phosphohomoserine or hydroxyproline or methionine.
[0953] [0073.0.1.1] Accordingly, in one embodiment, the process
according to the invention relates to a process which comprises:
[0954] (a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [0955] (b) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table IIA or IIB, columns 5 or 7, line 6 to 15, 339 to
355 or of a polypeptide being encoded by the nucleic acid molecule
of the present invention and described below, e.g. conferring an
increase of the fine chemical in the organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [0956] (c) growing the organism,
preferably a microorganism, a non-human animal, a plant or animal
cell, a plant or animal tissue or a plant under conditions which
permit the production of the fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and [0957] (d) if desired, revovering, optionally
isolating, the free and/or bound the fine chemical and, optionally
further free and/or bound amino acids synthetized by the organism,
the microorganism, the non-human animal, the plant or animal cell,
the plant or animal tissue or the plant.
[0958] [0074.0.0.1] to [0084.0.0.1]: see [0074.0.0.0] to
[0084.0.0.0]
[0959] [0085.0.1.1] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [0960] (a) a nucleic acid sequence as
indicated in Table IA or IB, columns 5 or 7, lines 6 to 15, 339 to
355 a derivative thereof, or [0961] (b) a genetic regulatory
element, for example a promoter, which is functionally linked to
the nucleic acid sequence as indicated in Table IA or IB, columns 5
or 7, lines 6 to 15, 339 to 355 or a derivative thereof, or [0962]
(c) (a) and (b) is/are not present in its/their natural genetic
environment or has/have been modified by means of genetic
manipulation methods, it being possible for the modification to be,
by way of example, a substitution, addition, deletion, inversion or
insertion of one or more nucleotide radicals. "Natural genetic
environment" means the natural chromosomal locus in the organism of
origin or the presence in a genomic library. In the case of a
genomic library, the natural, genetic environment of the nucleic
acid sequence is preferably at least partially still preserved. The
environment flanks the nucleic acid sequence at least on one side
and has a sequence length of at least 50 bp, preferably at least
500 bp, particularly preferably at least 1000 bp, very particularly
preferably at least 5000 bp.
[0963] [0086.0.0.1]: see [0086.0.0.0]
[0964] [0087.0.0.1]: see [0087.0.0.0]
[0965] [0088.0.1.1] In an advantageous embodiment of the invention,
the organism takes the form of a plant whose amino acid content is
modified advantageously owing to the nucleic acid molecule of the
present invention expressed. This is important for plant breeders
since, for example, the nutritional value of plants for monogastric
animals is limited by a few essential amino acids such as lysine,
threonine or methionine.
[0966] [0088.1.1.1] In one embodiment, after an activity of a
polypeptide of the present invention has been increased or
generated, or after the expression of a nucleic acid molecule or
polypeptide according to the invention has been generated or
increased, the transgenic plant generated can be grown on or in a
nutrient medium or else in the soil and subsequently harvested.
[0967] [0089.0.0.1] to [0097.0.0.1]: see [0089.0.0.0] to
[0097.0.0.0]
[0968] [0098.0.1.1] In a further embodiment, the fine chemical
threonine is produced in accordance with the invention and, if
desired, is isolated. The production of further amino acids such as
methionine, lysine and/or mixtures of amino acid by the process
according to the invention is advantageous.
[0969] [0099.0.0.1] to [0102.0.0.1]: see [0099.0.0.0] to
[0102.0.0.0]
[0970] [0103.0.1.1] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical threonine comprising or generating in an organism or a
part thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [0971] (a) nucleic acid molecule encoding,
preferably at least the mature form, of a polypeptide having a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines 6
to 15, 339 to 355; [0972] (b) nucleic acid molecule comprising,
preferably at least the mature form, of a nucleic acid molecule
having a sequence as indicated in Table IA or IB, columns 5 or 7,
lines 6 to 15, 339 to 355; [0973] (c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
fine chemical threonine in an organism or a part thereof; [0974]
(d) nucleic acid molecule encoding a polypeptide which has at least
50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical threonine in an
organism or a part thereof; [0975] (e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical threonine in an organism or a part
thereof; [0976] (f) nucleic acid molecule encoding a polypeptide,
the polypeptide being derived by substituting, deleting and/or
adding one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c) and conferring an increase in the amount
of the fine chemical threonine in an organism or a part thereof;
[0977] (g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and and
conferring an increase in the amount of the fine chemical threonine
in an organism or a part thereof; [0978] (h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers pairs having a sequence as indicated in Table
III, columns 7, lines 6 to 15, 339 to 355, and conferring an
increase in the amount of the fine chemical threonine in an
organism or a part thereof; [0979] (i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from an expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and and conferring an increase in
the amount of the fine chemical threonine in an organism or a part
thereof; [0980] (j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence having a sequences as
indicated in Table IV, column 7, lines 6 to 15, 339 to 355 and
conferring an increase in the amount of the fine chemical threonine
in an organism or a part thereof; [0981] (k) nucleic acid molecule
comprising one or more of the nucleic acid molecule encoding the
amino acid sequence of a polypeptide encoding a domain of a
polypeptide indicated in Table IIA or IIB, columns 5 or 7, lines 6
to 15, 339 to 355 and conferring an increase in the amount of the
fine chemical threonine in an organism or a part thereof; and
[0982] (l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the fine
chemical threonine in an organism or a part thereof; [0983] or
which comprises a sequence which is complementary thereto.
[0984] [0104.0.1.1] In one embodiment, the nucleic acid molecule of
the invention distinguishes over the sequence indicated in Table IA
or IB, columns 5 or 7, lines 6 to 15, 339 to 355 by one or more
nucleotides. In one embodiment, the nucleic acid molecule of the
present invention does not consist of the sequence shown in
indicated in Table IA or IB, columns 5 or 7, lines 6 to 15, 339 to
355. In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table IA or IB,
columns 5 or 7, lines 6 to 15, 339 to 355.
[0985] [0105.0.0.1] to [0107.0.0.1]: see [0105.0.0.0] to
[0107.0.0.0]
[0986] [0108.0.1.1] Nucleic acid molecules with the sequence as
indicated in Table IA or IB, columns 5 or 7, lines 6 to 15, 339 to
355, nucleic acid molecules which are derived from an amino acid
sequences as indicated in Table IIA or IIB, columns 5 or 7, lines 6
to 15, 339 to 355 or from polypeptides comprising the consensus
sequence as indicated in Table IV, column 7, lines 6 to 15, 339 to
355 or their derivatives or homologues encoding polypeptides with
the enzymatic or biological activity of a polypeptide as indicated
in Table IIA or IIB, column 3, 5 or 7, lines 6 to 15, 339 to 355 or
e.g. conferring a increase of the fine chemical threonine after
increasing its expression or activity are advantageously increased
in the process according to the invention.
[0987] [0109.0.0.1] see [0109.0.0.0]
[0988] [0110.0.1.1] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide of the invention can
be determined from generally accessible databases.
[0989] [0111.0.0.1] see [0111.0.0.0]
[0990] [0112.0.1.1] The nucleic acid molecules used in the process
according to the invention take the form of isolated nucleic acid
sequences, which encode polypeptides with an activity of a
polypeptide as indicated in Table Ila or IIB, column 3, lines 6 to
15, 339 to 355 or having the sequence of a polypeptide as indicated
in Table IIA or IIB, columns 5 and 7, lines 6 to 15, 339 to 355 and
conferring an increase of the fine chemical threonine.
[0991] [0113.0.0.1] to [0120.0.0.1]: see [0113.0.0.0] to
[0120.0.0.0]
[0992] [0121.0.1.1] However, it is also possible to use artificial
sequences, which differ in one or more bases from the nucleic acid
sequences found in organisms, or in one or more amino acid
molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table IIA or
IIB, columns 5 or 7, lines 6 to 15, 339 to 355 or the functional
homologues thereof as described herein, preferably conferring
above-mentioned activity, i.e. conferring a increase of the fine
chemical threonine after increasing its activity
[0993] [0122.0.0.1] to [127.0.0.1]: see [0122.0.0.0] to
[0127.0.0.0]
[0994] [0128.0.1.1] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, column 7,
lines 6 to 15, 339 to 355 by means of polymerase chain reaction can
be generated on the basis of a sequence as indicated in Table IA or
IB, columns 5 or 7, lines 6 to 15, 339 to 355 or the sequences
derived from sequences as indicated in Table IIA or IIB, columns 5
or 7, lines 6 to 15, 339 to 355.
[0995] [0129.0.1.1] Moreover, it is possible to identify conserved
regions from various organisms by carrying out protein sequence
alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequences indicated in Table IV,
column 7, lines 6 to 15, 339 to 355 are derived from said
alignments.
[0996] [0130.0.1.1] Degenerated primers can then be utilized by PCR
for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of the fine
chemical after increasing its expression or activity or further
functional homologs of the polypeptide of the invention from other
organisms.
[0997] [0131.0.0.1] to [0138.0.0.1]: see [0131.0.0.0] to
[0138.0.0.0]
[0998] [0139.0.1.1] Polypeptides having above-mentioned activity,
i.e. conferring a threonine increase, derived from other organisms,
can be encoded by other DNA sequences which hybridize to a
sequences indicated in Table IA or IB, columns 5 or 7, lines 6 to
15, 339 to 355 under relaxed hybridization conditions and which
code on expression for peptides having the threonine increasing
activity.
[0999] [0140.0.0.1] to [0146.0.0.1]: see [0140.0.0.0] to
[0146.0.0.0]
[1000] [0147.0.1.1] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
IA or IB, columns 5 or 7, lines 6 to 15, 339 to 355 is one which is
sufficiently complementary to one of said nucleotide sequences such
that it can hybridize to one of said nucleotide sequences thereby
forming a stable duplex. Preferably, the hybridisation is performed
under stringent hybrization conditions. However, a complement of
one of the herein disclosed sequences is preferably a sequence
complement thereto according to the base pairing of nucleic acid
molecules well known to the skilled person. For example, the bases
A and G undergo base pairing with the bases T and U or C, resp. and
visa versa. Modifications of the bases can influence the
base-pairing partner.
[1001] [0148.0.1.1] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table IA or IB, columns 5 or 7,
lines 6 to 15, 339 to 355 or a functional portion thereof and
preferably has above mentioned activity, in particular has
the--fine-chemical-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homog's.
[1002] [0149.0.1.1] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table IA or IB, columns 5 or
7, lines 6 to 15, 339 to 355 or a portion thereof and encodes a
protein having above-mentioned activity, e.g. conferring an
increase of the fine chemical.
[1003] [00149.1.1.1] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table IA
or IB, columns 5 or 7, lines 6 to 15, 339 to 355 has further one or
more of the activities annotated or known for the a protein as
indicated in Table IIA or IIB, column 3, lines 6 to 15, 339 to
355.
[1004] [0150.0.1.1] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table IA or IB, columns 5 or 7, lines
6 to 15, 339 to 355 for example a fragment which can be used as a
probe or primer or a fragment encoding a biologically active
portion of the polypeptide of the present invention or of a
polypeptide used in the process of the present invention, i.e.
having above-mentioned activity, e.g. conferring an increase of
fine chemical threonine if its activity is increased. The
nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences indicated in Table
[1005] IA or IB, columns 5 or 7, lines 6 to 15, 339 to 355, an
anti-sense sequence of one of the sequences indicated in Table IA
or IB, columns 5 or 7, lines 6 to 15, 339 to 355 or naturally
occurring mutants thereof. Primers based on a nucleotide of
invention can be used in PCR reactions to clone homologues of the
polypeptide of the invention or of the polypeptide used in the
process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primer pairs indicated in Table III, column 7, lines 6
to 15, 339 to 355 will result in a fragment of a polynucleotide
sequence as indicated in Table IA or IB, columns 5 or 7, lines 6 to
15, 339 to 355.
[1006] [0151.0.0.1] see [0151.0.0.0]
[1007] [0152.0.1.1] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines 6
to 15, 339 to 355 such that the protein or portion thereof
maintains the ability to participate in threonine production, in
particular a threonine increasing activity as mentioned above or as
described in the examples in plants or microorganisms is
comprised.
[1008] [0153.0.1.1] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 6 to 15, 339 to 355 such that the protein or portion thereof
is able to participate in the increase of threonine production. In
one embodiment, a protein or portion thereof as indicated in Table
IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to 355 has for
example an activity of a polypeptide indicated in Table IIA or IIB,
column 3, lines 6 to 15, 339 to 355.
[1009] [0154.0.1.1] In one embodiment, the nucleic acid molecule of
the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines 6
to 15, 339 to 355 and has above-mentioned activity, e.g. conferring
preferably the increase of the fine chemical.
[1010] [0155.0.0.1] see [0155.0.0.0]
[1011] [0156.0.0.1] see [0156.0.0.0]
[1012] [0157.0.1.1] The invention further relates to nucleic acid
molecules that differ from one of a nucleotide sequences as
indicated in Table IA or IB, columns 5 or 7, lines 6 to 15, 339 to
355 (and portions thereof) due to degeneracy of the genetic code
and thus encode a polypeptide of the present invention, in
particular a polypeptide having above mentioned activity, e.g.
conferring an increase in threonine in an organism, e.g. as that
polypeptides comprising the consensus sequences as indicated in
Table IV, columns 5 or 7, lines 6 to 15, 339 to 355 or of the
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
6 to 15, 339 to 355 or their functional homologues. Advantageously,
the nucleic acid molecule of the invention comprises, or in an
other embodiment has, a nucleotide sequence encoding a protein
comprising, or in another embodiment having, a consensus sequences
as indicated in Table IV, columns 5 or 7, lines 6 to 15, 339 to 355
or of the polypeptide as as indicated in Table IIA or IIB, columns
5 or 7, lines 6 to 15, 339 to 355 or the functional homologues. In
a still further embodiment, the nucleic acid molecule of the
invention encodes a full length protein which is substantially
homologous to an amino acid sequence comprising a consensus
sequence as indicated in Table IV, column 7, lines 6 to 15, 339 to
355, or of a polypeptide as indicated in Table IIA or IIB, columns
5 or 7, lines 6 to 15, 339 to 355 or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table IA columns 5 or 7, lines 6 to 15, 339 to
355.
[1013] [0158.0.0.1] to [0160.0.0.1]: see [0158.0.0.0] to
[0160.0.0.0]
[1014] [0161.0.1.1] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table IA or IB, columns 5 or 7, lines 6 to
15, 339 to 355. The nucleic acid molecule is preferably at least
20, 30, 50, 100, 250 or more nucleotides in length.
[1015] [0162.0.0.1]: see [0162.0.0.0]
[1016] [0163.0.1.1] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table IA or IB, columns 5 or 7, lines 6 to 15, 339
to 355 corresponds to a naturally-occurring nucleic acid molecule
of the invention. As used herein, a "naturally-occurring" nucleic
acid molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the fine chemical
increase after increasing the expression or activity thereof or the
activity of a protein of the invention or used in the process of
the invention.
[1017] [0164.0.0.1]: see [0164.0.0.0]
[1018] [0165.0.1.1] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table IA or IB, columns 5 or 7, lines 6 to 15, 339 to
355.
[1019] [0166.0.0.1]: see [0166.0.0.0]
[1020] [0167.0.0.1] see [0167.0.0.0]
[1021] [0168.0.1.1] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the fine chemical in an
organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 6 to 15, 339 to 355 yet retain said activity described
herein. The nucleic acid molecule can comprise a nucleotide
sequence encoding a polypeptide, wherein the polypeptide comprises
an amino acid sequence at least about 50% identical to an amino
acid sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 6 to 15, 339 to 355 and is capable of participation in the
increase of production of the fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines 6
to 15, 339 to 355 more preferably at least about 70% identical to
one of the sequences as indicated in Table IIA or IIB, columns 5 or
7, lines 6 to 15, 339 to 355 even more preferably at least about
80%, 90% or 95% homologous to a sequence as indicated in Table IIA
or IIB, columns 5 or 7, lines 6 to 15, 339 to 355 and most
preferably at least about 96%, 97%, 98%, or 99% identical to the
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines 6
to 15, 339 to 355.
[1022] [0169.0.0.1] to [0172.0.0.1]: see [0169.0.0.0] to
[0172.0.0.0]
[1023] [0173.0.1.1] For example a sequence which has a 80% homology
with sequence SEQ ID NO: 40199 at the nucleic acid level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 40199 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[1024] [0174.0.0.1]: see [0174.0.0.0]
[1025] [0175.0.1.1] For example a sequence which has a 80% homology
with sequence SEQ ID NO: 40200 at the protein level is understood
as meaning a sequence which, upon comparison with the sequence SEQ
ID NO: 40200 by the above program algorithm with the above
parameter set, has a 80% homology.
[1026] [0176.0.1.1] Functional equivalents derived from one of the
polypeptides as indicated in Table IIA or IIB, columns 5 or 7,
lines 6 to 15, 339 to 355 according to the invention by
substitution, insertion or deletion have at least least 30%, 35%,
40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by
preference at least 80%, especially preferably at least 85% or 90%,
91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%,
98% or 99% homology with one of the polypeptides as indicated in
Table IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to 355
according to the invention and are distinguished by essentially the
same properties as a polypeptide as indicated in Table IIA or IIB,
columns 5 or 7, lines 6 to 15, 339 to 355.
[1027] [0177.0.1.1] Functional equivalents derived from a nucleic
acid sequence as indicated in Table IA or IB, columns 5 or 7, lines
6 to 15, 339 to 355 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptides as indicated in Table IIA or
IIB, columns 5 or 7, lines 6 to 15, 339 to 355 according to the
invention and encode polypeptides having essentially the same
properties as a polypeptide as indicated in Table IIA or IIB,
columns 5 or 7, lines 6 to 15, 339 to 355.
[1028] [0178.0.0.1] see [0178.0.0.0]
[1029] [0179.0.1.1] A nucleic acid molecule encoding an homologous
to a protein sequence of as indicated in Table IIA or IIB, columns
5 or 7, lines 6 to 15, 339 to 355 can be created by introducing one
or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table IA or IB, columns 5
or 7, lines 6 to 15, 339 to 355 such that one or more amino acid
substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced into the encoding
sequences of sequences as indicated in Table IA or IB, columns 5 or
7, lines 6 to 15, 339 to 355 by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
[1030] [0180.0.0.1] to [0183.0.0.1]: see [0180.0.0.0] to
[0183.0.0.0]
[1031] [0184.0.1.1] Homologues of the nucleic acid sequences used,
with a sequence as indicated in Table IA or IB, columns 5 or 7,
lines 6 to 15, 339 to 355, or of the nucleic acid sequences derived
from a sequences as indicated in Table IIA or IIB, columns 5 or 7,
lines 6 to 15, 339 to 355 comprise also allelic variants with at
least approximately 30%, 35%, 40% or 45% homology, by preference at
least approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
IA or IB, columns 5 or 7, lines 6 to 15, 339 to 355 or from the
derived nucleic acid sequences, the intention being, however, that
the enzyme activity or the biological activity of the resulting
proteins synthesized is advantageously retained or increased.
[1032] [0185.0.1.1] In one embodiment of the present invention, the
nucleic acid molecule of the invention or used in the process of
the invention comprises one more sequence as indicated in Table IA
or IB, columns 5 or 7, lines 6 to 15, 339 to 355. In one embodiment
it is preferred that the nucleic acid molecule comprises as little
as possible other nucleotide sequences not shown in any one of
sequences as indicated in Table IA or IB, columns 5 or 7, lines 6
to 15, 339 to 355. In one embodiment, the nucleic acid molecule
comprises less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or
40 further nucleotides. In a further embodiment, the nucleic acid
molecule comprises less than 30, 20 or 10 further nucleotides. In
one embodiment, a nucleic acid molecule use in the process of the
invention is identical to a sequence as indicated in Table IA or
IB, columns 5 or 7, lines 6 to 15, 339 to 355.
[1033] [0186.0.1.1] Also preferred is that one or more nucleic acid
molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 6 to 15, 339 to 355. In one embodiment, the
nucleic acid molecule encodes less than 150, 130, 100, 80, 60, 50,
40 or 30 further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polynucleotide used in
the process of the invention is identical to the sequences as
indicated in Table IIA or IIB, columns 5 or 7, lines 6 to 15, 339
to 355.
[1034] [0187.0.1.1] In one embodiment, the nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 6 to 15, 339 to 355 comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a sequences as indicated in Table IIA or IIB,
columns 5 or 7, lines 6 to 15, 339 to 355.
[1035] [0188.0.1.1] Polypeptides (=proteins), which still have the
essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide as indicated in Table IIA or IIB, columns 5 or 7,
lines 6 to 15, 339 to 355, preferably compared to a sequence as
indicated in Table IIA or IIB, column 3 and 5, lines 6 to 15, 339
to 355 and expressed under identical conditions.
[1036] [0189.0.1.1] Homologues of sequences as indicated in Table
IA or IB, columns 5 or 7, lines 6 to 15, 339 to 355 or of derived
sequences as indicated in Table IIA or IIB, columns 5 or 7, lines 6
to 15, 339 to 355 also mean truncated sequences, cDNA,
single-stranded DNA or RNA of the coding and noncoding DNA
sequence. Homologues of said sequences are also understood as
meaning derivatives which comprise noncoding regions such as, for
example, UTRs, terminators, enhancers or promoter variants. The
promoters upstream of the nucleotide sequences stated can be
modified by one or more nucleotide substitution(s), insertion(s)
and/or deletion(s) without, however, interfering with the
functionality or activity either of the promoters, the open reading
frame (=ORF) or with the 3'-regulatory region such as terminators
or other 3' regulatory regions, which are far away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[1037] [0190.0.0.1] to [0203.0.0.1]: see [0190.0.0.0] to
[0203.0.0.0]
[1038] [0204.0.1.1] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule which comprises a nucleic acid
molecule selected from the group consisting of: [1039] (a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, columns 5 or 7, lines 6 to
15, 339 to 355, preferably of Table IIB, column 7, lines 6 to 15,
lines 339 to 355 or a fragment thereof conferring an increase in
the amount of the respective fine chemical threonine in an organism
or a part thereof [1040] (b) nucleic acid molecule comprising,
preferably at least the mature form, of a nucleic acid molecule as
indicated in Table I, columns 5 or 7, lines 6 to 15, 339 to 355,
preferably of Table IB, column 7, lines 6 to 15, lines 339 to 355
or a fragment thereof conferring an increase in the amount of the
respective fine chemical threonine in an organism or a part
thereof; [1041] (c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical threonine in an organism or a part thereof; [1042]
(d) nucleic acid molecule encoding a polypeptide whose sequence has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical threonine in an organism or a part thereof; [1043] (e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under under stringent hybridisation conditions and
conferring an increase in the amount of the respective fine
chemical threonine in an organism or a part thereof; [1044] (f)
nucleic acid molecule encoding a polypeptide, the polypeptide being
derived by substituting, deleting and/or adding one or more amino
acids of the amino acid sequence of the polypeptide encoded by the
nucleic acid molecules (a) to (d), preferably to (a) to (c), and
conferring an increase in the amount of the respective fine
chemical threonine in an organism or a part thereof; [1045] (g)
nucleic acid molecule encoding a fragment or an epitope of a
polypeptide which is encoded by one of the nucleic acid molecules
of (a) to (e), preferably to (a) to (c) and conferring an increase
in the amount of the respective fine chemical threonine in an
organism or a part thereof; [1046] (h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table IIIA or IIIB, column 7, lines 6 to 15, 339 to
355 and conferring an increase in the amount of the respective fine
chemical threonine in an organism or a part thereof; [1047] (i)
nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from a expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (g), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical threonine in
an organism or a part thereof; [1048] (j) nucleic acid molecule
which encodes a polypeptide comprising the consensus sequence as
indicated in Table IV, column 7, lines 6 to 15, 339 to 355 and
conferring an increase in the amount of the respective fine
chemical threonine in an organism or a part thereof; [1049] (k)
nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domaine of a polypeptide as indicated in
Table II, columns 5 or 7, lines 6 to 15, 339 to 355, preferably of
Table IIB, column 7, lines 6 to 15, lines 339 to 355 and conferring
an increase in the amount of the respective fine chemical threonine
in an organism or a part thereof; and [1050] (l) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment of at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (h) or of a nucleic acid molecule as
indicated in Table I, columns 5 or 7, lines 6 to 15, 339 to 355,
preferably of Table IB, column 7, lines 6 to 15, lines 339 to 355
or a nucleic acid molecule encoding, preferably at least the mature
form of, the polypeptide as indicated in Table II, columns 5 or 7,
lines 6 to 15, 339 to 355, preferably of Table IIB, column 7, lines
6 to 15, lines 339 to 355 and conferring an increase in the amount
of the respective fine chemical threonine in an organism or a part
thereof; or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over a sequence depicted in as
indicated in Table IA or IB, columns 5 or 7, lines 6 to 15, 339 to
355 by one or more nucleotides. In one embodiment, the nucleic acid
molecule of the invention does not consist of a sequence as
indicated in Table IA or IB, columns 5 or 7, lines 6 to 15, 339 to
355. In one embodiment, the nucleic acid molecule is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table I A or I B, columns 5 or 7, lines 6 to 15, 339
to 355. In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table II A or II B,
columns 5 or 7, lines 6 to 15, 339 to 355. In an other embodiment,
the nucleic acid molecule of the present invention is at least 30%,
40%, 50%, or 60% identical and less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table I A or I B,
columns 5 or 7, lines 6 to 15, 339 to 355. In a further embodiment
the nucleic acid molecule does not encode a polypeptide sequence as
indicated in Table II A or II B, columns 5 or 7, lines 6 to 15, 339
to 355. Accordingly, in one embodiment, the nucleic acid molecule
of the differs at least in one or more residues from a nucleic acid
molecule indicated in Table I A or I B, columns 5 or 7, lines 6 to
15, 339 to 355. Accordingly, in one embodiment, the nucleic acid
molecule of the present invention encodes a polypeptide, which
differs at least in one or more amino acids from a polypeptide
indicated in Table II A or I B, columns 5 or 7, lines 6 to 15, 339
to 355. In another embodiment, a nucleic acid molecule indicated in
Table I A or I B, columns 5 or 7, lines 6 to 15, 339 to 355 does
not encode a protein of a sequence indicated in Table II A or II B,
columns 5 or 7, lines 6 to 15, 339 to 355. Accordingly, in one
embodiment, the protein encoded by a sequences of a nucleic acid
accoriding to (a) to (l) does not consist of a sequence as
indicated in Table II A or II B, columns 5 or 7, lines 6 to 15, 339
to 355. In a further embodiment, the protein of the present
invention is at least 30%, 40%, 50%, or 60% identical to a protein
sequence indicated in Table II A or II B, columns 5 or 7, lines 6
to 15, 339 to 355 and less than 100%, preferably less than 99.999%,
99.99% or 99.9%, more preferably less than 99%, 985, 97%, 96% or
95% identical to a sequence as indicated in Table I A or II B,
columns 5 or 7, lines 6 to 15, 339 to 355.
[1051] [0205.0.0.1] to [0226.0.0.1]: see [0205.0.0.0] to
[0226.0.0.0]
[1052] [0227.0.1.1] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorgansims.
[1053] In addition to a sequence as indicated in Table IA or IB,
columns 5 or 7, lines 6 to 15, 339 to 355 or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the amino acid biosynthetic pathway such as for
L-lysine, L-methionine and/or L-threonine is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acid
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine sequences as indicated in Table IA or IB,
columns 5 or 7, lines 6 to 15, 339 to 355 with genes which
generally support or enhances to growth or yield of the target
organisms, for example genes which lead to faster growth rate of
microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[1054] [0228.0.0.1] to [0230.0.0.1]: see [0228.0.0.0] to
[0230.0.0.0]
[1055] [0231.0.1.1] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a threonine degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[1056] [0232.0.0.1] to [0282.0.0.1]: see [0232.0.0.0] to
[0282.0.0.0]
[1057] [0283.0.1.1] Moreover, a native polypeptide conferring the
increase of the fine chemical threonine in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an antibody against a protein as indicated in
Table IIA or IIB, column 3, lines 6 to 15, 339 to 355. E.g. an
antibody against a polypeptide as indicated in Table IIA or IIB,
columns 5 or 7, lines 6 to 15, 339 to 355 which can be produced by
standard techniques utilizing polypeptides comprising or consisting
of above mentioned sequences, e.g. the polypeptid of the present
invention or fragment thereof. Preferred are monoclonal
antibodies.
[1058] [0284.0.0.1]: see [0284.0.0.0]
[1059] [0285.0.1.1] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to 355 or as coded
by a nucleic acid molecule as indicated in Table IIA or IIB,
columns 5 or 7, lines 6 to 15, 339 to 355 or functional homologues
thereof.
[1060] [0286.0.1.1] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 6 to 15, 339 to 355 and in one other
embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table IV, column 7, lines 6 to 15, 339 to 355 whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid.
[1061] [0287.0.0.1] to [0290.0.0.1]: see [0287.0.0.0] to
[0290.0.0.0]
[1062] [0291.0.1.1] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of
plant or microorganism specific consensus sequences. In one
embodiment, said polypeptide of the invention distinguishes over a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines 6
to 15, 339 to 355 by one or more amino acids. In one embodiment,
the polypeptide distinguishes from a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to 355 by more
than1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, even more preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 6 to 15, 339
to 355 by not more than 80% or 70% of the amino acids, preferably
not more than 60% or 50%, more preferred not more than 40% or 30%,
even more preferred not more than 20% or 10%. In another
embodiment, said polypeptide of the invention does not consist of a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines 6
to 15, 339 to 355.
[1063] [0292.0.0.1]: see [0292.0.0.0]
[1064] [0293.0.1.1]
[1065] In one embodiment, the invention relates to a polypeptide
conferring an increase in the fine chemical threonine in an
organism or part being encoded by the nucleic acid molecule of the
invention or by the nucleic acid molecule of the invention used in
the process of the invention.
[1066] In one embodiment, the polypeptide of the invention is
having a sequence which distinguishes from a sequence as indicated
in Table IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to 355 by
one or more amino acids. In another embodiment, said polypeptide of
the invention does not consist of the sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to 355. In a
further embodiment, said polypeptide of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical. In one
embodiment, said polypeptide does not consist of the sequence
encoded by a nucleic acid molecules as indicated in Table IA or IB,
columns 5 or 7, lines 6 to 15, 339 to 355.
[1067] [0294.0.1.1]
[1068] In one embodiment, the present invention relates to a
polypeptide having an activity of a protein as indicated in Table
IIA or IIB, column 3, lines 6 to 15, 339 to 355, which
distinguishes over a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 6 to 15, 339 to 355 by one or more amino
acids, preferably by more than 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, even more
preferred are more than 40, 50, or 60 amino acids but even more
preferred by less than 70% of the amino acids, more preferred by
less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[1069] [0295.0.0.1] to [0297.0.0.1]: see [0295.0.0.0] to
[0297.0.0.0]
[1070] [0297.1.0.1] Non-polypeptide of the invention-chemicals are
e.g. polypeptides having not the activity of a polypeptide
indicated in Table IIA or IIB, columns 3, 5 or 7, lines 6 to 15,
339 to 355.
[1071] [0298.0.1.1] A polypeptide of the invention can participate
in the process of the present invention. The polypeptide or a
portion thereof comprises preferably an amino acid sequence which
is sufficiently homologous to an amino acid sequence as indicated
in Table IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to 355 such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to 355.
[1072] [0299.0.1.1] Further, the polypeptide can have an amino acid
sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table IA or IB, columns 5 or 7,
lines 6 to 15, 339 to 355. The preferred polypeptide of the present
invention preferably possesses at least one of the activities
according to the invention and described herein. A preferred
polypeptide of the present invention includes an amino acid
sequence encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions, to a nucleotide
sequence as indicated in Table IA or IB, columns 5 or 7, lines 6 to
15, 339 to 355 or which is homologous thereto, as defined
above.
[1073] [0300.0.1.1] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 6 to 15, 339 to 355 in the amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprises an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to 355.
[1074] [0301.0.0.1] see [0301.0.0.0]
[1075] [0302.0.1.1] Biologically active portions of a polypeptide
of the present invention include peptides comprising amino acid
sequences derived from the amino acid sequence of the polypeptide
of the present invention or used in the process of the present
invention, e.g., an amino acid sequence as indicated in Table IIA
or IIB, columns 5 or 7, lines 6 to 15, 339 to 355 or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to a polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of a polypeptide of the
present invention or used in the process of the present
invention.
[1076] [0303.0.0.1]: see [0303.0.0.0]
[1077] [0304.0.1.1] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
IIA or IIB, column 3, lines 6 to 15, 339 to 355 but having
differences in the sequence from said wild-type protein. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity in relation to the wild type protein.
[1078] [0305.0.0.1] to [0308.0.0.1]: see [0305.0.0.0] to
[0308.0.0.0]
[1079] [0309.0.1.1] In one embodiment, an reference to a "protein
(=polypeptide)" of the invention or as indicated in Table IIA or
IIB, columns 5 or 7, lines 6 to 15, 339 to 355 refers to a
polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas a "non-polypeptide of the invention" or "other
polypeptide" not being indicated in Table IIA or IIB, columns 5 or
7, lines 6 to 15, 339 to 355 refers to a polypeptide having an
amino acid sequence corresponding to a protein which is not
substantially homologous to a polypeptide of the invention,
preferably which is not substantially homologous to a polypeptide
as indicated in Table IIA or IIB, columns 5 or 7, lines 6 to 15,
339 to 355 e.g., a protein which does not confer the activity
described herein or annotated or known for as indicated in Table
IIA or IIB, column 3, lines 6 to 15, 339 to 355 and which is
derived from the same or a different organism. In one embodiment a
"non-polypeptide of the invention" or "other polypeptide" not being
indicate in Table IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to
355 does not confer an increase of the fine chemical in an organism
or part thereof.
[1080] [0310.0.0.1] to [0334.0.0.1]: see [0310.0.0.0] to
[0334.0.0.0]
[1081] [0335.0.1.1] The dsRNAi method has proved to be particularly
effective and advantageous for reducing the expression of a nucleic
acid sequences as indicated in Table II A or IIB, columns 5 or 7,
lines 6 to 15, 339 to 355 and/or homologs thereof. As described
inter alia in WO 99/32619, dsRNAi approaches are clearly superior
to traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table IA or
IB, columns 5 or 7, lines 6 to 15, 339 to 355 and/or homologs
thereof. In a double-stranded RNA molecule for reducing the
expression of an protein encoded by a nucleic acid sequence of one
of the sequences as indicated in Table IIA or IIB, columns 5 or 7,
lines 6 to 15, 339 to 355 and/or homologs thereof, one of the two
RNA strands is essentially identical to at least part of a nucleic
acid sequence, and the respective other RNA strand is essentially
identical to at least part of the complementary strand of a nucleic
acid sequence.
[1082] [0336.0.0.1] to [0342.0.0.1]: see [0336.0.0.0] to
[0342.0.0.0]
[1083] [0343.0.1.1] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table IA or IB, columns 5 or 7, lines 6 to
15, 339 to 355 or its homolog is not necessarily required in order
to bring about effective reduction in the expression. The advantage
is, accordingly, that the method is tolerant with regard to
sequence deviations as may be present as a consequence of genetic
mutations, polymorphisms or evolutionary divergences. Thus, for
example, using the dsRNA, which has been generated starting from a
sequence as indicated in Table IA or IB, columns 5 or 7, lines 6 to
15, 339 to 355 or homologs thereof of the one organism, may be used
to suppress the corresponding expression in another organism.
[1084] [0344.0.0.1] to [0361.0.0.1]: see [0344.0.0.0] to
[0361.0.0.0]
[1085] [0362.0.1.1] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the fine chemical
threonine in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to 355. Due to
the above mentioned activity the fine chemical content in a cell or
an organism is increased. For example, due to modulation or
manupulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. In one embodiment transgenic for a polypeptide having an
activity of a polypeptide as indicated in Table IIA or IIB, columns
5 or 7, lines 6 to 15, 339 to 355 means herein that due to
modulation or manipulation of the genome, an activity as annotated
for a polypeptide as indicated in Table IIA or IIB, columns 3,
lines 6 to 15, 339 to 355, e.g. having a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 6 to 15, 339 to 355 is
increased in a cell or an organism or a part thereof. Examples are
described above in context with the process of the invention.
[1086] [0363.0.0.1]: see [0363.0.0.0]
[1087] [0364.0.1.1] A naturally occurring expression cassette--for
example the naturally occurring combination of a promoter of a
polypeptide of the invention with the corresponding
protein-encoding-sequence--becomes a transgenic expression cassette
when it is modified by non-natural, synthetic "artificial" methods
such as, for example, mutagenization. Such methods have been
described (U.S. Pat. No. 5,565,350; WO 00/15815; also see
above).
[1088] [0365.0.0.1] to [0382.0.0.1]: see [0365.0.0.0] to
[0382.0.0.0]
[1089] [0383.0.1.1] For preparing hydroxy containing fine
chemicals, in particular the fine chemical threonine, it is
possible to use as hydroxy source organic hydroxy-containing
compounds such as, for example, alcohols, hydroxy-containing
organic acids, acetals, or compounds containing carbonyl or
carboxyl groups to be reduced by known methods of the art.
[1090] [0384.0.0.1] to [0392.0.0.1]: see [0384.0.0.0] to
[0392.0.0.0]
[1091] [0393.0.1.1] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [1092] (a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical threonine after expression, with the
nucleic acid molecule of the present invention; [1093] (b)
identifying the nucleic acid molecules, which hybridize under
relaxed stringent conditions with the nucleic acid molecule of the
present invention in particular to the nucleic acid molecule
sequence as indicated in Table IA or IB, columns 5 or 7, lines 6 to
15,339 to 355 and, optionally, isolating the full length cDNA clone
or complete genomic clone; [1094] (c) introducing the candidate
nucleic acid molecules in host cells, preferably in a plant cell or
a microorganism, appropriate for producing the fine chemical
threonine; [1095] (d) expressing the identified nucleic acid
molecules in the host cells; [1096] (e) assaying the the fine
chemical level in the host cells; and [1097] (f) identifying the
nucleic acid molecule and its gene product which expression confers
an increase in the the fine chemical level in the host cell after
expression compared to the wild type.
[1098] [0394.0.0.1] to [0398.0.0.1]: see [0394.0.0.0] to
[0398.0.0.0]
[1099] [0399.0.1.1] Furthermore, in one embodiment, the present
invention relates to a process for the identification of a compound
conferring increased in the fine chemical production in a plant or
microorganism, comprising the steps: [1100] (a) culturing a cell or
tissue or microorganism or maintaining a plant expressing the
polypeptide according to the invention or a nucleic acid molecule
encoding said polypeptide and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and the
polypeptide of the present invention or used in the process of the
invention; and [1101] (b) identifying if the compound is an
effective agonist by detecting the presence or absence or increase
of a signal produced by said readout system.
[1102] The screen for a gene product or an agonist conferring an
increase in the fine chemical production can be performed by growth
of an organism for example a microorganism in the presence of
growth reducing amounts of an inhibitor of the synthesis of the
fine chemical. Better growth, e.g. higher dividing rate or high dry
mass in comparison to the control under such conditions would
identify a gene or gene product or an agonist conferring an
increase in fine chemical production.
[1103] [0399.1.1.1] One can think to screen for increased
production of the fine chemical threonine by for example searching
for a resistance to a drug blocking the synthesis of the fine
chemical threonine and looking whether this effect is dependent on
the activity or expression of a polypeptide as indicated in Table
IIA or IIB, columns 5 or 7, lines 6 to 15,339 to 355 or a homolog
thereof, e.g. comparing the phenotyp of nearly identical organisms
with low and high activity of a protein as indicated in Table IIA
or IIB, columns 5 or 7, lines 6 to 15,339 to 355 after incubation
with the drug.
[1104] [0400.0.0.1] to [0430.0.0.1]: see [0400.0.0.1] to
[0430.0.0.0]
[0431.0.1.1] Example 1
Cloning SEQ ID NO: 81 or Another DNA Polynucleotide According the
Enclosed Sequence Listing Encoding an ORF as Shown in the Below
Table in Escherichia coli
[1105] [0432.0.1.1] SEQ ID NO: 81 or another DNA polynucleotide
according the enclosed sequence listing encoding an ORF as shown in
the below table was cloned into the plasmids pBR322 (Sutcliffe, J.
G. (1979) Proc. Natl Acad. Sci. USA, 75: 3737-3741); pACYC177
(Change & Cohen (1978) J. Bacteriol. 134: 1141-1156); plasmids
of the pBS series (pBSSK+, pBSSK- and others; Stratagene, LaJolla,
USA) or cosmids such as SuperCosi (Stratagene, LaJolla, USA) or
Lorist6 (Gibson, T. J. Rosenthal, A., and Waterson, R. H. (1987)
Gene 53: 283-286) for expression in E. coli using known,
well-established procedures (see, for example, Sambrook, J. et al.
(1989) "Molecular Cloning: A Laboratory Manual". Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons).
[1106] [0433.0.0.1] to [0460.0.0.1]: see [0433.0.0.0] to
[0460.0.0.0]
[0461.0.1.1] Example 10
Cloning SEQ ID NO: 81 for the Expression in Plants
[1107] [0462.0.0.1]: see [0462.0.0.0]
[1108] [0463.0.1.1] SEQ ID NO: 81 is amplified by PCR as described
in the protocol of the Pfu Turbo or DNA Herculase polymerase
(Stratagene).
[1109] [0464.0.0.1] to [0466.0.0.1]: see [0464.0.0.0] to
[0466.0.0.0]
[1110] [0467.0.1.1] The following primer sequences were selected
for the gene SEQ ID NO: 81:
TABLE-US-00008 i) forward primer SEQ ID NO: 83:
ATGTCGTCCTTATCCACTTCATTTG ii) reverse primer SEQ ID NO: 84:
TTAATTGTAACGGCTATATCTACTGG
[1111] [0468.0.0.1] to [0479.0.0.1]: see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.1.1] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 81
[1112] [0481.0.0.1] to [0513.0.0.1]: see [0481.0.0.0] to
[0513.0.0.0]
[1113] [0514.0.1.1] As an alternative, the amino acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55).
[1114] Arabidopsis thaliana plants were engineered as described in
Example 11.
[1115] The results of the different Arabidopsis plants analysed can
be seen from table 1 which follows:
TABLE-US-00009 TABLE 1 ORF Annotation Metabolite Min Max Methode
YFL050C di-trivalent inorganic threonine 1.193 1.557 GC cation
transporter YKR057W ribosomal protein, threonine 1.34 2.413 GC
similar to S21 ribosomal proteins, involved in ribosome biogenesis
and translation YIL150C chromatin binding threonine 1.256 4.186 GC
protein, required for S-phase (DNA synthesis) initiation or
completion YNL046W probable membrane threonine 1.178 1.526 GC
protein of the endoplasmatic reticulum YNL120C not been threonine
1.44 1.44 LC characterized yet b0186 lysine decarboxylase threonine
1.495 3.277 GC b0730 transcriptional threonine 1.531 2.772 LC
regulator of succinylCoA synthetase operon and fatty acyl response
regulator b1829 defined as a heat threonine 1.174 2.135 GC shock
protein with protease activity b2170 sugar efflux threonine 1.359
1.792 LC transporter B b0019 Na+/H+ antiporter threonine 1.244 1.44
GC
[1116] Metabolite Profiling Info:
TABLE-US-00010 ORF Metabolite Method Min Max b0464 Threonine GC
1.23 1.43 b1360 Threonine GC 1.16 1.38 b1738 Threonine LC 1.27 4.61
b1830 Threonine LC 1.24 1.43 b1896 Threonine LC + GC 1.46 2.08
b2414 Threonine GC 1.24 1.46 b2552 Threonine GC 1.17 1.37 b4004
Threonine GC 1.17 1.37 b2664 Threonine LC + GC 1.29 2.84 b3074
Threonine LC 1.31 1.59 b2270 Threonine LC 1.31 1.59 b3160 Threonine
LC 1.25 1.56 b3231 Threonine GC 1.17 1.32 b3462 Threonine GC 1.18
1.51 b3791 Threonine LC 1.38 1.44 b3966 Threonine GC 1.19 1.47
YOR245C Threonine GC 1.18 1.81
[1117] [0515.0.0.1]: to [0552.0.0.1]: see [0515.0.0.0] to
[0552.0.0.0]
[0552.1.1.1]: Example 15
Metabolite Profiling Info from Zea mays
[1118] Zea mays plants were engineered, grown and analyzed as
described in Example 14c.
[1119] The results of the different Zea mays plants analysed can be
seen from Table 2 which follows:
TABLE-US-00011 TABLE 2 ORF_NAME Metabolite Min Max b1829 Threonine
1.44 1.96 b2664 Threonine 1.77 3.94 YIL150C Threonine 1.68 2.98
YKR057W Threonine 1.59 5.59
[1120] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in threonine in
genetically modified corn plants expressing the
[1121] Saccharomyces cerevisiae nucleic acid sequence YIL150C or
YKR057Wor E. coli nucleic acid sequence b1829 or b2664 resp.
[1122] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YIL150C or its homologs, e.g. "a chromatin
binding protein, required for S-phase (DNA synthesis) initiation or
completion" or its homologs, is increased in corn plants,
preferably, an increase of the fine chemical threonine between 68%
and 198% is conferred.
[1123] In case the activity of the Saccharomyces cerevisiae protein
YKR057W or a ribosomal protein, similar to S21 ribosomal proteins,
involved in ribosome biogenesis and translation or its homolog, is
increased in corn plants, preferably, an increase of the fine
chemical threonine between 59% and 459%, is conferred.
[1124] In one embodiment, in case the activity of the E. coli
protein b1829 or its homologs, e.g. "the activity of a protease is
increased, preferably, the activity of a heat shock protein is
increased, more preferred the activity of a htpX protein", is
increased in corn plants, preferably, an increase of the fine
chemical threonine between 44% and 96% is conferred.
[1125] In one embodiment, in case the activity of the E. coli
protein b2664 or its homologs, e.g. "the activity defined as
putative transcriptional repressor with DNA-binding Winged helix
domain (GntR familiy)", is increased in corn plants, preferably, an
increase of the fine chemical threonine between 77% and 294% is
conferred.
[1126] [00552.2.0.1] see [00552.2.0.0]
[1127] [0553.0.1.1] [1128] 1. A process for the production of
threonine, which comprises [1129] (a) increasing or generating the
activity of a protein as indicated in Table IIA or IIB, columns 5
or 7, lines 6 to 15, 339 to 355 or a functional equivalent thereof
in a non-human organism, or in one or more parts thereof; and
[1130] (b) growing the organism under conditions which permit the
production of threonine in said organism. [1131] 2. A process for
the production of threonine, comprising the increasing or
generating in an organism or a part thereof the expression of at
least one nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [1132] (a) nucleic acid
molecule encoding of a polypeptide as indicated in Table IIA or
IIB, columns 5 or 7, lines 6 to 15, 339 to 355 or a fragment
thereof, which confers an increase in the amount of threonine in an
organism or a part thereof; [1133] (b) nucleic acid molecule
comprising of the nucleic acid molecule as indicated in Table IA or
IB, columns 5 or 7, lines 6 to 15, 339 to 355; [1134] (c) nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as a
result of the degeneracy of the genetic code and conferring an
increase in the amount of threonine in an organism or a part
thereof; [1135] (d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of threonine in
an organism or a part thereof; [1136] (e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
under stringent hybridisation conditions and conferring an increase
in the amount of threonine in an organism or a part thereof; [1137]
(f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table III, columns 5 or 7, lines 6 to 15, 339 to 355
and conferring an increase in the amount of the fine chemical
threonine in an organism or a part thereof; [1138] (g) nucleic acid
molecule encoding a polypeptide which is isolated with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (f) and conferring an increase in
the amount of threonine in an organism or a part thereof; [1139]
(h) nucleic acid molecule encoding a polypeptide comprising a
consensus sequence as indicated in Table IV, columns 5 or 7, lines
6 to 15, 339 to 355 and conferring an increase in the amount of the
fine chemical threonine in an organism or a part thereof; and
[1140] (i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of the fine chemical
threonine in an organism or a part thereof. [1141] or comprising a
sequence which is complementary thereto. [1142] 3. The process of
claim 1 or 2, comprising recovering of the free or bound
threonine.
[1143] 4. The process of any one of claims 1 to 3, comprising the
following steps: [1144] (a) selecting an organism or a part thereof
expressing a polypeptide encoded by the nucleic acid molecule
characterized in claim 2; [1145] (b) mutagenizing the selected
organism or the part thereof; [1146] (c) comparing the activity or
the expression level of said polypeptide in the mutagenized
organism or the part thereof with the activity or the expression of
said polypeptide of the selected organisms or the part thereof;
[1147] (d) selecting the mutated organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
[1148] (e) optionally, growing and cultivating the organisms or the
parts thereof; and [1149] (f) recovering, and optionally isolating,
the free or bound threonine produced by the selected mutated
organisms or parts thereof. [1150] 5. The process of any one of
claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. [1151] 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [1152] (a) nucleic acid molecule encoding a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
6 to 15, 339 to 355 or a fragment thereof, which confers an
increase in the amount of threonine in an organism or a part
thereof; [1153] (b) nucleic acid molecule comprising a nucleic acid
as indicated in Table IA or IB, columns 5 or 7, lines 6 to 15, 339
to 355; [1154] (c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of threonine in an
organism or a part thereof; [1155] (d) nucleic acid molecule which
encodes a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
threonine in an organism or a part thereof; [1156] (e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of threonine in an organism or a part
thereof; [1157] (f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers in Table III, column 8, lines 6 to 15, 339-355 and
conferring an increase in the amount of the fine chemical threonine
in an organism or a part thereof; [1158] (g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
threonine in an organism or a part thereof; [1159] (h) nucleic acid
molecule encoding a polypeptide comprising the consensus sequence
shown in Table IV, column 8, lines 6 to 15, 339 to 355 and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; and [1160] (i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof.
[1161] whereby the nucleic acid molecule distinguishes over the
sequence as indicated in Table IA or IB, columns 5 or 7, lines 6 to
15, 339 to 355 by one or more nucleotides. [1162] 7. A nucleic acid
construct which confers the expression of the nucleic acid molecule
of claim 6, comprising one or more regulatory elements. [1163] 8. A
vector comprising the nucleic acid molecule as claimed in claim 6
or the nucleic acid construct of claim 7. [1164] 9. The vector as
claimed in claim 8, wherein the nucleic acid molecule is in
operable linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [1165] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 9 or 10 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [1166] 11. The host cell of claim 10, which is a
transgenic host cell. [1167] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [1168] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [1169] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a Sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 6 to 15,339 to 355 by one or more amino acids
[1170] 15. An antibody, which binds specifically to the polypeptide
as claimed in claim 14. [1171] 16. A plant tissue, propagation
material, harvested material or a plant comprising the host cell as
claimed in claim 12 which is plant cell or an Agrobacterium. [1172]
17. A method for screening for agonists and antagonists of the
activity of a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of threonine in an
organism or a part thereof comprising: [1173] (a) contacting cells,
tissues, plants or microorganisms which express the a polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of threonine in an organism or a part
thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; [1174] (b) assaying the threonine level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [1175] (c)
identifying a agonist or antagonist by comparing the measured
threonine level or polypeptide expression level with a standard
threonine or polypeptide expression level measured in the absence
of said candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. [1176] 18. A process for the
identification of a compound conferring increased threonine
production in a plant or microorganism, comprising the steps:
[1177] (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of threonine in an organism or a part thereof and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
threonine in an organism or a part thereof; [1178] (b) identifying
if the compound is an effective agonist by detecting the presence
or absence or increase of a signal produced by said readout system.
[1179] 19. A method for the identification of a gene product
conferring an increase in threonine production in a cell,
comprising the following steps: [1180] (a) contacting the nucleic
acid molecules of a sample, which can contain a candidate gene
encoding a gene product conferring an increase in threonine after
expression with the nucleic acid molecule of claim 6; [1181] (b)
identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; [1182] (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing threonine; [1183]
(d) expressing the identified nucleic acid molecules in the host
cells; [1184] (e) assaying the threonine level in the host cells;
and [1185] (f) identifying nucleic acid molecule and its gene
product which expression confers an increase in the threonine level
in the host cell after expression compared to the wild type. [1186]
20. A method for the identification of a gene product conferring an
increase in threonine production in a cell, comprising the
following steps: [1187] (a) identifiying in a data bank nucleic
acid molecules of an organism; which can contain a candidate gene
encoding a gene product conferring an increase in the threonine
amount or level in an organism or a part thereof after expression,
and which are at least 20% homolog to the nucleic acid molecule of
claim 6; [1188] (b) introducing the candidate nucleic acid
molecules in host cells appropriate for producing threonine; [1189]
(c) expressing the identified nucleic acid molecules in the host
cells; [1190] (d) assaying the threonine level in the host cells;
and [1191] (e) identifying the nucleic acid molecule and its gene
product which expression confers an increase in the threonine level
in the host cell after expression compared to the wild type. [1192]
21. A method for the production of an agricultural composition
comprising the steps of the method of any one of claims 17 to 20
and formulating the compound identified in any one of claims 17 to
20 in a form acceptable for an application in agriculture. [1193]
22. A composition comprising the nucleic acid molecule of claim 6,
the polypeptide of claim 14, the nucleic acid construct of claim 7,
the vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [1194] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
threonine after expression. [1195] 24. Use of the polypeptide of
claim 14 or the nucleic acid construct claim 7 or the gene product
identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of
threonine levels in an organism. [1196] 25. Food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claims 10 to 12 or the gene product identified according to
the method of claim 19 or 20.
[1197] [0554.0.0.1] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[1198] [0000.0.0.2]: In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and corresponding embodiments as described herein
as follows.
[1199] [0001.0.0.2] to [0009.0.0.2]: see [0.0.01.0.0.0] to
[0008.0.0.0]
[1200] [0009.0.2.2] As described above, the essential amino acids
are necessary for humans and many mammals, for example for
livestock. Tryptophane (L-tryptophane) is one of the most reactive
amino acids. At pH 4.0-6.0 tryptophane amino group reacts with
aldehydes producing Schiff-bases. On the other hand if the amino
group is blocked by acetylation, tryptophane reacts with aldehydes
yielding carboline derivatives (carboline
1,2,3,4-tetrahydro-carboline-3-carboxylic acid). Tryptophane plays
a unique role in defense against infection because of its relative
scarcity compared to other amino acids. During infection, the body
induces tryptophane-catabolizing enzymes which increase
tryptophane's scarcity in an attempt to starve the infecting
organisms [R. R. Brown, Y. Ozaki, S. P. Datta, et al., Implications
of interferon-induced tryptophane catabolism in cancer, auto-immune
diseases and AIDS. In: Kynurenine and Serotonin Pathways, R.
Schwarcz, et al., (Eds.), Plenum Press, New York, 1991]. In most
proteins, tryptophane is the least abundant essential amino acid,
comprising approximately 1% of plant proteins and 1.5% of animal
proteins. Although the minimum daily requirement for tryptophane is
160 mg for women and 250 mg for men, 500-700 mg are recommended to
ensure high-quality protein intake. Actual tryptophane utilization
is substantially higher. Men use approximately 3.5 grams of
tryptophane to make one days's worth of protein [J. C. Peters,
Tryptophane Nutrition and Metabolism: an Overview. In: Kynurenine
and Serotonin Pathways, R. Schwarcz, et al., (Eds.), Plenum Press,
New York, 1991]. The balance is obtained by hepatic recycling of
tryptophane from used (catabolized) proteins.
[1201] Dietary tryptophane is well absorbed intestinally. About 10%
of the tryptophane circulating in the bloodstream is free, and 90%
is bound to the protein albumin. The tryptophane binding site on
albumin also has affinity for free fatty acids (FFAs), so
tryptophane is displaced when FFAs rise, as when fasting.
[1202] Although tryptophane is not usually the limiting amino acid
in protein synthesis, tryptophane may become insufficient for the
normal functioning of other tryptophane-dependent pathways.
Numerous lines of research point to tryptophane's central role in
regulation of feeding and other behaviors. Tryptophane is not only
typically the least abundant amino acid in the livers free amino
acid pool, but liver tryptophane-tRNA levels fall faster during
food deprivation than other indispensable amino acids [Q. R.
Rogers, The nutritional and metabolic effects of amino acid
imbalances. In: Protein Metabolism and Nutrition, D. J. A. Cole
(Ed.), Butterworths, London, 1976]. Under fasting conditions, and
possibly in wasting syndromes, tryptophane may become the
rate-limiting amino acid for protein synthesis [Peters, 1991].
[1203] [0010.0.0.2] and [0011.0.0.2]: see [0010.0.0.0] and
[0011.0.0.0]
[1204] [0012.0.2.2] It is an object of the present invention to
develop an inexpensive process for the synthesis of tryptophane,
preferably L-tryptophane. Tryptophane is together with methionine,
lysine and threonine (depending on the organism) one of the amino
acids which are most frequently limiting.
[1205] [0013.0.0.2]: see [0013.0.0.0]
[1206] [0014.0.2.2] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is tryptophane, preferably
L-tryptophane. Accordingly, in the present invention, the term "the
fine chemical" as used herein relates to "tryptophane". Further,
the term "the fine chemicals" as used herein also relates to fine
chemicals comprising tryptophane.
[1207] [0015.0.2.2] In one embodiment, the term "the fine chemical"
means tryptophane, preferably L-tryptophane. Throughout the
specification the term "the fine chemical" means tryptophane,
preferably L-tryptophane, its salts, ester or amids in free form or
bound to proteins. In a preferred embodiment, the term "the fine
chemical" means tryptophane, preferably L-tryptophane, in free form
or its salts or bound to proteins.
[1208] In one embodiment, the term "the fine chemical" and the term
"the respective fine chemical" mean at least one chemical compound
with an activity of the above mentioned fine chemical.
[1209] [0016.0.2.2] Accordingly, the present invention relates to a
process comprising
(a) increasing or generating the activity of one or more
[1210] YER173W, YGR104C, b0186, b0161, b0486, b1318, b2270, b3074,
b3983 and/or YHR189W--protein(s) or of a protein having the
sequence of a polypeptide encoded by a nucleic acid molecule
indicated in Table I, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362
in a non-human organism in one or more parts thereof and (b)
growing the organism under conditions which permit the production
of the fine chemical, thus, tryptophane or fine chemicals
comprising tryptophane, in said organism.
[1211] Accordingly, the present invention relates to a process for
the production of a fine chemical comprising
(a) increasing or generating the activity of one or more proteins
having the activity of a protein indicated in Table II, column 3,
lines 16 to 18 and/or lines 356 to 362 or having the sequence of a
polypeptide encoded by a nucleic acid molecule indicated in Table
I, column 5 or 7, lines 16 to 18 and/or lines 356 to 362, in a
non-human organism in one or more parts thereof and (b) growing the
organism under conditions which permit the production of the fine
chemical, in particular tryptophane.
[1212] [0017.0.0.2] and [0018.0.0.2]: see [0017.0.0.0] and
[0018.0.0.0]
[1213] [0019.0.2.2] Advantageously the process for the production
of the fine chemical leads to an enhanced production of the fine
chemical. The terms "enhanced" or "increase" mean at least a 10%,
20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or
100%, more preferably 150%, 200%, 300%, 400% or 500% higher
production of the fine chemical in comparison to the reference as
defined below, e.g. that means in comparison to an organism without
the aforementioned modification of the activity of a protein having
the activity of a protein indicated in Table II, column 3, lines 16
to 18 and/or lines 356 to 362 or encoded by nucleic acid molecule
indicated in Table I, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362.
[1214] [0020.0.2.2] Surprisingly it was found, that the transgenic
expression of at least one of the Saccaromyces cerevisiae
protein(s) indicated in Table II, Column 3, lines 16 to 17 and/or
362 and/or at least one of the Escherichia coli K12 proteins
indicated in Table II, Column 3, line 18 and/or lines 356 to 361 in
Arabidopsis thaliana conferred an increase in the threonine (or
fine chemical) content of the transformed plants.
[1215] [0021.0.0.2]: see [0021.0.0.0]
[1216] [0022.0.2.2] The sequence of YER173W from Saccharomyces
cerevisiae has been published in Dietrich, Nature 387 (6632 Suppl),
78-81, 1997, and Goffeau, Science 274 (5287), 546-547, 1996, and
its activity is beeing defined as an "Checkpoint protein, involved
in the activation of the DNA damage and meiotic pachytene
checkpoints; subunit of a clamp loader that loads Rad17p-Mec3p-Dc1p
onto DNA, homolog of the human and S. pompe Rad17 protein; Rad24p".
Accordingly, in one embodiment, the process of the present
invention comprises the use of a "Checkpoint protein, involved in
the activation of the DNA damage and meiotic pachytene checkpoints"
or its "subunit of a clamp loader that loads Rad17p-Mec3p-Dc1p onto
DNA" or a Rad24p from Saccaromyces cerevisiae or a Rad17 protein or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of tryptophane, in particular for increasing the
amount of tryptophane in free or bound form in an organism or a
part thereof, as mentioned.
[1217] The sequence of YGR104C from Saccharomyces cerevisiae has
been published in Thompson et al., Cell 73:1361-1375, 1993, and its
activity is beeing defined as an "RNA polymerase II suppressor
protein SRB5--yeast". Accordingly, in one embodiment, the process
of the present invention comprises the use of a "RNA polymerase II
suppressor protein (SRB5--yeast)" or its homolog, for the
production of the fine chemical, meaning of tryptophane, in
particular for increasing the amount of tryptophane in free or
bound form in an organism or a part thereof, as mentioned.
[1218] The sequence of b0186 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is beeing defined as a lysine decarboxylase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a lysine decarboxylase from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of tryptophane, in particular for increasing
the amount of tryptophane, preferably tryptophane in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a lysine decarboxylase is increased or generated, e.g. from E. coli
or a homolog thereof.
[1219] The sequence of b0161 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a periplasmic serine protease
(heat shock protein). Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of the helicobacter serine proteinase superfamily,
preferably a protein with a periplasmic serine protease (heat shock
protein) activity or its homolog, e.g. as shown herein, from
Escherichia coli K12 or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of tryptophane in free or
bound form in an organism or a part thereof, as mentioned.
[1220] The sequence of b0486 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is beeing defined as a amino-acid/amine transport protein
(of the APC family). Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with the
activity of the membrane protein ybaT superfamily, preferably a
protein with a amino-acid/amine transport protein (of the APC
family) activity from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of tryptophane, in
particular for increasing the amount of tryptophane in free or
bound form in an organism or a part thereof, as mentioned.
[1221] The sequence of b1318 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is beeing defined as a sugar transport protein (of the ABC
superfamily). Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with the
activity of the inner membrane protein malK (with ATP-binding
cassette homology) superfamily, preferably a protein with a sugar
transport protein (of the ABC superfamily) activity from E. coli or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of tryptophane, in particular for increasing the
amount of tryptophane in free or bound form in an organism or a
part thereof, as mentioned.
[1222] The sequence of b2270 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has not been characterized yet. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a protein b2270 from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
tryptophane, in particular for increasing the amount of tryptophane
in free or bound form in an organism or a part thereof, as
mentioned.
[1223] The sequence of b3074 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a tRNA synthetase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with the activity of
the secretion chaperone CsaA and/or methionyl-tRNA synthetase
(dimer-forming) superfamily, preferably a protein with a tRNA
synthetase activity from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
tryptophane, in particular for increasing the amount of tryptophane
in free or bound form in an organism or a part thereof, as
mentioned.
[1224] The sequence of b3983 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a 50S ribosomal subunit
protein L12. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with the
activity of the Escherichia coli ribosomal protein L11 superfamily,
preferably a protein with a 50S ribosomal subunit protein L12
activity from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of tryptophane, in
particular for increasing the amount of tryptophane in free or
bound form in an organism or a part thereof, as mentioned.
[1225] The sequence of YHR189W from Saccharomyces cerevisiae has
been published in and Goffeau, Science 274 (5287), 546-547, 1996
and Johnston, Nature 387 (6632 Suppl), 87-90, 1997, and its
activity is beeing defined as a peptidyl-tRNA hydrolase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with the activity of
the peptidyl-tRNA hydrolase superfamily, preferably a protein with
a t peptidyl-tRNA hydrolase activity from Saccharomyces cerevisiae
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of tryptophane, in particular for increasing
the amount of tryptophane in free or bound form in an organism or a
part thereof, as mentioned.
[1226] [0023.0.2.2] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the fine chemical amount or content. Further, in the
present invention, the term "homologue" relates to the sequence of
an organism having the highest sequence homology to the herein
mentioned or listed sequences of all expressed sequences of said
organism.
[1227] However, the person skilled in the art knows, that,
preferably, the homologue has said the--fine-chemical-increasing
activity and, if known, the same biological function or activity in
the organism as at least one of the protein(s) indicated in Table
I, Column 3, lines 16 to 18 and/or lines 356 to 362, e.g. having
the sequence of a polypeptide encoded by a nucleic acid molecule
comprising the sequence indicated in indicated in Table I, Column 5
or 7, lines 16 to 18 and/or lines 356 to 362.
[1228] In one embodiment, the homolog of any one of the
polypeptides indicated in Table II, lines 16 to 17 and/or line 362
is a homolog having the same or a similar activity, in particular
an increase of activity confers an increase in the content of the
fine chemical in the organsims and being derived from an Eukaryot.
In one embodiment, the homolog of a polypeptide indicated in Table
II, column 3, line 18 and/or lines 356 to 361 is a homolog having
the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms or part thereof, and being derived from bacteria. In
one embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 16 to 17 and/or line 362 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in an
organisms or part thereof, and being derived from Fungi. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 18 and/or lines 356 to 361 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organsims or part thereof and being derived from Proteobacteria. In
one embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 16 to 17 and/or line 362 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organsims or a part thereof and being derived from Ascomycota. In
one embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 18 and/or lines 356 to 361 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from
Gammaproteobacteria. In one embodiment, the homolog of a
polypeptide polypeptide indicated in Table II, column 3, lines 16
to 17 and/or line 362 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or part
thereof, and being derived from Saccharomycotina. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 18 and/or lines 356 to 361 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 16 to 17 and/or line 362 is
a homolog having the same or a similar activity, in particular an
increase of activity confers an increase in the content of the fine
chemical in the organisms or a part thereof, and being derived from
Saccharomycetes. In one embodiment, the homolog of the a
polypeptide indicated in Table II, column 3, line 18 and/or lines
356 to 361 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or part thereof, and
being derived from Enterobacteriaceae. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 16
to 17 and/or line 362 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms, and being
derived from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 18 and/or line
356 to 361 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof,
and being derived from Escherichia. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 16 to 17
and/or line 362 is a homolog having the same or a similar activity,
in particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof,
and being derived from Saccharomycetaceae. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 16
to 17 and/or line 362 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or a part
thereof, and being derived from Saccharomycetes.
[1229] [0023.1.2.2] Homologs of the polypeptides indicated in Table
II, column 3, lines 16 to 17 and/or line 362 may be the polypetides
encoded by the nucleic acid molecules polypeptide indicated in
Table I, column 7, lines 16 to 17 and/or line 362 or may be the
polypeptides indicated in Table II, column 7, lines 16 to 17 and/or
line 362. Homologs of the polypeptides polypeptide indicated in
Table II, column 3, line 18 and/or lines 356 to 361 may be the
polypetides encoded by the nucleic acid molecules polypeptide
indicated in Table I, column 7, lines 18 and/or lines 356 to 361 or
may be the polypeptides indicated in Table II, column 7, lines 18
and/or lines 356 to 361.
[1230] [0024.0.0.2]: see [0024.0.0.0]
[1231] [0025.0.2.2] In accordance with the invention, a protein or
polypeptide has the "activity of an protein of the invention", e.g.
the activity of a protein indicated in Table II, column 3, lines 16
to 18 and/or lines 356 to 362 if its de novo activity, or its
increased expression directly or indirectly leads to an increased
tryptophane level in the organism or a part thereof, preferably in
a cell of said organism. In a preferred embodiment, the protein or
polypeptide has the above-mentioned additional activities of a
protein indicated in Table II, column 3, lines 16 to 18 and/or
lines 356 to 362. Throughout the specification the activity or
preferably the biological activity of such a protein or polypeptide
or an nucleic acid molecule or sequence encoding such protein or
polypeptide is identical or similar if it still has the biological
or enzymatic activity of any one of the proteins indicated in Table
II, column 3, lines 16 to 18 and/or lines 356 to 362, i.e. or which
has at least 10% of the original enzymatic activity, preferably
20%, particularly preferably 30%, most particularly preferably 40%
in comparison to an any one of the proteins indicated in Table II,
column 3, lines 16 to 17 and/or line 362 of Saccharomyces and/or
any one of the proteins indicated in Table II, column 3, line 18
and/or lines 356 to 361 of E. coli K12.
[1232] [0025.1.0.2]: see [0025.1.0.0]
[1233] [0025.2.0.2]: see [0025.2.0.0]
[1234] [0026.0.0.2] to [0033.0.0.2]: see [0026.0.0.0] to
[0033.0.0.0]
[1235] [0034.0.2.2] Preferably, the reference, control or wild type
differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. as result of
an increase in the level of the nucleic acid molecule of the
present invention or an increase of the specific activity of the
polypeptide of the invention or the polypeptide used in the method
of the invention. E.g., it differs by or in the expression level or
activity of an protein having the activity of a protein as
indicated in Table II, column 3, Hines 16 to 18 and/or lines 356 to
362 or being encoded by a nucleic acid molecule indicated in Table
I, column 5, lines 16 to 18 and/or lines 356 to 362 or its
homologs, e.g. as indicated in Table I, column 7, lines 16 to 18
and/or lines 356 to 362, its biochemical or genetical causes and
therefore shows the increased amount of the fine chemical.
[1236] [0035.0.0.2] to [0044.0.0.2]: see [0035.0.0.0] to
[0044.0.0.0]
[1237] [0045.0.2.2] In case the activity of the Saccaromyces
cerevisiae protein YER173W or its homologs, e.g. a checkpoint
protein, involved in the activation of the DNA damage and meiotic
pachytene checkpoints; subunit of a clamp loader that loads
Rad17p-Mec3p-Ddc1p onto DNA or Rad24p or its homologs, e.g. the
human or S. pombe Rad17, e.g. as indicated in Table I, columns 5 or
7, line 16 is increased, preferably, an increase of the fine
chemical between 27% and 178% or more is conferred.
[1238] In case the activity of the Saccaromyces cerevisiae protein
YGR104C or its homologs, e.g. a RNA polymerase II suppressor
protein (SRB5--yeast) e.g. as indicated in Table I, columns 5 or 7,
line 17 is increased, preferably, an increase of the fine chemical
between 32% and 84% or more is conferred. (s.o.)
[1239] In case the activity of the Escherichia coli K12 protein
b0186 or a lysine decarboxylase or its homologs, e.g. as indicated
in Table I, columns 5 or 7, line 18 is increased, preferably, an
increase of the fine chemical between 32% and 146% is conferred.
S.o.
[1240] In case the activity of the Escherichia coli K12 protein
b0161 or its homologs, e.g. the activity of a protein of the
Helicobacter serine proteinase superfamily is increased,
preferably, of a protein having a periplasmic serine protease (heat
shock protein) activity, e.g. as indicated in Table I, columns 5 or
7, line 356, is increased conferring an increase of the respective
fine chemical, preferably tryptophane between 93% and 278% or
more.
[1241] In case the activity of the Escherichia coli K12 protein
b0486 or its homologs, e.g. the activity of a protein of the
membrane protein ybaT superfamily, preferably a protein with a
amino-acid/amine transport protein (of the APC family) activity,
e.g. as indicated in Table I, columns 5 or 7, line 357, is
increased conferring an increase of the respective fine chemical,
preferably tryptophane between 42% and 335% or more.
[1242] In case the activity of the Escherichia coli K12 protein
b1318 or its homologs, e.g. the activity of the inner membrane
protein malK (with ATP-binding cassette homology) superfamily,
preferably a protein with a sugar transport protein (of the ABC
superfamily) activity, e.g. as indicated in Table I, columns 5 or
7, line 358, is increased conferring an increase of the respective
fine chemical, preferably tryptophane between 136% and 330% or
more.
[1243] In case the activity of the Escherichia coli K12 protein
b2270 or its homologs, e.g. the activity of a b2270 protein of E.
coli, e.g. as indicated in Table I, columns 5 or 7, line 359, is
increased conferring an increase of the respective fine chemical,
preferably tryptophane between 33% and 79% or more.
[1244] In case the activity of the Escherichia coli K12 protein
b3074 or its homologs, e.g. the activity of a protein of the
secretion chaperone CsaA and/or methionyl-tRNA synthetase
(dimer-forming) superfamily, preferably a protein with a tRNA
synthetase activity, e.g. as indicated in Table I, columns 5 or 7,
line 360, is increased conferring an increase of the respective
fine chemical, preferably tryptophane between 33% and 79% or
more.
[1245] In case the activity of the Escherichia coli K12 protein
b3983 or its homologs, e.g. the activity of a Escherichia coli
ribosomal protein L11 superfamily, preferably a protein with a 50S
ribosomal subunit protein L12 activity, e.g. as indicated in Table
I, columns 5 or 7, line 361, is increased conferring an increase of
the respective fine chemical, preferably tryptophane between 33%
and 387% or more.
[1246] In case the activity of the Saccaromyces cerevisiae protein
YHR189W or its homologs, e.g. the activity of a peptidyl-tRNA
hydrolase superfamily, preferably a protein with a peptidyl-tRNA
hydrolase activity, e.g. as indicated in Table I, columns 5 or 7,
line 362, is increased conferring an increase of the respective
fine chemical, preferably tryptophane between 31% and 66% or
more.
[1247] [0046.0.2.2] In one embodiment, in case the activity of the
Saccaromyces cerevisiae protein YER173W or its homologs, e.g. a
checkpoint protein, involved in the activation of the DNA damage
and meiotic pychtene checkpoints; subunit of a clamp loader that
loads Rad17p-Mec3p-Ddc1p onto DNA or or Rad24p or its homologs,
e.g. the human or S. pombe Rad17, e.g. as indicated in Table I,
columns 5 or 7, line 16, is increased, preferably, an increase of
the fine chemical and of proline is conferred.
[1248] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YGR104C or its homologs, e.g. a RNA polymerase
II suppressor protein (SRB5--yeast), e.g. as indicated in Table I,
columns 5 or 7, line 17, is increased, preferably, an increase of
the fine chemical and glutamic acid is conferred.
[1249] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0186 or its homologs, e.g. a lysine decarboxylase
or its homologs,e.g. as indicated in Table I, columns 5 or 7, line
18, is increased preferably, an increase of the fine chemical and
of methionine is conferred.
[1250] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0161 or its homologs is increased, e.g. the
activity of a protein of the Helicobacter serine proteinase
superfamily is increased, preferably, of a protein having a
periplasmic serine protease (heat shock protein) activity, e.g. as
indicated in Table I, columns 5 or 7, line 356, is increased an
increase of the respective fine chemical, preferably of tryptophane
and of further amino acid(s) is conferred.
[1251] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0486 or its homologs is increased, e.g. the
activity of a protein of the membrane protein ybaT superfamily,
preferably a protein with a amino-acid/amine transport protein (of
the APC family) activity, e.g. as indicated in Table I, columns 5
or 7, line 357, is increased an increase of the respective fine
chemical, preferably of tryptophane and of further amino acid(s) is
conferred.
[1252] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1318 or its homologs is increased, e.g. the
activity of the inner membrane protein malK (with ATP-binding
cassette homology) superfamily, preferably a protein with a sugar
transport protein (of the ABC superfamily) activity, e.g. as
indicated in Table I, columns 5 or 7, line 358, is increased an
increase of the respective fine chemical, preferably of tryptophane
and of further amino acid(s) is conferred.
[1253] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2270 or its homologs is increased, e.g. the
activity of a transcriptional regulator, is increased, e.g. as
indicated in Table I, columns 5 or 7, line 359, is increased an
increase of the respective fine chemical, preferably of tryptophane
and of further amino acid(s) is conferred.
[1254] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3074 or its homologs is increased, e.g. the
activity of a tRNA synthetase or its homologs, e.g. transcriptional
regulator, e.g. as indicated in Table I, columns 5 or 7, line 360,
is increased an increase of the respective fine chemical,
preferably of tryptophane and of further amino acid(s) is
conferred.
[1255] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3983 or its homologs is increased, e.g. the
activity of a Escherichia coli ribosomal protein L11 superfamily,
preferably a protein with a 50S ribosomal subunit protein L12
activity, e.g.
[1256] as indicated in Table I, columns 5 or 7, line 361, is
increased an increase of the respective fine chemical, preferably
of tryptophane and of further amino acid(s) is conferred.
[1257] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YHR189W or its homologs is increased, e.g. the
activity of a peptidyl-tRNA hydrolase superfamily, preferably a
protein witha peptidyl-tRNA hydrolase activity, e.g. as indicated
in Table I, columns 5 or 7, line 362, is increased an increase of
the respective fine chemical, preferably of tryptophane and of
further amino acid(s) is conferred.
[1258] [0047.0.0.2] and [0048.0.0.2]: see [0047.0.0.0] and
[0048.0.0.0]
[1259] [0049.0.2.2] A protein having an activity conferring an
increase in the amount or level of the fine chemical preferably has
the structure of the polypeptide described herein, in particular of
a polypeptides comprising a consensus sequence as indicated in
Table IV, columns 7, lines 16 to 18 and/or lines 356 to 362 or of a
polypeptide as indicated in Table II, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362 or the functional homologues thereof as
described herein, or is encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 or
its herein described functional homologues and has the herein
mentioned activity.
[1260] [0050.0.2.2] For the purposes of the present invention, the
term "tryptophane" and "L-tryptophane" also encompass the
corresponding salts, such as, for example, tryptophane
hydrochloride or tryptophane sulfate. Preferably the term
tryptophane is intended to encompass the term L-tryptophane.
[1261] [0051.0.0.2] and [0052.0.0.2]: see [0051.0.0.0] and
[0052.0.0.0]
[1262] [0053.0.2.2] In one embodiment, the process of the present
invention comprises one or more of the following steps [1263] (a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention or the nucleic acid molecule or
the polypeptide used in the method of the invention, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 16 to 18 and/or lines 356 to 362 or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362, having herein-mentioned the
fine chemical-increasing activity; [1264] (b) stabilizing a mRNA
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, e.g. of a polypeptide having
an activity of a protein as indicated in Table II, column 3, lines
16 to 18 and/or lines 356 to 362 or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362, or of a mRNA encoding the polypeptide of the present
invention having herein-mentioned tryptophane increasing activity;
[1265] (c) increasing the specific activity of a protein conferring
the increased expression of a protein encoded by the nucleic acid
molecule of the invention or of the polypeptide of the present
invention or the polypeptide used in the method of the invention
having herein-mentioned tryptophane increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 16 to 18 and/or lines 356 to 362 or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362, or decreasing the
inhibiitory regulation of the polypeptide of the invention or the
polypeptide used in the method of the invention; [1266] (d)
generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention or of the polypeptide
of the invention or the polypeptide used in the method of the
invention having herein-mentioned tryptophane increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 16 to 18 and/or lines 356 to 362, or
its homologs activity, e.g. as indicated in Table II, columns 5 or
7, lines 16 to 18 and/or lines 356 to 362; [1267] (e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned tryptophane increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 16 to 18 and/or lines 356 to 362, or its and/or
lines 356 to 362, by adding one or more exogenous inducing factors
to the organisms or parts thereof; [1268] (f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned tryptophane increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 16 to 18 and/or lines 356 to 362, or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362; [1269] (g) increasing the
copy number of a gene conferring the increased expression of a
nucleic acid molecule encoding a polypeptide encoded by the nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention or the polypeptide of the invention or
the polypeptide used in the method of the invention having
herein-mentioned tryptophane increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 16 to 18 and/or lines 356 to 362, or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362; [1270] (h) Increasing the
expression of the endogenous gene encoding the polypeptide of the
invention or the polypeptide used in the method of the invention,
e.g. a polypeptide having an activity of a protein as indicated in
Table II, column 3, lines 16 to 18 and/or lines 356 to 362, or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362, copy by adding positive
expression or removing negative expression elements; e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; [1271] (i)
Modulating growth conditions of an organism in such a manner, that
the expression or activity of the gene encoding the protein of the
invention or the protein itself is enhanced for example
microorganisms or plants can be grown for of heat shock proteins,
which can lead an enhanced the fine chemical production; and/or
[1272] (j) selecting of organisms with expecially high activity of
the proteins of the invention from natural or from mutagenized
resources and breeding them into the target organisms, eg the elite
crops.
[1273] [0054.0.2.2] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of tryptophane after
increasing the expression or activity of the encoded polypeptide or
having the activity of a polypeptide having an activity of a
protein as indicated in Table II, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362 or its homologs activity, e.g. as indicated
in Table II, columns 5 or 7, lines 16 to 18 and/or lines 356 to
362.
[1274] [0055.0.0.2] to [0071.0.0.2]: see [0055.0.0.0] to
[0071.0.0.0]
[1275] [0072.0.2.2] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to tryptophane chorismic acid, anthralinic acid,
N-5'-Phosphoribosyl-anthranilate,
1-(o-Carboxyphenylamino)-1-deoxyribulose 5-phosphate.,
1-(Indol-3-yl)-glycerin-3-phosphate, and 5-hydroxytrytophane.
[1276] [0073.0.2.2] Accordingly, in one embodiment, the process
according to the invention relates to a process which
comprises:
[1277] providing a non-human organism, preferably a microorganism,
a non-human animal, a plant or animal cell, a plant or animal
tissue or a plant; [1278] (a) increasing an activity of a
polypeptide of the invention or the polypeptide used in the method
of the invention or a homolog thereof, e.g. as indicated in Table
II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the respective fine chemical in the organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [1279] (b) growing the organism,
preferably the microorganism, the non-human animal, the plant or
animal cell, the plant or animal tissue or the plant under
conditions which permit the production of the respective fine
chemical in the organism, preferably the microorganism, the plant
cell, the plant tissue or the plant; and [1280] (c) if desired,
recovering, optionally isolating, the free and/or bound the fine
chemical and, optionally further free and/or bound amino acids
synthetized by the organism, the microorganism, the non-human
animal, the plant or animal cell, the plant or animal tissue or the
plant.
[1281] [0074.0.0.2] to [0084.0.0.2]: see [0074.0.0.0] to
[0084.0.0.0]
[1282] [0085.0.2.2] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [1283] a) a nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362 a derivative thereof, or [1284] b) a genetic regulatory
element, for example a promoter, which is functionally linked to
the nucleic acid sequence as indicated in Table I, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362, or a derivative thereof, or
[1285] c) (a) and (b) is/are not present in its/their natural
genetic environment or has/have been modified by means of genetic
manipulation methods, it being possible for the modification to be,
by way of example, a substitution, addition, deletion, inversion or
insertion of one or more nucleotide. "Natural genetic environment"
means the natural chromosomal locus in the organism of origin or
the presence in a genomic library. In the case of a genomic
library, the natural, genetic environment of the nucleic acid
sequence is preferably at least partially still preserved. The
environment flanks the nucleic acid sequence at least on one side
and has a sequence length of at least 50 bp, preferably at least
500 bp, particularly preferably at least 1000 bp, very particularly
preferably at least 5000 bp.
[1286] [0086.0.0.2] and [0087.0.0.2]: see [0086.0.0.0] and
[0087.0.0.0]
[1287] [0088.0.2.2] In an advantageous embodiment of the invention,
the organism takes the form of a plant whose amino acid content is
modified advantageously owing to the nucleic acid molecule of the
present invention expressed. This is important for plant breeders
since, for example, the nutritional value of plants for monogastric
animals is limited by a few essential amino acids such as lysine,
threonine or methionine or tryptophane.
[1288] [0088.1.0.2] to [0097.0.0.2]: see [0088.1.0.0] to
[0097.0.0.0]
[1289] [0098.0.2.2] In a preferred embodiment, the fine chemical
(tryptophane) is produced in accordance with the invention and, if
desired, is isolated. The production of further amino acids such as
methionine, lysine and/or threonine mixtures of amino acid by the
process according to the invention is advantageous.
[1290] [0099.0.0.2] to [0102.0.0.2]: see [0099.0.0.0] to
[0102.0.0.0]
[1291] [0103.0.2.2] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [1292]
(a) nucleic acid molecule encoding, preferably at least the mature
form, of a polypeptide having a sequence as indicated in Table II,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362; [1293] (b)
nucleic acid molecule comprising, preferably at least the mature
form, of a nucleic acid molecule having a sequence as indicated in
Table I, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362;
[1294] (c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [1295] (d) nucleic acid molecule encoding a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [1296] (e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under r stringent hybridisation conditions and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [1297] (f) nucleic acid molecule encoding a
polypeptide, the polypeptide being derived by substituting,
deleting and/or adding one or more amino acids of the amino acid
sequence of the polypeptide encoded by the nucleic acid molecules
(a) to (d), preferably to (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[1298] (g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [1299] (h) nucleic acid molecule comprising a nucleic
acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers pairs having a sequence as indicated in Table III, columns
7, lines 16 to 18 and/or lines 356 to 362 and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [1300] (i) nucleic acid molecule encoding a
polypeptide which is isolated, e.g. from an expression library,
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (h), preferably to
(a) to (c), and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [1301] (j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequences as indicated in Table IV, column 7,
lines 16 to 18 and/or lines 356 to 362, and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; [1302] (k) nucleic acid molecule comprising one or more of
the nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of a polypeptide indicated in Table
II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362, and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; and [1303] (l) nucleic acid molecule
which is obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably to (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; or which comprises a sequence which is complementary
thereto.
[1304] [00103.1.0.0.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362, by one or more nucleotides. In one
embodiment, the nucleic acid molecule used in the process of the
invention does not consist of the sequence indicated in Table I A,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362: In one
embodiment, the nucleic acid molecule used in the process of the
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I A, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II A, columns 5 or 7, lines 16 to 18 and/or
lines 356 to 362.
[1305] [00103.2.0.0.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362, by one or more nucleotides. In one
embodiment, the nucleic acid molecule used in the process of the
invention does not consist of the sequence shown in indicated in
Table I B, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362:
In one embodiment, the nucleic acid molecule used in the process of
the invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I B, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II B, columns 5 or 7, lines 16 to 18 and/or
lines 356 to 362.
[1306] [0104.0.2.2] In one embodiment, the nucleic acid molecule of
the invention or used in the process of the invention distinguishes
over the sequence indicated in Table I, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362, by one or more nucleotides. In one
embodiment, the nucleic acid molecule of the present invention or
used in the process of the invention does not consist of the
sequence n indicated in Table I, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362.: In one embodiment, the nucleic acid
molecule of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to a sequence indicated in Table I,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 In another
embodiment, the nucleic acid molecule does not encode a polypeptide
of a sequence indicated in Table I, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362.
[1307] [0105.0.0.2] to [0107.0.0.2]: see [0105.0.0.0] to
[0107.0.0.0]
[1308] [0108.0.2.2] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362 nucleic acid molecules which are derived from a amino
acid sequences as indicated in Table II, columns 5 or 7, lines 16
to 18 and/or lines 356 to 362 or from polypeptides comprising the
consensus sequence as indicated in Table IV, column 7, lines 16 to
18 and/or lines 356 to 362 or their derivatives or homologues
encoding polypeptides with the enzymatic or biological activity of
a polypeptide as indicated in Table II, column 3, 5 or 7, lines 16
to 18 and/or lines 356 to 362 or e.g. conferring a increase of the
fine chemical after increasing its expression or activity are
advantageously increased in the process according to the
invention.
[1309] [0109.0.0.2]: see [0109.0.0.0]
[1310] [0110.0.2.2] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide of the invention or
the polypeptide used in the method of the invention or used in the
process of the invention, e.g. of a protein as indicated in Table
II, column 5, lines 16 to 18 and/or lines 356 to 362 or being
encoded by a nucleic acid molecule indicated in Table I, column 5,
lines 16 to 18 and/or lines 356 to 362 or of its homologs, e.g. as
indicated in Table II, column 7, lines 16 to 18 and/or lines 356 to
362 can be determined from generally accessible databases.
[1311] [0111.0.0.2]: see [0111.0.0.0]
[1312] [0112.0.2.2] The nucleic acid molecules used in the process
according to the invention take the form of isolated nucleic acid
sequences, which encode polypeptides with an activity of a
polypeptide as indicated in Table II, column 3, lines 16 to 18
and/or lines 356 to 362 or having the sequence of a polypeptide as
indicated in Table II, columns 5 and 7, lines 16 to 18 and/or lines
356 to 362 and conferring an tryptophane increase.
[1313] [0113.0.0.2] to [120.0.0.2]: see [0113.0.0.0] to
[0120.0.0.0]
[1314] [0121.0.2.2] However, it is also possible to use artificial
sequences, which differ in one or more bases from the nucleic acid
sequences found in organisms, or in one or more amino acid
molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 or the
functional homologues thereof as described herein, preferably
conferring above-mentioned activity, i.e. conferring a increase of
the fine chemical after increasing its activity.
[1315] [0122.0.0.2] to [0127.0.0.2]: see [0122.0.0.0] to
[0127.0.0.0]
[1316] [0128.0.2.2] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, column 7,
lines 16 to 18 and/or lines 356 to 362 by means of polymerase chain
reaction can be generated on the basis of a sequence as indicated
in Table I, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362
or the sequences derived from sequences as indicated in Table II,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362.
[1317] [0129.0.2.2] Moreover, it is possible to identify conserved
regions from various organisms by carrying out protein sequence
alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention or the polypeptide used in the method of the invention,
from which conserved regions, and in turn, degenerate primers can
be derived. Conserved region for the polypeptide of the invention
or the polypeptide used in the method of the invention are
indicated in the alignments shown in the figures. Conserved regions
are those, which show a very little variation in the amino acid in
one particular position of several homologs from different origin.
The consensus sequences indicated in Table IV, column 7, lines 16
to 18 and/or lines 356 to 362 are derived from said alignments.
[1318] [0130.0.2.2] to [0138.0.0.2]: see [0131.0.0.0] to
[0138.0.0.0]
[1319] [0139.0.2.2] Polypeptides having above-mentioned activity,
i.e. conferring a tryptophane increase, derived from other
organisms, can be encoded by other DNA sequences which hybridize to
a sequences indicated in Table I, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362 under relaxed hybridization conditions and
which code on expression for peptides having the tryptophane
increasing activity.
[1320] [0140.0.0.2] to [0146.0.0.2]: see [0140.0.0.0] to
[0146.0.0.0]
[1321] [0147.0.2.2] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 is one
which is sufficiently complementary to one of said nucleotide
sequences such that it can hybridize to one of said nucleotide
sequences thereby forming a stable duplex. Preferably, the
hybridisation is performed under stringent hybrization conditions.
However, a complement of one of the herein disclosed sequences is
preferably a sequence complement thereto according to the base
pairing of nucleic acid molecules well known to the skilled person.
For example, the bases A and G undergo base pairing with the bases
T and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[1322] [0148.0.2.2] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines
16 to 18 and/or lines 356 to 362, preferably of Table I B, columns
5 or 7, lines 16 to 18 and/or lines 356 to 362 or a functional
portion thereof and preferably has above mentioned activity, in
particular has the--fine-chemical-increasing activity after
increasing its activity or an activity of a product of a gene
encoding said sequence or its homologs.
[1323] [0149.0.2.2] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362, preferably of Table I B,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 or a portion
thereof and encodes a protein having above-mentioned activity and
as indicated in indicated in Table II, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362, preferably of Table II B, columns 5 or
7, lines 16 to 18 and/or lines 356 to 362, e.g. conferring an
increase of the fine chemical.
[1324] [0149.1.2.2] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362, preferably
of Table I B, columns 5 or 7, lines 16 to 18 and/or lines 356 to
362 has further one or more of the activities annotated or known
for a protein as indicated in Table II, column 3, lines 16 to 18
and/or lines 356 to 362.
[1325] [0150.0.2.2] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table I, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362,
preferably of Table I B, columns 5 or 7, lines 16 to 18 and/or
lines 356 to 362, for example a fragment which can be used as a
probe or primer or a fragment encoding a biologically active
portion of the polypeptide of the present invention or of a
polypeptide used in the process of the present invention, i.e.
having above-mentioned activity, e.g. conferring an increase of
tryptophane if its activity is increased. The nucleotide sequences
determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences indicated in Table I, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362, an anti-sense sequence of one of the
sequences indicated in Table I, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362, or naturally occurring mutants thereof.
Primers based on a nucleotide sequence of the invention can be used
in PCR reactions to clone homologues of the polypeptide of the
invention or of the polypeptide used in the process of the
invention, e.g. as the primers described in the examples of the
present invention, e.g. as shown in the examples. A PCR with the
primer pairs indicated in Table III, column 7, lines 16 to 18
and/or lines 356 to 362 will result in a fragment of a
polynucleotide sequence as indicated in Table I, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362.
[1326] [0151.0.0.2]: [see 0151.0.0.0]
[1327] [0152.0.2.2] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362 such that the protein or portion thereof
maintains the ability to participate in tryptophane production, in
particular a tryptophane increasing activity as mentioned above or
as described in the examples in plants or microorganisms is
comprised.
[1328] [0153.0.2.2] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362 such that the protein or portion thereof
is able to participate in the increase of tryptophane production.
In one embodiment, a protein or portion thereof as indicated in
Table II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362
has for example an activity of a polypeptide indicated in Table II,
column 3, lines 16 to 18 and/or lines 356 to 362.
[1329] [0154.0.2.2] In one embodiment, the nucleic acid molecule of
the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362 and has above-mentioned activity, e.g.
conferring preferably the increase of the fine chemical.
[1330] [0155.0.0.2] and [0156.0.0.2]: see [0155.0.0.0] to
[0156.0.0.0]
[1331] [0157.0.2.2] The invention further relates to nucleic acid
molecules that differ from one of a nucleotide sequences as
indicated in Table I, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362 (and portions thereof) due to degeneracy of the genetic
code and thus encode a polypeptide of the present invention, in
particular a polypeptide having above mentioned activity, e.g.
conferring an increase in tryptophane in a organism, e.g. as that
polypeptides comprising the consensus sequences as indicated in
Table IV, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 or
of the polypeptide as indicated in Table II, columns 5 or 7, lines
16 to 18 and/or lines 356 to 362 or their functional homologues.
Advantageously, the nucleic acid molecule of the invention or the
nucleic acid molecule used in the method of the invention
comprises, or in an other embodiment has, a nucleotide sequence
encoding a protein comprising, or in an other embodiment having, a
consensus sequences as indicated in Table IV, columns 5 or 7, lines
16 to 18 and/or lines 356 to 362 or of the polypeptide as indicated
in Table II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362
or the functional homologues. In a still further embodiment, the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention encodes a full length protein
which is substantially homologous to an amino acid sequence
comprising a consensus sequence as indicated in Table IV, column 7,
lines 16 to 18 and/or lines 356 to 362, or of a polypeptide as
indicated in Table II, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362 or the functional homologues thereof. However, in a
preferred embodiment, the nucleic acid molecule of the present
invention does not consist of a sequence as indicated in Table I,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362. Preferably
the nucleic acid molecule of the invention is a functional
homologue or identical to a nucleic acid molecule indicated in
Table I B, columns 5 or 7, lines 16 to 18 and/or lines 356 to
362.
[1332] [0158.0.0.2] to [0160.0.0.2]: see [0158.0.0.0] to
[0160.0.0.0]
[1333] [0161.0.2.2] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362. The nucleic acid molecule is preferably at
least 20, 30, 50, 100, 250 or more nucleotides in length.
[1334] [0162.0.0.2]: see [0162.0.0.0]
[1335] [0163.0.2.2] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 16 to 18 and/or
lines 356 to 362 corresponds to a naturally-occurring nucleic acid
molecule of the invention. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein). Preferably, the nucleic acid molecule encodes a natural
protein having above-mentioned activity, e.g. conferring the fine
chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[1336] [0164.0.0.2]: see [0164.0.0.0]
[1337] [0165.0.2.2] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362.
[1338] [0166.0.0.2] and [0167.0.0.2]: see [0166.0.0.0] and
[0167.0.0.0][0168.0.2.2]
[1339] Accordingly, the invention relates to nucleic acid molecules
encoding a polypeptide having above-mentioned activity, e.g.
conferring an increase in the fine chemical in an organisms or
parts thereof that contain changes in amino acid residues that are
not essential for said activity. Such polypeptides differ in amino
acid sequence from a sequence contained in as sequence as indicated
in Table II, columns 5 or 7, lines 16 to 18 and/or lines 356 to
362, preferably of Table II B, column 7, lines 16 to 18 and/or
lines 356 to 362 yet retain said activity described herein. The
nucleic acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table II, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362, preferably of Table II B, column 7, lines 16 to 18
and/or lines 356 to 362 and is capable of participation in the
increase of production of the fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table II, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362, preferably of Table II B, column 7, lines
16 to 18 and/or lines 356 to 362 more preferably at least about 70%
identical to one of the sequences as indicated in Table II, columns
5 or 7, lines 16 to 18 and/or lines 356 to 362, preferably of Table
II B, column 7, lines 16 to 18 and/or lines 356 to 362 even more
preferably at least about 80%, 90% or 95% homologous to a sequence
as indicated in Table II, columns 5 or 7, lines 16 to 18 and/or
lines 356 to 362, preferably of Table II B, column 7, lines 16 to
18 and/or lines 356 to 362 and most preferably at least about 96%,
97%, 98%, or 99% identical to the sequence as indicated in Table
II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362,
preferably of Table II B, column 7, lines 16 to 18 and/or lines 356
to 362.
[1340] [0169.0.0.2] to [0172.0.0.2]: see [0169.0.0.0] to
[0172.0.0.0]
[1341] [0173.0.2.2] For example a sequence which has a 80% homology
with sequence SEQ ID No: 732 at the nucleic acid level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID No: 732 by the above Gap program algorithm with the
above parameter set, has a 80% homology.
[1342] [0174.0.0.2]: see [0174.0.0.0]
[1343] [0175.0.2.2] For example a sequence which has a 80% homology
with sequence SEQ ID No: 733 at the protein level is understood as
meaning a sequence which, upon comparison with the sequence SEQ ID
No: 733 by the above program algorithm with the above parameter
set, has a 80% homology.
[1344] [0176.0.2.2] Functional equivalents derived from one of the
polypeptides as indicated in Table II, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362 according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 according to
the invention and are distinguished by essentially the same
properties as a polypeptide as indicated in Table II, columns 5 or
7, lines 16 to 18 and/or lines 356 to 362.
[1345] [0177.0.2.2] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362, preferably of Table I B, column 7,
lines 16 to 18 and/or lines 356 to 362 according to the invention
by substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of a polypeptides as indicated in Table II,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362, preferably
of Table I B, column 7, lines 16 to 18 and/or lines 356 to 362
according to the invention and encode polypeptides having
essentially the same properties as a polypeptide as indicated in
Table II, columns 5 or 7, Hines 16 to 18 and/or lines 356 to 362,
preferably of Table I B, column 7, lines 16 to 18 and/or lines 356
to 362.
[1346] [0178.0.0.2]: see [0178.0.0.0]
[1347] [0179.0.2.2] A nucleic acid molecule encoding an homologous
to a protein sequence of as indicated in Table II, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362, preferably of Table II B,
column 7, lines 16 to 18 and/or lines 356 to 362 can be created by
introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of the nucleic acid molecule
of the present invention, in particular as indicated in Table I,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 such that
one or more amino acid substitutions, additions or deletions are
introduced into the encoded protein. Mutations can be introduced
into the encoding sequences of a sequences as indicated in Table I,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[1348] [0180.0.0.2] to [0183.0.0.2]: see [0180.0.0.0] to
[0183.0.0.0]
[1349] [0184.0.2.2] Homologues of the nucleic acid sequences used,
with a sequence as indicated in Table I, columns 5 or 7, lines 16
to 18 and/or lines 356 to 362, preferably of Table I B, column 7,
lines 16 to 18 and/or lines 356 to 362, or of the nucleic acid
sequences derived from a sequences as indicated in Table II,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362, preferably
of Table I B, column 7, lines 16 to 18 and/or lines 356 to 362
comprise also allelic variants with at least approximately 30%,
35%, 40% or 45% homology, by preference at least approximately 50%,
60% or 70%, more preferably at least approximately 90%, 91%, 92%,
93%, 94% or 95% and even more preferably at least approximately
96%, 97%, 98%, 99% or more homology with one of the nucleotide
sequences shown or the abovementioned derived nucleic acid
sequences or their homologues, derivatives or analogues or parts of
these. Allelic variants encompass in particular functional variants
which can be obtained by deletion, insertion or substitution of
nucleotides from the sequences shown, preferably from a sequence as
indicated in Table I, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362 or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[1350] [0185.0.2.2] In one embodiment of the present invention, the
nucleic acid molecule of the invention or used in the process of
the invention comprises one or more sequences as indicated in Table
I, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362,
preferably of Table I B, column 7, lines 16 to 18 and/or lines 356
to 36. In one embodiment it is preferred that the nucleic acid
molecule comprises as little as possible other nucleotide sequences
not shown in any one of sequences as indicated in Table I, columns
5 or 7, lines 16 to 18 and/or lines 356 to 362, preferably of Table
I B, column 7, lines 16 to 18 and/or lines 356 to 362. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule use in the process of the invention is identical to a
sequences as indicated in Table I, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362, preferably of Table I B, column 7, lines
16 to 18 and/or lines 356 to 362.
[1351] [0186.0.2.2] Also preferred is that one or more nucleic acid
molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 16 to 18 and/or lines 356 to 362, preferably of Table
II B, column 7, lines 16 to 18 and/or lines 356 to 362. In one
embodiment, the nucleic acid molecule encodes less than 150, 130,
100, 80, 60, 50, 40 or 30 further amino acids. In a further
embodiment, the encoded polypeptide comprises less than 20, 15, 10,
9, 8, 7, 6 or 5 further amino acids. In one embodiment, the encoded
polypeptide used in the process of the invention is identical to
the sequences as indicated in Table II, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362, preferably of Table II B, column 7,
lines 16 to 18 and/or lines 356 to 362.
[1352] [0187.0.2.2] In one embodiment, the nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table II, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362, preferably of Table II B,
column 7, lines 16 to 18 and/or lines 356 to 362 and comprises less
than 100 further nucleotides. In a further embodiment, said nucleic
acid molecule comprises less than 30 further nucleotides. In one
embodiment, the nucleic acid molecule used in the process is
identical to a coding sequence encoding a sequences as indicated in
Table II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362,
preferably of Table II B, column 7, lines 16 to 18 and/or lines 356
to 362.
[1353] [0188.0.2.2] Polypeptides (=proteins), which still have the
essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide as indicated in Table II, columns 5 or 7, lines 16
to 18 and/or lines 356 to 362, preferably compared to a sequence as
indicated in Table II, column 3 and 5, lines 16 to 18 and/or lines
356 to 362, and expressed under identical conditions.
[1354] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, columns 7, lines 16 to 18 and/or lines 356 to 362
[1355] [0189.0.2.2] Homologues of a sequences as indicated in Table
I, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 or of a
derived sequences as indicated in Table II, columns 5 or 7, lines
16 to 18 and/or lines 356 to 362 also mean truncated sequences,
cDNA, single-stranded DNA or RNA of the coding and noncoding DNA
sequence. Homologues of said sequences are also understood as
meaning derivatives which comprise noncoding regions such as, for
example, UTRs, terminators, enhancers or promoter variants. The
promoters upstream of the nucleotide sequences stated can be
modified by one or more nucleotide substitution(s), insertion(s)
and/or deletion(s) without, however, interfering with the
functionality or activity either of the promoters, the open reading
frame (=ORF) or with the 3'-regulatory region such as terminators
or other 3' regulatory regions, which are far away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[1356] [0190.0.0.2] to [0203.0.0.2]: see [0190.0.0.0] to
[0203.0.0.0]
[1357] [0204.0.2.2] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule which comprises a nucleic acid
molecule selected from the group consisting of: [1358] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362, preferably of Table II B, column 7,
lines 16 to 18 and/or lines 356 to 362 or a fragment thereof
conferring an increase in the amount of the fine chemical in an
organism or a part thereof [1359] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule as indicated in Table II, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362, preferably of Table II B, column 7, lines
16 to 18 and/or lines 356 to 362 or a fragment thereof conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [1360] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [1361] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[1362] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [1363] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [1364] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [1365] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table III, column 7, lines 16 to 18 and/or lines
356 to 362 and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [1366] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[1367] j) nucleic acid molecule which encodes a polypeptide
comprising the consensus sequence as indicated in Table IV, column
7, lines 16 to 18 and/or lines 356 to 362 and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [1368] k) nucleic acid molecule encoding the amino
acid sequence of a polypeptide encoding a domain of a polypeptide
as indicated in Table II, columns 5 or 7, lines 16 to 18 and/or
lines 356 to 362, preferably of Table II B, column 7, lines 16 to
18 and/or lines 356 to 362 and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; and [1369]
l) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment of at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (h) or of a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362, preferably of Table II B,
column 7, lines 16 to 18 and/or lines 356 to 362 or a nucleic acid
molecule encoding, preferably at least the mature form of, the
polypeptide as indicated in Table II, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362, preferably of Table II B, column 7,
lines 16 to 18 and/or lines 356 to 362 and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over the sequence indicated in Table IA
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362, by one or
more nucleotides. In one embodiment, the nucleic acid molecule does
not consist of the sequence shown and in indicated in Table I A or
I B, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362: In one
embodiment, the nucleic acid molecule is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to a sequence indicated in Table I A
or I B, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362. In
another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II A or II B, columns
5 or 7, lines 16 to 18 and/or lines 356 to 362. In an other
embodiment, the nucleic acid molecule of the present invention is
at least 30%, 40%, 50%, or 60% identical and less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence indicated in
Table I A or I B, columns 5 or 7, lines 16 to 18 and/or lines 356
to 362. In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table II A or II B,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362.
Accordingly, in one embodiment, the nucleic acid molecule of the
differs at least in one or more residues from a nucleic acid
molecule indicated in Table I A or I B, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362. Accordingly, in one embodiment, the
nucleic acid molecule of the present invention encodes a
polypeptide, which differs at least in one or more amino acids from
a polypeptide indicated in Table II A or I B, columns 5 or 7, lines
16 to 18 and/or lines 356 to 362. In another embodiment, a nucleic
acid molecule indicated in Table I A or I B, columns 5 or 7, lines
16 to 18 and/or lines 356 to 362 does not encode a protein of a
sequence indicated in Table II A or II B, columns 5 or 7, lines 16
to 18 and/or lines 356 to 362. Accordingly, in one embodiment, the
protein encoded by a sequences of a nucleic acid according to (a)
to (l) does not consist of a sequence as indicated in Table II A or
II B, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362. In a
further embodiment, the protein of the present invention is at
least 30%, 40%, 50%, or 60% identical to a protein sequence
indicated in Table II A or II B, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362 and less than 100%, preferably less than
99.999%, 99.99% or 99.9%, more preferably less than 99%, 985, 97%,
96% or 95% identical to a sequence as indicated in Table I A or II
B, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362.
[1370] [0205.0.0.2] to [0226.0.0.2]: see [0205.0.0.0] to
[0226.0.0.0]
[1371] [0227.0.2.2] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorgansim.
[1372] In addition to a sequence as indicated in Table I, columns 5
or 7, lines 16 to 18 and/or lines 356 to 362 or its derivatives, it
is advantageous additionally to express and/or mutate further genes
in the organisms. Especially advantageously, additionally at least
one further gene of the amino acid biosynthetic pathway such as for
L-lysine, L-threonine and/or L-methionine or L-tryptophane is
expressed in the organisms such as plants or microorganisms. It is
also possible that the regulation of the natural genes has been
modified advantageously so that the gene and/or its gene product is
no longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all.
[1373] In addition it might be advantageously to combine a
sequences as indicated in Table I, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362 with genes which generally support or
enhances to growth or yield of the target organismn, for example
genes which lead to faster growth rate of microorganisms or genes
which produces stress-, pathogen, or herbicide resistant
plants.
[1374] [0228.0.0.2] to [0230.0.0.2]: see [0228.0.0.0] to
[0230.0.0.0]
[1375] [0231.0.0.2] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a tryptophane degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[1376] [0232.0.0.2] to [0282.0.0.2]: see [0232.0.0.0] to
[0282.0.0.0]
[1377] [0283.0.2.2] Moreover, a native polypeptide conferring the
increase of the fine chemical in an organism or part thereof can be
isolated from cells (e.g., endothelial cells), for example using
the antibody of the present invention as described below, in
particular, an antibody against a protein as indicated in Table II,
column 3, lines lines 16 to 18 and/or lines 356 to 362 E.g. an
antibody against a polypeptide as indicated in Table II, columns 5
or 7, lines 16 to 18 and/or lines 356 to 362, which can be produced
by standard techniques utilizing polypeptides comprising or
consisting of above mentioned sequences, e.g. the polypeptide of
the present invention or fragment thereof, Preferred are monoclonal
antibodies, specifically binding to polypeptide as indicated in
Table II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362.
[0284.0.0.2] see [0284.0.0.0]
[1378] [0285.0.2.2] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 or as
encoded by a nucleic acid molecule as indicated in Table I, columns
5 or 7, lines 16 to 18 and/or lines 356 to 362 or functional
homologues thereof.
[1379] [0286.0.2.2] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 16 to 18 and/or lines 356 to 362 and in
one another embodiment, the present invention relates to a
polypeptide comprising or consisting of a consensus sequence as
indicated in Table IV, column 7, lines 16 to 18 and/or lines 356 to
362 whereby 20 or less, preferably 15 or 10, preferably 9, 8, 7, or
6, more preferred 5 or 4, even more preferred 3, even more
preferred 2, even more preferred 1, most preferred 0 of the amino
acids positions indicated can be replaced by any amino acid or, in
an further embodiment, can be replaced and/or absent. In one
embodiment, the present invention relates to the method of the
present invention comprising a polypeptide or to a polypeptide
comprising more than one consensus sequences as indicated in Table
IV, column 7, lines 16 to 18 and/or lines 356 to 362.
[1380] amino acidamino acid[0287.0.0.2] to [290.0.0.2]: see
[0287.0.0.0] to [0290.0.0.0]
[1381] [0291.0.2.2] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[1382] Accordingly, in one embodiment, the present invention
relates to a polypeptide comprising or consisting of plant or
microorganism specific consensus sequences. In one embodiment, said
polypeptide of the invention distinguishes over a sequence as
indicated in Table II A or IIB, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362 by one or more amino acids. In one
embodiment, polypeptide distinguishes form a sequence as indicated
in Table II A or IIB, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362 by more than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, even more
preferred are more than 40, 50, or 60 amino acids and, preferably,
the sequence of the polypeptide of the invention distinguishes from
a sequence as indicated in Table II A or II B, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362 by not more than 80% or 70%
of the amino acids, preferably not more than 60% or 50%, more
preferred not more than 40% or 30%, even more preferred not more
than 20% or 10%. In an other embodiment, said polypeptide of the
invention does not consist of a sequence as indicated in Table II A
or II B, columns 5 or 7, lines 16 to 18 and/or lines 356 to
362.
[1383] [0292.0.0.2]: see [0292.0.0.0]
[1384] [0293.0.2.2] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the process
of the invention.
[1385] In one embodiment, the polypeptide of the invention has a
sequence which distinguishes from a sequence as indicated in Table
II A or II B, columns 5 or 7, lines 16 to 18 and/or lines 356 to
362 by one or more amino acids. In an other embodiment, said
polypeptide of the invention does not consist of the sequence as
indicated in Table II A or II B, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362. In a further embodiment, said polypeptide
of the present invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical. In one embodiment, said polypeptide does not
consist of the sequence encoded by a nucleic acid molecules as
indicated in Table I A or IB, columns 5 or 7, lines 16 to 18 and/or
lines 356 to 362.
[1386] [0294.0.2.2] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 16 to 18 and/or lines 356 to
362, which distinguishes over a sequence as indicated in Table II A
or II B, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 by
one or more amino acids, preferably by more than 5, 6, 7, 8 or 9
amino acids, preferably by more than 10, 15, 20, 25 or 30 amino
acids, even more preferred are more than 40, 50, or 60 amino acids
but even more preferred by less than 70% of the amino acids, more
preferred by less than 50%, even more preferred my less than 30% or
25%, more preferred are 20% or 15%, even more preferred are less
than 10%.
[1387] [0295.0.0.2] to [0297.0.0.2]: see [0295.0.0.0] to
[0297.0.0.0]
[1388] [0297.1.0.2] Non-polypeptide of the invention-chemicals are
e.g. polypeptides having not the activity and/or amino acid
sequence of a polypeptide indicated in Table II, columns 3, 5 or 7,
lines 16 to 18 and/or lines 356 to 362.
[1389] [0298.0.2.2] A polypeptide of the invention can participate
in the process of the present invention. The polypeptide or a
portion thereof comprises preferably an amino acid sequence which
is sufficiently homologous to an amino acid sequence as indicated
in Table II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362
such that the protein or portion thereof maintains the ability to
confer the activity of the present invention. The portion of the
protein is preferably a biologically active portion as described
herein. Preferably, the polypeptide used in the process of the
invention has an amino acid sequence identical to a sequence as
indicated in Table II, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362.
[1390] [0299.0.2.2] Further, the polypeptide can have an amino acid
sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, Hines
16 to 18 and/or lines 356 to 362. The preferred polypeptide of the
present invention preferably possesses at least one of the
activities according to the invention and described herein. A
preferred polypeptide of the present invention includes an amino
acid sequence encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions, to a nucleotide
sequence as indicated in Table I, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362 or which is homologous thereto, as defined
above.
[1391] [0300.0.2.2] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 in amino
acid sequence due to natural variation or mutagenesis, as described
in detail herein. Accordingly, the polypeptide comprise an amino
acid sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%,
65% or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 16 to 18 and/or lines 356 to
362.
[1392] [0301.0.0.2]: see [0301.0.0.0]
[1393] [0302.0.2.2] Biologically active portions of an polypeptide
of the present invention include peptides comprising amino acid
sequences derived from the amino acid sequence of the polypeptide
of the present invention or used in the process of the present
invention, e.g., an amino acid sequence as indicated in Table II,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 or the amino
acid sequence of a protein homologous thereto, which include fewer
amino acids than a full length polypeptide of the present invention
or used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[1394] [0303.0.0.2]: see [0303.0.0.0]
[1395] [0304.0.2.2] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 16 to 18 and/or lines 356 to 362 but having
differences in the sequence from said wild-type protein. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity in relation to the wild type protein.
[1396] [0305.0.0.2] to [0306.0.0.2]: see [0305.0.0.0] to
[0306.0.0.0]
[1397] [00306.1.2.2] Preferrably, the compound is a composition
comrising the tryptophane or a recovered tryptophane, in
particular, the fine chemical, free or in protein-bound form.
[1398] [0307.0.0.2]: to [0308.0.0.2]: see [0305.0.0.0] to
[03086.0.0.0]
[1399] [0309.0.2.2] In one embodiment, a reference to a "protein
(=polypeptide)" of the invention or as indicated in Table II,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 refers to a
polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas a "non-polypeptide of the invention" or "other
polypeptide"--not being indicated in Table II, columns 5 or 7,
lines 16 to 18 and/or lines 356 to 362 refers to a polypeptide
having an amino acid sequence corresponding to a protein which is
not substantially homologous a polypeptide of the invention,
preferably which is not substantially homologous to a as indicated
in Table II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362
e.g., a protein which does not confer the activity described herein
or annotated or known for as indicated in Table II, column 3, lines
16 to 18 and/or lines 356 to 362 and which is derived from the same
or a different organism. In one embodiment a "non-polypeptide of
the invention" or "other polypeptide" not being indicate in Table
II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 does not
confer an increase of the fine chemical in an organism or part
thereof.
[1400] [0310.0.0.2] to [334.0.0.2]: see [0310.0.0.0] to
[0334.0.0.0]
[1401] [0335.0.2.2] The dsRNAi method has proved to be particularly
effective and advantageous for reducing the expression of a nucleic
acid sequences as indicated in Table II, columns 5 or 7, lines 16
to 18 and/or lines 356 to 362 and/or homologs thereof. As described
inter alia in WO 99/32619, dsRNAi approaches are clearly superior
to traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table I,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 and/or
homologs thereof. In a double-stranded RNA molecule for reducing
the expression of an protein encoded by a nucleic acid sequence of
one of the sequences as indicated in Table I, columns 5 or 7, lines
16 to 18 and/or lines 356 to 362 and/or homologs thereof, one of
the two RNA strands is essentially identical to at least part of a
nucleic acid sequence, and the respective other RNA strand is
essentially identical to at least part of the complementary strand
of a nucleic acid sequence.
[1402] [0336.0.0.2] to [0342.0.0.2]: see [0336.0.0.0] to
[0342.0.0.0]
[1403] [0343.0.2.2] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 16 to 18
and/or lines 356 to 362 or its homolog is not necessarily required
in order to bring about effective reduction in the expression. The
advantage is, accordingly, that the method is tolerant with regard
to sequence deviations as may be present as a consequence of
genetic mutations, polymorphisms or evolutionary divergences. Thus,
for example, using the dsRNA, which has been generated starting
from a sequence as indicated in Table I, columns 5 or 7, lines 16
to 18 and/or lines 356 to 362 or homologs thereof of the one
organism, may be used to suppress the corresponding expression in
another organism.
[1404] [0344.0.0.2] to [0361.0.0.2]: see [0344.0.0.0] to
[0361.0.0.0]
[1405] [0362.0.2.2] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the fine chemical in
a cell or an organism or a part thereof, e.g. the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention, the nucleic acid construct of the
invention, the antisense molecule of the invention, the vector of
the invention or a nucleic acid molecule encoding the polypeptide
of the invention, e.g. the polypeptide as indicated in Table II,
columns 5 or 7, lines 16 to 18 and/or lines 356 to 362. Due to the
above mentioned activity the fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or the polypeptide used in the method of the invention or
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof. In one
embodiment transgenic for a polypeptide having an activity of a
polypeptide as indicated in Table II, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362 means herein that due to modulation or
manipulation of the genome, an activity as annotated for a
polypeptide as indicated in Table II, columns 3, lines 16 to 18
and/or lines 356 to 362, e.g. having a sequence as indicated in
Table II, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 is
increased in a cell or an organism or a part thereof. Examples are
described above in context with the process of the invention.
[1406] [0363.0.0.2] to [0382.0.0.2]: see [0365.0.0.0] to
[0382.0.0.0]
[1407] [0383.0.2.2] For preparing aromatic compound-containing fine
chemicals, in particular the fine chemical, it is possible to use
as aromat source organic aromatic-containing compounds such as, for
example, benzene, naphthaline, indole, pyrrole, furen, oxazole,
imidazole, thiophene, pyrridin, pyrrimidine or else organic
aromatic compounds such as benzoic acid and chorismic, shikimic,
aminobenzoic, kynurenic acids or pyridoxidal.
[1408] [0384.0.0.2]: see [0384.0.0.0]
[1409] [0385.0.2.2] The fermentation broths obtained in this way,
containing in particular L-tryptophane, L-methionine, L-threonine
and/or L-lysine, normally have a dry matter content of from 7.5 to
25% by weight. Sugar-limited fermentation is additionally
advantageous, at least at the end, but especially over at least 30%
of the fermentation time. This means that the concentration of
utilizable sugar in the fermentation medium is kept at, or reduced
to, 0 to 3 g/l during this time. The fermentation broth is then
processed further. Depending on requirements, the biomass can be
removed entirely or partly by separation methods, such as, for
example, centrifugation, filtration, decantation or a combination
of these methods, from the fermentation broth or left completely in
it. The fermentation broth can then be thickened or concentrated by
known methods, such as, for example, with the aid of a rotary
evaporator, thin-film evaporator, falling film evaporator, by
reverse osmosis or by nanofiltration. This concentrated
fermentation broth can then be worked up by freeze-drying, spray
drying, spray granulation or by other processes.
[1410] [0386.0.0.2] to [392.0.0.2]: see [0386.0.0.0] to
[0392.0.0.0]
[1411] [0393.0.2.2] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [1412] (a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [1413] (b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table I, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362,
preferably of Table I B, column 7, lines 16 to 18 and/or lines 356
to 362 and, optionally, isolating the full length cDNA clone or
complete genomic clone; [1414] (c) introducing the candidate
nucleic acid molecules in host cells, preferably in a plant cell or
a microorganism, appropriate for producing the fine chemical;
[1415] (d) expressing the identified nucleic acid molecules in the
host cells; [1416] (e) assaying the fine chemical level in the host
cells; and [1417] (f) identifying the nucleic acid molecule and its
gene product which expression confers an increase in the fine
chemical level in the host cell after expression compared to the
wild type.
[1418] [0394.0.0.2] to [0399.0.0.2]: see [0394.0.0.0] to
[0399.0.0.0]
[1419] [0399.1.2.2] One can think to screen for increased
production of the fine chemical by for example searching for a
resistance to a drug blocking the synthesis of the fine chemical
and looking whether this effect is dependent on the activity or
expression of a polypeptide as indicated in Table II, columns 5 or
7, lines 16 to 18 and/or lines 356 to 362 or a homolog thereof,
e.g. comparing the phenotype of nearly identical organisms with low
and high activity of a protein as indicated in Table II, columns 5
or 7, lines 16 to 18 and/or lines 356 to 362 after incubation with
the drug.
[1420] [0400.0.0.2] to [0416.0.0.2]: see [0400.0.0.0] to
[0416.0.0.0]
[1421] [0417.0.2.2] The nucleic acid molecule of the invention, the
vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the amino acid production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect plants against herbicides, which
block the amino acid, in particular the fine chemical, synthesis in
said plant. Examples of herbicides blocking the amino acid
synthesis in plants are for example sulfonylurea and imidazolinone
herbicides which catalyze the first step in branched-chain amino
acid biosynthesis.
[1422] [0418.0.0.2] to [0423.0.0.2]: see [0418.0.0.0] to
[0423.0.0.0][0424.0.2.2]
[1423] Accordingly, the nucleic acid of the invention, the
polypeptide of the invention, the nucleic acid construct of the
invention, the organisms, the host cell, the microorgansims, the
plant, plant tissue, plant cell, or the part thereof of the
invention, the vector of the invention, the agonist identified with
the method of the invention, the nucleic acid molecule identified
with the method of the present invention, can be used for the
production of the fine chemical or of the fine chemical and one or
more other amino acids, in particular methionine, threonine,
alanine, glutamine, glutamic acid, valine, asparagine,
phenylalanine, leucine, proline, Tryptophan tyrosine, isoleucine
and arginine.
[1424] Accordingly, the nucleic acid of the invention, or the
nucleic acid molecule identified with the method of the present
invention or the complement sequences thereof, the polypeptide of
the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the fine chemical in a organism or part thereof, e.g.
in a cell.
[1425] [0425.0.0.2] to [0430.0.0.2]: see [0425.0.0.0] to
[0430.0.0.0]
[0431.0.2.2] Example 1
Cloning SEQ ID No: 732 in Escherichia coli
[1426] [0432.0.0.0] to [0460.0.0.2]: see [0433.0.0.0] to
[0460.0.0.0]
[0461.0.2.2] Example 10
Cloning SEQ ID NO: 732 for the Expression in Plants
[1427] [0462.0.0.2] see [0462.0.0.0]
[1428] [0463.0.2.2] SEQ ID NO: 732 is amplified by PCR as described
in the protocol of the Pfu Turbo or DNA Herculase polymerase
(Stratagene).
[1429] [0464.0.0.2] to [0466.0.0.2]: see [0464.0.0.0] to
[0466.0.0.0]
[1430] [0467.0.2.2] The following primer sequences were selected
for the gene SEQ ID No: 732:
TABLE-US-00012 i) forward primer (SEQ ID No: 734)
ATGGATAGTACGAATTTGAACAAACG ii) reverse primer (SEQ ID No: 735)
TTAGAGTATTTCCAGATCTGAATCTG
[1431] [0468.0.0.2] to [0479.0.0.2]: see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.2.2] Example 11
Generation of Transgenic Plants which Express SEQ ID No: 732
[1432] [0481.0.0.2] to [0513.0.0.2]: see [0481.0.0.0] to
[0513.0.0.0]
[1433] [0514.0.2.2] As an alternative, the amino acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55).
[1434] The results of the different plant analyses can be seen from
the table which follows:
TABLE-US-00013 TABLE 1 ORF Annotation Metabolite Min Max Method
YER173W Checkpoint protein, Tryptophane 1.27 2.78 LC involved in
the activation of the DNA damage and meiotic pachytene checkpoints
YGR104C RNA polymerase II Tryptophane 1.32 1.84 LC suppressor
protein SRB5 - yeast; Suppressor of RNA polymerase B SRB5 b0186
lysine decarboxylase Tryptophane 1.32 2.46 LC
TABLE-US-00014 TABLE 1b ORF MetChemID Metabolite Method Min Max
b0161 10000035 Tryptophane LC 1.93 3.78 b0486 10000035 Tryptophane
LC 1.42 4.35 b1318 10000035 Tryptophane LC 2.36 4.30 b2270 10000035
Tryptophane LC 1.33 1.79 b3074 10000035 Tryptophane LC 1.33 1.79
b3983 10000035/ Tryptophane LC + GC 1.33 4.87 30000016 YHR189W
10000035 Tryptophane LC 1.31 1.66
[1435] [0515.0.0.2] to [0552.2.0.2]: see [0515.0.0.0] to
[0552.2.0.0]
[1436] [0553.0.2.2]
1. A process for the production of tryptophane, which comprises
[1437] (a) increasing or generating the activity of a protein as
indicated in Table II, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362 or a functional equivalent thereof in a non-human
organism, or in one or more parts thereof; and [1438] (b) growing
the organism under conditions which permit the production of
tryptophane in said organism. 2. A process for the production of
tryptophane, comprising the increasing or generating in an organism
or a part thereof the expression of at least one nucleic acid
molecule comprising a nucleic acid molecule selected from the group
consisting of: [1439] (a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362 or a fragment thereof, which confers an
increase in the amount of tryptophane in an organism or a part
thereof; [1440] (b) nucleic acid molecule comprising of the nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362; [1441] (c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as a result of the degeneracy
of the genetic code and conferring an increase in the amount of
tryptophane in an organism or a part thereof; [1442] (d) nucleic
acid molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of tryptophane in an organism or a part thereof;
[1443] (e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of tryptophane
in an organism or a part thereof; [1444] (f) nucleic acid molecule
which encompasses a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers or primer pairs as indicated in Table
III, column 7, lines 16 to 18 and/or lines 356 to 362 and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [1445] (g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
tryptophane in an organism or a part thereof; [1446] (h) nucleic
acid molecule encoding a polypeptide comprising a consensus
sequence as indicated in Table IV, column 7, lines 16 to 18 and/or
lines 356 to 362 and conferring an increase in the amount of the
fine chemical in an organism or a part thereof; [1447] (i) nucleic
acid molecule which is obtainable by screening a suitable nucleic
acid library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof.
or comprising a sequence which is complementary thereto. 3. The
process of claim 1 or 2, comprising recovering of the free or bound
tryptophane. 4. The process of any one of claims 1 to 3, comprising
the following steps: [1448] (a) selecting an organism or a part
thereof expressing a polypeptide encoded by the nucleic acid
molecule characterized in claim 2; [1449] (b) mutagenizing the
selected organism or the part thereof; [1450] (c) comparing the
activity or the expression level of said polypeptide in the
mutagenized organism or the part thereof with the activity or the
expression of said polypeptide of the selected organisms or the
part thereof; [1451] (d) selecting the mutated organisms or parts
thereof, which comprise an increased activity or expression level
of said polypeptide compared to the selected organism or the part
thereof; [1452] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [1453] (f) recovering, and
optionally isolating, the free or bound tryptophane produced by the
selected mutated organisms or parts thereof. 5. The process of any
one of claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [1454] (a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 16 to
18 and/or lines 356 to 362 or a fragment thereof, which confers an
increase in the amount of tryptophane in an organism or a part
thereof; [1455] (b) nucleic acid molecule comprising of a nucleic
acid as indicated in Table I, columns 5 or 7, lines 16 to 18 and/or
lines 356 to 362; [1456] (c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as a result of the degeneracy of the
genetic code and conferring an increase in the amount of
tryptophane in an organism or a part thereof; [1457] (d) nucleic
acid molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of tryptophane in an organism or a part thereof;
[1458] (e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of tryptophane
in an organism or a part thereof; [1459] (f) nucleic acid molecule
which encompasses a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers or primer pairs as indicated in Table
III, 7, lines 16 to 18 and/or lines 356 to 362 and conferring an
increase in the amount of tryptophane in an organism or a part
thereof; [1460] (g) nucleic acid molecule encoding a polypeptide
which is isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of tryptophane in an
organism or a part thereof; [1461] (h) nucleic acid molecule
encoding a polypeptide comprising the consensus sequence as
indicated in Table IV, column 7, lines 16 to 18 and/or lines 356 to
362 and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; and [1462] (i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof.
whereby the nucleic acid molecule distinguishes over the sequence
as indicated in Table IA, columns 5 or 7, lines 16 to 18 and/or
lines 356 to 362 by one or more nucleotides. 7. A nucleic acid
construct which confers the expression of the nucleic acid molecule
of claim 6, comprising one or more regulatory elements. 8. A vector
comprising the nucleic acid molecule as claimed in claim 6 or the
nucleic acid construct of claim 7. 9. The vector as claimed in
claim 8, wherein the nucleic acid molecule is in operable linkage
with regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic host. 10. A host
cell, which has been transformed stably or transiently with the
vector as claimed in claim 9 or 10 or the nucleic acid molecule as
claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 2 to 5. 11. The
host cell of claim 10, which is a transgenic host cell. 12. The
host cell of claim 10 or 11, which is a plant cell, an animal cell,
a microorganism, or a yeast cell, a fungus cell, a prokaryotic
cell, an eukaryotic cell or an archaebacterium. 13. A process for
producing a polypeptide, wherein the polypeptide is expressed in a
host cell as claimed in any one of claims 10 to 12. 14. A
polypeptide produced by the process as claimed in claim 13 or
encoded by the nucleic acid molecule as claimed in claim 6 whereby
the polypeptide distinguishes over a sequence as indicated in Table
II A, columns 5 or 7, lines 16 to 18 and/or lines 356 to 362 by one
or more amino acids. 15. An antibody, which binds specifically to
the polypeptide as claimed in claim 14. 16. A plant tissue,
propagation material, harvested material or a plant comprising the
host cell as claimed in claim 12 which is plant cell or an
Agrobacterium. 17. A method for screening for agonists and
antagonists of the activity of a polypeptide encoded by the nucleic
acid molecule of claim 6 conferring an increase in the amount of
tryptophane in an organism or a part thereof comprising: [1463] (a)
contacting cells, tissues, plants or microorganisms which express
the a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of tryptophane in an organism
or a part thereof with a candidate compound or a sample comprising
a plurality of compounds under conditions which permit the
expression the polypeptide; [1464] (b) assaying the tryptophane
level or the polypeptide expression level in the cell, tissue,
plant or microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [1465] (c)
identifying a agonist or antagonist by comparing the measured
tryptophane level or polypeptide expression level with a standard
tryptophane or polypeptide expression level measured in the absence
of said candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. 18. A method for the identification
of a compound conferring increased tryptophane production in a
plant or microorganism, comprising the steps:
[1466] a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of tryptophane in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with dais readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
tryptophane in an organism or a part thereof;
[1467] b) identifying if the compound is an effective agonist by
detecting the presence or absence or increase of a signal produced
by said readout system.
19. A method for the identification of a gene product conferring an
increase in tryptophane production in a cell, comprising the
following steps: [1468] a) contacting the nucleic acid molecules of
a sample, which can contain a candidate gene encoding a gene
product conferring an increase in tryptophane after expression with
the nucleic acid molecule of claim 6; [1469] b) identifying the
nucleic acid molecules, which hybridise under relaxed stringent
conditions with the nucleic acid molecule of claim 6; [1470] c)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing tryptophane; d) expressing the identified
nucleic acid molecules in the host cells; [1471] e) assaying the
tryptophane level in the host cells; and [1472] f) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the tryptophane level in the host cell in the host
cell after expression compared to the wild type. 20. A method for
the identification of a gene product conferring an increase in
tryptophane production in a cell, comprising the following steps:
[1473] a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the tryptophane amount or level
in an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; [1474]
b) introducing the candidate nucleic acid molecules in host cells
appropriate for producing tryptophane; [1475] c) expressing the
identified nucleic acid molecules in the host cells; [1476] d)
assaying the tryptophane level in the host cells; and [1477] e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the tryptophane level in the host
cell after expression compared to the wild type. 21. A method for
the production of an agricultural composition comprising the steps
of the method of any one of claims 17 to 20 and formulating the
compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. 22. A composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of any
one of claim 8 or 9, an antagonist or agonist identified according
to claim 17, the compound of claim 18, the gene product of claim 19
or 20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. 23. Use of the nucleic acid molecule as claimed
in claim 6 for the identification of a nucleic acid molecule
conferring an increase of tryptophane after expression. 24. Use of
the polypeptide of claim 14 or the nucleic acid construct claim 7
or the gene product identified according to the method of claim 19
or 20 for identifying compounds capable of conferring a modulation
of tryptophane levels in an organism. 25. Food or feed composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 17, the host cell of
claims 10 to 12 or the gene product identified according to the
method of claim 19 or 20. 26. Use of the nucleic acid molecule of
claim 6, the polypeptide of claim 14, the nucleic acid construct of
claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the host cell of claims 10 to 12
or the gene product identified according to the method of claim 19
or 20 for the protection of a plant against a tryptophane synthesis
inhibiting herbicide.
[1478] [0554.0.0.2] Abstract: see [0554.0.0.0]:
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[1479] [0000.0.0.3] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and corresponding embodiments as described herein
as follows.
[1480] [0001.0.0.3] to [0008.0.0.3]: see [0001.0.0.0] to
[0008.0.0.0]
[1481] [0009.0.3.3] As described above, the essential amino acids
are necessary for humans and many mammals, for example for
livestock. The branched-chain amino acids (BCAA) leucine,
isoleucine and valine are among the nine dietary indispensable
amino acids for humans. BCAA accounts for 35-40% of the dietary
indispensable amino acids in body protein and 14% of the total
amino acids in skeletal muscle (Ferrando et al., (1995) Oral
branched chain amino acids decrease whole-body proteolysis. J.
Parenter. Enteral Nutr. 19: 47-54.13). They share a common membrane
transport system and enzymes for their transamination and
irreversible oxidation (Block, K. P. (1989) Interactions among
leucine, isoleucine, and valine with special reference to the
branched chain amino acid antagonism. In: Absorption and
Utilization of Amino Acids (Friedman, M., ed.), pp. 229-244, CRC
Press, Boca Raton, Fla. and Champe, P. C. & Harvey, R. A.
(1987) Amino acids: metabolism of carbon atoms. In: Biochemistry
(Champ, P. C. & Harvery, P. A., eds.), pp. 242-252, J. B.
Lippincott, Philadelphia, Pa.). Further, for patient suffering from
Maple Syrup Urine Disease (MSUD) a reduced uptake of those
branched-chain amino acids is essential. Dietary sources of the
branched-chain amino acids are principally derived from animal and
vegetable proteins. The branched-chain amino acids (BCAA) leucine,
isoleucine and valine are limiting for the growth of many mammals.
Therefore the branched-chain amino acids are supplemented in the
feed of broiler, leg hens, turkey, swine or cattle diets.
[1482] [0010.0.0.3] see [0010.0.0.0]
[1483] [0011.0.0.3] see [0011.0.0.0]
[1484] [0012.0.3.3] It is an object of the present invention to
develop an inexpensive process for the synthesis of leucine and/or
isoleucine and/or valine, preferably L-leucine and/or L-isoleucine
and/or L-valine.
[1485] [0013.0.0.3] see [0013.0.0.0]
[1486] [0014.0.3.3] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is leucine and/or isoleucine
and/or valine, preferably L-leucine and/or L-isoleucine and/or
L-valine. Accordingly, in the present invention, the term "the fine
chemical" as used herein relates to "leucine and/or isoleucine
and/or valine". Further, the term "the fine chemicals" as used
herein also relates to fine chemicals comprising leucine and/or
isoleucine and/or valine.
[1487] [0015.0.3.3] In one embodiment, the term "the fine chemical"
means leucine and/or isoleucine and/or valine, preferably L-leucine
and/or L-isoleucine and/or L-valine. Throughout the specification
the term "the fine chemical" means leucine and/or isoleucine and/or
valine, preferably L-leucine and/or L-isoleucine and/or L-valine,
its salts, ester or amids in free form or bound to proteins. In a
preferred embodiment, the term "the fine chemical" means leucine
and/or isoleucine and/or valine, preferably L-leucine and/or
L-isoleucine and/or L-valine, in free form or its salts or bound to
proteins.
[1488] [0016.0.3.3] Accordingly, the present invention relates to a
process comprising [1489] (a) increasing or generating the activity
of a YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C,
b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124,
b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664,
b3117, b3256, b3938, b3983, b4054, and/or b4327 protein in a
non-human organism in one or more parts thereof and [1490] (b)
growing the organism under conditions which permit the production
of the fine chemical, thus, leucine and/or isoleucine and/or valine
or fine chemicals comprising leucine and/or isoleucine and/or
valine, in said organism. [1491] and/or [1492] (a) increasing or
generating the activity of one or more proteins having the activity
of one or more YDL127W, YDR245W, YDR271C, YER173W, YGR101W,
YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C,
YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151,
b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022,
b2414, b2664, b3117, b3256, b3938, b3983, b4054, and/or b4327
protein(s) or of a protein having the sequence of a polypeptide
indicated in Table IIA or IIB, column 3, lines 19 to 29, 29a to
29u, 363 to 385 or having the sequence of a polypeptide encoded by
a nucleic acid molecule indicated in Table IA or IB, column 5 or 7,
lines 19 to 29, 29a to 29u, 363 to 385, in a non-human organism in
one or more parts thereof and [1493] (b) growing the organism under
conditions which permit the production of the fine chemical, in
particular isoleucine, valine and/or leucine in said organism
[1494] and/or [1495] (a) increasing or generating the activity of
one or more YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C,
YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W,
YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827,
b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414,
b2664, b3117, b3256, b3938, b3983, b4054, and/or b4327 protein(s)
or of a protein having the sequence of a polypeptide encoded by a
nucleic acid molecule indicated in Table II, columns 5 or 7, lines
19 to 29, 29a to 29u, 363 to 385 [1496] in a non-human organism in
one or more parts thereof and [1497] (b) growing the organism under
conditions which permit the production of the fine chemical,
meaning of leucine, valine and/or isoleucine or fine chemicals
comprising leucine, valine and/or isoleucine in said organism;
[1498] and/or [1499] (a) increasing or generating the activity of
one or more [1500] YDL127W, YDR245W, YDR271C, YER173W, YGR101W,
YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C,
YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151,
b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022,
b2414, b2664, b3117, b3256, b3938, b3983, b4054, and/or b4327
protein(s) or of a protein having the sequence of a polypeptide
encoded by a nucleic acid molecule indicated in Table II, columns 5
or 7, lines 19 to 29, 29a to 29u, 363 to 385 [1501] in a non-human
organism in one or more parts thereof and [1502] (b) growing the
organism under conditions which permit the production of the fine
chemical, meaning of valine, leucine and/or isoleucine or fine
chemicals comprising valine, leucine and/or isoleucine in said
organism.
[1503] Accordingly, the present invention relates to a process for
the production of a fine chemical comprising [1504] (a) increasing
or generating the activity of one or more proteins having the
activity of a protein indicated in Table II, column 3, lines 19 to
29, 29a to 29u, 363 to 385 or having the sequence of a polypeptide
encoded by a nucleic acid molecule indicated in Table I, column 5
or 7, lines 19 to 29, 29a to 29u, 363 to 385, in a non-human
organism in one or more parts thereof and [1505] (b) growing the
organism under conditions which permit the production of the fine
chemical, in particular arginine and/or glutamate and/or glutamine
and/or proline resp.
[1506] [0016.1.3.3] Accordingly, the term "the fine chemical" means
in one embodiment "isoleucine" in relation to all sequences listed
in Table I to IV, lines 19 to 29, 29a, 363, 367, 373, 377, 379, 384
or homologs thereof and means in one embodiment "valine" in
relation to all sequences listed in Tables I to IV, lines 29a to
29j, 365, 369 to 372, 375, 376, 380, 381, 382, 385 or homologs
thereof and means in one embodiment "leucine" in relation to all
sequences listed in Table I to IV, lines 29k to 29u, 364, 366, 368,
374, 378, 383 or homologs thereof.
[1507] Accordingly, in one embodiment the term "the fine chemical"
means "valine, leucine and isoleucine" in relation to all sequences
listed in Table I to IV, lines363, 364, 365, 367, 368 and 369, in
one embodiment the term "the fine chemical" means "leucine and
isoleucine" in relation to all sequences listed in Table I to IV,
lines373 and 374, 377 and 378.
[1508] Accordingly, the term "the fine chemical" can mean "leucine"
and/or "isoleucine" and/or "valine" owing to circumstances and the
context. In order to illustrate that the meaning of the term "the
fine chemical" means "arginine", and/or "glutamate" and/or
"glutamine" and/or "proline" the term "the respective fine
chemical" is also used.
[1509] [0017.0.0.3] see [0017.0.0.0]
[1510] [0018.0.0.3] see [0018.0.0.0]
[1511] [0019.0.3.3] Advantageously the process for the production
of the fine chemical leads to an enhanced production of the fine
chemical. The terms "enhanced" or "increase" mean at least a 10%,
20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or
100%, more preferably 150%, 200%, 300%, 400% or 500% higher
production of the respective fine chemical in comparison to the
reference as defined below, e.g. that means in comparison to an
organism without the aforementioned modification of the activity
of. a protein indicated in Table II, column 3, lines 19 to 29, 29a
to 29u, 363 to 385 or encoded by nucleic acid molecule indicated in
Table I, columns 5 or 7, linesl9 to 29, 29a to 29u, 363 to 385.
[1512] [0020.0.3.3] Surprisingly it was found, that the transgenic
expression of the Saccharomyces cerevisiae protein YDL127W,
YDR245W, YDR271C, YER173W,
[1513] YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C,
YIL150C, YOR350C, YFL019C and/or YFR042W, and/or the Escherichia
coli K12 protein b1708, b1829, b2957, b3366, b0828, b3966, b4151,
b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022,
b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 in
Arabidopsis thaliana conferred an increase in the leucine and/or
isoleucine and/or valine (or fine chemical) content of the
transformed plants.
[1514] [0021.0.0.3] see [0021.0.0.0]
[1515] [0022.0.3.3] The sequence of YDL127W from Saccharomyces
cerevisiae has been published in Jacq et al., Nature 387 (6632
Suppl), 75-78, 1997 and Goffeau, Science 274 (5287), 546-547, 1996,
and its activity is beeing defined as a "G1/S-specific cyclin PCL2
(Cyclin HCS26 homolog) protein". Accordingly, in one embodiment,
the process of the present invention comprises the use of a
"G1/S-specific cyclin PCL2 (Cyclin HCS26 homolog) protein" or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of leucine and/or isoleucine and/or valine, in
particular for increasing the amount of leucine and/or isoleucine
and/or valine, preferably leucine and/or isoleucine and/or valine
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a "G1/S-specific cyclin PCL2 (Cyclin
HCS26 homolog) protein" is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[1516] The sequence of YDR245W from Saccharomyces cerevisiae has
been published in Jacq et al., Nature 387 (6632 Suppl), 75-78, 1997
and Goffeau, Science 274 (5287), 546-547, 1996, and its activity is
beeing defined as an galactosyl-(mannosyl)-transferase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a galactosyl-(mannosyl)-transferase
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of leucine and/or isoleucine and/or valine,
in particular for increasing the amount of leucine and/or
isoleucine and/or valine, preferably leucine and/or isoleucine
and/or valine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a
galactosyl-(mannosyl)-transferase is increased or generated, e.g.
from Saccharomyces cerevisiae or a homolog thereof. The sequence of
YDR271C was submitted by Le T., Johnston M., (March-1996) to the
EMBL/GenBank/DDBJ databases, by Waterston R.; (MAY-1996) and Jia
Y., (JUNE-1997) to the EMBL/GenBank/DDBJ databases and its cellular
activity has not been characterized yet. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a YDR271C activity from Saccaromyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of leucine and/or isoleucine and/or valine, in particular
for increasing the amount of leucine and/or isoleucine and/or
valine, preferably leucine and/or isoleucine and/or valine in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a YDR271C protein is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof. The sequence of
YER173w from Saccharomyces cerevisiae has been published in
Dietrich, Nature 387 (6632 Suppl), 78-81, 1997, and Goffeau,
Science 274 (5287), 546-547, 1996, and its activity is beeing
defined as an "Checkpoint protein, involved in the activation of
the DNA damage and meiotic pachytene checkpoints;". Accordingly, in
one embodiment, the process of the present invention comprises the
use of a "Checkpoint protein, involved in the activation of the DNA
damage and meiotic pachytene checkpoints" or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
leucine and/or isoleucine and/or valine, in particular for
increasing the amount of leucine and/or isoleucine and/or valine,
preferably leucine and/or isoleucine and/or valine in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a "Checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints" is increased or generated, e.g.
from Saccharomyces cerevisiae or a homolog thereof.
[1517] The sequence of YGR101W from Saccharomyces cerevisiae has
been published in Tettelin, H., Nature 387 (6632 Suppl), 81-84,
1997 and Goffeau, A., Science 274 (5287), 546-547, 1996, and its
activity is beeing defined as a rhomboid protease. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a rhomboid protease or its homolog, for the production of
the fine chemical, meaning of leucine and/or isoleucine and/or
valine, in particular for increasing the amount of leucine and/or
isoleucine and/or valine, preferably leucine and/or isoleucine
and/or valine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a rhomboid protease is increased
or generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof. The sequence of YJL072C from Saccharomyces cerevisiae has
been published in Goffeau, A., Science 274 (5287), 546-547, 1996
and Galibert, F., EMBO J. 15 (9), 2031-2049, 1996, and its activity
is beeing defined as a "subunit of the GINS complex required for
chromosomal DNA replication". Accordingly, in one embodiment, the
process of the present invention comprises the use of a "subunit of
the GINS complex required for chromosomal DNA replication" or its
homolog, for the production of the fine chemical, meaning of
leucine and/or isoleucine and/or valine, in particular for
increasing the amount of leucine and/or isoleucine and/or valine,
preferably leucine and/or isoleucine and/or valine in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a "subunit of the GINS complex required for chromosomal DNA
replication" is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof. The sequence of YKR057W from
Saccharomyces cerevisiae has been published in Dujon et al., Nature
369 (6479), 371-378, 1994 and Goffeau et al., Science 274 (5287),
546-547, 1996 and its activity is beeing defined as a ribosomal
protein, similar to S21A, S26A and/or YS25 ribosomal proteins,
involved in ribosome biogenesis and translation. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a ribosomal protein, similar to S21A, S26A and/or YS25
ribosomal proteins, involved in ribosome biogenesis and translation
from Saccaromyces cerevisiae or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a ribosomal
protein, similar to S21A, S26A and/or YS25 ribosomal proteins,
involved in ribosome biogenesis and translation is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[1518] The sequence of YNL135C from Saccharomyces cerevisiae has
been published in Philippsen, P., Nature 387 (6632 Suppl), 93-98,
1997 and Goffeau, A., Science 274 (5287), 546-547, 1996, and its
activity is beeing defined as a peptidylprolyl isomerase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a peptidylprolyl isomerase or its
homolog, for the production of the fine chemical, meaning of
leucine and/or isoleucine and/or valine, in particular for
increasing the amount of leucine and/or isoleucine and/or valine,
preferably leucine and/or isoleucine and/or valine in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a peptidylprolyl isomerase is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[1519] The sequence of YFL013C from Saccharomyces cerevisiae has
been published in Goffeau, A., Science 274 (5287), 546-547, 1996
and Murakami, Y., Nat. Genet. 10 (3), 261-268, 1995, and its
activity is beeing defined as a "subunit of the INO80 chromatin
remodeling complex". Accordingly, in one embodiment, the process of
the present invention comprises the use of a "subunit of the INO80
chromatin remodeling complex" or its homolog, for the production of
the fine chemical, meaning of leucine and/or isoleucine and/or
valine, in particular for increasing the amount of leucine and/or
isoleucine and/or valine, preferably leucine and/or isoleucine
and/or valine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a "subunit of the INO80 chromatin
remodeling complex" protein is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[1520] The sequence of YGR104C from Saccharomyces cerevisiae has
been published in Thompson et al., Cell 73:1361-1375, 1993, and its
activity is beeing defined as an "RNA polymerase II suppressor
protein SRB5--yeast and/or suppressor of RNA polymerase B SRB5"".
Accordingly, in one embodiment, the process of the present
invention comprises the use of a "RNA polymerase II suppressor
protein SRB5--yeast and/or suppressor of RNA polymerase B SRB5" or
its homolog, for the production of the fine chemical, meaning of
leucine and/or isoleucine and/or valine, in particular for
increasing the amount of leucine and/or isoleucine and/or valine,
preferably leucine and/or isoleucine and/or valine in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a "RNA polymerase II suppressor protein SRB5--yeast and/or
suppressor of RNA polymerase B SRB5" is increased or generated,
e.g. from Saccharomyces cerevisiae or a homolog thereof.
[1521] The sequence of YIL150C from Saccharomyces cerevisiae has
been published in Goffeau et al., Science 274 (5287), 546-547, 1996
and Churcher et al., Nature 387 (6632 Suppl), 84-87, 1997 and its
activity is beeing defined as a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a chromatin binding protein, required for S-phase (DNA
synthesis) initiation or completion, from Saccaromyces cerevisiae
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of leucine and/or isoleucine and/or valine,
in particular for increasing the amount of leucine and/or
isoleucine and/or valine, preferably leucine and/or isoleucine
and/or valine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a chromatin binding protein,
required for S-phase (DNA synthesis) initiation or completion is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[1522] The sequence of YOR350C from Saccharomyces cerevisiae has
been published in Goffeau, A., Science 274 (5287), 546-547, 1996
and Dujon, B., Nature 387 (6632 Suppl), 98-102, 1997, and its
cellular activity has not been characterized yet. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a YOR350C protein or its homolog, for the production of the
fine chemical, meaning of leucine and/or isoleucine and/or valine,
in particular for increasing the amount of leucine and/or
isoleucine and/or valine, preferably leucine and/or isoleucine
and/or valine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a YOR350C protein is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog thereof.
The sequence of YFR042W from Saccharomyces cerevisiae has been
published in Goffeau st al., Science 274 (5287), 546-547, 1996 and
Murakami, Y., Nat. Genet. 10 (3), 261-268, 1995 and its activity is
beeing defined as a "protein required for cell viability in yeast".
Accordingly, in one embodiment, the process of the present
invention comprises the use of a "protein required for cell
viability in yeast", from Saccaromyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of leucine and/or isoleucine and/or valine, in particular
for increasing the amount of leucine and/or isoleucine and/or
valine, preferably leucine and/or isoleucine and/or valine in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a "protein required for cell viability in yeast" is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[1523] The sequence of YFL019C from Saccharomyces cerevisiae has
been published in Murakami, Y., Nat. Genet. 10 (3), 261-268, 1995
and its activity is beeing defined as a hypothetical 13.7 kDa
protein in the PAU5-LPD1 intergenic region. Accordingly, in one
embodiment, the process of the present invention comprises the use
of the protein encoded by the nucleic acid sequence YFL019C, from
Saccaromyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a protein encoded
by the nucleic acid sequence YFL019C is increased or generated,
e.g. from Saccharomyces cerevisiae or a homolog thereof.
[1524] The sequence of b1708 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is beeing defined as a lipoprotein. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a lipoprotein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of leucine
and/or isoleucine and/or valine, in particular for increasing the
amount of leucine and/or isoleucine and/or valine, preferably
leucine and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a lysine
decarboxylase is increased or generated, e.g. from E. coli or a
homolog thereof.
[1525] The sequence of b1829 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is beeing defined as a heat shock protein with
protease activity (htpx). Accordingly, in one embodiment, the
process of the present invention comprises the use of a heat shock
protein with protease activity (htpx) from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of leucine and/or isoleucine and/or valine, in particular
for increasing the amount of leucine and/or isoleucine and/or
valine, preferably leucine and/or isoleucine and/or valine in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a heat shock protein with protease (htpx) activity is
increased or generated, e.g. from
[1526] E. coli or a homolog thereof.
[1527] The sequence of b2957 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is beeing defined as a periplasmic L-asparaginase
II. Accordingly, in one embodiment, the process of the present
invention comprises the use of a periplasmic L-asparaginase II from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of leucine and/or isoleucine and/or
valine, in particular for increasing the amount of leucine and/or
isoleucine and/or valine, preferably leucine and/or isoleucine
and/or valine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of periplasmic L-asparaginase II is
increased or generated, e.g. from E. coli or a homolog thereof. The
sequence of b3366 from Escherichia coli K12 has been published in
Blattner et al.,
[1528] Science 277(5331), 1453-1474, 1997, and its activity is
beeing defined as the a small subunit of a nitrite reductase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a small subunit of a nitrite
reductase from E. coli or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the small subunit
of a nitrite reductase, preferably of the small subunit of a
nitrite reductase is increased or generated, e.g. from E. coli or a
homolog thereof.
[1529] The sequence of b0828 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is beeing defined as the a probable asparaginase
(EC:3.5.1.1) and/or ybiK protein (L-asparagine amidohydrolase).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a probable asparaginase (EC:3.5.1.1)
and/or ybiK protein (L-asparagine amidohydrolase) from E. coli or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of leucine and/or isoleucine and/or valine, in
particular for increasing the amount of leucine and/or isoleucine
and/or valine, preferably leucine and/or isoleucine and/or valine
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a probable asparaginase (EC:3.5.1.1)
and/or ybiK protein (L-asparagine amidohydrolase) is increased or
generated, e.g. from E. coli or a homolog thereof.
[1530] The sequence of b3966 from Escherichia coli K12 has been
published in R; Heller, J. Bacteriol. 161, 904-908, 1985; R;
Doublet, J. Bacteriol. 174, 5772-5779, 1992 and R; Gustafsson, J.
Bacteriol. 173, 1757-1764, 1991, and its activity is beeing defined
as a outer membrane porin. Accordingly, in one embodiment, the
process of the present invention comprises the use of a outer
membrane porin from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a outer membrane
porin is increased or generated, e.g. from E. coli or a homolog
thereof.
[1531] The sequence of b4151 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is beeing defined as a fumarate reductase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a fumarate reductase from E. coli or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of leucine and/or isoleucine and/or valine, in
particular for increasing the amount of leucine and/or isoleucine
and/or valine, preferably leucine and/or isoleucine and/or valine
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a fumarate reductase is increased or
generated, e.g. from E. coli or a homolog thereof.
[1532] The sequence of b1827 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is beeing defined as a repressor protein with a
DNA-binding Winged helix domain (IcIR family). Accordingly, in one
embodiment, the process of the present invention comprises the use
of a repressor protein with a DNA-binding Winged helix domain (IcIR
family) from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a repressor
protein with a DNA-binding Winged helix domain (IcIR family) is
increased or generated, e.g. from E. coli or a homolog thereof.
[1533] The sequence of b0124 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a protein having glucose
dehydrogenase activity. Accordingly, in one embodiment, the process
of the present invention comprises the use of a glucose
dehydrogenase from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a glucose
dehydrogenase is increased or generated, e.g. from E. coli or a
homolog thereof.
[1534] The sequence of b0149 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a bifunctional penicillin
g-binding protein 1 b: glycosyl transferase (N-terminal);
transpeptidase (C-terminal). Accordingly, in one embodiment, the
process of the present invention comprises the use of a
bifunctional penicillin g-binding protein 1 b: glycosyl transferase
(N-terminal); transpeptidase (C-terminal) from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of leucine and/or isoleucine and/or valine, in
particular for increasing the amount of leucine and/or isoleucine
and/or valine, preferably leucine and/or isoleucine and/or valine
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a protein with a bifunctional penicillin
g-binding protein 1 b: glycosyl transferase (N-terminal);
transpeptidase (C-terminal) is increased or generated, e.g. from E.
coli or a homolog thereof.
[1535] The sequence of b0161 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a periplasmic serine protease
Do, heat shock protein; periplasmic serine protease Do; heat shock
protein HtrA. Accordingly, in one embodiment, the process of the
present invention comprises the use of a periplasmic serine
protease Do, heat shock protein; periplasmic serine protease Do;
heat shock protein HtrA from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of leucine
and/or isoleucine and/or valine, in particular for increasing the
amount of leucine and/or isoleucine and/or valine, preferably
leucine and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a periplasmic
serine protease Do, heat shock protein; periplasmic serine protease
Do; heat shock protein HtrA is increased or generated, e.g. from E.
coli or a homolog thereof.
[1536] The sequence of b0486 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative amino acid/amine
transport protein. Accordingly, in one embodiment, the process of
the present invention comprises the use of a putative amino
acid/amine transport protein from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
leucine and/or isoleucine and/or valine, in particular for
increasing the amount of leucine and/or isoleucine and/or valine,
preferably leucine and/or isoleucine and/or valine in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a putative amino acid/amine transport protein is increased or
generated, e.g. from E. coli or a homolog thereof.
[1537] The sequence of b1313 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative dehydrogenase with
NAD(P)-binding and GroES domains. Accordingly, in one embodiment,
the process of the present invention comprises the use of a
putative dehydrogenase with NAD(P)-binding and GroES domainsprotein
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a dehydrogenase
with NAD(P)-binding and GroES domainsprotein is increased or
generated, e.g. from E. coli or a homolog thereof.
[1538] The sequence of b1343 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as an ATP-dependent RNA helicase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein with an ATP-dependent RNA
helicase from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of an ATP-dependent
RNA helicase is increased or generated, e.g. from E. coli or a
homolog thereof.
[1539] The sequence of b1463 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as N-hydroxyarylamine
O-acetyltransferase. Accordingly, in one embodiment, the process of
the present invention comprises the use of a N-hydroxyarylamine
O-acetyltransferase from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of leucine
and/or isoleucine and/or valine, in particular for increasing the
amount of leucine and/or isoleucine and/or valine, preferably
leucine and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a
N-hydroxyarylamine O-acetyltransferase is increased or generated,
e.g. from E. coli or a homolog thereof.
[1540] The sequence of b2022 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a bifunctional:
histidinol-phosphatase (N-terminal); imidazoleglycerol-phosphate
dehydratase (C-terminal); imidazoleglycerolphosphate dehydratase
and histidinol-phosphate phosphatase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a bifunctional: histidinol-phosphatase (N-terminal);
imidazoleglycerol-phosphate dehydratase (C-terminal);
imidazoleglycerolphosphate dehydratase and histidinol-phosphate
phosphatase from E. coli or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a bifunctional:
histidinol-phosphatase (N-terminal); imidazoleglycerol-phosphate
dehydratase (C-terminal); imidazoleglycerol-phosphate dehydratase
and histidinol-phosphate phosphatase is increased or generated,
e.g. from E. coli or a homolog thereof.
[1541] The sequence of b2414 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a subunit of cysteine synthase
A and O-acetylserine sulfhydrolase A, a PLP-dependent enzyme.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a subunit of cysteine synthase A and
O-acetylserine sulfhydrolase A, a PLP-dependent enzyme from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of leucine and/or isoleucine and/or valine,
in particular for increasing the amount of leucine and/or
isoleucine and/or valine, preferably leucine and/or isoleucine
and/or valine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a subunit of cysteine synthase A
and O-acetylserine sulfhydrolase A, a PLP-dependent enzyme is
increased or generated, e.g. from E. coli or a homolog thereof.
[1542] The sequence of b2664 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as putative transcriptional
repressor with DNA-binding Winged helix domain (GntR familiy)
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein with a putative
transcriptional repressor with DNA-binding Winged helix domain
(GntR familiy) activity from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of leucine
and/or isoleucine and/or valine, in particular for increasing the
amount of leucine and/or isoleucine and/or valine, preferably
leucine and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a putative
transcriptional repressor with DNA-binding Winged helix domain
(GntR familiy) is increased or generated, e.g. from E. coli or a
homolog thereof.
[1543] The sequence of b3117 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a threonine dehydratase,
catabolic, PLP-dependent enzyme. Accordingly, in one embodiment,
the process of the present invention comprises the use of a
threonine dehydrotase, catabolic, PLP-dependent enzyme from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of leucine and/or isoleucine and/or valine,
in particular for increasing the amount of leucine and/or
isoleucine and/or valine, preferably leucine and/or isoleucine
and/or valine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a threonine dehydratase,
catabolic, PLP-dependent enzyme is increased or generated, e.g.
from E. coli or a homolog thereof.
[1544] The sequence of b3256 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as an acetyl CoA carboxylase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of an acetyl CoA carboxylase from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of leucine and/or isoleucine and/or
valine, in particular for increasing the amount of leucine and/or
isoleucine and/or valine, preferably leucine and/or isoleucine
and/or valine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of an acetyl CoA carboxylase enzyme
is increased or generated, e.g. from E. coli or a homolog
thereof.
[1545] The sequence of b3938 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a transcriptional repressor
for methionine biosynthesis. Accordingly, in one embodiment, the
process of the present invention comprises the use of a
transcriptional repressor activity for methionine biosynthesis from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of leucine and/or isoleucine and/or
valine, in particular for increasing the amount of leucine and/or
isoleucine and/or valine, preferably leucine and/or isoleucine
and/or valine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a transcriptional repressor for
methionine biosynthesis is increased or generated, e.g. from E.
coli or a homolog thereof.
[1546] The sequence of b3983 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a 50S ribosomal subunit
protein L12. Accordingly, in one embodiment, the process of the
present invention comprises the use of a 50S ribosomal subunit
protein L12 from E. coli or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a 50S ribosomal
subunit protein L12 is increased or generated, e.g. from E. coli or
a homolog thereof.
[1547] The sequence of b4054 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a tyrosine aminotransferase,
tyrosine repressible. Accordingly, in one embodiment, the process
of the present invention comprises the use of a tyrosine
aminotransferase, tyrosine repressible from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of leucine and/or isoleucine and/or valine, in particular
for increasing the amount of leucine and/or isoleucine and/or
valine, preferably leucine and/or isoleucine and/or valine in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a tyrosine aminotransferase, tyrosine repressible is
increased or generated, e.g. from E. coli or a homolog thereof.
[1548] The sequence of b4327 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a HTH-type transcriptional
regulator with periplasmic binding protein domain (LysR
family).
[1549] Accordingly, in one embodiment, the process of the present
invention comprises the use of a HTH-type transcriptional regulator
with periplasmic binding protein domain (LysR family) from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of leucine and/or isoleucine and/or valine,
in particular for increasing the amount of leucine and/or
isoleucine and/or valine, preferably leucine and/or isoleucine
and/or valine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a HTH-type transcriptional
regulator with periplasmic binding protein domain (LysR family) is
increased or generated, e.g. from E. coli or a homolog thereof.
[1550] [0023.0.3.3] In one embodiment, the homolog of the YDL127W,
YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C,
YFL013C, YGR104C, YIL150C, YOR350C, YFR042W and/or YFL019C is a
homolog having said activity and being derived from an eukaryotic.
In one embodiment, the homolog of the b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327 is a homolog having said activity and being
derived from bacteria. In one embodiment, the homolog of the
YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W
is a homolog having said activity and being derived from Fungi. In
one embodiment, the homolog of the b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327 is a homolog having said activity and being
derived from Proteobacteria. In one embodiment, the homolog of the
YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W
is a homolog having said activity and being derived from
Ascomyceta. In one embodiment, the homolog of the b1708, b1829,
b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161,
b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256,
b3938, b3983, b4054 and/or b4327 is a homolog having said activity
and being derived from Gammaproteobacteria. In one embodiment, the
homolog of the YDL127W, YDR245W, YDR271C, YER173W, YGR101W,
YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C,
YFL019C and/or YFR042W is a homolog having said activity and being
derived from Saccharomycotina. In one embodiment, the homolog of
the b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124,
b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664,
b3117, b3256, b3938, b3983, b4054 and/or b4327 is a homolog having
said activity and being derived from Enterobacteriales. In one
embodiment, the homolog of the YDL127W, YDR245W, YDR271C, YER173W,
YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C,
YOR350C, YFL019C and/or YFR042W is a homolog having said activity
and being derived from Saccharomycetes. In one embodiment, the
homolog of the b1708, b1829, b2957, b3366, b0828, b3966, b4151,
b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022,
b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 is a
homolog having said activity and being derived from
Enterobacteriaceae. In one embodiment, the homolog of the YDL127W,
YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C,
YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W is a
homolog having said activity and being derived from
Saccharomycetales. In one embodiment, the homolog of the b1708,
b1829, b2957, b3366, b0828, b3966, b4151 b1827, b0124, b0149,
b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117,
b3256, b3938, b3983, b4054 and/or b4327 is a homolog having said
activity and being derived from Escherichia. In one embodiment, the
homolog of the YDL127W, YDR245W, YDR271C, YER173W, YGR101W,
YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C,
YFL019C and/or YFR042W is a homolog having said activity and being
derived from Saccharomycetaceae. In one embodiment, the homolog of
the YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W
is a homolog having said activity and being derived from
Saccharomycetes.
[1551] [0023.1.0.3] Homologs of the polypeptide indicated in Table
II, column 3, lines 19 to 29, 29a to 29u, 363 to 385 may be the
polypetides encoded by the nucleic acid molecules indicated in
Table I, column 7, linesl9 to 29, 29a to 29u, 363 to 385, resp., or
may be the polypeptides indicated in Table II, column 7, lines19 to
29, 29a to 29u, 363 to 385 resp.
[1552] [0024.0.0.3] Further homologs of are described herein
below.
[1553] [0025.0.3.3] In accordance with the invention, a protein or
polypeptide has the "activity of an YDL127W, YDR245W, YDR271C,
YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C,
YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327 protein" if its de novo activity, or its
increased expression directly or indirectly leads to an increased
leucine and/or isoleucine and/or valine level in the organism or a
part thereof, preferably in a cell of said organism and the protein
has the above mentioned activities of a YDL127W, YDR245W, YDR271C,
YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C,
YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327 protein. Throughout the specification the
activity or preferably the biological activity of such a protein or
polypeptide or an nucleic acid molecule or sequence encoding such
protein or polypeptide is identical or similar if it still has the
biological or enzymatic activity of an YDL127W, YDR245W, YDR271C,
YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C,
YIL150C, YOR350C, YFR042W, YFL 019C, b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327 protein, or which has at least 10% of the
original enzymatic activity, preferably 20%, particularly
preferably 30%, most particularly preferably 40% in comparison to
an YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFL019C and/or YFR042W
of Saccharomyces cerevisiae and/or b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327 protein of E. coli K12.
[1554] [0025.1.0.3] In one embodiment, the polypeptide of the
invention or the polypeptide used in the method of the invention
confers said activity, e.g. the increase of the fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary distant to the organism in which it is
expressed. For example origin and expressing organism are derived
from different families, orders, classes or phylums.
[1555] [0025.2.0.3] In one embodiment, the polypeptide of the
invention or the polypeptide used in the method of the invention
confers said activity, e.g. the increase of the fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organsim indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[1556] [0026.0.0.3] to [0033.0.0.3]: see [0026.0.0.0] to
[0033.0.0.0]
[1557] [0034.0.3.3] Preferably, the reference, control or wild type
differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention, e.g. by or in the expression
level or activity of an protein having the activity of an
YDL127W,
[1558] YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C,
b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124,
b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664,
b3117, b3256, b3938, b3983, b4054 and/or b4327 or being encoded by
a nucleic acid molecule indicated in Table I, column 5, 19 to 29,
29a to 29u, 363 to 385 or its homologs, e.g. as indicated in Table
I, column 7, 19 to 29, 29a to 29u, 363 to 385, its biochemical or
genetical causes and the increased amount of the fine chemical.
[1559] [0035.0.0.3] to [0044.0.0.3]: see [0035.0.0.0] to
[0044.0.0.0]
[1560] [0045.0.3.3] In case the activity of the Saccharomyces
cerevisiae protein YDL127W or its homologs, e.g. as indicated in
Table IA or IB, columns 5 or 7, line 19 or a "G1/S-specific cyclin
PCL2 (Cyclin HCS26 homolog) protein", involved in the mitotic cell
cycle and cell cycle control is increased, preferably, in one
embodiment an increase of the fine chemical, preferably of
isoleucine, of 33% or more is conferred. In case the activity of
the Saccharomyces cerevisiae protein YDR245W or its homologs, e.g.
as indicated in Table IA or IB, columns 5 or 7, line 29k or a
galactosyl-(mannosyl)-transferase, involved in the C-compound and
carbohydrate utilization, budding, cell polarity and filament
formation, protein modification is increased, preferably, in one
embodiment an increase of the fine chemical, preferably of leucine,
between 50% and 173% or more is conferred.
[1561] In case the activity of the Saccaromyces cerevisiae protein
YDR271C or its homologs is increased, preferably, in one embodiment
an increase of the fine chemical, preferably of isoleucine, between
30% and 74% or more is conferred.
[1562] In case the activity of the Saccharomyces cerevisiae protein
YER173w or its homologs, e.g. as indicated in Table IA or IB,
columns 5 or 7, line 29b or a checkpoint protein, involved in the
activation of the DNA damage and meiotic pachytene checkpoints; DNA
recombination and DNA repair, cell cycle checkpoints (checkpoints
of morphogenesis, DNA-damage,-replication, mitotic phase and
spindle), nucleic acid binding, DNA synthesis and replication is
increased, preferably, in one embodiment the increase of the fine
chemical between 18% and 187%, preferably of valine btween 18% and
82% or of leucine between 93% and 187% or more is conferred.
[1563] In case the activity of the Saccharomyces cerevisiae protein
YGR101W or its homologs, e.g. as indicated in Table IA or IB,
columns 5 or 7, line 29c or a rhomboid protease, involved in
cellular communication/signal transduction mechanism is increased,
preferably, in one embodiment an increase of the fine chemical,
preferably of valine, between 47% and 60% or more is conferred.
[1564] In case the activity of the Saccharomyces cerevisiae protein
YJL072C or its homologs, e.g. as indicated in Table IA or IB,
columns 5 or 7, line 21 or a "subunit of the GINS complex required
for chromosomal DNA replication" protein, involved in cell
differentiation, late embryonic development, systemic regulation
of/interaction with enviroment, directional cell growth
(morphogenesis) is increased, preferably, in one embodiment an
increase of the fine chemical, preferably of isoleucine, between
31% and 356% or more is conferred.
[1565] In case the activity of the Saccharomyces cerevisiae protein
YKR057W or its homologs, e.g. as indicated in Table IA or IB,
columns 5 or 7, line 29d or a ribosomal protein, similar to S21A,
S26A and/or YS25 ribosomal proteins, involved in ribosome
biogenesis, cell differentiation and translation is increased,
preferably, in one embodiment an increase of the fine chemical,
preferably of valine, between 19% and 100% or more is
conferred.
[1566] In case the activity of the Saccharomyces cerevisiae protein
YNL135C or its homologs, e.g. as indicated in Table IA or IB,
columns 5 or 7, line 29m or a peptidylprolyl isomerase, involved in
protein folding and stabilization, protein targeting, sorting and
translocation, protein synthesis is increased, preferably, in one
embodiment an increase of the fine chemical, preferably of leucine,
between 45% and 280% or more is conferred.
[1567] In case the activity of the Saccharomyces cerevisiae protein
YFL013C or its homologs, e.g. as indicated in Table IA or IB,
columns 5 or 7, line 29e or a "subunit of the INO80 chromatin
remodeling complex" is increased, preferably, in one embodiment an
increase of the fine chemical, preferably of valine, between 22%
and 58% or more is conferred.
[1568] In case the activity of the Saccharomyces cerevisiae protein
YGR104C or its homologs, e.g. as indicated in Table IA or IB,
columns 5 or 7, line 22 or a "RNA polymerase II suppressor protein
SRB5--yeast and/or suppressor of RNA polymerase B SRB5" involved in
transcription activities is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
isoleucine, between 33% and 102% or more is conferred.
[1569] In case the activity of the Saccharomyces cerevisiae protein
YIL150C or its homologs e.g. as indicated in Table IA or IB,
columns 5 or 7, line 23 or a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion, involved in
DNA synthesis and replication, mitotic cell cycle and cell cycle
control, is increased, preferably, in one embodiment the increase
of the fine chemical between 18% and 535%, preferably of valine
between 18% and 314% or of isoleucine of 535% is conferred.
[1570] In case the activity of the Saccharomyces cerevisiae protein
YOR350C or its homologs is increased, preferably, in one embodiment
the increase of the fine chemical between 101% and 348%, preferably
of leucine between 247% and 348% or of isoleucine between 101% and
178% is conferred.
[1571] In case the activity of the Saccharomyces cerevisiae protein
YFR042W or its homologs e.g. as indicated in Table IA or IB,
columns 5 or 7, line 29n or a "protein required for cell viability
in yeast" is increased, preferably, in one embodiment the increase
of the fine chemical, preferably of leucine, between 43% and 325%
is conferred.
[1572] In case the activity of the Saccharomyces cerevisiae protein
YFL019C or its homologs e.g. as indicated in Table IA or IB,
columns 5 or 7, line 385 or a hypothetical 13.7 kDa protein in
PAU5-LPD1 intergenic region is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of valine,
between 22% and 58% is conferred.
[1573] In case the activity of the Escherichia coli K12 protein
b1708 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 25 or a lipoprotein, involved in cell
growth/morphogenesis, cytokinesis (cell division)/septum formation
and sporulation, is increased, preferably, in one embodiment the
increase of the fine chemical between 33% and 333%, preferably of
valine between 33% and 149%, of isoleucine between 51% and 290% and
of leucine between 60% and 333% is conferred.
[1574] In case the activity of the Escherichia coli K12 protein
b1829 or its homologs is increased, e.g. as indicated in Table IA
or IB, columns 5 or 7, line 26 or the activity of a a heat shock
protein with protease activity (htpx), involved in stress response,
pheromone response, mating-type determination, protein
modification, proteolytic degradation is increased preferably, in
one embodiment the increase of the fine chemical between 16% and
1480%, preferably of isoleucine between 77% and 1480% and of
leucine between 60% and 1200% is conferred.
[1575] In case the activity of the Escherichia coli K12 protein
b2957 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 29 h or a periplasmic L-asparaginase II, involved in
biosynthesis of aspartate, biosynthesis of asparagine, nitrogen and
sulfur utilization, amino acid degradation (catabolism), is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of valine between 32% and 303% is
conferred.
[1576] In case the activity of the Escherichia coli K12 protein
b3366 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 29r or a small subunit of a nitrite reductase is
increased, preferably, in one embodiment the increase of the fine
chemical between 39% and 581%, preferably of isoleucine between 39%
and 327% and of leucine between 65% and 581% is conferred.
[1577] In case the activity of the Escherichia coli K12 protein
b0828 or its homologs e.g. a as indicated in Table IA or IB,
columns 5 or 7, line 28 or asparaginase (EC:3.5.1.1) and/or ybiK
protein (L-asparagine amidohydrolase), involved in amino acid
degradation (catabolism), proteolytic degradation, protein
synthesis, degradation of amino acids of the aspartate group,
biosynthesis of glycosides, is increased, preferably, in one
embodiment the increase of the fine chemical between 20% and 102%,
preferably of valine between 20% and 61% and of isoleucine between
60% and 102% is conferred.
[1578] In case the activity of the Escherichia coli K12 protein
b3966 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 29 or a outer membrane porin, is increased,
preferably, in one embodiment the increase of the fine chemical
between 59% and 647%, preferably of isoleucine between 79% and 392%
and of leucine between 59% and 647% is conferred.
[1579] In case the activity of the Escherichia coli K12 protein
b4151 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 29t or a repressor protein with a DNA-binding Winged
helix domain (IcIR family), involved in transcriptional control, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of leucine between 31% and 46% is
conferred.
[1580] In case the activity of the Escherichia coli K12 protein
b1827 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 29a or a fumarate reductase, is increased, preferably,
in one embodiment the increase of the fine chemical between 24% and
884%, preferably of valine between 24% and 192%, of isoleucine
between 81% and 582% and of leucine between 63% and 884% is
conferred.
[1581] In case the activity of the Escherichia coli K12 protein
b0124 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 363 or a glucose dehydrogenase, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of isoleucine between 43% and 638%, preferably of
leucine between 56% and 570%, preferably of valine between 24% and
268% is conferred.
[1582] In case the activity of the Escherichia coli K12 protein
b0149 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 366 or a bifunctional penicillin g-binding protein 1
b: glycosyl transferase (N-terminal); transpeptidase (C-terminal)
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of leucine between 62% and 99% is
conferred.
[1583] In case the activity of the Escherichia coli K12 protein
b0161 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 367 or a periplasmic serine protease, heat shock
protein, preferably, in one embodiment the increase of the fine
chemical, preferably of leucine between 71% and 1140%, preferably
of valine between 29% and 399%, preferably of isoleucine between
98% and 1030% is conferred.
[1584] In case the activity of the Escherichia coli K12 protein
b0486 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 370 or a putative amino acid/amine transport protein
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of valine between 22% and 181% is
conferred.
[1585] In case the activity of the Escherichia coli K12 protein
b1313 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 371 or a putative dehydrogenase, with NAD(P)-binding
and GroES domains is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of valine between 22% and
50% is conferred.
[1586] In case the activity of the Escherichia coli K12 protein
b1343 or its homologs, e.g. as indicated in Table IA or IB, columns
5 or 7, line 372 or an ATP-dependent RNA helicase, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of valine between 21% and 23% is conferred.
[1587] In case the activity of the Escherichia coli K12 protein
b1463 or its homologs e.g. a as indicated in Table IA or IB,
columns 5 or 7, line 373 or N-hydroxyarylamine 0-acetyltransferase
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of isoleucine between 39% and 119%,
preferably of leucine between 66% and 157% is conferred.
[1588] In case the activity of the Escherichia coli K12 protein
b2022 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 375 or a bifunctional: histidinol-phosphatase
(N-terminal); imidazoleglycerol-phosphate dehydratase (C-terminal);
imidazoleglycerolphosphate dehydratase and histidinol-phosphate
phosphatase is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of valine between 22% and
26% is conferred.
[1589] In case the activity of the Escherichia coli K12 protein
b2414 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 376 or a subunit of cysteine synthase A and
O-acetylserine sulfhydrolase A, PLP-dependent enzyme is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of valine between 35% and 139% is conferred.
[1590] In case the activity of the Escherichia coli K12 protein
b2664 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 377 or a putative transcriptional repressor with
DNA-binding Winged helix domain (GntR familiy) is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of leucine between 67% and 1050%, preferably of
isoleucine between 42% and 1170% is conferred.
[1591] In case the activity of the Escherichia coli K12 protein
b3117 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 379 or a threonine dehydratase, catabolic,
PLP-dependent is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of isoleucine between 47%
and 656% is conferred.
[1592] In case the activity of the Escherichia coli K12 protein
b3256 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 380 or an acetyl CoA carboxylase is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of valine between 27% and 36% is conferred.
[1593] In case the activity of the Escherichia coli K12 protein
b3938 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 381 or a transcriptional repressor for methionine
biosynthesis is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of valine between 24% and
42% is conferred.
[1594] In case the activity of the Escherichia coli K12 protein
b3983 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 382 or a 50S ribosomal subunit protein L12 is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of valine between 24% and 202% is
conferred.
[1595] In case the activity of the Escherichia coli K12 protein
b4054 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 383 or a tyrosine aminotransferase, tyrosine
repressible is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of leucine between 126%
and 163% is conferred.
[1596] In case the activity of the Escherichia coli K12 protein
b4327 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 384 or a HTH-type transcriptional regulator with
periplasmic binding protein domain (LysR family) is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of isoleucine between 42% and 54% is conferred.
[1597] [0046.0.3.3] In case the activity of the Saccharomyces
cerevisiae protein YDL127W YER173w or its homologs, e.g. a
"G1/S-specific cyclin PCL2 (Cyclin HCS26 homolog) protein" a
checkpoint protein is increased, preferably an increase of the fine
chemical and of tryptophane is conferred.
[1598] In case the activity of the Saccharomyces cerevisiae protein
YDR271C or its homologs is increased, preferably an increase of the
fine chemical and of proline is is conferred.
[1599] In case the activity of the Saccharomyces cerevisiae protein
YER173w or its homologs, e.g. a checkpoint protein is increased,
preferably an increase of the fine chemical and of tryptophane is
conferred.
[1600] In case the activity of the Saccharomyces cerevisiae protein
YGR101W or its homologs, e.g. a rhomboid protease is increased,
preferably an increase of the fine chemical and of phenylalanine is
conferred.
[1601] In case the activity of the Saccharomyces cerevisiae protein
YJL072C or its homologs, e.g. a "subunit of the GINS complex
required for chromosomal DNA replication" protein is increased,
preferably an increase of the fine chemical and of phenylalanine is
conferred.
[1602] In case the activity of the Saccharomyces cerevisiae protein
YKR057W or its homologs, e.g. a ribosomal protein, similar to S21A,
S26A and/or YS25 ribosomal proteins is increased, preferably an
increase of the fine chemical and of threonine.
[1603] In case the activity of the Saccharomyces cerevisiae protein
YFL013C or its homologs, e.g. a "subunit of the INO80 chromatin
remodeling complex" is increased, preferably an increase of the
fine chemical and of 2,3-Dimethyl-5-phytylquinolis conferred.
[1604] In case the activity of the Saccharomyces cerevisiae protein
YGR104C or its homologs, e.g. a "RNA polymerase II suppressor
protein SRB5--yeast and/or suppressor of RNA polymerase B SRB5''RNA
polymerase II suppressor protein (SRB5--yeast) is increased,
preferably an increase of the fine chemical and of glutamic acid is
conferred.
[1605] In case the activity of the Saccharomyces cerevisiae protein
YIL150C or its homologs e.g. a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion, involved in
DNA synthesis and replication, mitotic cell cycle and cell cycle
control is increased, preferably an increase of the fine chemical
and of threonine is conferred.
[1606] In case the activity of the Saccharomyces cerevisiae protein
YOR350C or its homologs is increased, preferably an increase of the
fine chemical and of tyrosine is conferred.
[1607] In case the activity of the Saccharomyces cerevisiae protein
YFR042W or its homologs e.g. a "protein required for cell viability
in yeast" is increased, preferably an increase of the fine chemical
and of glutamine is conferred.
[1608] In case the activity of the Escherichia coli K12 protein
b1708 or its homologs e.g. a lipoprotein, involved in cell
growth/morphogenesis, cytokinesis (cell division)/septum formation
and sporulation is increased, preferably an increase of the fine
chemical and of phenylalanine is conferred.
[1609] In case the activity of the Escherichia coli K12 protein
b1829 or its homologs is increased, e.g. the activity of a heat
shock protein with protease activity (htpx) is increased,
preferably an increase of the fine chemical and of threonine is
conferred.
[1610] In case the activity of the Escherichia coli K12 protein
b3966 or its homologs e.g. a outer membrane porin is increased,
preferably an increase of the fine chemical and of threonine is
conferred.
[1611] In case the activity of the Escherichia coli K12 protein
b1827 or its homologs e.g. a fumarate reductase is increased,
preferably an increase of the fine chemical and of proline is
conferred.
[1612] In case the activity of the Saccharomyces cerevisiae protein
YFL019C or its homologs e.g. as indicated in Table IA or IB,
columns 5 or 7, line 385 or a hypothetical 13.7 kDa protein in
PAU5-LPD1 intergenic region is increased, preferably, in one
embodiment an increase of the fine chemical and further amino
acid(s) in free or protein bound form is conferred.
[1613] In case the activity of the Escherichia coli K12 protein
b0124 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 363 or a glucose dehydrogenase, is increased,
preferably, in one embodiment an increase of the fine chemicals and
further amino acid(s) in free or protein bound form is
conferred.
[1614] In case the activity of the Escherichia coli K12 protein
b0149 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 366 or a bifunctional penicillin g-binding protein 1
b: glycosyl transferase (N-terminal); transpeptidase (C-terminal)
is increased, preferably, in one embodiment an increase of the fine
chemical and further amino acid(s) in free or protein bound form is
conferred.
[1615] In case the activity of the Escherichia coli K12 protein
b0161 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 367 or a periplasmic serine protease, heat shock
protein, preferably, in one embodiment an increase of the fine
chemicals and further amino acid(s) in free or protein bound form
is conferred
[1616] In case the activity of the Escherichia coli K12 protein
b0486 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 370 or a putative amino acid/amine transport protein
is increased, preferably, in one embodiment an increase of the fine
chemical and further amino acid(s) in free or protein bound form is
conferred.
[1617] In case the activity of the Escherichia coli K12 protein
b1313 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 371 or a putative dehydrogenase, with NAD(P)-binding
and GroES domains is increased, preferably, in one embodiment an
increase of the fine chemical and further amino acid(s) in free or
protein bound form is conferred.
[1618] In case the activity of the Escherichia coli K12 protein
b1343 or its homologs, e.g. as indicated in Table IA or IB, columns
5 or 7, line 372 or an ATP-dependent RNA helicase, is increased,
preferably, in one embodiment an increase of the fine chemical and
further amino acid(s) in free or protein bound form is
conferred.
[1619] In case the activity of the Escherichia coli K12 protein
b1463 or its homologs e.g. a as indicated in Table IA or IB,
columns 5 or 7, line 373 or N-hydroxyarylamine 0-acetyltransferase
is increased, preferably, in one embodiment an increase of the fine
chemical and further amino acid(s) in free or protein bound form is
conferred.
[1620] In case the activity of the Escherichia coli K12 protein
b2022 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 375 or a bifunctional: histidinol-phosphatase
(N-terminal); imidazoleglycerol-phosphate dehydratase is increased,
preferably, in one embodiment an increase of the fine chemical and
further amino acid(s) in free or protein bound form is
conferred.
[1621] In case the activity of the Escherichia coli K12 protein
b2414 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 376 or a subunit of cysteine synthase A and
O-acetylserine sulfhydrolase A, PLP-dependent enzyme is increased,
preferably, in one embodiment an increase of the fine chemical and
further amino acid(s) in free or protein bound form is
conferred.
[1622] In case the activity of the Escherichia coli K12 protein
b2664 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 377 or a putative transcriptional repressor with
DNA-binding Winged helix domain (GntR familiy) is increased,
preferably, in one embodiment an increase of the fine chemicals and
further amino acid(s) in free or protein bound form is
conferred.
[1623] In case the activity of the Escherichia coli K12 protein
b3117 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 379 or a threonine dehydratase, catabolic,
PLP-dependent is increased, preferably, in one embodiment an
increase of the fine chemical and further amino acid(s) in free or
protein bound form is conferred.
[1624] In case the activity of the Escherichia coli K12 protein
b3256 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 380 or an acetyl CoA carboxylase is increased,
preferably, in one embodiment an increase of the fine chemical and
further amino acid(s) in free or protein bound form is
conferred.
[1625] In case the activity of the Escherichia coli K12 protein
b3938 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 381 or a transcriptional repressor for methionine
biosynthesis is increased, preferably, in one embodiment an
increase of the fine chemical and further amino acid(s) in free or
protein bound form is conferred.
[1626] In case the activity of the Escherichia coli K12 protein
b3983 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 382 or a 50S ribosomal subunit protein L12 is
increased, preferably, in one embodiment an increase of the fine
chemical and further amino acid(s) in free or protein bound form is
conferred.
[1627] In case the activity of the Escherichia coli K12 protein
b4054 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 383 or a tyrosine aminotransferase, tyrosine
repressible is increased, preferably, in one embodiment an increase
of the fine chemical and further amino acid(s) in free or protein
bound form is conferred.
[1628] In case the activity of the Escherichia coli K12 protein
b4327 or its homologs e.g. as indicated in Table IA or IB, columns
5 or 7, line 384 or a HTH-type transcriptional regulator with
periplasmic binding protein domain (LysR family) is increased,
preferably, in one embodiment the increase of the fine chemical and
further amino acid(s) in free or protein bound form is
conferred.
[1629] [0047.0.0.3] see [0047.0.0.0]
[1630] [0048.0.0.3] see [0048.0.0.0]
[1631] [0049.0.3.3] A protein having an activity conferring an
increase in the amount or level of the fine chemical preferably has
the structure of the polypeptide described herein, in particular of
the polypeptides comprising the consensus sequence as indicated in
Table IV, column 7, line 19 to 29, 29a to 29u, 363 to 385 or of the
polypeptide as shown in Table IIA or IIB, columns 5 or 7, lines 19
to 29, 29a to 29u, 363 to 385 or the functional homologues thereof
as described herein, or is encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by the nucleic acid molecule as shown in
Table IA or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to
385 or its herein described functional homologues and has the
herein mentioned activity.
[1632] [0050.0.3.3] For the purposes of the present invention, the
term "leucine" and/or "isoleucine" and/or "valine" and "L-leucine"
and/or "L-isoleucine" and/or "L-valine" also encompass the
corresponding salts, such as, for example, leucine- and/or
isoleucine- and/or valine-hydrochloride or leucine and/or
isoleucine and/or valine sulfate. Preferably the term leucine
and/or isoleucine and/or valine is intended to encompass the term
L-leucine and/or L-isoleucine and/or L-valine.
[1633] [0051.0.0.3] see [0051.0.0.0]
[1634] [0052.0.0.3] see [0052.0.0.0]
[1635] [0053.0.3.3] In one embodiment, the process of the present
invention comprises one or more of the following steps [1636] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention, e.g. of a polypeptide as indicated
in table II, columns 5 and 7, lines 19 to 29, 29a to 29u, 363 to
385 having an YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C,
YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W,
YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827,
b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414,
b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or
its homologs activity having herein-mentioned leucine and/or
isoleucine and/or valine increasing activity; [1637] b) stabilizing
a mRNA conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention, e.g. of a polypeptide
having a YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C,
YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W,
YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827,
b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414,
b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or
its homologs activity or of a mRNA encoding the polypeptide of the
present invention having herein-mentioned leucine and/or isoleucine
and/or valine increasing activity; [1638] c) increasing the
specific activity of a protein conferring the increasd expression
of a protein encoded by the nucleic acid molecule of the invention
or of the polypeptide of the present invention having
herein-mentioned leucine and/or isoleucine and/or valine increasing
activity, e.g. of a polypeptide having a YDL127W, YDR245W, YDR271C,
YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C,
YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327 protein or its homologs activity, or decreasing
the inhibiitory regulation of the polypeptide of the invention;
[1639] d) generating or increasing the expression of an endogenous
or artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned leucine and/or isoleucine
and/or valine increasing activity, e.g. of a polypeptide having the
YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C,
b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124,
b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664,
b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or its
homologs activity; [1640] e) stimulating activity of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the present invention or a polypeptide of
the present invention having herein-mentioned leucine and/or
isoleucine and/or valine increasing activity, e.g. of a polypeptide
having the YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C,
YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W,
YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827,
b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414,
b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or
its homologs activity, by adding one or more exogenous inducing
factors to the organisms or parts thereof; [1641] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned leucine and/or isoleucine and/or valine
increasing activity, e.g. of a polypeptide having the YDL127W,
YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C,
YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829,
b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161,
b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256,
b3938, b3983, b4054 and/or b4327 protein or its homologs activity;
and/or [1642] g) increasing the copy number of a gene conferring
the increased expression of a nucleic acid molecule encoding a
polypeptide encoded by the nucleic acid molecule of the invention
or the polypeptide of the invention having herein-mentioned leucine
and/or isoleucine and/or valine increasing activity, e.g. of a
polypeptide having the YDL127W, YDR245W, YDR271C, YER173W, YGR101W,
YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C,
YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151,
b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022,
b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327
protein or its homologs activity. [1643] h) Increasing the
expression of the endogenous gene encoding the polypeptide of the
invention, e.g. a polypeptide having the YDL127W, YDR245W, YDR271C,
YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C,
YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327 protein or its homologs activity, by adding
positive expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced. and/or
[1644] i) Modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced the fine chemical
production. [1645] j) selecting of organisms with expecially high
activity of the proteins of the invention from natural or from
mutagenized resources and breeding them into the target organisms,
eg the elite crops.
[1646] [0054.0.2.3] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of leucine and/or
isoleucine and/or valine after increasing the expression or
activity of the encoded polypeptide or having the activity of a
polypeptide having an YDL127W, YDR245W, YDR271C, YER173W, YGR101W,
YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C,
YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151,
b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022,
b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327
protein or its homologs activity as indicated in table II, columns
5 and 7, lines 19 to 29, 29a to 29u, 363 to 385.
[1647] [0055.0.0.3] to [0064.0.0.3] see [0055.0.0.0] to
[0064.0.0.0]
[1648] [0065.0.3.3] The activation of an endogenous polypeptide
having above-mentioned activity, e.g. having the activity of a
YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C,
b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124,
b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664,
b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or of the
polypeptide of the invention, e.g. conferring the increase of
leucine and/or isoleucine and/or valine after increase of
expression or activity can also be increased by introducing a
synthetic transcription factor, which binds close to the coding
region of the YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C,
YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W,
YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827,
b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414,
b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein
encoding gene and activates its transcription. A chimeric zinc
finger protein can be construed, which comprises a specific
DNA-binding domain and an activation domain as e.g. the VP16 domain
of Herpes Simplex virus. The specific binding domain can bind to
the regulatory region of the YDL127W, YDR245W, YDR271C, YER173W,
YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C,
YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828,
b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343,
b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054
and/or b4327 protein encoding gene. The expression of the chimeric
transcription factor in a organism, in particular in a plant, leads
to a specific expression of YDL127W, YDR245W, YDR271C, YER173W,
YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C,
YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828,
b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343,
b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054
and/or b4327 protein, see e.g. in WO01/52620, Oriz, Proc. Natl.
Acad. Sci. USA, 2002, Vol. 99, 13290 or Guan, Proc. Natl. Acad.
Sci. USA, 2002, Vol. 99, 13296.
[1649] [0066.0.0.3] to [0069.0.0.3]: see [0066.0.0.0] to
[0069.0.0.0]
[1650] [0070.0.3.3] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding the
YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C,
b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124,
b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664,
b3117, b3256, b3938, b3983, b4054 and/or b4327 protein into an
organism alone or in combination with other genes, it is possible
not only to increase the biosynthetic flux towards the end product,
but also to increase, modify or create de novo an advantageous,
preferably novel metabolites composition in the organism, e.g. an
advantageous amino acid composition comprising a higher content of
(from a viewpoint of nutrional physiology limited) amino acids,
like tryptophane, methionine, lysine and/or threonine.
[1651] [0071.0.0.3] see [0071.0.0.0]
[1652] [0072.0.3.3] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to leucine and/or isoleucine and/or valine 2-Acetolactate,
2-3-Dihydroxyisovalerate, 2-Oxoisovalerate, 3-Hydroxyisobutyrate,
3-Hydroxy-lsobutyryl-CoA, Methylacrylyl-CoA, Isobutyryl-CoA,
2-Aceto-2-hydroxybutyrate, 2:3-Di-OH-3-methylvalerate,
2-Oxo-3-methylvalerate, 2-Methylacetoacetyl-CoA,
2-Methyl-3-hydroxybutyryl-CoA, Tiglyl-CoA, 2 Methylbutyryl-CoA,
22-lsopropylmalate, 3-lsopropylmalate, Oxoleucine, Isovaleryl-CoA,
3-Methylcrotonyl-CoA and/or 3-Methylglutaconyl-CoA.
[1653] [0073.0.3.3] Accordingly, in one embodiment, the process
according to the invention relates to a process which comprises:
[1654] e) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [1655] f) increasing the YDL127W,
YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C,
YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829,
b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161,
b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256,
b3938, b3983, b4054 and/or b4327 protein activity or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the fine chemical in the organism, preferably in the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue or the plant, [1656] g) growing the
organism, preferably the microorganism, the non-human animal, the
plant or animal cell, the plant or animal tissue or the plant under
conditions which permit the production of the fine chemical in the
organism, preferably the microorganism, the plant cell, the plant
tissue or the plant; and [1657] h) if desired, revovering,
optionally isolating, the free and/or bound the fine chemical and,
optionally further free and/or bound amino acids synthetized by the
organism, the microorganism, the non-human animal, the plant or
animal cell, the plant or animal tissue or the plant.
[1658] [0074.0.0.3] to [0084.0.0.3]: see [0074.0.0.0] to
[0084.0.0.0]
[1659] [0085.0.3.3] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [1660] a) the nucleic acid sequence as
depicted in Table IA or IB, columns 5 and 7, lines 19 to 29, 29a to
29u, 363 to 385 or a derivative thereof, or [1661] b) a genetic
regulatory element, for example a promoter, which is functionally
linked to the nucleic acid sequence as depicted in Table IA or IB,
columns 5 and 7, lines 19 to 29, 29a to 29u, 363 to 385 or a
derivative thereof, or [1662] c) (a) and (b) is/are not present in
its/their natural genetic environment or has/have been modified by
means of genetic manipulation methods, it being possible for the
modification to be, by way of example, a substitution, addition,
deletion, inversion or insertion of one or more nucleotide.
"Natural genetic environment" means the natural chromosomal locus
in the organism of origin or the presence in a genomic library. In
the case of a genomic library, the natural, genetic environment of
the nucleic acid sequence is preferably at least partially still
preserved. The environment flanks the nucleic acid sequence at
least on one side and has a sequence length of at least 50 bp,
preferably at least 500 bp, particularly preferably at least 1000
bp, very particularly preferably at least 5000 bp.
[1663] [0086.0.0.3]: see [0086.0.0.0]
[1664] [0087.0.0.3]: see [0087.0.0.0]
[1665] [0088.0.3.3] In an advantageous embodiment of the invention,
the organism takes the form of a plant whose amino acid content is
modified advantageously owing to the nucleic acid molecule of the
present invention expressed. This is important for plant breeders
since, for example, the nutritional value of plants for monogastric
animals is limited by a few essential amino acids such as lysine,
threonine or methionine or tryptophane. After the YDL127W, YDR245W,
YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C,
YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957,
b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486,
b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938,
b3983, b4054 and/or b4327 protein activity has been increased or
generated, or after the expression of nucleic acid molecule or
polypeptide according to the invention has been generated or
increased, the transgenic plant generated thus is grown on or in a
nutrient medium or else in the soil and subsequently harvested.
[1666] [0089.0.0.3] to [0097.0.0.3]: see [0089.0.0.0] to
[0097.0.0.0]
[1667] [0098.0.3.3] In a preferred embodiment, the fine chemical
(leucine and/or isoleucine and/or valine) is produced in accordance
with the invention and, if desired, is isolated. The production of
further amino acids such as methionine, lysine and/or threonine
mixtures of amino acid by the process according to the invention is
advantageous.
[1668] [0099.0.0.3] to [0102.0.0.3]: see [0099.0.0.0] to
[0102.0.0.0]
[1669] [0103.0.3.3] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [1670]
a) nucleic acid molecule encoding, preferably at least the mature
form, of the polypeptide shown in Table IIA or IIB, columns 5 and
7, lines 19 to 29, 29a to 29u, 363 to 385 or a fragment thereof,
which confers an increase in the amount of the fine chemical in an
organism or a part thereof; [1671] b) nucleic acid molecule
comprising, preferably at least the mature form, of the nucleic
acid molecule shown in Table IA or IB, columns 5 and 7, lines 19 to
29, 29a to 29u, 363 to 385 [1672] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
fine chemical in an organism or a part thereof; [1673] d) nucleic
acid molecule encoding a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; [1674] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [1675] f)
nucleic acid molecule encoding a polypeptide, the polypeptide being
derived by substituting, deleting and/or adding one or more amino
acids of the amino acid sequence of the polypeptide encoded by the
nucleic acid molecules (a) to (d), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [1676] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [1677] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers in Table III, column 7, lines 19 to 29,
29a to 29u, 363 to 385 and conferring an increase in the amount of
the fine chemical in an organism or a part thereof; [1678] i)
nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [1679] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence shown in
Table IV, column 7, lines 19 to 29, 29a to 29u, 363 to 385 and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [1680] k) nucleic acid molecule
comprising one or more of the nucleic acid molecule encoding the
amino acid sequence of a polypeptide encoding a domain of the
polypeptide shown in Table IA or IB, columns 5 and 7, lines 19 to
29, 29a to 29u, 363 to 385 and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; and [1681]
l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; or which comprises a
sequence which is complementary thereto.
[1682] [0104.0.3.3] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence depicted in
Table IA or IB, columns 5 and 7, lines 19 to 29, 29a to 29u, 363 to
385 preferably over the sequences as shown in Table IA, columns 5
or 7, lines 19 to 29, 29a to 29u, 363 to 385 by one or more
nucleotides or does not consist of the sequence shown in Table IA
or IB, columns 5 and 7, lines 19 to 29, 29a to 29u, 363 to 385
preferably not of the sequences as shown in Table IA, columns 5 or
7, lines 19 to 29, 29a to 29u, 363 to 385. In one embodiment, the
nucleic acid molecule of the present invention is less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to the sequence shown in
Table IA or IB, lines 19 to 29, 29a to 29u, 363 to 385, preferably
to the sequences as shown in Table IA, columns 5 or 7, lines 19 to
29, 29a to 29u, 363 to 385. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of the sequence shown in
Table IIA or IIB, lines 19 to 29, 29a to 29u, 363 to 385,
preferably of sequences as shown in Table IA, columns 5 or 7, lines
19 to 29, 29a to 29u, 363 to 385.
[1683] [0105.0.0.3] to [0107.0.0.3]: see [0105.0.0.0] to
[0107.0.0.0]
[1684] [0108.0.3.3] Nucleic acid molecules with the sequence shown
in Table IA or IB, lines 19 to 29, 29a to 29u, 363 to 385, nucleic
acid molecules which are derived from the amino acid sequences
shown in Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a to
29u, 363 to 385 or from polypeptides comprising the consensus
sequence shown in Table IV, columns 7, lines 19 to 29, 29a to 29u,
363 to 385, or their derivatives or homologues encoding
polypeptides with the enzymatic or biological activity of an
YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C,
b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124,
b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664,
b3117, b3256, b3938, b3983, b4054 and/or b4327 protein or
conferring a leucine and/or isoleucine and/or valine increase after
increasing its expression or activity are advantageously increased
in the process according to the invention.
[1685] [0109.0.0.3] see [0109.0.0.0]
[1686] [0110.0.3.3] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with YDL127W, YDR245W, YDR271C, YER173W, YGR101W,
YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C,
YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151,
b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022,
b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327
protein activity can be determined from generally accessible
databases.
[1687] [0111.0.0.3] see [0111.0.0.0]
[1688] [0112.0.3.3] The nucleic acid molecules used in the process
according to the invention take the form of isolated nucleic acid
sequences, which encode polypeptides with YDL127W, YDR245W,
YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C,
YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957,
b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486,
b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938,
b3983, b4054 and/or b4327 protein activity and conferring a leucine
and/or isoleucine and/or valine increase.
[1689] [0113.0.0.3] to [0120.0.0.3]: see [0113.0.0.0] to
[0120.0.0.0]
[1690] [0121.0.3.3] However, it is also possible to use artificial
sequences, which differ in one or more bases from the nucleic acid
sequences found in organisms, or in one or more amino acid
molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences shown in Table IIA or
IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 or the
functional homologues thereof as described herein, preferably
conferring above-mentioned activity, i.e. conferring a leucine
and/or isoleucine and/or valine increase after increasing its
activity, e.g. having the activity of an YDL127W, YDR245W, YDR271C,
YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C,
YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327 protein.
[1691] [0122.0.0.3] to [127.0.0.3]: see [0122.0.0.0] to
[0127.0.0.0]
[1692] [0128.0.3.3] Synthetic oligonucleotide primers for the
amplification, e.g. as shown in Table III, column 7, lines 19 to
29, 29a to 29u, 363 to 385 by means of polymerase chain reaction
can be generated on the basis of a sequence shown herein, for
example the sequence shown in Table IA or IB, columns 5 or 7, lines
19 to 29, 29a to 29u, 363 to 385 or the sequences derived from
Table IIA or IIB, lines 19 to 29, 29a to 29u, 363 to 385.
[1693] [0129.0.3.3] Moreover, it is possible to identify conserved
regions from various organisms by carrying out protein sequence
alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence shown in Table IV, column
7, lines 19 to 29, 29a to 29u, 363 to 385 is derived from said
alignments.
[1694] [0130.0.3.3] Degenerated primers can then be utilized by PCR
for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of leucine
and/or isoleucine and/or valine after increasing the expression or
activity or having an YDL127W, YDR245W, YDR271C, YER173W, YGR101W,
YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C,
YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151,
b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022,
b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327
activity or further functional homologs of the polypeptide of the
invention from other organisms. [0131.0.0.3] to [0138.0.0.3]: see
[0131.0.0.0] to [0138.0.0.0]
[1695] [0139.0.3.3] Polypeptides having above-mentioned activity,
i.e. conferring the fine chemical increase, derived from other
organisms, can be encoded by other DNA sequences which hybridize to
the sequences shown in Table IA or IB, columns 5 or 7, lines 19 to
29, 29a to 29u, 363 to 385 under relaxed hybridization conditions
and which code on expression for peptides having the leucine and/or
isoleucine and/or valine increasing activity.
[1696] [0140.0.0.3] to [0146.0.0.3]: see [0140.0.0.0] to
[0146.0.0.0]
[1697] [0147.0.3.3] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences shown in Table IA
or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385,
preferably in Table I B, column 7, lines 19 to 29, 29a to 29u, 363
to 385 is one which is sufficiently complementary to one of the
nucleotide sequences shown in Table IA or IB, lines 19 to 29, 29a
to 29u, 363 to 385 such that it can hybridize to one of the
nucleotide sequences shown in Table IA or IB, lines 19 to 29, 29a
to 29u, 363 to 385 thereby forming a stable duplex. Preferably, the
hybridisation is performed under stringent hybrization conditions.
However, a complement of one of the herein disclosed sequences is
preferably a sequence complement thereto according to the base
pairing of nucleic acid molecules well known to the skilled person.
For example, the bases A and G undergo base pairing with the bases
T and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner [0148.0.3.3] The nucleic acid
molecule of the invention comprises a nucleotide sequence which is
at least about 30%, 35%, 40% or 45%, preferably at least about 50%,
55%, 60% or 65%, more preferably at least about 70%, 80%, or 90%,
and even more preferably at least about 95%, 97%, 98%, 99% or more
homologous to a nucleotide sequence shown in Table IA or IB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385, preferably
in Table I B, column 7, lines 19 to 29, 29a to 29u, 363 to 385, or
a portion thereof and preferably has above mentioned activity, in
particular having a leucine and/or isoleucine and/or valine
increasing activity after increasing the activity or an activity of
an YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C,
b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124,
b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664,
b3117, b3256, b3938, b3983, b4054 and/or b4327 gene product.
[1698] [0149.0.3.3] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences shown in Table IA or IB, columns 5 or 7,
lines 19 to 29, 29a to 29u, 363 to 385, preferably in Table I B,
column 7, lines 19 to 29, 29a to 29u, 363 to 385 or a portion
thereof and encodes a protein having above-mentioned activity, e.g.
conferring a leucine and/or isoleucine and/or valine increase, and
optionally, the activity of YDL127W, YDR245W, YDR271C, YER173W,
YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C,
YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828,
b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343,
b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054
and/or b4327.
[1699] [0150.0.3.3] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences in Table IA or IB, columns 5 or 7, lines 19 to 29,
29a to 29u, 363 to 385, preferably in Table I B, column 7, lines 19
to 29, 29a to 29u, 363 to 385 for example a fragment which can be
used as a probe or primer or a fragment encoding a biologically
active portion of the polypeptide of the present invention or of a
polypeptide used in the process of the present invention, i.e.
having above-mentioned activity, e.g. conferring an increase of
leucine and/or isoleucine and/or valine if its activity is
increased. The nucleotide sequences determined from the cloning of
the present protein-according-to-the-invention-encoding gene allows
for the generation of probes and primers designed for use in
identifying and/or cloning its homologues in other cell types and
organisms. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, 15 preferably about 20 or 25, more
preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one of the sequences set forth, e.g., in Table IA or IB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385, an
anti-sense sequence of one of the sequences, e.g., set forth in
Table IA or IB, lines 19 to 29, 29a to 29u, 363 to 385, or
naturally occurring mutants thereof. Primers based on a nucleotide
of invention can be used in PCR reactions to clone homologues of
the polypeptide of the invention or of the polypeptide used in the
process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primers shown in Table III, column 7, lines 19 to 29,
29a to 29u, 363 to 385 will result in a fragment of YDL127W,
YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C,
YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829,
b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161,
b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256,
b3938, b3983, b4054 and/or b4327 gene product.
[1700] [0151.0.0.3] see [0151.0.0.0]
[1701] [0152.0.3.3] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to the amino acid
sequence of Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a
to 29u, 363 to 385 such that the protein or portion thereof
maintains the ability to participate in the fine chemical
production, in particular a leucine and/or isoleucine and/or valine
increasing the activity as mentioned above or as described in the
examples in plants or microorganisms is comprised.
[1702] [0153.0.3.3] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence shown in Table IIA or IIB, columns 5 or 7, lines 19
to 29, 29a to 29u, 363 to 385 such that the protein or portion
thereof is able to participate in the increase of the fine chemical
production. For examples having an YDL127W, YDR245W, YDR271C,
YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C,
YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327 activity are described herein.
[1703] [0154.0.3.3] In one embodiment, the nucleic acid molecule of
the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence of Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a
to 29u, 363 to 385 and having above-mentioned activity, e.g.
conferring preferably the increase of the fine chemical.
[1704] [0155.0.0.3] and [0156.0.0.3]: see [0155.0.0.0] and
[0156.0.0.0]
[1705] [0157.0.3.3] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences shown in
Table IA or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to
385 (and portions thereof) due to degeneracy of the genetic code
and thus encode a polypeptide of the present invention, in
particular a polypeptide having above mentioned activity, e.g.
conferring an increase in the fine chemical in a organism, e.g. as
that polypeptides encoded by the sequence shown in Table IIA or
IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 or the
functional homologues. However, in a preferred embodiment, the
nucleic acid molecule of the present invention does not consist of
a sequence as indicated in Table I, columns 5 or 7, lines 19 to 29,
29a to 29u, 363 to 385, preferably as indicated in Table I A,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385. Preferably
the nucleic acid molecule of the invention is a functional
homologue or identical to a nucleic acid molecule indicated in
Table I B, column 7, lines 19 to 29, 29a to 29u, 363 to 385.
[1706] [0158.0.0.3] to [0160.0.0.3]: see [0158.0.0.0] to
[0160.0.0.0]
[1707] [0161.0.3.3] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising the
sequence shown in Table IA or IB, columns 5 or 7, lines 19 to 29,
29a to 29u, 363 to 385. The nucleic acid molecule is preferably at
least 20, 30, 50, 100, 250 or more nucleotides in length.
[1708] [0162.0.0.3]: see [0162.0.0.0]
[1709] [0163.0.3.3] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
of Table IA or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363
to 385 corresponds to a naturally-occurring nucleic acid molecule
of the invention. As used herein, a "naturally-occurring" nucleic
acid molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the fine chemical
increase after increasing the expression or activity thereof or the
activity of a protein of the invention or used in the process of
the invention.
[1710] [0164.0.0.3]: see [0164.0.0.0]
[1711] [0165.0.3.3] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. in Table IA
or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385
[1712] [0166.0.0.3] and [0167.0.0.3]: see [0166.0.0.0] and
[0167.0.0.0]
[1713] [0168.0.3.3] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the the fine chemical in
an organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a
to 29u, 363 to 385 yet retain said activity described herein. The
nucleic acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence of
Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363
to 385 and is capable of participation in the increase of
production of the fine chemical after increasing its activity, e.g.
its expression. Preferably, the protein encoded by the nucleic acid
molecule is at least about 60% identical to the sequence in Table
IIA or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385
more preferably at least about 70% identical to one of the
sequences in Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a
to 29u, 363 to 385, even more preferably at least about 80%, 90%,
95% homologous to the sequence in Table IIA or IIB, columns 5 or 7,
lines 19 to 29, 29a to 29u, 363 to 385, and most preferably at
least about 96%, 97%, 98%, or 99% identical to the sequence in
Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363
to 385.
[1714] Accordingly, the invention relates to nucleic acid molecules
encoding a polypeptide having above-mentioned activity, e.g.
conferring an increase in the the respective fine chemical in an
organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 19 to 29, 29a to 29u, 363 to 385, preferably of Table II B,
column 7, 19 to 29, 29a to 29u, 363 to 385 yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table II, columns 5 or 7, lines
19 to 29, 29a to 29u, 363 to 385, preferably of Table II B, column
7, lines 19 to 29, 29a to 29u, 363 to 385 and is capable of
participation in the increase of production of the respective fine
chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table II,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385, preferably
of Table II B, column 7, lines 19 to 29, 29a to 29u, 363 to 385,
more preferably at least about 70% identical to one of the
sequences as indicated in Table II, columns 5 or 7, lines 19 to 29,
29a to 29u, 363 to 385, preferably of Table II B, column 7, lines
19 to 29, 29a to 29u, 363 to 385, even more preferably at least
about 80%, 90%, or 95% homologous to a sequence as indicated in
Table II, columns 5 or 7, 19 to 29, 29a to 29u, 363 to 385,
preferably of Table II B, column 7, lines 19 to 29, 29a to 29u, 363
to 385, and most preferably at least about 96%, 97%, 98%, or 99%
identical to the sequence as indicated in Table II, columns 5 or 7,
lines 19 to 29, 29a to 29u, 363 to 385, preferably of Table II B,
column 7, lines 19 to 29, 29a to 29u, 363 to 385.
[1715] [0169.0.0.3] to [0172.0.0.3]: see [0169.0.0.0] to
[0172.0.0.0]
[1716] [0173.0.3.3] For example a sequence which has a 80% homology
with sequence SEQ ID No 899 at the nucleic acid level is understood
as meaning a sequence which, upon comparison with the sequence SEQ
ID No 899 by the above Gap program algorithm with the above
parameter set, has a 80% homology.
[1717] [0174.0.0.3]: see [0174.0.0.0]
[1718] [0175.0.3.3] For example a sequence which has a 80% homology
with sequence SEQ ID No 900 at the protein level is understood as
meaning a sequence which, upon comparison with the sequence SEQ ID
No 900 by the above program algorithm with the above parameter set,
has a 80% homology.
[1719] [0176.0.3.3] Functional equivalents derived from one of the
polypeptides as shown in Table IIA or IIB, columns 5 or 7, lines 19
to 29, 29a to 29u, 363 to 385 according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as shown in Table IIA or
IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385
according to the invention and are distinguished by essentially the
same properties as the polypeptide as shown in Table IIA or IIB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385.
[1720] [0177.0.3.3] Functional equivalents derived from the nucleic
acid sequence as shown in Table IA or IB, columns 5 or 7, lines 19
to 29, 29a to 29u, 363 to 385 according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as shown in Table IIA or
IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385
according to the invention and encode polypeptides having
essentially the same properties as the polypeptide as shown in
Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363
to 385.
[1721] [0178.0.0.3] see [0178.0.0.0]
[1722] [0179.0.3.3] A nucleic acid molecule encoding an homologous
to a protein sequence of Table IIA or IIB, columns 5 or 7, lines 19
to 29, 29a to 29u, 363 to 385, preferably of Table II B, column 7,
lines 19 to 29, 29a to 29u, 363 to 385, can be created by
introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of the nucleic acid molecule
of the present invention, in particular of Table IA or IB, columns
5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 such that one or
more amino acid substitutions, additions or deletions are
introduced into the encoded protein. Mutations can be introduced
into the encoding sequences of Table IA or IB, columns 5 or 7,
lines 19 to 29, 29a to 29u, 363 to 385 by standard techniques, such
as site-directed mutagenesis and PCR-mediated mutagenesis.
[1723] [0180.0.0.3] to [0183.0.0.3]: see [0180.0.0.0] to
[0183.0.0.0]
[1724] [0184.0.3.3] Homologues of the nucleic acid sequences used,
with the sequence shown in Table IA or IB, columns 5 or 7, lines 19
to 29, 29a to 29u, 363 to 385 preferably of Table I B, column 7,
lines 19 to 29, 29a to 29u, 363 to 385, or of the nucleic acid
sequences derived from the sequences Table IA or IB, columns 5 or
7, lines 19 to 29, 29a to 29u, 363 to 385, preferably of Table I B,
column 7, lines 19 to 29, 29a to 29u, 363 to 385, comprise also
allelic variants with at least approximately 30%, 35%, 40% or 45%
homology, by preference at least approximately 50%, 60% or 70%,
more preferably at least approximately 90%, 91%, 92%, 93%, 94% or
95% and even more preferably at least approximately 96%, 97%, 98%,
99% or more homology with one of the nucleotide sequences shown or
the abovementioned derived nucleic acid sequences or their
homologues, derivatives or analogues or parts of these. Allelic
variants encompass in particular functional variants which can be
obtained by deletion, insertion or substitution of nucleotides from
the sequences shown, preferably from Table IA or IB, columns 5 or
7, lines 19 to 29, 29a to 29u, 363 to 385, or from the derived
nucleic acid sequences, the intention being, however, that the
enzyme activity or the biological activity of the resulting
proteins synthesized is advantageously retained or increased.
[1725] [0185.0.3.3] In one embodiment of the present invention, the
nucleic acid molecule of the invention or used in the process of
the invention comprises the sequences shown in any of the Table IA
or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385,
preferably of Table I B, column 7, lines 19 to 29, 29a to 29u, 363
to 385. It is preferred that the nucleic acid molecule comprises as
little as possible other nucleotides not shown in any one of Table
IA or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385,
preferably of Table I B, column 7, lines 19 to 29, 29a to 29u, 363
to 385. In one embodiment, the nucleic acid molecule comprises less
than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or 40 further
nucleotides. In a further embodiment, the nucleic acid molecule
comprises less than 30, 20 or 10 further nucleotides. In one
embodiment, the nucleic acid molecule use in the process of the
invention is identical to the sequences shown in Table IA or IB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385, preferably
of Table I B, column 7, lines 19 to 29, 29a to 29u, 363 to 385.
[1726] [0186.0.3.3] Also preferred is that the nucleic acid
molecule used in the process of the invention encodes a polypeptide
comprising the sequence shown in Table IIA or IIB, lines columns 5
or 7, lines 19 to 29, 29a to 29u, 363 to 385, preferably of Table
II B, column 7, 19 to 29, 29a to 29u, 363 to 385. In one
embodiment, the nucleic acid molecule encodes less than 150, 130,
100, 80, 60, 50, 40 or 30 further amino acids. In a further
embodiment, the encoded polypeptide comprises less than 20, 15, 10,
9, 8, 7, 6 or 5 further amino acids. In one embodiment used in the
inventive process, the encoded polypeptide is identical to the
sequences shown in Table IIA or IIB, columns 5 or 7, lines 19 to
29, 29a to 29u, 363 to 385, preferably of Table II B, column 7,
lines 19 to 29, 29a to 29u, 363 to 385.
[1727] [0187.0.3.3] In one embodiment, the nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence shown in Table IIA or IIB, columns 5 or 7,
lines 19 to 29, 29a to 29u, 363 to 385, preferably of Table II B,
column 7, lines 19 to 29, 29a to 29u, 363 to 385 comprises less
than 100 further nucleotides. In a further embodiment, said nucleic
acid molecule comprises less than 30 further nucleotides. In one
embodiment, the nucleic acid molecule used in the process is
identical to a coding sequence of the sequences shown in Table IA
or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385,
preferably of Table I B, column 7, lines 19 to 29, 29a to 29u, 363
to 385.
[1728] [0188.0.3.3] Polypeptides (=proteins), which still have the
essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide shown in Table IIA or IIB, columns 5 or 7, lines
19 to 29, 29a to 29u, 363 to 385 expressed under identical
conditions.
[1729] [0189.0.3.3] Homologues of Table IA or IB, columns 5 or 7,
lines 19 to 29, 29a to 29u, 363 to 385 or of the derived sequences
of Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u,
363 to 385 also mean truncated sequences, cDNA, single-stranded DNA
or RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives which comprise
noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[1730] [0190.0.0.3] to [0203.0.0.3]: see [0190.0.0.0] to
[0203.0.0.0]
[1731] [0204.0.0.3] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule which comprises a nucleic acid
molecule selected from the group consisting of: [1732] a) nucleic
acid molecule encoding, preferably at least the mature form, of the
polypeptide shown in Table IIA or IIB, columns 5 or 7, lines 19 to
29, 29a to 29u, 363 to 385, preferably of Table IIB, column 7,
lines 19 to 29, 29a to 29u, 363 to 385; or a fragment thereof
conferring an increase in the amount of the fine chemical in an
organism or a part thereof [1733] b) nucleic acid molecule
comprising, preferably at least the mature form, of the nucleic
acid molecule shown in Table IA or IB, columns 5 or 7, lines 19 to
29, 29a to 29u, 363 to 385, preferably of Table IB, column 7, lines
19 to 29, 29a to 29u, 363 to 385 or a fragment thereof conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [1734] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [1735] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[1736] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [1737] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [1738] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [1739] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using the primers in Table III,
column 7, lines 19 to 29, 29a to 29u, 363 to 385 and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [1740] i) nucleic acid molecule encoding a
polypeptide which is isolated, e.g. from a expression library, with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (g), preferably to (a)
to (c) and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [1741] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence shown in Table IV, column 7, lines 19 to 29, 29a to 29u,
363 to 385 and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [1742] k) nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domaine of the polypeptide shown in Table IIA or IIB, columns 5
or 7, lines 19 to 29, 29a to 29u, 363 to 385 and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; and [1743] l) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of the nucleic acid molecule shown in Table IA or IB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 or a nucleic
acid molecule encoding, preferably at least the mature form of, the
polypeptide shown in Table IIA or IIB, columns 5 or 7, lines 19 to
29, 29a to 29u, 363 to 385, and conferring an increase in the
amount of the fine chemical in an organism or a part thereof; or
which encompasses a sequence which is complementary thereto;
whereby, preferably, the nucleic acid molecule according to (a) to
(l) distinguishes over the sequence depicted in Table IA, columns 5
or 7, lines 19 to 29, 29a to 29u, 363 to 385 by one or more
nucleotides. In one embodiment, the nucleic acid molecule of the
invention does not consist of the sequence shown in Table IA or IB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385. In another
embodiment, the nucleic acid molecule of the present invention is
at least 30% identical and less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to the sequence shown in Table IA or IB, columns 5
or 7, lines 19 to 29, 29a to 29u, 363 to 385. In a further
embodiment the nucleic acid molecule does not encode the
polypeptide sequence shown in Table IIA or IIB, columns 5 or 7,
lines 19 to 29, 29a to 29u, 363 to 385. Accordingly, in one
embodiment, the nucleic acid molecule of the present invention
encodes in one embodiment a polypeptide which differs at least in
one or more amino acids from the polypeptide depicted in Table IIA
or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 In
another embodiment, the nucleic acid molecule depicted in Table IA
or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 does
not encode a protein of the sequence shown in Table IIA or IIB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385.
Accordingly, in one embodiment, the protein encoded by a sequences
of a nucleic acid according to (a) to (l) does not consist of the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 19 to 29,
29a to 29u, 363 to 385. In a further embodiment, the protein of the
present invention is at least 30% identical to protein sequence
depicted in Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a
to 29u, 363 to 385 and less than 100%, preferably less than
99.999%, 99.99% or 99.9%, more preferably less than 99%, 98%, 97%,
96% or 95% identical to the sequence shown in Table IIA or IIB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385.
[1744] [0205.0.0.3] to [0226.0.0.3]: see [0205.0.0.0] to
[0226.0.0.0]
[1745] [0227.0.3.3] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorgansims.
[1746] In addition to the sequence mentioned in Table IA or IB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 or its
derivatives, it is advantageous additionally to express and/or
mutate further genes in the organisms. Especially advantageously,
additionally at least one further gene of the amino acid
biosynthetic pathway such as for L-lysine, L-threonine and/or
L-methionine or L-leucine and/or isoleucine and/or valine is
expressed in the organisms such as plants or microorganisms. It is
also possible that the regulation of the natural genes has been
modified advantageously so that the gene and/or its gene product is
no longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine the sequences shown in Table IA or IB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385, with genes
which generally support or enhances to growth or yield of the
target organisms, for example genes which lead to faster growth
rate of microorganisms or genes which produces stress-, pathogen,
or herbicide resistant plants.
[1747] [0228.0.0.3] to [0230.0.0.3]: see [0228.0.0.0] to
[0230.0.0.0]
[1748] [0231.0.0.3] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a leucine and/or isoleucine and/or
valine degrading protein is attenuated, in particular by reducing
the rate of expression of the corresponding gene.
[1749] [0232.0.0.3] to [0282.0.0.3]: see [0232.0.0.0] to
[0282.0.0.0]
[1750] [0283.0.3.3] Moreover, native polypeptide conferring the
increase of the fine chemical in an organism or part thereof can be
isolated from cells (e.g., endothelial cells), for example using
the antibody of the present invention as described below, in
particular, an anti-YDL127W, YDR245W, YDR271C, YER173W, YGR101W,
YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C,
YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151,
b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022,
b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327
protein antibody or an antibody against polypeptides as shown in
Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363
to 385, which can be produced by standard techniques utilizing the
polypeptid of the present invention or fragment thereof, i.e., the
polypeptide of this invention. Preferred are monoclonal
antibodies.
[1751] [0284.0.0.3]: see [0284.0.0.0]
[1752] [0285.0.3.3] In one embodiment, the present invention
relates to a polypeptide having the sequence shown in Table IIA or
IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 or as
coded by the nucleic acid molecule shown in Table IA or IB, columns
5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 or functional
homologues thereof.
[1753] [0286.0.3.3] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
comprising or consisting of the consensus sequence shown in Table
IV, lines 19 to 29, 29a to 29u, 363 to 385 and in one another
embodiment, the present invention relates to a polypeptide
comprising or consisting of the consensus sequence shown in Table
IV, lines 19 to 29, 29a to 29u, 363 to 385,
[1754] whereby 20 or less, preferably 15 or 10, preferably 9, 8, 7,
or 6, more preferred 5 or 4, even more preferred 3, even more
preferred 2, even more preferred 1, most preferred 0 of the amino
acids positions indicated can be replaced by any amino acid or, in
an further embodiment, can be replaced and/or absent.
[1755] [0287.0.0.3] to [0290.0.0.3]: see [0287.0.0.0] to
[0290.0.0.0]
[1756] [0291.0.3.3] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[1757] In one embodiment, said polypeptide of the invention
distinguishes over the sequence depicted in Table IIA or IIB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 by one or
more amino acids. In one embodiment, polypeptide distinguishes form
the sequence shown in Table IIA or IIB, columns 5 or 7, lines 19 to
29, 29a to 29u, 363 to 385 by more than 5, 6, 7, 8 or 9 amino
acids, preferably by more than 10, 15, 20, 25 or 30 amino acids,
evenmore preferred are more than 40, 50, or 60 amino acids and,
preferably, the sequence of the polypeptide of the invention
distinguishes from the sequence shown in Table IIA or IIB, columns
5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 by not more than 80%
or 70% of the amino acids, preferably not more than 60% or 50%,
more preferred not more than 40% or 30%, even more preferred not
more than 20% or 10%. In another embodiment, said polypeptide of
the invention does not consist of the sequence shown in Table IIA
or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385.
[1758] [0292.0.0.3]: see [0292.0.0.0]
[1759] [0293.0.3.3] In one embodiment, the invention relates to
polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or used in the process of the invention and having a
sequence which distinguishes from the sequence as shown in Table
IIA or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385
by one or more amino acids. In an other embodiment, said
polypeptide of the invention does not consist of the sequence shown
in Table IIA or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u,
363 to 385. In a further embodiment, said polypeptide of the
present invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by the nucleic acid molecules shown in Table
IA or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to
385.
[1760] [0294.0.3.3] In one embodiment, the present invention
relates to a polypeptide having YDL127W, YDR245W, YDR271C, YER173W,
YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C,
YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828,
b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343,
b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054
and/or b4327 protein activity, which distinguishes over the
sequence depicted in Table IIA or IIB, columns 5 or 7, lines 19 to
29, 29a to 29u, 363 to 385 by one or more amino acids, preferably
by more than 5, 6, 7, 8 or 9 amino acids, preferably by more than
10, 15, 20, 25 or 30 amino acids, even more preferred are more than
40, 50, or 60 amino acids but even more preferred by less than 70%
of the amino acids, more preferred by less than 50%, even more
preferred my less than 30% or 25%, more preferred are 20% or 15%,
even more preferred are less than 10%.
[1761] [0295.0.0.3] to [0297.0.0.3]: see [0295.0.0.0] to
[0297.0.0.0]
[1762] [0297.0.3.3] The language "substantially free of cellular
material" includes preparations of the polypeptide of the invention
in which the protein is separated from cellular components of the
cells in which it is naturally or recombinantly produced. In one
embodiment, the language "substantially free of cellular material"
includes preparations having less than about 30% (by dry weight) of
"contaminating protein", more preferably less than about 20% of
"contaminating protein", still more preferably less than about 10%
of "contaminating protein", and most preferably less than about 5%
"contaminating protein". The term "Contaminating protein" relates
to polypeptides, which are not polypeptides of the present
invention. When the polypeptide of the present invention or
biologically active portion thereof is recombinantly produced, it
is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less
than about 10%, and most preferably less than about 5% of the
volume of the protein preparation. The language "substantially free
of chemical precursors or other chemicals" includes preparations in
which the polypeptide of the present invention is separated from
chemical precursors or other chemicals which are involved in the
synthesis of the protein. The language "substantially free of
chemical precursors or other chemicals" includes preparations
having less than about 30% (by dry weight) of chemical precursors
or non-YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C,
YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W,
YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827,
b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414,
b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 chemicals,
more preferably less than about 20% chemical precursors or
non-YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C,
b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124,
b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664,
b3117, b3256, b3938, b3983, b4054 and/or b4327 chemicals, still
more preferably less than about 10% chemical precursors or
non-YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W,
YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C,
b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827, b0124,
b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414, b2664,
b3117, b3256, b3938, b3983, b4054 and/or b4327 chemicals, and most
preferably less than about 5% chemical precursors or non-YDL127W,
YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C,
YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829,
b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161,
b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256,
b3938, b3983, b4054 and/or b4327 chemicals. In preferred
embodiments, isolated proteins or biologically active portions
thereof lack contaminating proteins from the same organism from
which the polypeptide of the present invention is derived.
Typically, such proteins are produced by recombinant techniques,
[00297.1.0.3] Non-polypeptide of the invention-chemicals are e.g.
polypeptides having not the activity and/or the amino acid sequence
of a polypeptide indicated in Table II, columns 3, 5 or 7, lines 19
to 29, 29a to 29u, 363 to 385.
[1763] [0298.0.3.3] A polypeptide of the invention can participate
in the process of the present invention. The polypeptide or a
portion thereof comprises preferably an amino acid sequence which
is sufficiently homologous to an amino acid sequence shown in Table
IIA or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385
such that the protein or portion thereof maintains the ability to
confer the activity of the present invention. The portion of the
protein is preferably a biologically active portion as described
herein. Preferably, the polypeptide used in the process of the
invention has an amino acid sequence identical as shown in Table
IIA or IIB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to
385.
[1764] [0299.0.3.3] Further, the polypeptide can have an amino acid
sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences of Table IIA or IIB, columns 5 or 7, lines 19
to 29, 29a to 29u, 363 to 385. The preferred polypeptide of the
present invention preferably possesses at least one of the
activities according to the invention and described herein. A
preferred polypeptide of the present invention includes an amino
acid sequence encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions, to a nucleotide
sequence of Table IA or IB, columns 5 or 7, lines 19 to 29, 29a to
29u, 363 to 385 or which is homologous thereto, as defined
above.
[1765] [0300.0.3.3] Accordingly the polypeptide of the present
invention can vary from Table IIA or IIB, columns 5 or 7, lines 19
to 29, 29a to 29u, 363 to 385 in amino acid sequence due to natural
variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprise an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90%, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence of Table IIA or IIB, columns 5 or 7, lines 19
to 29, 29a to 29u, 363 to 385.
[1766] [0301.0.0.3]: see [0301.0.0.0]
[1767] [0302.0.3.3] Biologically active portions of an polypeptide
of the present invention include peptides comprising amino acid
sequences derived from the amino acid sequence of the polypeptide
of the present invention or used in the process of the present
invention, e.g., the amino acid sequence shown in Table IIA or IIB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 or the amino
acid sequence of a protein homologous thereto, which include fewer
amino acids than a full length polypeptide of the present invention
or used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[1768] [0303.0.0.3]: see [0303.0.0.0]
[1769] [0304.0.3.3] Manipulation of the nucleic acid molecule of
the invention may result in the production of YDL127W, YDR245W,
YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C,
YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957,
b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486,
b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938,
b3983, b4054 and/or b4327 protein having differences from the
wild-type YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C,
YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W,
YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827,
b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414,
b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein.
These proteins may be improved in efficiency or activity, may be
present in greater numbers in the cell than is usual, or may be
decreased in efficiency or activity in relation to the wild type
protein.
[1770] [0305.0.0.3]: see [0305.0.0.0]
[1771] [0305.0.3.3] Any mutagenesis strategies for the polypeptide
of the present invention or the polypeptide used in the process of
the present invention to result in increasing said activity are not
meant to be limiting; variations on these strategies will be
readily apparent to one skilled in the art. Using such strategies,
and incorporating the mechanisms disclosed herein, the nucleic acid
molecule and polypeptide of the invention may be utilized to
generate plants or parts thereof, expressing wildtype YDL127W,
YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C,
YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829,
b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161,
b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256,
b3938, b3983, b4054 and/or b4327 proteins or mutated YDL127W,
YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C,
YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829,
b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161,
b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256,
b3938, b3983, b4054 and/or b4327 protein encoding nucleic acid
molecules and polypeptide molecules of the invention such that the
yield, production, and/or efficiency of production of a desired
compound is improved.
[1772] [0306.0.0.3] to [0308.0.0.3]: see [0306.0.0.0] to
[0308.0.0.0]
[1773] [0309.0.3.3] In one embodiment, an "YDL127W, YDR245W,
YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C,
YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957,
b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486,
b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938,
b3983, b4054 and/or b4327 protein (=polypeptide)" refers to a
polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas a "non-YDL127W, YDR245W, YDR271C, YER173W,
YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C,
YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828,
b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313, b1343,
b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983, b4054
and/or b4327 polypeptide" or "other polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous a polypeptide of the
invention, preferably which is not substantially homologous to a
polypeptide having an YDL127W, YDR245W, YDR271C, YER173W, YGR101W,
YJL072C, YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C,
YFR042W, YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151,
b1827, b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022,
b2414, b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327
protein activity, e.g., a protein which does not confer the
activity described herein and which is derived from the same or a
different organism.
[1774] [0310.0.0.3] to [0334.0.0.3]: see [0310.0.0.0] to
[0334.0.0.0]
[1775] [0335.0.3.3] The dsRNAi method has proved to be particularly
effective and advantageous for reducing the expression of the
nucleic acid sequences of the Table IA or IB, columns 5 or 7, lines
19 to 29, 29a to 29u, 363 to 385 and/or homologs thereof. As
described inter alia in WO 99/32619, dsRNAi approaches are clearly
superior to traditional antisense approaches. The invention
therefore furthermore relates to double-stranded RNA molecules
(dsRNA molecules) which, when introduced into an organism,
advantageously into a plant (or a cell, tissue, organ or seed
derived therefrom), bring about altered metabolic activity by the
reduction in the expression of the nucleic acid sequences of the
Table IA or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to
385 and/or homologs thereof. In a double-stranded RNA molecule for
reducing the expression of an protein encoded by a nucleic acid
sequence of one of the Table IA or IB, columns 5 or 7, lines 19 to
29, 29a to 29u, 363 to 385 and/or homologs thereof, one of the two
RNA strands is essentially identical to at least part of a nucleic
acid sequence, and the respective other RNA strand is essentially
identical to at least part of the complementary strand of a nucleic
acid sequence.
[1776] [0336.0.0.3] to [0342.0.0.3]: see [0336.0.0.0] to
[0342.0.0.0]
[1777] [0343.0.3.3] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence of one of the Table IA or IB, columns 5 or 7, lines 19 to
29, 29a to 29u, 363 to 385 or its homolog is not necessarily
required in order to bring about effective reduction in the
expression. The advantage is, accordingly, that the method is
tolerant with regard to sequence deviations as may be present as a
consequence of genetic mutations, polymorphisms or evolutionary
divergences. Thus, for example, using the dsRNA, which has been
generated starting from a sequence of one of Table IA or IB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 or homologs
thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[1778] [0344.0.0.3] to [0361.0.0.3]: see [0344.0.0.0] to
[0361.0.0.0]
[1779] [0362.0.3.3] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the fine chemical in
a cell or an organism or a part thereof, e.g. the nucleic acid
molecule of the invention, the nucleic acid construct of the
invention, the antisense molecule of the invention, the vector of
the invention or a nucleic acid molecule encoding the polypeptide
of the invention, e.g. encoding a polypeptide having an YDL127W,
YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C,
YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829,
b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161,
b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256,
b3938, b3983, b4054 and/or b4327 protein activity. Due to the above
mentioned activity the fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manupulation, the cellular activity is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an YDL127W, YDR245W, YDR271C,
YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C,
YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327 protein or activity means herein that due to
modulation or manipulation of the genome, the activity of YDL127W,
YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C,
YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829,
b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161,
b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256,
b3938, b3983, b4054 and/or b4327 or a YDL127W, YDR245W, YDR271C,
YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C, YGR104C,
YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957, b3366,
b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486, b1313,
b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938, b3983,
b4054 and/or b4327-like activity is increased in the cell or
organism or part thereof. Examples are described above in context
with the process of the invention.
[1780] [0363.0.0.3]: see [0363.0.0.0]
[1781] [0364.0.3.3] A naturally occurring expression cassette--for
example the naturally occurring combination of the YDL127W,
YDR245W, YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C,
YFL013C, YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829,
b2957, b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161,
b0486, b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256,
b3938, b3983, b4054 and/or b4327 protein promoter with the
corresponding YDL127W, YDR245W, YDR271C, YER173W, YGR101W, YJL072C,
YKR057W, YNL135C, YFL013C, YGR104C, YIL150C, YOR350C, YFR042W,
YFL019C, b1708, b1829, b2957, b3366, b0828, b3966, b4151, b1827,
b0124, b0149, b0161, b0486, b1313, b1343, b1463, b2022, b2414,
b2664, b3117, b3256, b3938, b3983, b4054 and/or b4327 protein
gene--becomes a transgenic expression cassette when it is modified
by non-natural, synthetic "artificial" methods such as, for
example, mutagenization. Such methods have been described (U.S.
Pat. No. 5,565,350; WO 00/15815; also see above).
[1782] [0365.0.0.3] to [0382.0.0.3]: see [0365.0.0.0] to
[0382.0.0.0]
[1783] [0383.0.3.3] For preparing branched-chain amino acid
compound-containing fine chemicals, in particular the fine
chemical, it is possible to use as branched-chain amino acid source
organic branched-chain amino acid-containing compounds such as, for
example, isovalerate, isopropylmalate, oxoisocaproate,
isovaleryl-compounds, methylvalerate, isobutyrate,
methyl-butyryl-compounds, isopropyrate, isopropyl-compounds or else
organic branched-chain amino acid precursor compounds.
[1784] [0384.0.0.3] to [0392.0.0.3]: see [0384.0.0.0] to
[0392.0.0.0]
[1785] [0393.0.3.3] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [1786] (g) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [1787] (h) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence shown in Table
IA or IB, columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385,
preferably in Table I B, column 7, lines 19 to 29, 29a to 29u, 363
to 385 and, optionally, isolating the full length cDNA clone or
complete genomic clone; [1788] (i) introducing the candidate
nucleic acid molecules in host cells, preferably in a plant cell or
a microorganism, appropriate for producing the fine chemical;
[1789] (j) expressing the identified nucleic acid molecules in the
host cells; [1790] (k) assaying the the fine chemical level in the
host cells; and [1791] (l) identifying the nucleic acid molecule
and its gene product which expression confers an increase in the
the fine chemical level in the host cell after expression compared
to the wild type.
[1792] [0394.0.0.3] to [0398.0.0.3]: see [0394.0.0.0] to
[0398.0.0.0]
[1793] [0399.0.3.3] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increased the fine chemical production in a plant or
microorganism, comprising the steps: [1794] (c) culturing a cell or
tissue or microorganism or maintaining a plant expressing the
polypeptide according to the invention or a nucleic acid molecule
encoding said polypeptide and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and the
polypeptide of the present invention or used in the process of the
invention; and [1795] (d) identifying if the compound is an
effective agonist by detecting the presence or absence or increase
of a signal produced by said readout system.
[1796] The screen for a gene product or an agonist conferring an
increase in the fine chemical production can be performed by growth
of an organism for example a microorganism in the presence of
growth reducing amounts of an inhibitor of the synthesis of the
fine chemical. Better growth, eg higher dividing rate or high dry
mass in comparison to the control under such conditions would
identify a gene or gene product or an agonist conferring an
increase in fine chemical production.
[1797] One can think to screen for increased fine chemical
production by for example resistance to drugs blocking isoleucine
synthesis and looking whether this effect is YDL127W dependent eg
comparing near identical organisms with low and high YDL127W
activity.
[1798] [0400.0.0.3] to [0430.0.0.3]: see [0400.0.0.1] to
[0430.0.0.0]
[0431.0.3.3] Example 1
Cloning SEQ ID No: 899 in Escherichia coli
[1799] [0432.0.3.3] SEQ ID No: 899 was cloned into the plasmids
pBR322 (Sutcliffe, J. G. (1979) Proc. Natl Acad. Sci. USA, 75:
3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol. 134:
1141-1156); plasmids of the pBS series (pBSSK+, pBSSK- and others;
Stratagene, LaJolla, USA) or cosmids such as SuperCosi
(Stratagene,
[1800] LaJolla, USA) or Lorist6 (Gibson, T. J. Rosenthal, A., and
Waterson, R. H. (1987) Gene 53: 283-286) for expression in E. coli
using known, well-established procedures (see, for example,
Sambrook, J. et al. (1989) "Molecular Cloning: A Laboratory
Manual". Cold Spring Harbor Laboratory Press or Ausubel, F. M. et
al. (1994) "Current Protocols in Molecular Biology", John Wiley
& Sons).
[1801] [0433.0.0.3] to [0460.0.0.3]: see [0433.0.0.0] to
[0460.0.0.0]
[0461.0.3.3] Example 10
Cloning SEQ ID NO: 899 for the Expression in Plants
[1802] [0462.0.0.3]: see [0462.0.0.0]
[1803] [0463.0.3.3] SEQ ID NO: 899 is amplified by PCR as described
in the protocol of the Pfu Turbo or DNA Herculase polymerase
(Stratagene).
[1804] [0464.0.0.3] to [0466.0.0.3]: see [0464.0.0.0] to
[0466.0.0.0]
[1805] [0467.0.3.3] The following primer sequences were selected
for the gene SEQ ID No: 899:
TABLE-US-00015 i) forward primer (SEQ ID No: 911) atgtcaaact
acgaagcctt gctg ii) reverse primer (SEQ ID No: 912) tcacagggcg
cgctttacta aaat
[1806] [0468.0.0.3] to [0479.0.0.3]: see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.3.3] Example 11
Generation of Transgenic Plants which Express SEQ ID No: 899
[1807] [0481.0.0.3] to [0513.0.0.3]: see [0481.0.0.0] to
[0513.0.0.0]
[1808] [0514.0.3.3] As an alternative, the amino acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55). [1809] The results of the different plant analyses
can be seen from the table which follows:
TABLE-US-00016 [1809] TABLE 1 ORF Metabolite Method Min Max YDL127W
Isoleucine GC 1.33 1.33 YDR245W Leucine GC 1.5 2.73 YDR271C
Isoleucine GC 1.3 1.74 YER173W Valine GC 1.18 1.82 YER173W Leucine
GC 1.93 2.87 YGR101W Valine GC 1.47 1.6 YJL072C Isoleucine GC 1.31
4.56 YKR057W Valine GC 1.19 2 YNL135C Leucine GC 1.45 3.8 YFL013C
Valine GC 1.22 1.58 YGR104C Isoleucine GC 1.33 2.02 YIL150C Valine
GC 1.18 4.14 YIL150C Isoleucine GC 6.35 6.35 YOR350C Isoleucine GC
2.01 2.78 YOR350C Leucine GC 3.47 4.48 YFR042W Leucine GC 1.43 4.25
YFR019C Valine GC 1.22 1.58 b1708 Valine GC 1.33 2.49 b1708
Isoleucine GC 1.51 3.9 b1708 Leucine GC 1.6 4.33 b1829 Isoleucine
GC 1.77 15.8 b1829 Leucine GC 1.6 13 b2957 Valine GC 1.32 4.03
b3366 Isoleucine GC 1.39 4.27 b3366 Leucine GC 1.65 6.81 b0828
Valine GC 1.2 1.61 b0828 Isoleucine GC 1.6 2.02 b3966 Isoleucine GC
1.79 4.92 b3966 Leucine GC 1.59 7.47 b4151 Leucine GC 1.31 1.46
b1827 Valine GC 1.24 2.92 b1827 Isoleucine GC 1.81 6.82 b1827
Leucine GC 1.63 9.84 b0124 Isoleucine GC 1.43 7.38 b0124 Leucine GC
1.56 6.7 b0124 Valine GC 1.24 3.68 b0149 Leucine GC 1.62 1.99 b0161
Valine GC 1.29 4.99 b0161 Isoleucine GC 1.98 11.3 b0161 Leucine GC
1.71 12.4 b0486 Valine GC 1.22 2.81 b1313 Valine GC 1.22 1.5 b1343
Valine GC 1.21 1.23 b1463 Isoleucine GC 1.39 2.19 b1463 Leucine GC
1.66 2.57 b2022 Valine GC 1.22 1.26 b2414 Valine GC 1.35 2.39 b2664
Isoleucine GC 1.42 12.7 b2664 Leucine GC 1.67 11.5 b3117 Isoleucine
GC 1.47 7.56 b3256 Valine GC 1.27 1.36 b3938 Valine GC 1.24 1.42
b3983 Valine GC 1.24 3.02 b4054 Leucine GC 2.26 2.63 b4327
Isoleucine GC 1.42 1.54
[1810] [0515.0.0.3]: to [0552.0.0.3]: see [0515.0.0.0] to
[0552.0.0.0]
[0552.1.3.3]: Example 15
Metabolite Profiling Info from Zea mays
[1811] Zea mays plants were engineered, grown and analyzed as
described in Example 14c.
[1812] The results of the different Zea mays plants analysed can be
seen from Table 2 which follows:
TABLE-US-00017 TABLE 2 ORF_NAME Metabolite Min Max b1829 Isoleucine
1.28 1.37 b1829 Leucine 1.37 1.97 b2414 Valine 1.26 1.52 b2664
Isoleucine 1.91 4.81 b2664 Leucine 2.17 5.29 YDR271C Isoleucine
1.36 3.73 YKR057W Valine 1.29 1.62 YIL150C Valine 1.89 3.26 YIL150C
Isoleucine 1.79 2.98
[1813] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in valine and/or
isoleucine and/or leucine in genetically modified corn plants
expressing the Saccharomyces cerevisiae nucleic acid sequence
YKR057W, YDR271C or YIL150C and/or E. coli nucleic acid sequence
b1829, b2414 or b2664 resp.
[1814] In case the activity of the Saccharomyces cerevisiae protein
YKR057W or a ribosomal protein, similar to S21 ribosomal proteins,
involved in ribosome biogenesis and translation or its homolog, is
increased in corn plants, preferably, an increase of the fine
chemical valine between 29% and 62% is conferred.
[1815] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YIL150C or its homologs, e.g. "a chromatin
binding protein, required for S-phase (DNA synthesis) initiation or
completion" or its homologs, is increased in corn plants,
preferably, an increase of the fine chemical valine between 89% and
226% and/or of the fine chemical isoleucine between 79% and 198% is
conferred.
[1816] In one embodiment, in case the activity of the E. coli
protein b1829 or its homologs, e.g. "the activity of a a heat shock
protein with protease activity (htpx), involved in stress response,
pheromone response, mating-type determination, protein
modification, proteolytic degradation", is increased in corn
plants, preferably, an increase of the fine chemical isoleucine
between 28% and 37% is conferred and/or an increase of the fine
chemical leucine between 37% and 97% is conferred.
[1817] In one embodiment, in case the activity of the E. coli
protein b2414 or its homologs, e.g. "a subunit of cysteine synthase
A and O-acetylserine sulfhydrolase A, PLP-dependent enzyme", is
increased in corn plants, preferably, an increase of the fine
chemical valine between 26% and 52% is conferred.
[1818] In one embodiment, in case the activity of the E. coli
protein b2664 or its homologs, e.g. "a putative transcriptional
repressor with DNA-binding Winged helix domain (GntR familiy)", is
increased in corn plants, preferably, an increase of the fine
chemical isoleucine between 91% and 381% is conferred. and/or an
increase of the fine chemical leucine between 117% and 429% is
conferred.
[1819] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein
[1820] YDR271C or its homologs, its activity has not been
characterized yet, is increased in corn plants, preferably, an
increase of the fine chemical isoleucine between 36% and 273% is
conferred.
[1821] [00552.2.0.3]: see [00552.2.0.0]
[1822] [00553.0.3.3] [1823] 1. A process for the production of
leucine and/or isoleucine and/or valine, which comprises [1824] (a)
increasing or generating the activity of a YDL127W, YDR245W,
YDR271C, YER173W, YGR101W, YJL072C, YKR057W, YNL135C, YFL013C,
YGR104C, YIL150C, YOR350C, YFR042W, YFL019C, b1708, b1829, b2957,
b3366, b0828, b3966, b4151, b1827, b0124, b0149, b0161, b0486,
b1313, b1343, b1463, b2022, b2414, b2664, b3117, b3256, b3938,
b3983, b4054 and/or b4327 protein in a non-human organism, or in
one or more parts thereof; and [1825] (b) growing the organism
under conditions which permit the production of leucine and/or
isoleucine and/or valine in said organism. [1826] 2. A process for
the production of leucine and/or isoleucine and/or valine,
comprising the increasing or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [1827] a) nucleic acid molecule encoding the
polypeptide shown in Table IIA or IIB, columns 5 or 7, lines 19 to
29, 29a to 29u, 363 to 385 or a fragment thereof, which confers an
increase in the amount of leucine and/or isoleucine and/or valine
in an organism or a part thereof; [1828] b) nucleic acid molecule
comprising of the nucleic acid molecule shown in Table IA or IB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385; [1829] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of leucine and/or isoleucine
and/or valine in an organism or a part thereof; [1830] d) nucleic
acid molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of leucine and/or isoleucine and/or valine in an
organism or a part thereof; [1831] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of leucine and/or isoleucine and/or valine in an
organism or a part thereof; [1832] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers shown in Table III, column 7, lines 19 to 29, 29a
to 29u, 363 to 385 and conferring an increase in the amount of
leucine and/or isoleucine and/or valine in an organism or a part
thereof; [1833] g) nucleic acid molecule encoding a polypeptide
which is isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of leucine and/or
isoleucine and/or valine in an organism or a part thereof; [1834]
h) nucleic acid molecule encoding a polypeptide comprising the
consensus sequence shown in Table IV, column 7, lines 19 to 29, 29a
to 29u, 363 to 385 and conferring an increase in the amount of the
fine chemical in an organism or a part thereof; and [1835] i)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment thereof having at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (k) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof. [1836] or comprising a sequence which
is complementary thereto. [1837] 3. The process of claim 1 or 2,
comprising recovering of the free or bound leucine and/or
isoleucine and/or valine. [1838] 4. The process of any one of
claims 1 to 3, comprising the following steps: [1839] (a) selecting
an organism or a part thereof expressing a polypeptide encoded by
the nucleic acid molecule characterized in claim 2; [1840] (b)
mutagenizing the selected organism or the part thereof; [1841] (c)
comparing the activity or the expression level of said polypeptide
in the mutagenized organism or the part thereof with the activity
or the expression of said polypeptide of the selected organisms or
the part thereof; [1842] (d) selecting the mutated organisms or
parts thereof, which comprise an increased activity or expression
level of said polypeptide compared to the selected organism or the
part thereof; [1843] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [1844] (f) recovering, and
optionally isolating, the free or bound leucine and/or isoleucine
and/or valine produced by the selected mutated organisms or parts
thereof. [1845] 5. The process of any one of claims 1 to 4, wherein
the activity of said protein or the expression of said nucleic acid
molecule is increased or generated transiently or stably. [1846] 6.
An isolated nucleic acid molecule comprising a nucleic acid
molecule selected from the group consisting of: [1847] a) nucleic
acid molecule encoding the polypeptide shown in Table IIA or IIB,
columns 5 or 7, lines 19 to 29, 29a to 29u, 363 to 385 or a
fragment thereof, which confers an increase in the amount of
leucine and/or isoleucine and/or valine in an organism or a part
thereof; b) nucleic acid molecule comprising the nucleic acid
molecule shown in Table [1848] IA or IB, columns 5 or 7, lines 19
to 29, 29a to 29u, 363 to 385; c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as a result of the degeneracy
of the genetic code and conferring an increase in the amount of
leucine and/or isoleucine and/or valine in an organism or a part
thereof; [1849] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of leucine
and/or isoleucine and/or valine in an organism or a part thereof;
[1850] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of leucine
and/or isoleucine and/or valine in an organism or a part thereof;
[1851] f) nucleic acid molecule which encompasses a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers shown in
Table III, column 7, lines 19 to 29, 29a to 29u, 363 to 385 and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [1852] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
leucine and/or isoleucine and/or valine in an organism or a part
thereof; [1853] h) nucleic acid molecule encoding a polypeptide
comprising the consensus sequence shown in Table IV, column 7,
lines 19 to 29, 29a to 29u, 363 to 385 and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; and [1854] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof.
[1855] whereby the nucleic acid molecule distinguishes over the
sequence as shown in Table IA, columns 5 or 7, lines 19 to 29, 29a
to 29u, 363 to 385 by one or more nucleotides. [1856] 7. A nucleic
acid construct which confers the expression of the nucleic acid
molecule of claim 6, comprising one or more regulatory elements.
[1857] 8. A vector comprising the nucleic acid molecule as claimed
in claim 6 or the nucleic acid construct of claim 7. [1858] 9. The
vector as claimed in claim 8, wherein the nucleic acid molecule is
in operable linkage with regulatory sequences for the expression in
a prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [1859] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [1860] 11. The host cell of claim 10, which is a
transgenic host cell. [1861] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [1862] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [1863] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over the sequence as shown in Table IIA, columns 5 or
7, lines 19 to 29, 29a to 29u, 363 to 385 by one or more amino
acids 15. An antibody, which binds specifically to the polypeptide
as claimed in claim 14. [1864] 16. A plant tissue, propagation
material, harvested material or a plant comprising the host cell as
claimed in claim 12 which is plant cell or an Agrobacterium. [1865]
17. A method for screening for agonists and antagonists of the
activity of a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of leucine and/or
isoleucine and/or valine in an organism or a part thereof
comprising: [1866] (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of leucine and/or isoleucine and/or valine in an organism or
a part thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; [1867] (b) assaying the leucine and/or isoleucine
and/or valine level or the polypeptide expression level in the
cell, tissue, plant or microorganism or the media the cell, tissue,
plant or microorganisms is cultured or maintained in; and [1868]
(c) identifying a agonist or antagonist by comparing the measured
leucine and/or isoleucine and/or valine level or polypeptide
expression level with a standard leucine and/or isoleucine and/or
valine or polypeptide expression level measured in the absence of
said candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. [1869] 18. A process for the
identification of a compound conferring increased leucine and/or
isoleucine and/or valine production in a plant or microorganism,
comprising the steps: [1870] (a) culturing a plant cell or tissue
or microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of leucine and/or isoleucine and/or valine
in an organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with dais readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of leucine and/or isoleucine
and/or valine in an organism or a part thereof; [1871] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [1872] 19. A method for the identification of a
gene product conferring an increase in leucine and/or isoleucine
and/or valine production in a cell, comprising the following steps:
[1873] (a) contacting the nucleic acid molecules of a sample, which
can contain a candidate gene encoding a gene product conferring an
increase in leucine and/or isoleucine and/or valine after
expression with the nucleic acid molecule of claim 6; [1874] (b)
identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; [1875] (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing leucine and/or
isoleucine and/or valine; [1876] (d) expressing the identified
nucleic acid molecules in the host cells; [1877] (e) assaying the
leucine and/or isoleucine and/or valine level in the host cells;
and [1878] (f) identifying nucleic acid molecule and its gene
product which expression confers an increase in the leucine and/or
isoleucine and/or valine level in the host cell in the host cell
after expression compared to the wild type. [1879] 20. A method for
the identification of a gene product conferring an increase in
leucine and/or isoleucine and/or valine production in a cell,
comprising the following steps: [1880] (a) identifiying in a data
bank nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the leucine and/or isoleucine and/or valine amount or level in an
organism or a part thereof after expression, and which are at least
20% homolog to the nucleic acid molecule of claim 6; [1881] (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing leucine and/or isoleucine and/or valine;
[1882] (c) expressing the identified nucleic acid molecules in the
host cells; [1883] (d) assaying the leucine and/or isoleucine
and/or valine level in the host cells; and [1884] (e) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the leucine and/or isoleucine and/or valine level in
the host cell after expression compared to the wild type. [1885]
21. A method for the production of an agricultural composition
comprising the steps of the method of any one of claims 17 to 20
and formulating the compound identified in any one of claims 17 to
20 in a form acceptable for an application in agriculture. [1886]
22. A composition comprising the nucleic acid molecule of claim 6,
the polypeptide of claim 14, the nucleic acid construct of claim 7,
the vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier.
[1887] 23. Use of the nucleic acid molecule as claimed in claim 6
for the identification of a nucleic acid molecule conferring an
increase of leucine and/or isoleucine and/or valine after
expression. [1888] 24. Use of the polypeptide of claim 14 or the
nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of leucine and/or isoleucine
and/or valine levels in an organism. [1889] 25. Food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claims 10 to 12 or the gene product identified according to
the method of claim 19 or 20. [1890] 26. Use of the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the host cell of claims 10
to 12 or the gene product identified according to the method of
claim 19 or 20 for the protection of a plant against a leucine
and/or isoleucine and/or valine synthesis inhibiting herbicide.
[1891] [00554.0.0.3] Abstract: see [00554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[1892] [0000.0.0.4] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and corresponding embodiments as described herein
as follows.
[1893] [0001.0.0.4] to [0008.0.0.4]: see [0001.0.0.0] to
[0008.0.0.0]
[1894] [0009.0.4.4] As described above, the essential amino acids
are necessary for humans and many mammals, for example for
livestock. Arginine is a semi-essential amino acid involved in
multiple areas of human physiology and metabolism. It is not
considered essential because humans can synthesize it de novo from
glutamine, glutamate, and proline. However, dietary intake remains
the primary determinant of plasma arginine levels, since the rate
of arginine biosynthesis does not increase to compensate for
depletion or inadequate supply. Dietary arginine intake regulates
whole body arginine synthesis from proline in the neonatal piglet.
The maximal rate of arginine synthesis (0.68 g/kg/d) is not enough
to supply the whole body metabolic requirement for arginine in the
young pig. In animals, glutamate functions as a neurotransmitter
and activates glutamate receptor cation channels (iGluRs), which
trigger electrical or Ca.sup.2+ signal cascades. In plants, amino
acids are involved in signalling of both plant nitrogen status and
plant nitrogen:carbon ratios. Endogenous glutamine has been
implicated in feedback inhibition of root N uptake, via the
suppression of transcription of genes encoding inorganic nitrogen
transporters (Rawat et al., Plant Journal 19: 143-152, 1999; Zhuo
et al., Plant Journal 17: 563-568, 1999). The nonessential amino
acid, proline, is synthesized from L-ornithine or L-glutamate. The
proline from L-ornithine is linked to protein metabolism in the
urea cycle and the proline from L-glutamate is linked to
carbohydrate metabolism. Collagen is the major reservoir for
proline in the body. Vitamin C should be used with proline for
collagen problems.
[1895] [0010.0.0.4] to [0011.0.0.4]: see [0010.0.0.0] to
[0011.0.0.0]:
[1896] [0012.0.4.4] It is an object of the present invention to
develop an inexpensive process for the synthesis of arginine and/or
glutamate and/or glutamine and/or proline, preferably L-arginine
and/or L-glutamate and/or L-glutamine and/or L-proline.
[1897] [0013.0.0.4]: see [0013.0.0.0]
[1898] [0014.0.4.4] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is arginine and/or glutamate
and/or glutamine and/or proline, preferably L-arginine and/or
L-glutamate and/or L-glutamine and/or L-proline. Accordingly, in
the present invention, the term "the fine chemical" as used herein
relates to "arginine and/or glutamate and/or glutamine and/or
proline". Further, the term "the fine chemicals" as used herein
also relates to fine chemicals comprising arginine and/or glutamate
and/or glutamine and/or proline.
[1899] [0015.0.4.4] In one embodiment, the term "the fine chemical"
means arginine and/or glutamate and/or glutamine and/or proline,
preferably L-arginine and/or L-glutamate and/or L-glutamine and/or
L-proline. Throughout the specification the term "the fine
chemical" means arginine and/or glutamate and/or glutamine and/or
proline, preferably
[1900] L-arginine and/or L-glutamate and/or L-glutamine and/or
L-proline, its salts, ester or amids in free form or bound to
proteins. In a preferred embodiment, the term "the fine chemical"
means arginine and/or glutamate and/or glutamine and/or proline,
preferably L-arginine and/or L-glutamate and/or L-glutamine and/or
L-proline, in free form or its salts or bound to proteins.
[1901] [0016.0.4.4] Accordingly, the present invention relates to a
process comprising [1902] (a) increasing or generating the activity
of one or more [1903] YDR316W, YHR130C, YKR057W, YNL090W, b1829,
b0695, b1284, b2095, b0161, b2307 and/or b3936-protein(s) or of a
protein having the sequence of a polypeptide encoded by a nucleic
acid molecule indicated in Table II, columns 5 or 7, lines 30 to
37, 390, 405 and/or 430; [1904] in a non-human organism in one or
more parts thereof and [1905] (b) growing the organism under
conditions which permit the production of the fine chemical,
meaning of arginine or fine chemicals comprising arginine in said
organism; [1906] or [1907] (a) increasing or generating the
activity of one or more [1908] YBR204C, YFL013C, YGR104C, YPRO24W,
YPR133W-A, b0730, b0050, b0057, b0161, b1343, b1693, b1736, b1738,
b1896, b2307, b2710, b2818, b3074, b3116, b3169, b3619, b3791,
b4346, and/or YFL019C--protein(s) or of a protein having the
sequence of a polypeptide encoded by a nucleic acid molecule
indicated in Table II, columns 5 or 7, lines 38 to 43, 386, 387,
391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427,
434 and/or 435; [1909] in a non-human organism in one or more parts
thereof and [1910] (b) growing the organism under conditions which
permit the production of the fine chemical, meaning of glutamate or
fine chemicals comprising glutamate in said organism;
[1911] or [1912] (a) increasing or generating the activity of one
or more [1913] YBRO30W, YDL106C, YDR271C, YEL045C, YER173W,
YFL050C, YGR135W, YIL150C, YNL090W, YPR138C, b0730, b2699, b1827,
b0138, b0149, b1360, b2553, b2664, b3644 and/or b3919-protein(s) or
of a protein having the sequence of a polypeptide encoded by a
nucleic acid molecule indicated in Table II, columns 5 or 7, lines
44 to 56, 388, 389, 398, 411, 412, 425 and/or 429; [1914] in a
non-human organism in one or more parts thereof and [1915] (b)
growing the organism under conditions which permit the production
of the fine chemical, meaning of proline or fine chemicals
comprising proline in said organism; [1916] or [1917] (a)
increasing or generating the activity of one or more [1918]
YER173W, YFR042W, YKR057W, b1829, b1852, b4265, b0161, b0486,
b0849, b0970, b1343, b1886, b1926, b2414, b2426, b2489, b2553,
b2818, b3064, b3160, b3166, b3169, b3231, b3680, b3719, b4004,
b4074 and/or b4133-protein(s) or of a protein having the sequence
of a polypeptide encoded by a nucleic acid molecule indicated in
Table II, columns 5 or 7, lines 57 to 62, 392 to 395, 397, 402,
404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431
to 433; [1919] in a non-human organism in one or more parts thereof
and [1920] (b) growing the organism under conditions which permit
the production of the fine chemical, meaning glutamine or fine
chemicals comprising glutamine in said organism.
[1921] Accordingly, the present invention relates to a process for
the production of a fine chemical comprising [1922] (a) increasing
or generating the activity of one or more proteins having the
activity of a protein indicated in Table II, column 3, lines 30 to
62 and/or lines 386 to 435 or having the sequence of a polypeptide
encoded by a nucleic acid molecule indicated in Table I, column 5
or 7, lines 30 to 62 and/or lines 386 to 435, in a non-human
organism in one or more parts thereof and [1923] (b) growing the
organism under conditions which permit the production of the fine
chemical, in particular arginine and/or glutamate and/or glutamine
and/or proline resp.
[1924] [0016.1.4.4] Accordingly, the term "the fine chemical" means
in one embodiment "arginine" in relation to all sequences listed in
Table I to IV, lines 30 to 37, 390, 405 and/or 430 or homologs
thereof and means in one embodiment "glutamate" in relation to all
sequences listed in Tables I to IV, lines 38 to 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434
and/or 435 or homologs thereof and means in one embodiment
"proline" in relation to all sequences listed in Table I to IV,
lines 44 to 56, 388, 389, 398, 411, 412, 425 and/or 429 or homologs
thereof and means in one embodiment "glutamine" in relation to all
sequences listed in Tables I to IV, lines 57 to 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 or homologs thereof.
[1925] Accordingly, in one embodiment the term "the fine chemical"
means "glutamate" and "proline" in relation to all sequences listed
in Table I to IV, lines 43 and 54, in one embodiment the term "the
fine chemical" means "arginine" and "glutamine" in relation to all
sequences listed in Table I to IV, lines 32 and 59, and, lines 34
and 60, and, 390 and 392,
[1926] in one embodiment the term "the fine chemical" means
"glutamine" and "proline" in relation to all sequences listed in
Table I to IV, lines 57 and 48, and, 410 and 411,
[1927] in one embodiment the term "the fine chemical" means
"arginine" and "glutamate" in relation to all sequences listed in
Table I to IV, lines 390 and 391, and, 405 and 406. in one
embodiment the term "the fine chemical" means "glutamate" and
"glutamine" in relation to all sequences listed in Table I to IV,
lines 391 and 392, and, 396 and 397, and, 414 and 415, and, 421 and
422, and, 427 and 428, in one embodiment the term "the fine
chemical" means "arginine" and "glutamate" and "glutamine" in
relation to all sequences listed in Table I to IV, lines 390 and
391 and 392.
[1928] Accordingly, the term "the fine chemical" can mean
"arginine" and/or "glutamate" and/or "glutamine" and/or "proline",
owing to circumstances and the context. In order to illustrate that
the meaning of the term "the fine chemical" means "arginine",
and/or "glutamate" and/or "glutamine" and/or "proline" the term
"the respective fine chemical" is also used.
[1929] [0017.0.0.4] to [0018.0.0.4]: see [0017.0.0.0] to
[0018.0.0.0]
[1930] [0019.0.4.4] Advantageously the process for the production
of the fine chemical leads to an enhanced production of the fine
chemical. The terms "enhanced" or "increase" mean at least a 10%,
20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or
100%, more preferably 150%, 200%, 300%, 400% or 500% higher
production of the respective fine chemical in comparison to the
reference as defined below, e.g. that means in comparison to an
organism without the aforementioned modification of the activity of
a protein indicated in Table II, column 3, lines 30 to 62 and/or
lines 386 to 435 or encoded by nucleic acid molecule indicated in
Table I, columns 5 or 7, lines 30 to 62 and/or lines 386 to
435.
[1931] [0020.0.4.4] Surprisingly it was found, that the transgenic
expression of at least one of the Saccaromyces cerevisiae
protein(s) indicated in Table II, Column 3, lines 30 to 33 for
arginine
[1932] and/or lines 38 to 42 and/or 435 for glutamate
[1933] and/or lines 44 to 53 for proline
[1934] and/or lines 57 to 59 for glutamine
[1935] in Arabidopsis thaliana conferred an increase in the
respective fine chemical content of the transformed plants
[1936] and/or
[1937] at least one of the Escherichia coli K12 proteins indicated
in Table II, Column 3, lines 34 to 37, 390, 405 and/or 430 for
arginine
[1938] and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427 and/or 434 for glutamate
[1939] and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or
429 for proline
[1940] and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415,
416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for
glutamine
[1941] in Arabidopsis thaliana conferred an increase in the
respective fine chemical content of the transformed plants.
[1942] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein as indicated in
Table II, column 5, lines 43 and 54 in Arabidopsis thaliana
conferred an increase in glutamate and/or proline (or the
respective fine chemical) content of the transformed plants. Thus,
in one embodiment, said protein or its homologs are used for the
production of glutamate; in one embodiment, said protein or its
homologs are used for the production of proline; in one embodiment,
said protein or its homologs are used for the production of one or
more fine chemical selected from the group consisting of: glutamate
and/or proline.
[1943] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein as indicated in
Table II, column 5, lines 43 and 54 and/or lines 390 and 392 and/or
the Saccharomyces cerevisiae protein as indicated in Table II,
column 5, lines 32 and 59 in Arabidopsis thaliana conferred an
increase in arginine and/or glutamine (or the respective fine
chemical) content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of arginine; in one embodiment, said protein or its
homologs are used for the production of glutamine; in one
embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of: arginine and/or glutamine.
[1944] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein as indicated in Table II,
column 5, lines 48 and 57 and/or the Escherichia coli K12 protein
as indicated in Table II, column 5, lines 411 and 410 in
Arabidopsis thaliana conferred an increase in proline and/or
glutamine (or the respective fine chemical) content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of proline; in one embodiment,
said protein or its homologs are used for the production of
glutamine, in one embodiment, said protein or its homologs are used
for the production of one or more fine chemical selected from the
group consisting of: proline and/or glutamine.
[1945] Surprisingly it was found, that the transgenic expression of
the the Escherichia coli K12 protein as indicated in Table II,
column 5, lines 391 and 392 and/or lines 396 and 397 and/or lines
414 and 415 and/or lines 421 and 422 and/or lines 427 and 428 in
Arabidopsis thaliana conferred an increase in glutamate and/or
glutamine (or the respective fine chemical) content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of glutamate; in one
embodiment, said protein or its homologs are used for the
production of glutamine, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of: glutamate and/or
glutamine.
[1946] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 390 and 391, and, 405 and 406 in Arabidopsis thaliana
conferred an increase in arginine and/or glutamate (or the
respective fine chemical) content of the transformed plants. Thus,
in one embodiment, said protein or its homologs are used for the
production of arginine, in one embodiment, said protein or its
homologs are used for the production of glutamate, in one
embodiment, said protein or its homologs are used for the
production of arginine and glutamate.
[1947] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 390 and 391 and 392 in Arabidopsis thaliana conferred an
increase in arginine and/or glutamate and/or glutamine (or the
respective fine chemical) content of the transformed plants. Thus,
in one embodiment, said protein or its homologs are used for the
production of arginine, in one embodiment, said protein or its
homologs are used for the production of glutamate; in one
embodiment, said protein or its homologs are used for the
production of glutamine, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of: arginine and/or glutamate
and/or glutamine.
[1948] [0021.0.0.4]: see [0021.0.0.0]
[1949] [0022.0.4.4] The sequence of b0695 from Escherichia coli K12
has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as sensory
histidine kinase in two-component regulatory system. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product with an activity of the sensor histidine
kinase homology superfamily, preferably a protein with a sensory
histidine kinase in two-component regulatory system activity from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of arginine, in particular for
increasing the amount of arginine, preferably arginine in free or
bound form in an organism or a part thereof, as mentioned.
[1950] The sequence of b0730 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as transcriptional regulator of
succinylCoA synthetase operon and fatty acyl response regulator.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the transcription regulator GntR superfamily, preferably a protein
with a transcriptional regulator of succinylCoA synthetase operon
or a fatty acid response regulator activity from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glutamate and/or proline, in particular for
increasing the amount of glutamate and/or proline, in particular
for increasing the amount of glutamate, in particular for
increasing the amount of proline, in particular for increasing the
amount of glutamate and proline, preferably glutamate and/or
proline in free or bound form in an organism or a part thereof, as
mentioned.
[1951] The sequence of b1284 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative transcriptional
regulator with DNA-binding Winged helix domain (DeoR family).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the regulatory protein gutR superfamily, preferably a protein with
transcriptional regulator with DNA-binding Winged helix domain
(DeoR family) activity from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
arginine, iin particular for increasing the amount of arginine,
preferably arginine in free or bound form in an organism or a part
thereof, as mentioned.
[1952] The sequence of b1827 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative transcriptional
repressor protein with a DNA-binding Winged helix domain (IcIR
family). Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the acetate operon repressor superfamily, preferably a protein with
a transcriptional repressor protein with a DNA-binding Winged helix
domain (IcIR family) activity from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
arginine, in particular for increasing the amount of arginine,
preferably arginine in free or bound form in an organism or a part
thereof, as mentioned.
[1953] The sequence of b1829 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a heat shock protein with
protease activity. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of heat-shock protein htpX superfamily, preferably a
protein with a "heat shock protein with protease activity" from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of glutamine and/or proline, in
particular for increasing the amount of arginine and/or glutamine,
in particular for increasing the amount of proline, in particular
for increasing the amount of glutamine, in particular for
increasing the amount of proline and glutamine, preferably
increasing the amount of proline and/or glutamine, in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a heat shock protein with protease activity is increased or
generated, e.g. from E. coli or a homolog thereof.
[1954] The sequence of b1852 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a glucose-6-phosphate
dehydrogenase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of glucose-6-phosphate dehydrogenase superfamily,
preferably a protein with a glucose-6-phosphate dehydrogenase
activity from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of glutamine, in
particular for increasing the amount of glutamine, preferably
increasing the amount of glutamine in free or bound form in an
organism or a part thereof, as mentioned.
[1955] The sequence of b2095 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a tagatose-6-phosphate kinase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
Escherichia probable tagatose 6-phosphate kinase gatZ superfamily,
preferably a protein with a tagatose-6-phosphate kinase activity
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of arginine, in particular
for increasing the amount of arginine, preferably increasing the
amount of arginine in free or bound form in an organism or a part
thereof, as mentioned.
[1956] The sequence of b2699 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a DNA strand exchange and
recombination protein with protease and nuclease activity.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
recombination protein recA superfamily, preferably a protein with a
DNA strand exchange and recombination protein with protease and
nuclease activity from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
proline, in particular for increasing the amount of proline,
preferably increasing the amount of proline in free or bound form
in an organism or a part thereof, as mentioned.
[1957] The sequence of b4265 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a L-idonate transport protein.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
D-serine permease superfamily, preferably a protein with a
L-idonate transport protein activity from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of glutamine, in particular for increasing the amount of
glutamine, preferably increasing the amount of glutamine in free or
bound form in an organism or a part thereof, as mentioned.
[1958] The sequence of YBR030W from Saccharomyces cerevisiae has
been published in Feldmann et al., EMBO J., 13 (24), 5795-5809
(1994) and Goffeau, Science 274 (5287), 546-547, 1996, and its
cellular activity has not been characterized yet. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of Saccharomyces cerevisiae
hypothetical protein YBR030w superfamily, preferably a protein with
a YBR030W activity from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of proline, in particular for increasing the amount of
proline, preferably proline in free or bound form in an organism or
a part thereof, as mentioned.
[1959] The sequence of YDL106C from Saccharomyces cerevisiae has
been published in Jacq et al., Nature 387 (6632 Suppl), 75-78,
1997, and Goffeau, Science 274 (5287), 546-547, 1996, and its
activity is being defined as homeobox transcription factor.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
unassigned homeobox proteins, homeobox homology proteins
superfamily, preferably a protein with a "homeobox transcription
factor" activity or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of proline, in particular
for increasing the amount of proline, preferably proline in free or
bound form in an organism or a part thereof, as mentioned.
[1960] The sequence of YFR042W from Saccharomyces cerevisiae has
been published in Goffeau st al., Science 274 (5287), 546-547, 1996
and Murakami, Y., Nat. Genet. 10 (3), 261-268, 1995 and its
activity is being defined as a "protein required for cell viability
in yeast". Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of Saccharomyces cerevisiae probable membrane protein
YFR042w superfamily, preferably a protein with a "protein required
for cell viability in yeast" activity, from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the fine chemical, meaning of glutamine, in particular for
increasing the amount of glutamine, preferably glutamine in free or
bound form in an organism or a part thereof, as mentioned.
[1961] The sequence of YGR135W from Saccharomyces cerevisiae has
been published in Goffeau st al., Science 274 (5287), 546-547, 1996
and Tettelin et al., Nature 387 (6632 Suppl), 81-84 (1997) and its
activity is being defined as a "proteasome component Y13".
Accordingly, in one embodiment, the process of the present
invention comprises the use of a a gene product with an activity of
multicatalytic endopeptidase complex chain C9 superfamily,
preferably a protein with proteasome component Y13 activity, from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of proline, in
particular for increasing the amount of proline, preferably proline
in free or bound form in an organism or a part thereof, as
mentioned.
[1962] The sequence of YHR130C from Saccharomyces cerevisiae has
been published in Johnston et al., Science 265:2077-2082 (1994),
and its cellular activity has not been characterized yet.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a YHR130C activity from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of arginine, in
particular for increasing the amount of arginine, preferably
arginine in free or bound form in an organism or a part thereof, as
mentioned.
[1963] The sequence of YIL150C from Saccharomyces cerevisiae has
been published in Goffeau st al., Science 274 (5287), 546-547, 1996
and Churcher et al., Nature 387 (6632 Suppl), 84-87, 1997 and its
activity is being defined as a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a chromatin binding protein, required for S-phase (DNA
synthesis) initiation or completion, from Saccharomyces cerevisiae
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of proline, in particular for increasing the
amount of proline, preferably proline in free or bound form in an
organism or a part thereof, as mentioned.
[1964] The sequence of YPR024W from Saccharomyces cerevisiae has
been published in Goffeau st al., Science 274 (5287), 546-547, 1996
and Bussey et al., Nature 387 (6632 Suppl), 103-105 (1997) and its
activity is being defined as a mitochondrial protein of the
CDC48/PAS1/SEC18 family of ATPases. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of FtsH/SEC18/CDC48-type ATP-binding
domain homology; cell division protein ftsH superfamily, preferably
a protein with a mitochondrial protein of the CDC48/PAS1/SEC18
family of ATPases activity, from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glutamate, in particular for increasing the
amount of glutamate, preferably glutamate in free or bound form in
an organism or a part thereof, as mentioned.
[1965] The sequence of YPR133W-A from Saccharomyces cerevisiae has
been published in Goffeau st al., Science 274 (5287), 546-547, 1996
and Bussey et al., Nature 387 (6632 Suppl), 103-105 (1997) and its
activity is being defined as a translocase of the outer
mitochondrial membrane. Accordingly, in one embodiment, the process
of the present invention comprises the use of a translocase of the
outer mitochondrial membrane, from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glutamate, in particular for increasing the
amount of glutamate, preferably glutamate in free or bound form in
an organism or a part thereof, as mentioned.
[1966] The sequence of YPR138C from Saccharomyces cerevisiae has
been published in Goffeau st al., Science 274 (5287), 546-547, 1996
and Bussey et al., Nature 387 (6632 Suppl), 103-105 (1997) and its
activity is being defined as a NH.sup.4+ transporter. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product with an activity of ammonium transport
protein; ammonium transporter nrgA superfamily, preferably a
protein with a NH.sup.4+ transporter activity, from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the fine chemical, meaning of proline, in particular for
increasing the amount of proline, preferably proline in free or
bound form in an organism or a part thereof, as mentioned.
[1967] The sequence of YBR204C from Saccharomyces cerevisiae has
been published in Goffeau st al., Science 274 (5287), 546-547, 1996
and Feldmann et al., EMBO J. 13 (24), 5795-5809 (1994) and its
activity is being defined as a peroxisomal lipase. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a peroxisomal lipase, from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glutamate, in particular for increasing the
amount of glutamate, preferably glutamate in free or bound form in
an organism or a part thereof, as mentioned.
[1968] The sequence of YDR271C was submitted by Le T., Johnston M.,
(March-1996) to the EMBL/GenBank/DDBJ databases, by Waterston R.;
(MAY-1996) and Jia Y., (JUNE-1997) to the EMBL/GenBank/DDBJ
databases and its cellular activity has not been characterized yet.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a YDR271C activity from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of proline, in
particular for increasing the amount of proline, preferably proline
in free or bound form in an organism or a part thereof, as
mentioned. I
[1969] The sequence of YDR316W from Saccharomyces cerevisiae has
been published in Goffeau st al., Science 274 (5287), 546-547, 1996
and Jacq et al., Nature 387 (6632 Suppl), 75-78 (1997), and its
activity is being defined as a putative
S-adenosylmethionine-dependent methyltransferase of the seven
beta-strand family. Accordingly, in one embodiment, the process of
the present invention comprises the use of a a gene product with an
activity of bioC homology superfamily, preferably a protein with
putative S-adenosylmethionine-dependent methyltransferase of the
seven beta-strand family activity, from Saccharomyces cerevisiae or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of arginine in particular for increasing the
amount of arginine, preferably arginine in free or bound form in an
organism or a part thereof, as mentioned.
[1970] The sequence of YEL045C from Saccharomyces cerevisiae was
published by Dietrich et al., Nature 387:78-81(1997) and its
cellular activity has not been characterized yet. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of Saccharomyces
hypothetical protein YEL045c superfamily, preferably a protein with
a YEL045C activity from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of proline, in particular for increasing the amount of
proline, preferably proline in free or bound form in an organism or
a part thereof, as mentioned.
[1971] The sequence of YER173w from Saccharomyces cerevisiae has
been published in Dietrich, Nature 387 (6632 Suppl), 78-81, 1997,
and Goffeau, Science 274 (5287), 546-547, 1996, and its activity is
being defined as an "Checkpoint protein, involved in the activation
of the DNA damage and meiotic pachytene checkpoints;". Accordingly,
in one embodiment, the process of the present invention comprises
the use of a "Checkpoint protein, involved in the activation of the
DNA damage and meiotic pachytene checkpoints" or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
the amount of glutamine and/or proline, in particular for
increasing the amount of glutamine and/or proline, in particular
for increasing the amount of glutamine, in particular for
increasing the amount of proline, in particular for increasing the
amount of glutamine and proline, preferably glutamine and/or
proline in free or bound form in an organism or a part thereof, as
mentioned.
[1972] The sequence of YFL013C from Saccharomyces cerevisiae has
been published in Goffeau, A., Science 274 (5287), 546-547, 1996
and Murakami, Y., Nat. Genet. 10 (3), 261-268, 1995, and its
activity is being defined as a "subunit of the INO80 chromatin
remodeling complex". Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of Saccharomyces cerevisiae probable membrane protein
YFL013c superfamily, preferably a protein with a "subunit of the
INO80 chromatin remodeling complex" activity or its homolog, for
the production of the fine chemical, meaning of glutamate, in
particular for increasing the amount of glutamate, preferably
glutamate in free or bound form in an organism or a part thereof,
as mentioned.
[1973] The sequence of YFL050C from Saccharomyces cerevisiae has
been published in Murakami et al., Nat. Genet. 10 (3), 261-268,
1995, and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is defined as a di- trivalent inorganic cation
transporter. Accordingly, in one embodiment, the process of the
present invention comprises the use of a a gene product with an
activity of magnesium and cobalt transport protein superfamily,
preferably a protein with with a di- trivalent inorganic cation
transporter activity from Saccaromyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning proline, in particular for increasing the amount of
proline, preferably proline in free or bound form in an organism or
a part thereof, as mentioned.
[1974] The sequence of YGR104C from Saccharomyces cerevisiae has
been published in Thompson et al., Cell 73:1361-1375, 1993, and its
activity is being defined as an "RNA polymerase II suppressor
protein SRB5--yeast and/or suppressor of RNA polymerase B SRB5"".
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
RNA polymerase II suppressor protein SRB5--yeast superfamily,
preferably a protein with a "RNA polymerase II suppressor protein
SRB5--yeast and/or suppressor of RNA polymerase B SRB5" activity or
its homolog, for the production of the fine chemical, meaning of
glutamate, in particular for increasing the amount of glutamate,
preferably glutamate in free or bound form in an organism or a part
thereof, as mentioned.
[1975] The sequence of YKR057W from Saccharomyces cerevisiae has
been published in Dujon et al., Nature 369 (6479), 371-378, 1994
and Goffeau et al., Science 274 (5287), 546-547, 1996 and its
activity is being defined as a ribosomal protein, similar to S21
ribosomal proteins, involved in ribosome biogenesis and
translation. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of rat ribosomal protein S21 superfamily, preferably a
protein with a ribosomal protein, similar to S21 ribosomal
proteins, involved in ribosome biogenesis and translation activity
from Saccharomyces cerevisiae or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of arginine and/or
glutamine, in particular for increasing the amount of arginine
and/or glutamine, in particular for increasing the amount of
arginine, in particular for increasing the amount of glutamine, in
particular for increasing the amount of arginine and glutamine,
preferably arginine and/or glutamine in free or bound form in an
organism or a part thereof, as mentioned.
[1976] The sequence of b0050 (Accession number NP.sub.--414592)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a conserved protein potentially involved in protein
protein interaction. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of apaG protein superfamily, preferably a protein with the
activity of a conserved protein potentially involved in
protein-protein interaction from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
glutamate in particular for increasing the amount of glutamate,
preferably glutamate in free or bound form in an organism or a part
thereof, as mentioned.
[1977] The sequence of b0057 (Accession number NP.sub.--414599)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is not been
characterized yet. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of b0057 protein from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
glutamate in particular for increasing the amount of glutamate,
preferably glutamate in free or bound form in an organism or a part
thereof, as mentioned.
[1978] The sequence of b0138 (Accession number NP.sub.--414680)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is is being
defined as a fimbrial-like adhesin protein. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of b0138 protein from E. coli or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of proline in particular for increasing the
amount of proline, preferably proline in free or bound form in an
organism or a part thereof, as mentioned.
[1979] The sequence of b0149 (Accession number NP.sub.--414691)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a bifunctional penicillin-binding protein lb: glycosyl
transferase (N-terminal); transpeptidase (C-terminal). Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product with an activity of penicillin-binding
protein 1B superfamily, preferably a protein with the activity of a
bifunctional penicillin-binding protein ib: glycosyl transferase
(N-terminal); transpeptidase (C-terminal) from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of proline, in particular for increasing the
amount of proline, preferably proline in free or bound form in an
organism or a part thereof, as mentioned.
[1980] The sequence of b0161 (Accession number NP.sub.--414691)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a periplasmic serine protease (heat shock protein).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
Helicobacter serine proteinase superfamily, preferably a protein
with the activity of a periplasmic serine protease (heat shock
protein) from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of arginine and/or
glutamate and/or glutamine, in particular for increasing the amount
of arginine, in particular for increasing the amount of glutamate,
in particular for increasing the amount of glutamine, in particular
for increasing the amount of arginine and glutamate, in particular
for increasing the amount of arginine and glutamine, in particular
for increasing the amount of glutamine and glutamate, in particular
for increasing the amount of arginine and glutamine and glutamate,
preferably arginine and/or glutamate and/or glutamine in free or
bound form in an organism or a part thereof, as mentioned.
[1981] The sequence of b0486 (Accession number NP.sub.--415019)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a amino-acid/amine transport protein (APC family).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
membrane protein ybaT superfamily, preferably a protein with the
activity of a amino-acid/amine transport protein (APC family) from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of glutamine, in particular for
increasing the amount of glutamine, preferably glutamine in free or
bound form in an organism or a part thereof, as mentioned.
[1982] The sequence of b0849 (Accession number NP.sub.--415370)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a glutaredoxin 1 redox coenzyme for
glutathione-dependent ribonucleotide reductase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of glutaredoxin superfamily,
preferably a protein with the activity of a glutaredoxin 1 redox
coenzyme for glutathione-dependent ribonucleotide reductase protein
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of glutamine, in
particular for increasing the amount of glutamine, preferably
glutamine in free or bound form in an organism or a part thereof,
as mentioned.
[1983] The sequence of b0970 (Accession number NP.sub.--415490)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a glutamate receptor. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of Escherichia coli ybhL protein
superfamily, preferably a protein with the activity of a glutamate
receptor protein from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of glutamine, in
particular for increasing the amount of glutamine, preferably
glutamine in free or bound form in an organism or a part thereof,
as mentioned.
[1984] The sequence of b1343 (Accession number NP.sub.--415490)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a ATP-dependent RNA helicase, stimulated by 23S rRNA.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
Escherichia coli b1343 protein, preferably a protein with the
activity of a ATP-dependent RNA helicase, stimulated by 23S rRNA
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of glutamine and/or
glutamate, in particular for increasing the amount of glutamine, in
particular for increasing the amount of glutamate, in particular
for increasing the amount of glutamine and glutamate, preferably
glutamine and/or glutamate in free or bound form in an organism or
a part thereof, as mentioned.
[1985] The sequence of b1360 (Accession number NP.sub.--415878)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a DNA replication protein. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of DNA replication protein dnaC
superfamily, preferably a protein with the activity of a DNA
replication protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
proline, in particular for increasing the amount of proline,
preferably proline in free or bound form in an organism or a part
thereof, as mentioned.
[1986] The sequence of b1693 (Accession number NP.sub.--416208)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a 3-dehydroquinate dehydratase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of 3-dehydroquinate dehydratase
superfamily, preferably a protein with the activity of a DNA
replication protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
glutamate, in particular for increasing the amount of glutamate,
preferably glutamate in free or bound form in an organism or a part
thereof, as mentioned.
[1987] The sequence of b1736 (Accession number NP.sub.--416250)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a PEP-dependent phosphotransferase enzyme,
cellobiose/arbutin/salicin sugar-specific protein. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of phosphotransferase system
lactose-specific enzyme II, factor III superfamily, preferably a
protein with the activity of a PEP-dependent phosphotransferase
enzyme, cellobiose/arbutin/salicin sugar-specific protein from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of glutamate, in particular for
increasing the amount of glutamate, preferably glutamate in free or
bound form in an organism or a part thereof, as mentioned.
[1988] The sequence of b1738 (Accession number NP.sub.--416252)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a PEP-dependent phosphotransferase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of phosphotransferase system
enzyme II cellobiose-specific factor IIB superfamily, preferably a
protein with the activity of a PEP-dependent phosphotransferase
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of glutamate, in
particular for increasing the amount of glutamate, preferably
glutamate in free or bound form in an organism or a part thereof,
as mentioned
[1989] The sequence of b1886 (Accession number NP.sub.--416400)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a methyl-accepting chemotaxis protein II, aspartate
sensor receptor. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of methyl-accepting chemotaxis protein superfamily,
preferably a protein with the activity of a methyl-accepting
chemotaxis protein II, aspartate sensor receptor from E. coli or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glutamine, in particular for increasing the
amount of glutamine, preferably glutamine in free or bound form in
an organism or a part thereof, as mentioned.
[1990] The sequence of b1896 (Accession number NP.sub.--416410)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a trehalose-6-phosphate synthase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of alpha-trehalose-phosphate
synthase (UdP-forming) superfamily, preferably a protein with the
activity of a trehalose-6-phosphate synthase from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glutamate, in particular for increasing the
amount of glutamate, preferably glutamate in free or bound form in
an organism or a part thereof, as mentioned
[1991] The sequence of b1926 (Accession number NP.sub.--416436)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a flagellar protein fliT. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of flagellar protein fliT from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of glutamine, in particular for
increasing the amount of glutamine, preferably glutamine in free or
bound form in an organism or a part thereof, as mentioned.
[1992] The sequence of b2307 (Accession number NP.sub.--416810)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a histidine and lysine/arginine/ornithine transport
protein (ABC superfamily, membrane). Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of histidine permease protein M
superfamily, preferably a protein with the activity of a histidine
and lysine/arginine/ornithine transport protein (ABC superfamily,
membrane) from E. coli or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of glutamate and/or
arginine, in particular for increasing the amount of glutamate, in
particular for increasing the amount of arginine, in particular for
increasing the amount of glutamate and arginine, preferably
glutamate and/or arginine in free or bound form in an organism or a
part thereof, as mentioned.
[1993] The sequence of b2414 (Accession number NP.sub.--416909)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a subunit of cysteine synthase A and O-acetylserine
sulfhydrolase A, PLP-dependent enzyme. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of threonine dehydratase
superfamily, preferably a protein with the activity of a subunit of
cysteine synthase A and O-acetylserine sulfhydrolase A,
PLP-dependent enzyme from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
glutamine, in particular for increasing the amount of glutamine,
preferably glutamine in free or bound form in an organism or a part
thereof, as mentioned.
[1994] The sequence of b2426 (Accession number NP.sub.--416921)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a putative oxidoreductase with NAD(P)-binding domain.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
ribitol dehydrogenase, short-chain alcohol dehydrogenase homology
superfamily, preferably a protein with the activity of a putative
oxidoreductase with NAD(P)-binding domain from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glutamine, in particular for increasing the
amount of glutamine, preferably glutamine in free or bound form in
an organism or a part thereof, as mentioned.
[1995] The sequence of b2489 (Accession number NP.sub.--416984)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a hydrogenase Fe--S subunit. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of psbG protein superfamily,
preferably a protein with the activity of a hydrogenase Fe--S
subunit from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of glutamine, in
particular for increasing the amount of glutamine, preferably
glutamine in free or bound form in an organism or a part thereof,
as mentioned.
[1996] The sequence of b2553 (Accession number NP.sub.--417048)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a regulatory protein P-II for glutamine synthetase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
regulatory protein P-II superfamily, preferably a protein with the
activity of a regulatory protein P-II for glutamine synthetase from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of proline and/or glutamine, in
particular for increasing the amount of proline, in particular for
increasing the amount of glutamine, in particular for increasing
the amount of proline and glutamine, preferably proline and/or
glutamine in free or bound form in an organism or a part thereof,
as mentioned.
[1997] The sequence of b2664 (Accession number NP.sub.--417150)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a transcriptional repressor with DNA-binding Winged
helix domain (GntR familiy). Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of transcription regulator gabP
superfamily, preferably a protein with the activity of
transcriptional repressor with DNA-binding Winged helix domain
(GntR familiy) from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of proline, in
particular for increasing the amount of proline, preferably proline
in free or bound form in an organism or a part thereof, as
mentioned.
[1998] The sequence of b2710 (Accession number NP.sub.--417190)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a flavorubredoxin (FIRd) bifunctional NO and O.sub.2
reductase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of Escherichia coli hypothetical protein b2710, rubredoxin
homology, Methanobacterium flavoprotein A superfamily, preferably a
protein with the activity of a a flavorubredoxin (FIRd)
bifunctional NO and O.sub.2 reductase from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of glutamate, in particular for increasing the amount of
glutamate, preferably glutamate in free or bound form in an
organism or a part thereof, as mentioned.
[1999] The sequence of b2818 (Accession number NP.sub.--417295)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a N-acetylglutamate synthase (amino acid
N-acetyltransferase). Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of amino-acid acetyltransferase, acetylglutamate kinase
superfamily, preferably a protein with the activity of a a
N-acetylglutamate synthase (amino acid N-acetyltransferase) from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of glutamine and/or glutamate, in
particular for increasing the amount of glutamine, in particular
for increasing the amount of glutamate, in particular for
increasing the amount of glutamine and glutamte, preferably
glutamate and/or glutamine in free or bound form in an organism or
a part thereof, as mentioned.
[2000] The sequence of b3064 (Accession number NP.sub.--417536)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a O-sialoglycoprotein endopeptidase, with actin-like
ATPase domain. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of O-sialoglycoprotein endopeptidase superfamily,
preferably a protein with the activity of a 0-sialoglycoprotein
endopeptidase, with actin-like ATPase domain from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glutamine, in particular for increasing the
amount of glutamine, preferably glutamine in free or bound form in
an organism or a part thereof, as mentioned.
[2001] The sequence of b3074 (Accession number NP.sub.--417545)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a tRNA synthetase. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of secretion chaperone CsaA,
methionyl-tRNA synthetase, dimer-forming superfamily, preferably a
protein with the activity of a tRNA synthetase from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glutamate, in particular for increasing the
amount of glutamate, preferably glutamate in free or bound form in
an organism or a part thereof, as mentioned.
[2002] The sequence of b3116 (Accession number NP.sub.--417586)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a L-threonine/L-serine permease, anaerobically inducible
(HAAAP family). Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of threonine-serine permease superfamily, preferably a
protein with the activity of a L-threonine/L-serine permease,
anaerobically inducible (HAAAP family) from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of glutamate, in particular for increasing the amount of
glutamate, preferably glutamate in free or bound form in an
organism or a part thereof, as mentioned.
[2003] The sequence of b3160 (Accession number NP.sub.--417629)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as monooxygenase with luciferase-like ATPase activity.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
ynbW protein superfamily, preferably a protein with the activity of
a monooxygenase with luciferase-like ATPase activity from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of glutamine, in particular for increasing
the amount of glutamine, preferably glutamine in free or bound form
in an organism or a part thereof, as mentioned.
[2004] The sequence of b3166 (Accession number NP.sub.--417635)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as tRNA pseudouridine 5S synthase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of Escherichia coli protein P35
superfamily, preferably a protein with the activity of a tRNA
pseudouridine 5S synthase from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
glutamine, in particular for increasing the amount of glutamine,
preferably glutamine in free or bound form in an organism or a part
thereof, as mentioned.
[2005] The sequence of b3169 (Accession number NP.sub.--417638)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a transcription termination-antitermination factor.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
Escherichia coli transcription factor nusA superfamily, preferably
a protein with the activity of a transcription
termination-antitermination factor from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of glutamine and/or glutamate, in particular for increasing
the amount of glutamine, in particular for increasing the amount of
glutamate, in particular for increasing the amount of glutamine and
glutamte, preferably glutamate and/or glutamine in free or bound
form in an organism or a part thereof, as mentioned.
[2006] The sequence of b3231 (Accession number NP.sub.--417698)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a 50S ribosomal subunit protein L13. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of Escherichia coli ribosomal
protein L13 superfamily, preferably a protein with the activity of
a 50S ribosomal subunit protein L13 from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of glutamine, in particular for increasing the amount of
glutamine, preferably glutamine in free or bound form in an
organism or a part thereof, as mentioned.
[2007] The sequence of b3619 (Accession number NP.sub.--418076)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a ADP-L-glycero-D-mannoheptose-6-epimerase,
NAD(P)-binding. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of ADPglyceromanno-heptose 6-epimerase, UDPglucose
4-epimerase homology superfamily, preferably a protein with the
activity of a ADP-L-glycero-D-mannoheptose-6-epimerase,
NAD(P)-binding from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of glutamate, in
particular for increasing the amount of glutamate, preferably
glutamate in free or bound form in an organism or a part thereof,
as mentioned.
[2008] The sequence of b3644 (Accession number NP.sub.--418101)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a uncharacterized stress-induced protein. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product with an activity of protein H10467
superfamily, preferably a protein with the activity of a
Uncharacterized stress-induced protein from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of proline, in particular for increasing the amount of
proline, preferably proline in free or bound form in an organism or
a part thereof, as mentioned.
[2009] The sequence of b3680 (Accession number NP.sub.--418136)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a transcriptional regulator with homeodomain-like DNA
binding domain (AraC/XylS family). Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of Escherichia coli b3680 protein,
preferably a protein with the activity of a transcriptional
regulator with homeodomain-like DNA binding domain (AraC/XylS
family) from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of glutamine, in
particular for increasing the amount of glutamine, preferably
glutamine in free or bound form in an organism or a part thereof,
as mentioned.
[2010] The sequence of b3791 (Accession number NP.sub.--418238)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a transaminase involved in lipopolysaccharide
biosynthesis. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of erythromycin resistance protein superfamily, preferably
a protein with the activity of a transaminase involved in
lipopolysaccharide biosynthesis from E. coli or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
of glutamine and/or glutamate, in particular for increasing the
amount of glutamine, in particular for increasing the amount of
glutamate, in particular for increasing the amount of glutamine and
glutamate, preferably glutamate and/or glutamine in free or bound
form in an organism or a part thereof, as mentioned.
[2011] The sequence of b3919 (Accession number NP.sub.--418354)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a triosephosphate isomerase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of triose-phosphate isomerase
superfamily, preferably a protein with the activity of a
triosephosphate isomerase from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
proline, in particular for increasing the amount of proline,
preferably proline in free or bound form in an organism or a part
thereof, as mentioned.
[2012] The sequence of b3936 (Accession number NP.sub.--418371)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a 50S ribosomal subunit protein L32. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of Escherichia coli ribosomal
protein L31 superfamily, preferably a protein with the activity of
a 50S ribosomal subunit protein L32 from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of arginine, in particular for increasing the amount of
arginine, preferably arginine in free or bound form in an organism
or a part thereof, as mentioned.
[2013] The sequence of b4004 (Accession number NP.sub.--418432)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a transcriptional regulatory protein. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of nitrogen assimilation
regulatory protein ntrC or response regulator homology, RNA
polymerase sigma factor interaction domain homology superfamily,
preferably a protein with the activity of a transcriptional
regulatory protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
glutamine, in particular for increasing the amount of glutamine,
preferably glutamine in free or bound form in an organism or a part
thereof, as mentioned.
[2014] The sequence of b4074 (Accession number NP.sub.--418498)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a cytochrome c-type biogenesis protein. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of nrfE protein superfamily,
preferably a protein with the activity of a Cytochrome c-type
biogenesis protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
glutamine, in particular for increasing the amount of glutamine,
preferably glutamine in free or bound form in an organism or a part
thereof, as mentioned.
[2015] The sequence of b4133 (Accession number NP.sub.--418557)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a transcriptional activator of pH response (OmpR
family). Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
b4133 protein, preferably a protein with the activity of a
transcriptional activator of pH response (OmpR family) from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of glutamine, in particular for increasing
the amount of glutamine, preferably glutamine in free or bound form
in an organism or a part thereof, as mentioned.
[2016] The sequence of b4346 (Accession number NP.sub.--418766)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a component of 5-methylcytosine-specific restriction
enzyme McrBC. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of 5-methylcytosine-specific restriction enzyme B
superfamily, preferably a protein with the activity of a component
of 5-methylcytosine-specific restriction enzyme McrBC from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of glutamate, in particular for increasing
the amount of glutamate, preferably glutamate in free or bound form
in an organism or a part thereof, as mentioned.
[2017] The sequence of YFL019C (Accession number S48324.) from
Saccharomyces cerevisiae has been published in Murakami et al.,
Nat. Genet. 10:261-268(1995) and its activity is not been
characterized yet. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of a YFL019C protein from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glutamate, in particular for increasing the
amount of glutamate, preferably glutamate in free or bound form in
an organism or a part thereof, as mentioned.
[2018] [0023.0.4.4] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[2019] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 30 to 33 for
arginine
[2020] and/or lines 38 to 42 and/or 435 for glutamate
[2021] and/or lines 44 to 53 for proline
[2022] and/or lines 57 to 59 for glutamine, resp. is a homolog
having the same or a similar activity, resp. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms. In one embodiment, the
homolog is a homolog with a sequence as indicated in Table I or II,
column 7, lines 30 to 33 for arginine
[2023] and/or lines 38 to 42 and/or 435 for glutamate
[2024] and/or lines 44 to 53 for proline
[2025] and/or lines 57 to 59 for glutamine, resp. In one
embodiment, the homolog of one of the polypeptides indicated in
Table II, column 3, lines 30 to 33 for arginine
[2026] and/or lines 38 to 42 and/or 435 for glutamate
[2027] and/or lines 44 to 53 for proline
[2028] and/or lines 57 to 59 for glutamine resp., is derived from
an eukaryotic. In one embodiment, the homolog is derived from
Fungi. In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, lines 30 to 33 for arginine
[2029] and/or lines 38 to 42 and/or 435 for glutamate
[2030] and/or lines 44 to 53 for proline
[2031] and/or lines 57 to 59 for glutamine, resp., is derived from
Ascomyceta. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 30 to 33 for arginine
[2032] and/or lines 38 to 42 and/or 435 for glutamate
[2033] and/or lines 44 to 53 for proline
[2034] and/or lines 57 to 59 for glutamine, resp., is derived from
Saccharomycotina. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 30 to 33 for arginine
[2035] and/or lines 38 to 42 and/or 435 for glutamate
[2036] and/or lines 44 to 53 for proline
[2037] and/or lines 57 to 59 for glutamine, resp., is derived from
Saccharomycetes. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 30 to 33 for arginine
[2038] and/or lines 38 to 42 and/or 435 for glutamate
[2039] and/or lines 44 to 53 for proline
[2040] and/or lines 57 to 59 for glutamine, resp., is a homolog
being derived from Saccharomycetales. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 30
to 33 for arginine
[2041] and/or lines 38 to 42 and/or 435 for glutamate
[2042] and/or lines 44 to 53 for proline
[2043] and/or lines 57 to 59 for glutamine, resp., is a homolog
having the same or a similar activity being derived from
Saccharomycetaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 30 to 33 for arginine
[2044] and/or lines 38 to 42 and/or 435 for glutamate
[2045] and/or lines 44 to 53 for proline
[2046] and/or lines 57 to 59 for glutamine, resp., is a homolog
having the same or a similar activity being derived from
Saccharomycetes.
[2047] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 34 to 37, 390,
405 and/or 430 for arginine
[2048] and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427 and/or 434 for glutamate
[2049] and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or
429 for proline
[2050] and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415,
416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for glutamine
resp. is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms. In one
embodiment, the homolog is a homolog with a sequnence as indicated
in Table I or II, column 7, lines 34 to 37, 390, 405 and/or 430 for
arginine
[2051] and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427 and/or 434 for glutamate
[2052] and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or
429 for proline
[2053] and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415,
416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for glutamine,
resp. In one embodiment, the homolog of one of the polypeptides
indicated in Table II, column 3, lines 34 to 37, 390, 405 and/or
430 for arginine
[2054] and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427 and/or 434 for glutamate
[2055] and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or
429 for proline
[2056] and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415,
416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for glutamine
is derived from an bacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 34 to 37, 390,
405 and/or 430 for arginine
[2057] and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427 and/or 434 for glutamate
[2058] and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or
429 for proline
[2059] and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415,
416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for glutamine
is derived from Proteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 34 to 37, 390,
405 and/or 430 for arginine
[2060] and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427 and/or 434 for glutamate
[2061] and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or
429 for proline
[2062] and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415,
416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for glutamine
is a homolog having the same or a similar activity being derived
from Gammaproteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 34 to 37, 390,
405 and/or 430 for arginine
[2063] and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427 and/or 434 for glutamate
[2064] and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or
429 for proline
[2065] and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415,
416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for glutamine
is derived from Enterobacteriales. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 34 to 37,
390, 405 and/or 430 for arginine
[2066] and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427 and/or 434 for glutamate
[2067] and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or
429 for proline
[2068] and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415,
416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for glutamine
is a homolog being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 34 to 37, 390, 405 and/or 430 for arginine
[2069] and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427 and/or 434 for glutamate
[2070] and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or
429 for proline
[2071] and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415,
416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for glutamine
is a homolog having the same or a similar activity and being
derived from Escherichia.
[2072] [0023.1.4.4] Homologs of the polypeptide indicated in Table
II, column 3, lines 30 to 62 and/or lines 386 to 435 may be the
polypetides encoded by the nucleic acid molecules indicated in
Table I, column 7, lines 30 to 62 and/or lines 386 to 435, resp.,
or may be the polypeptides indicated in Table II, column 7, lines
30 to 62 and/or lines 386 to 435, resp.
[2073] [0024.0.0.4]: see [0024.0.0.0]
[2074] [0025.0.4.4] In accordance with the invention, a protein or
polypeptide has the "activity of an protein of the invention", e.g.
the activity of a protein indicated in Table II, column 3, lines 34
to 37, 390, 405 and/or 430 for arginine
[2075] and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427, 434 and/or 435 for glutamate
[2076] and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or
429 for proline
[2077] and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415,
416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for glutamine
resp., if its de novo activity, or its increased expression
directly or indirectly leads to an increased arginine and/or
glutamate and/or proline and/or glutamine, resp., in the organism
or a part thereof, preferably in a cell of said organism. In a
preferred embodiment, the protein or polypeptide has the
above-mentioned additional activities of a protein indicated in
Table II, column 3, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434 and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp. Throughout the specification the activity or
preferably the biological activity of such a protein or polypeptide
or an nucleic acid molecule or sequence encoding such protein or
polypeptide is identical or similar if it still has the biological
or enzymatic activity of any one of the proteins indicated in Table
II, column 3, lines 34 to 37, 390, 405 and/or 430 and/or lines 43,
386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421,
424, 427, 434 and/or 435 and/or lines 54 to 56, 388, 389, 398, 411,
412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407
to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433
resp. or which has at least 10% of the original enzymatic activity,
preferably 20%, particularly preferably 30%, most particularly
preferably 40% in comparison to any one of the proteins indicated
in Table II, column 3, lines 30 to 33 and/or lines 38 to 42 and/or
435 and/or lines 44 to 53 and/or lines 57 to 59 of Saccharomyces
cerevisiae and/or any one of the proteins indicated in Table II,
column 3, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386,
387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424,
427 and/or 434 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 of E. coli
K12.
[2078] [0025.1.0.4] to [0033.0.0.4]: see [0025.1.0.0] to
[0033.0.0.0]
[2079] [0034.0.4.4] Preferably, the reference, control or wild type
differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of an protein having the activity of a
protein as indicated in Table II, column 3, lines 34 to 37, 390,
405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401,
403, 406, 413, 414, 417, 418, 421, 424, 427 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp.,
[2080] or being encoded by a nucleic acid molecule indicated in
Table I, column 5, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434 and/or 435 and/or lines 54 to 56, 388, 389, 398,
411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402,
404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431
to 433 resp.,
[2081] or its homologs, e.g. as indicated in Table I, column 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.
its biochemical or genetical causes and therefore shows the
increased amount of the respective fine chemical.
[2082] [0035.0.0.4] to [0044.0.0.4]: see [0035.0.0.0] to
[0044.0.0.0]
[2083] [0045.0.4.4] In case the activity of the Escherichia coli
K12 protein b0695 or its homologs, as indicated in Table I, columns
5 or 7, line 35, e.g. a sensory histidine kinase in two-component
signal transduction system (sensor kinase component), modification
by phosphorylation, dephosphorylation, unspecified signal
transduction, regulation of respiration, aerobic respiration, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of arginine between 51% and 319% or more is
conferred.
[2084] In case the activity of the Escherichia coli K12 protein
b0730 or its homologs, as indicated in Table I, columns 5 or 7,
line 43 or 54, e.g. a transcriptional regulator for regulation of
C-compound and carbohydrate utilization, transcriptional control,
prokaryotic nucleotide, transcriptional repressor, DNA binding, is
increased, preferably, in one embodiment the increase of the fine
chemical between 35% and 272%, preferably of glutamate between 55%
and 115% and/or of proline between 35% and 272%, or more is
conferred.
[2085] In case the activity of the Escherichia coli K12 protein
b1284 or its homologs, as indicated in Table I, columns 5 or 7,
line 36, e.g. a transcriptional regulator for regulation of
C-compound and carbohydrate utilization, transcriptional control,
transcriptional repressor, DNA binding, is increased, preferably,
in one embodiment the increase of the fine chemical, preferably of
arginine between 47% and 183% or more is conferred.
[2086] In case the activity of the Escherichia coli K12 protein
b1827 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 56, e.g. a transcriptional repressor for transcriptional
control, is increased, preferably, in one embodiment the increase
of the fine chemical, preferably of proline between 42% and 126%,
or more is conferred.
[2087] In case the activity of the Escherichia coli K12 protein
b1829 or its homologs, e.g.as indicated in Table I, columns 5 or 7,
line 34 or 60, is increased, e.g. the activity of a heat shock
protein with protease activity (htpx), involved in stress response,
pheromone response, mating-type determination, sex-specific
proteins, protein modification, proteolytic degradation is
increased, preferably, in one embodiment the increase of the fine
chemical between 45% and 1141%, preferably of glutamine between 50%
and 68% and/or of arginine between 45% and 1141% or more is
conferred.
[2088] In case the activity of the Escherichia coli K12 protein
b1852 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 61, is increased, e.g. the activity of a
glucose-6-phosphate dehydrogenase, involved in pentose-phosphate
pathway oxidative branch, C-compound and carbohydrate utilization,
NAD/NADP binding, nucleotide metabolism, metabolism of vitamins,
cofactors, and prosthetic groups, energy is increased, preferably,
in one embodiment the increase of the fine chemical, preferably of
glutamine between 40% and 42% or more is conferred.
[2089] In case the activity of the Escherichia coli K12 protein
b2095 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 37, is increased, e.g. the activity of a
tagatose-6-phosphate kinase is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
arginine between 55% and 59% or more is conferred.
[2090] In case the activity of the Escherichia coli K12 protein
b2699 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 55, is increased, e.g. the activity of a recombination
protein recA, involved in DNA recombination and DNA repair,
pheromone response, mating-type determination, sex-specific
proteins, nucleotide binding is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of proline
between 32% and 141% or more is conferred.
[2091] In case the activity of the Escherichia coli K12 protein
b4265 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 62, is increased, e.g. the activity of a D-serine permease,
involved in C-compound and carbohydrate transports, C-compound and
carbohydrate utilization is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
glutamine between 32% and 47% or more is conferred.
[2092] In case the activity of the Saccharomyces cerevisiae protein
YBRO30W or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 44, e.g. a "uncharacterized protein YBRO30W", involved in
C-compound and carbohydrate utilization, pentose-phosphate pathway
and/or transcriptional control is increased, preferably, in one
embodiment an increase of the fine chemical, preferably of proline
between 51% and 282% or more is conferred.
[2093] In case the activity of the Saccharomyces cerevisiae protein
YDL106C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 45, e.g. a homeobox proteins, involved in regulation of
nucleotide metabolism, regulation of phosphate utilization,
transcriptional control, nucleus is increased, preferably, in one
embodiment an increase of the fine chemical, preferably of proline
between 51% and 99% or more is conferred.
[2094] In case the activity of the Saccaromyces cerevisiae protein
YFR042W or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 58, e.g. a "protein required for cell viability in yeast"
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of glutamine, between 41% and 43% or more
is conferred.
[2095] In case the activity of the Saccharomyces cerevisiae protein
YGR135W or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 50, e.g. a proteasome component Y13, involved in
cytoplasmic and nuclear degradation, endoplasmic reticulum,
nucleus, cell differentiation, proteasomal degradation is
increased, preferably, in one embodiment an increase of the fine
chemical, preferably of proline between 32% and 289% or more is
conferred.
[2096] In case the activity of the Saccharomyces cerevisiae protein
YHR130C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 31, e.g. a "hypothetical protein YBR030W" is increased,
preferably, in one embodiment an increase of the fine chemical,
preferably of arginine between 67% and 85% or more is
conferred.
[2097] In case the activity of the Saccharomyces cerevisiae protein
YIL150C or its homologs, as indicated in Table I, columns 5 or 7,
line 51, e.g. a chromatin binding protein, required for S-phase
(DNA synthesis) initiation or completion, involved in DNA synthesis
and replication, mitotic cell cycle and cell cycle control, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of proline between 33% and 304% or more is
conferred.
[2098] In case the activity of the Saccharomyces cerevisiae protein
YPRO24W or its homologs e.g. as indicated in Table I, columns 5 or
7, line 41, e.g. a mitochondrial protein of the CDC48/PAS1/SEC18
family of ATPases, required for assembly of protein complexes,
other proteolytic degradation, mitochondrion, protein folding and
stabilization, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glutamate between 26%
and 43% or more is conferred.
[2099] In case the activity of the Saccharomyces cerevisiae protein
YPR133W-A or its homologs, e.g. as indicated in Table I, columns 5
or 7, line 42, e.g. a translocase of the outer mitochondrial
membrane, is increased, preferably, in one embodiment the increase
of the fine chemical, preferably of glutamate between 34% and 68%
or more is conferred.
[2100] In case the activity of the Saccharomyces cerevisiae protein
YPR138C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 53, e.g. a ammonium transport protein, involved in anion
transports (Cl.sup.-, SO.sub.4.sup.2-, PO.sub.4.sup.3-, etc.),
other cation transports (Na.sup.+, K.sup.+, Ca.sup.2+,
NH.sub.4.sup.+, etc.), nitrogen and sulfur transport, cellular
import, transport through plasma membrane, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of proline between 54% and 520% or more is
conferred.
[2101] In case the activity of the Saccharomyces cerevisiae protein
YBR204C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 38, e.g. a peroxisomal lipase, involved in breakdown of
lipids, fatty acids and isoprenoids, peroxisome, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of glutamate between 55% and 76% or more is
conferred.
[2102] In case the activity of the Saccharomyces cerevisiae protein
YDR271C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 46, e.g. a "hypothetical protein YDR271C" is increased,
preferably, in one embodiment an increase of the fine chemical,
preferably of proline, between 36% and 482% or more is
conferred.
[2103] In case the activity of the Saccharomyces cerevisiae protein
YDR316W or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 30, e.g. a S-adenosylmethionine-dependent methyltransferase
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of arginine between 45% and 102% or more
is conferred.
[2104] In case the activity of the Saccharomyces cerevisiae protein
YEL045C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 47, e.g. a "hypothetical protein YBRO30W" is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of proline between 41% and 89% or more is conferred.
[2105] In case the activity of the Saccharomyces cerevisiae protein
YER173w or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 48 or 57, e.g. a checkpoint protein, involved in the
activation of the DNA damage and meiotic pachytene checkpoints; DNA
recombination and DNA repair, cell cycle checkpoints (checkpoints
of morphogenesis, DNA-damage,-replication, mitotic phase and
spindle), nucleic acid binding, DNA synthesis and replication is
increased, preferably, in one embodiment the increase of the fine
chemical between 34% and 285%, preferably of glutamine between 86%
and 285% and/or of proline between 34% and 191% or more is
conferred.
[2106] In case the activity of the Saccharomyces cerevisiae protein
YFL013C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 39, e.g. a "subunit of the INO80 chromatin remodeling
complex" is increased, preferably, in one embodiment an increase of
the fine chemical, preferably of glutamate, between 81% and 134% or
more is conferred.
[2107] In case the activity of the Saccharomyces cerevisiae protein
YFL050C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 49, e.g. a di-, tri-valent inorganic cation transporte,
involved in heavy metal ion transports (Cu, Fe, etc.), cellular
import, detoxification, homeostasis of metal ions (Na, K, Ca etc.),
transport through plasma membrane is increased, preferably, in one
embodiment an increase of the fine chemical, preferably of proline,
between 44% and 74% or more is conferred.
[2108] In case the activity of the Saccharomyces cerevisiae protein
YGR104C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 40, e.g. a "RNA polymerase II suppressor protein
SRB5--yeast and/or suppressor of RNA polymerase B SRB5" involved in
transcription activities is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
glutamate, between 64% and 96% or more is conferred.
[2109] In case the activity of the Saccharomyces cerevisiae protein
YKR057W or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 32 or 59, e.g. a ribosomal protein, similar to S21A, S26A
and/or YS25 ribosomal proteins, involved in ribosome biogenesis,
cell differentiation and translation is increased, preferably, in
one embodiment an increase of the fine chemical between 41% and
457%, preferably of glutamine between 41% and 284% and/or of
arginine between 57% and 457% or more is conferred. In case the
activity of the Escherichia coli K12 protein b0050 or its homologs
e.g. a conserved protein potentially involved in protein protein
interaction e.g. as indicated in Table II, columns 5 or 7, line
386, is increased, preferably, in one embodiment the increase of
the fine chemical, preferably of glutamate between 37% and 97% or
more is conferred.
[2110] In case the activity of the Escherichia coli K12 protein
b0057 or its homologs e.g. a protein with an activity as defined in
[0022.0.4.4],e.g. as indicated in Table II, columns 5 or 7, line
387, is increased, preferably, in one embodiment the increase of
the fine chemical, preferably of glutamate between 35% and 83% or
more is conferred.
[2111] In case the activity of the Escherichia coli K12 protein
b0138 or its homologs e.g. a fimbrial-like adhesin protein e.g. as
indicated in Table II, columns 5 or 7, line 388, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of proline between 50% and 180% or more is
conferred.
[2112] In case the activity of the Escherichia coli K12 protein
b0149 or its homologs e.g. a bifunctional penicillin-binding
protein 1 b: glycosyl transferase (N-terminal); transpeptidase
(C-terminal) e.g. as indicated in Table II, columns 5 or 7, line
389, is increased, preferably, in one embodiment the increase of
the fine chemical, preferably of proline between 33% and 120% or
more is conferred.
[2113] In case the activity of the Escherichia coli K12 protein
b0161 or its homologs e.g. a periplasmic serine protease e.g. as
indicated in Table II, columns 5 or 7, lines 390 to 392, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of arginine between 628% and 881% or more,
preferably of glutamate between 35% and 65% or more, preferably of
glutamine between 43% and 256% or more, preferably of arginine and
glutamate between 35% and 881% or more, preferably of arginine and
glutamine between 43% and 881% or more, preferably of glutamate and
glutamine between 35% and 256% or more, preferably of arginine and
glutamate and glutamine between 35% and 881% or more is
conferred.
[2114] In case the activity of the Escherichia coli K12 protein
b0486 or its homologs e.g. a amino-acid/amine transport protein
(APC family) e.g. as indicated in Table II, columns 5 or 7, line
393, is increased, preferably, in one embodiment the increase of
the fine respective chemical, preferably of glutamine between 51%
and 128% or more, is conferred.
[2115] In case the activity of the Escherichia coli K12 protein
b0849 or its homologs e.g. a glutaredoxin 1 redox coenzyme for
glutathione-dependent ribonucleotide reductase e.g. as indicated in
Table II, columns 5 or 7, line 394, is increased, preferably, in
one embodiment the increase of the fine chemical, preferably of
glutamine between 37% and 50% or more is conferred.
[2116] In case the activity of the Escherichia coli K12 protein
b0970 or its homologs e.g. a glutamate receptor e.g. as indicated
in Table II, columns 5 or 7, line 395, is increased, preferably, in
one embodiment the increase of the fine respective chemical,
preferably of glutamine between 59% and 380% or more, is
conferred.
[2117] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1343 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 396 and 397, is increased, e.g. the
activity of a protein involved in rRNA processing and/or
translation is increased, preferred the activity of a ATP-dependent
RNA helicase, stimulated by 23S rRNA or its homolog is increased.
Preferably, an increase of the respective fine chemical preferably
of glutamine between 37% and 39% or more is conferred, preferably
of glutamate between 48% and 99% or more is conferred, preferably
of glutamine and glutamate between 37% and 99% or more is
conferred.
[2118] In case the activity of the Escherichia coli K12 protein
b1360 or a protein with the activity defined as putative DNA
replication protein or its homologs, e.g. transcriptional
regulator, e.g. as indicated in Table II, columns 5 or 7, line 398
is increased, preferably, in one embodiment an increase of the fine
chemical, preferably of proline between 33% and 70% or more is
conferred.
[2119] In case the activity of the Escherichia coli K12 protein
b1693 or its homologs e.g. a 3-dehydroquinate dehydratase e.g. as
indicated in Table II, columns 5 or 7, line 399, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of glutamate between 39% and 149% or more is
conferred.
[2120] In case the activity of the Escherichia coli K12 protein
b1736 or its homologs e.g. a PEP-dependent phosphotransferase
enzyme, e.g. as indicated in Table II, columns 5 or 7, line 400, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of glutamate between 46% and 97% or more is
conferred.
[2121] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1738 or a protein with the activity defined as
PEP-dependent phosphotransferase or its homologs, e.g. as indicated
in Table II, columns 5 or 7, line 401, is increased, preferably, in
one embodiment an increase of the fine chemical preferably of
glutamate between 38% and 107% or more is conferred.
[2122] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1886 or a methyl-accepting chemotaxis protein II,
aspartate sensor receptor or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 402, is increased, preferably, in
one embodiment an increase of the fine chemical preferably of
glutamine between 36% and 124% or more is conferred.
[2123] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or a trehalose-6-phosphate synthase or its
homologs, e.g. as indicated in Table II, columns 5 or 7, line 403,
is increased, preferably, in one embodiment an increase of the fine
chemical preferably of glutamate between 67% and 162% or more is
conferred.
[2124] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1926 or a flagellar protein fliT or its homologs,
e.g. as indicated in Table II, columns 5 or 7, line 404, is
increased, preferably, in one embodiment an increase of the fine
chemical preferably of glutamine between 7% and 27% or morre is
conferred.
[2125] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2307 or a histidine and lysine/arginine/ornithine
transport protein (ABC superfamily, membrane) or its homologs, e.g.
as indicated in Table II, columns 5 or 7, line 405 and 406, is
increased, preferably, in one embodiment an increase of the fine
chemical, preferably of arginine between 95% and 247% or more,
preferably of glutamate between 35% and 89% or more, preferably of
arginine and glutamatne between 35 and 247% or more is
conferred.
[2126] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2414 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 407, is increased, e.g. the activity of a
protein of the threonine dehydratase-superfamily is increased
preferably the activity of a protein involved in amino acid
biosynthesis, biosynthesis of the cysteine-aromatic group,
degradation of amino acids of the cysteine-aromatic group, nitrogen
and sulfur utilizationbiosynthesis of the aspartate family,
degradation of amino acids of the aspartate group, biosynthesis of
sulfuric acid and L-cysteine derivatives, biosynthesis of secondary
products derived from primary amino acids, biosynthesis of
secondary products derived from glycine, L-serine and L-alanine,
pyridoxal phosphate binding is increased, preferred the activity of
a subunit of cysteine synthase A and 0-acetylserine sulfhydrolase
A, PLP-dependent enzyme or its homolog is increased. Preferably, an
increase of the respective fine chemical, preferably of glutamine
between 30% and 56% or more is conferred.
[2127] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2426 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 408, is increased, e.g. the activity of a
oxidoreductase with NAD(P)-binding domain is increased. Preferably,
an increase of the respective fine chemical, preferably of
glutamine between 31% and 62% or more is conferred.
[2128] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2489 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 409, is increased, e.g. the activity of a
hydrogenase Fe--S subunit is increased. Preferably, an increase of
the respective fine chemical, preferably of glutamine between 33%
and 44% or more is conferred.
[2129] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2553 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 410 and 411, is increased, e.g. the
activity of a regulatory protein P-II for glutamine synthetase is
increased. Preferably, an increase of the respective fine chemical,
preferably of glutamine between 55% and 90% or more, preferably of
proline between 49% and 68% or more, preferably of glutamine and
proline between 49% and 90% or more is conferred.
[2130] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2664 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 412, is increased, e.g. the activity of a
hydrogenase Fe--S subunit is increased. Preferably, an increase of
the respective fine chemical, preferably of proline between 35% and
853% or more is conferred.
[2131] In case the activity of the Escherichia coli K12 protein
b2710 or its homologs e.g. a flavorubredoxin (FIRd) bifunctional NO
and O.sub.2 reductase e.g. as indicated in Table II, columns 5 or
7, line 413, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glutamate between 35%
and 38% or more is conferred. In one embodiment, in case the
activity of the Escherichia coli K12 protein b2818 or its homologs,
e.g. as indicated in Table I, columns 5 or 7, line 414 and 415, is
increased, e.g. the activity of a N-acetylglutamate synthase (amino
acid N-acetyltransferase is increased. Preferably, an increase of
the respective fine chemical, preferably of glutamate between 50%
and 129% or more, preferably of glutamine between 45% and 519% or
more, preferably of glutamate and glutamine between 45% and 519% or
more is conferred.
[2132] In case the activity of the Escherichia coli K12 protein
b3064 or its homologs e.g. a putative O-sialoglycoprotein
endopeptidase, with actin-like ATPase domain e.g. as indicated in
Table II, columns 5 or 7, line 416, is increased, preferably, in
one embodiment the increase of the fine chemical, preferably of
glutamine between 72% and 141% or more is conferred.
[2133] In case the activity of the Escherichia coli K12 protein
b3074 or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 417, is increased, e.g. the activity of a tRNA synthetase
is increased, preferably, an increase of the respective fine
chemical, preferably of glutamate between 34% and 85% or more is
conferred.
[2134] In case the activity of the Escherichia coli K12 protein
b3116 or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 418, is increased, e.g. the activity of a
L-threonine/L-serine permease, anaerobically inducible (HAAAP
family) is increased, preferably, an increase of the respective
fine chemical, preferably of glutamate between 35% and 98% or more
is conferred.
[2135] In case the activity of the Escherichia coli K12 protein
b3160 or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 419, is increased, e.g. the activity of a monooxygenase
with luciferase-like ATPase activity is increased, preferably, an
increase of the respective fine chemical, preferably of glutamine
between 38% and 189% or more is conferred.
[2136] In case the activity of the Escherichia coli K12 protein
b3166 or its homologs e.g. a tRNA pseudouridine 5S synthase e.g. as
indicated in Table II, columns 5 or 7, line 420, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of glutamine between 29% and 40% or more is
conferred.
[2137] In case the activity of the Escherichia coli K12 protein
b3169 or its homologs e.g. a transcription
termination-antitermination factor e.g. as indicated in Table II,
columns 5 or 7, line 421 and 422, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
glutamine between 55% and 111% or more, preferably of glutamate
between 42% and 140% or more, preferably of glutamine and glutamate
between 42% and 140% or more is conferred.
[2138] In case the activity of the Escherichia coli K12 protein
b3231 or its homologs e.g. a 50S ribosomal subunit protein L13 e.g.
as indicated in Table II, columns 5 or 7, line 423, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of glutamine between 50% and 164% or more is
conferred.
[2139] In case the activity of the Escherichia coli K12 protein
b3619 or its homologs e.g. a
ADP-L-glycero-D-mannoheptose-6-epimerase, NAD(P)-binding e.g. as
indicated in Table II, columns 5 or 7, line 424, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of glutamate between 40% and 122% or more is
conferred.
[2140] In case the activity of the Escherichia coli K12 protein
b3644 or its homologs e.g. an uncharacterized stress-induced
protein e.g. as indicated in Table II, columns 5 or 7, line 425, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of proline between 32% and 241% or more is
conferred.
[2141] In case the activity of the Escherichia coli K12 protein
b3680 or its homologs e.g. an uncharacterized stress-induced
protein e.g. as indicated in Table II, columns 5 or 7, line 426, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of glutamine between 50% and 199% or more is
conferred.
[2142] In case the activity of the Escherichia coli K12 protein
b3791 or its homologs e.g. an uncharacterized stress-induced
protein e.g. as indicated in Table II, columns 5 or 7, line 427 and
428, is increased, preferably, in one embodiment the increase of
the fine chemical, preferably of glutamine between 28% and 57% or
more, preferably of glutamate between 39% and 57% or more,
preferably of glutamine and glutamate between 28% and 57% or more
is conferred.
[2143] In case the activity of the Escherichia coli K12 protein
b3919 or its homologs e.g. an triosephosphate isomerase e.g. as
indicated in Table II, columns 5 or 7, line 429, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of proline between 35% and 118% or more is
conferred.
[2144] In case the activity of the Escherichia coli K12 protein
b3936 or its homologs e.g. an 50S ribosomal subunit protein L32
e.g. as indicated in Table II, columns 5 or 7, line 430, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of arginine between 120% and 398% or more is
conferred.
[2145] In case the activity of the Escherichia coli K12 protein
b4004 or its homologs e.g. a transcriptional regulatory protein
e.g. as indicated in Table II, columns 5 or 7, line 431, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of glutamine between 30% and 36% or more is
conferred.
[2146] In case the activity of the Escherichia coli K12 protein
b4074 or its homologs e.g. a cytochrome c-type biogenesis protein
e.g. as indicated in Table II, columns 5 or 7, line 432, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of glutamine between 40% and 42% or more is
conferred.
[2147] In case the activity of the Escherichia coli K12 protein
b4133 or its homologs e.g. a transcriptional activator of pH
response (OmpR family) e.g. as indicated in Table II, columns 5 or
7, line 433, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glutamine between 59%
and 212% or more is conferred.
[2148] In case the activity of the Escherichia coli K12 protein
b4346 or its homologs e.g. a component of 5-methylcytosine-specific
restriction enzyme McrBC e.g. as indicated in Table II, columns 5
or 7, line 434, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glutamate between 38%
and 44% or more is conferred.
[2149] In case the activity of the Saccharomyces cerevisiae protein
YFL019C or its homologs e.g. a protein with the activity as
indicated in [0022.0.4.4] e.g. as indicated in Table II, columns 5
or 7, line 435, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glutamate between 81%
and 134% or more is conferred.
[2150] [0046.0.4.4] In case the activity of the Escherichia coli
K12 protein b0695 or its homologs, e.g. as indicated in Table I,
columns 5 or 7, line 35, e.g. a sensory histidine kinase is
increased, preferably an increase of the fine chemical and of
phenylalanine is conferred.
[2151] In case the activity of the Escherichia coli K12 protein
b0730 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 43 or 54, e.g. a transcriptional regulator is increased,
preferably an increase of the fine chemical and of fumerate is
conferred.
[2152] In case the activity of the Escherichia coli K12 protein
b1284 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 36, e.g. a transcriptional regulator is increased,
preferably an increase of the fine chemical and of fumaric acid is
conferred.
[2153] In case the activity of the Escherichia coli K12 protein
b1827 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 56, e.g. a transcriptional repressor is increased,
preferably an increase of the fine chemical and of isoleucince is
conferred.
[2154] In case the activity of the Escherichia coli K12 protein
b1829 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 34 or 60, is increased, e.g. the activity of a heat shock
protein with protease activity (htpx is increased, preferably an
increase of the fine chemical and of isoleucine is conferred.
[2155] In case the activity of the Escherichia coli K12 protein
b1852 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 61, is increased, e.g. the activity of a
glucose-6-phosphate dehydrogenase is increased, preferably an
increase of the fine chemical and of myoinositol is conferred.
[2156] In case the activity of the Escherichia coli K12 protein
b2095 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 37, is increased, e.g. the activity of a
tagatose-6-phosphate kinase is increased preferably an increase of
the fine chemical and of alanine is conferred.
[2157] In case the activity of the Escherichia coli K12 protein
b2699 or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 55, is increased, e.g. the activity of a recombination
protein recA is increased, preferably an increase of the fine
chemical and of fumerate is conferred.
[2158] In case the activity of the Saccaromyces cerevisiae protein
YFR042W or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 58, e.g. a "protein required for cell viability in yeast"
is increased, preferably an increase of the fine chemical and of
Leucine is conferred.
[2159] In case the activity of the Saccharomyces cerevisiae protein
YHR130C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 31, e.g. a "uncharacterized protein YHR130C" is increased,
preferably an increase of the fine chemical and of phenylalanine is
conferred.
[2160] In case the activity of the Saccharomyces cerevisiae protein
YIL150C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 51, e.g. a chromatin binding protein is increased,
preferably an increase of the fine chemical and of valine is
conferred.
[2161] In case the activity of the Saccharomyces cerevisiae protein
YPR024W or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 41, e.g. a mitochondrial protein of the CDC48/PAS1/SEC18
family of ATPases is increased, preferably an increase of the fine
chemical and of fumerate is conferred.
[2162] In case the activity of the Saccharomyces cerevisiae protein
YPR138C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 53, e.g. a ammonium transport protein is increased,
preferably an increase of the fine chemical and of phenylalanine is
conferred.
[2163] In case the activity of the Saccharomyces cerevisiae protein
YBR204C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 38, e.g. a peroxisomal lipase is increased, preferably an
increase of the fine chemical and of inositol is conferred.
[2164] In case the activity of the Saccharomyces cerevisiae protein
YDR271C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 46, e.g. a "uncharacterized protein YDR271C" is increased,
preferably an increase of the fine chemical and of isoleucine is
conferred.
[2165] In case the activity of the Saccharomyces cerevisiae protein
YER173W or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 48 or 57, e.g. a checkpoint protein is increased,
preferably an increase of the fine chemical and of valine is
conferred.
[2166] In case the activity of the Saccharomyces cerevisiae protein
YFL013C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 39, e.g. a "subunit of the INO80 chromatin remodeling
complex" is increased, preferably an increase of the fine chemical
and of valine is conferred.
[2167] In case the activity of the Saccharomyces cerevisiae protein
YFL050C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 49, e.g. a di-, tri-valent inorganic cation transporter is
increased, preferably an increase of the fine chemical and of
threonine is conferred.
[2168] In case the activity of the Saccharomyces cerevisiae protein
YGR104C or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 40, e.g. a "RNA polymerase II suppressor protein
SRB5--yeast and/or suppressor of RNA polymerase B SRB5" is
increased, preferably an increase of the fine chemical and of
isoleucine is conferred.
[2169] In case the activity of the Saccharomyces cerevisiae protein
YKR057W or its homologs, e.g. as indicated in Table I, columns 5 or
7, line 32 or 59, e.g. a ribosomal protein, similar to S21A, S26A
and/or YS25 ribosomal proteins is increased, preferably an increase
of the fine chemical and of threonine is conferred.
[2170] In case the activity of the Escherichia coli K12 protein
b0050 or its homologs e.g. a conserved protein potentially involved
in protein protein interaction e.g. as indicated in Table II,
columns 5 or 7, line 386, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
glutamate and of an other amino acid or more is conferred.
[2171] In case the activity of the Escherichia coli K12 protein
b0057 or its homologs e.g. a protein with an activity as defined in
[0022.0.4.4],e.g. as indicated in Table II, columns 5 or 7, line
387, is increased, preferably, in one embodiment the increase of
the fine chemical, preferably of glutamate and of an other amino
acid or more is conferred.
[2172] In case the activity of the Escherichia coli K12 protein
b0138 or its homologs e.g. a fimbrial-like adhesin protein e.g. as
indicated in Table II, columns 5 or 7, line 388, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of proline and of one or more other amino acid(s) or
more is conferred.
[2173] In case the activity of the Escherichia coli K12 protein
b0149 or its homologs e.g. a bifunctional penicillin-binding
protein 1 b: glycosyl transferase (N-terminal); transpeptidase
(C-terminal) e.g. as indicated in Table II, columns 5 or 7, line
389, is increased, preferably, in one embodiment the increase of
the fine chemical, preferably of proline and of one or more other
amino acid(s)o(s) is conferred.
[2174] In case the activity of the Escherichia coli K12 protein
b0161 or its homologs e.g. a periplasmic serine protease e.g. as
indicated in Table II, columns 5 or 7, lines 390 to 392, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of arginine and/or of glutamate and/or of
glutamine and of one or more other amino acid(s) is conferred.
[2175] In case the activity of the Escherichia coli K12 protein
b0486 or its homologs e.g. a amino-acid/amine transport protein
(APC family) e.g. as indicated in Table II, columns 5 or 7, line
393, is increased, preferably, in one embodiment the increase of
the fine respective chemical, preferably of glutamine and of one or
more other amino acid(s) is conferred.
[2176] In case the activity of the Escherichia coli K12 protein
b0849 or its homologs e.g. a glutaredoxin 1 redox coenzyme for
glutathione-dependent ribonucleotide reductase e.g. as indicated in
Table II, columns 5 or 7, line 394, is increased, preferably, in
one embodiment the increase of the fine chemical, preferably of
glutamine and of one or more other amino acid(s) is conferred.
[2177] In case the activity of the Escherichia coli K12 protein
b0970 or its homologs e.g. a glutamate receptor e.g. as indicated
in Table II, columns 5 or 7, line 395, is increased, preferably, in
one embodiment the increase of the fine respective chemical,
preferably of glutamine and of one or more other amino(s) acid(s)
is conferred.
[2178] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1343 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 396 and 397, is increased, e.g. the
activity of a protein involved in rRNA processing and/or
translation is increased, preferred the activity of a ATP-dependent
RNA helicase, stimulated by 23S rRNA or its homolog is increased.
Preferably, an increase of the respective fine chemical preferably
of glutamine and/or of glutamate and of one or more other amino
acid(s) is conferred.
[2179] In case the activity of the Escherichia coli K12 protein
b1360 or a protein with the activity defined as putative DNA
replication protein or its homologs, e.g. transcriptional
regulator, e.g. as indicated in Table II, columns 5 or 7, line 398
is increased, preferably, in one embodiment an increase of the fine
chemical, preferably of proline between and of one or more other
amino acid(s) conferred.
[2180] In case the activity of the Escherichia coli K12 protein
b1693 or its homologs e.g. a 3-dehydroquinate dehydratase e.g. as
indicated in Table II, columns 5 or 7, line 399, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of glutamate and of one or more other amino acid(s) is
conferred.
[2181] In case the activity of the Escherichia coli K12 protein
b1736 or its homologs e.g. a PEP-dependent phosphotransferase
enzyme, e.g. as indicated in Table II, columns 5 or 7, line 400, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of glutamate and of one or more other amino
acid(s) is conferred.
[2182] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1738 or a protein with the activity defined as
PEP-dependent phosphotransferase or its homologs, e.g. as indicated
in Table II, columns 5 or 7, line 401, is increased, preferably, in
one embodiment an increase of the fine chemical preferably of
glutamate and of one or more other amino acid(s) is conferred.
[2183] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1886 or a methyl-accepting chemotaxis protein II,
aspartate sensor receptor or its homologs, e.g.
[2184] as indicated in Table II, columns 5 or 7, line 402, is
increased, preferably, in one embodiment an increase of the fine
chemical preferably of glutamine and of one or more other amino
acid(s) is conferred.
[2185] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or a trehalose-6-phosphate synthase or its
homologs, e.g. as indicated in Table II, columns 5 or 7, line 403,
is increased, preferably, in one embodiment an increase of the fine
chemical preferably of glutamate and of one or more other amino
acid(s) is conferred.
[2186] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1926 or a flagellar protein fliT or its homologs,
e.g. as indicated in Table II, columns 5 or 7, line 404, is
increased, preferably, in one embodiment an increase of the fine
chemical preferably of glutamine and of one or more other amino
acid(s) is conferred.
[2187] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2307 or a flagellar protein fliT or its homologs,
e.g. as indicated in Table II, columns 5 or 7, line 405 and 406, is
increased, preferably, in one embodiment an increase of the fine
chemical, preferably of arginine and/or of glutamate and of one or
more other amino acid(s) is conferred.
[2188] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2414 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 407, is increased, e.g. the activity of a
protein of the threonine dehydratase-superfamily is increased
preferably the activity of a protein involved in amino acid
biosynthesis, biosynthesis of the cysteine-aromatic group,
degradation of amino acids of the cysteine-aromatic group, nitrogen
and sulfur utilizationbiosynthesis of the aspartate family,
degradation of amino acids of the aspartate group, biosynthesis of
sulfuric acid and L-cysteine derivatives, biosynthesis of secondary
products derived from primary amino acids, biosynthesis of
secondary products derived from glycine, L-serine and L-alanine,
pyridoxal phosphate binding is increased, preferred the activity of
a subunit of cysteine synthase A and 0-acetylserine sulfhydrolase
A, PLP-dependent enzyme or its homolog is increased.
[2189] Preferably, an increase of the respective fine chemical,
preferably of glutamine and of one or more other amino acid(s) is
conferred.
[2190] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2426 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 408, is increased, e.g. the activity of a
oxidoreductase with NAD(P)-binding domain is increased. Preferably,
an increase of the respective fine chemical, preferably of
glutamine and of one or more other amino acid(s) is conferred.
[2191] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2489 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 409, is increased, e.g. the activity of a
hydrogenase Fe--S subunit is increased. Preferably, an increase of
the respective fine chemical, preferably of glutamine and of one or
more other amino acid(s) is conferred.
[2192] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2553 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 410 and 411, is increased, e.g. the
activity of a regulatory protein P-II for glutamine synthetase is
increased. Preferably, an increase of the respective fine chemical,
preferably of glutamine and/or of proline and of one or more other
amino acid(s) is conferred.
[2193] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2644 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 412, is increased, e.g. the activity of a
hydrogenase Fe--S subunit is increased. Preferably, an increase of
the respective fine chemical, preferably of proline and of one or
more other amino acid(s) is conferred.
[2194] In case the activity of the Escherichia coli K12 protein
b2710 or its homologs e.g. a flavorubredoxin (FIRd) bifunctional NO
and O.sub.2 reductase e.g. as indicated in Table II, columns 5 or
7, line 413, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glutamate and of one
or more other amino acid(s) is conferred.
[2195] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2818 or its homologs, e.g. as indicated in Table
I, columns 5 or 7, line 414 and 415, is increased, e.g. the
activity of a N-acetylglutamate synthase (amino acid
N-acetyltransferase is increased. Preferably, an increase of the
respective fine chemical, preferably of glutamate and/or of
glutamine and of one or more other amino acid(s) is conferred.
[2196] In case the activity of the Escherichia coli K12 protein
b3064 or its homologs e.g. a putative O-sialoglycoprotein
endopeptidase, with actin-like ATPase domain e.g. as indicated in
Table II, columns 5 or 7, line 416, is increased, preferably, in
one embodiment the increase of the fine chemical, preferably of
glutamine and of one or more other amino acid(s) is conferred.
[2197] In case the activity of the Escherichia coli K12 protein
b3074 or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 417, is increased, e.g. the activity of a tRNA synthetase
is increased, preferably, an increase of the respective fine
chemical, preferably of glutamate and of one or more other amino
acid(s) is conferred.
[2198] In case the activity of the Escherichia coli K12 protein
b3116 or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 418, is increased, e.g. the activity of a
L-threonine/L-serine permease, anaerobically inducible (HAAAP
family) is increased, preferably, an increase of the respective
fine chemical, preferably of glutamate and of one or more other
amino acid(s) is conferred.
[2199] In case the activity of the Escherichia coli K12 protein
b3160 or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 419, is increased, e.g. the activity of a monooxygenase
with luciferase-like ATPase activity is increased, preferably, an
increase of the respective fine chemical, preferably of glutamine
and of one or more other amino acid(s) is conferred.
[2200] In case the activity of the Escherichia coli K12 protein
b3166 or its homologs e.g. a tRNA pseudouridine 5S synthase e.g. as
indicated in Table II, columns 5 or 7, line 420, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of glutamine and of one or more other amino acid(s) is
conferred.
[2201] In case the activity of the Escherichia coli K12 protein
b3169 or its homologs e.g. a transcription
termination-antitermination factor e.g. as indicated in Table II,
columns 5 or 7, line 421 and 422, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
glutamine and/or of glutamate and of one or more other amino
acid(s) is conferred.
[2202] In case the activity of the Escherichia coli K12 protein
b3231 or its homologs e.g. a 50S ribosomal subunit protein L13 e.g.
as indicated in Table II, columns 5 or 7, line 423, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of glutamine and of one or more other amino acid(s) is
conferred.
[2203] In case the activity of the Escherichia coli K12 protein
b3619 or its homologs e.g. a
ADP-L-glycero-D-mannoheptose-6-epimerase, NAD(P)-binding e.g. as
indicated in Table II, columns 5 or 7, line 424, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of glutamate and of one or more other amino acid(s) is
conferred.
[2204] In case the activity of the Escherichia coli K12 protein
b3644 or its homologs e.g. an uncharacterized stress-induced
protein e.g. as indicated in Table II, columns 5 or 7, line 425, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of proline and of one or more other amino
acid(s) is conferred.
[2205] In case the activity of the Escherichia coli K12 protein
b3680 or its homologs e.g. an uncharacterized stress-induced
protein e.g. as indicated in Table II, columns 5 or 7, line 426, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of glutamine and of one or more other amino
acid(s) is conferred.
[2206] In case the activity of the Escherichia coli K12 protein
b3791 or its homologs e.g. an uncharacterized stress-induced
protein e.g. as indicated in Table II, columns 5 or 7, line 427 and
428, is increased, preferably, in one embodiment the increase of
the fine chemical, preferably of glutamine and/or of glutamate
between and of one or more other amino acid(s) is conferred.
[2207] In case the activity of the Escherichia coli K12 protein
b3919 or its homologs e.g. an triosephosphate isomerase e.g. as
indicated in Table II, columns 5 or 7, line 429, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of proline and of one or more other amino acid(s) is
conferred.
[2208] In case the activity of the Escherichia coli K12 protein
b3936 or its homologs e.g. an 50S ribosomal subunit protein L32
e.g. as indicated in Table II, columns 5 or 7, line 430, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of arginine and of one or more other amino
acid(s) is conferred.
[2209] In case the activity of the Escherichia coli K12 protein
b4004 or its homologs e.g. a transcriptional regulatory protein
e.g. as indicated in Table II, columns 5 or 7, line 431, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of glutamine and of one or more other amino
acid(s) is conferred.
[2210] In case the activity of the Escherichia coli K12 protein
b4074 or its homologs e.g. a cytochrome c-type biogenesis protein
e.g. as indicated in Table II, columns 5 or 7, line 432, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of glutamine and of one or more other amino
acid(s) is conferred.
[2211] In case the activity of the Escherichia coli K12 protein
b4133 or its homologs e.g. a transcriptional activator of pH
response (OmpR family) e.g. as indicated in Table II, columns 5 or
7, line 433, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glutamine and of one
or more other amino acid(s) is conferred.
[2212] In case the activity of the Escherichia coli K12 protein
b4346 or its homologs e.g. a component of 5-methylcytosine-specific
restriction enzyme McrBC e.g. as indicated in Table II, columns 5
or 7, line 434, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glutamate and of one
or more other amino acid(s) is conferred.
[2213] In case the activity of the Saccharomyces cerevisiae protein
YFL019C or its homologs e.g. a protein with the activity as
indicated in [0022.0.4.4] e.g. as indicated in Table II, columns 5
or 7, line 435, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glutamate and of one
or more other amino acid(s) is conferred.
[2214] [0047.0.0.4] and [0048.0.0.4]: see [0047.0.0.0] and
[0048.0.0.0]
[2215] [0049.0.4.4] A protein having an activity conferring an
increase in the amount or level of arginine chemical preferably has
the structure of the polypeptide described herein, in particular of
a polypeptides comprising a consensus sequence as indicated in
Table IV, columns 7, lines 30 to 37, 390, 405 and/or 430 or of a
polypeptide as indicated in Table II, columns 5 or 7, lines 30 to
37, 390, 405 and/or 430 or the functional homologues thereof as
described herein, or is encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 30 to 37, 390, 405 and/or 430 or its
herein described functional homologues and has the herein mentioned
activity.
[2216] A protein having an activity conferring an increase in the
amount or level of glutamate preferably has the structure of the
polypeptide described herein, in particular of a polypeptides
comprising a consensus sequence as indicated in Table IV, column 7,
lines 38 to 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414,
417, 418, 421, 424, 427, 434 and/or 435 or of a polypeptide as
indicated in Table II, columns 5 or 7, lines 38 to 43, 386, 387,
391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427,
434 and/or 435 or the functional homologues thereof as described
herein, or is encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, lines 38 to 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427, 434 and/or 435 or its herein
described functional homologues and has the herein mentioned
activity.
[2217] A protein having an activity conferring an increase in the
amount or level of proline preferably has the structure of the
polypeptide described herein, in particular of a polypeptides
comprising a consensus sequence as indicated in Table IV, column 7,
lines 44 to 56, 388, 389, 398, 411, 412, 425 and/or 429 or of a
polypeptide as indicated in Table II, columns 5 or 7, lines 44 to
56, 388, 389, 398, 411, 412, 425 and/or 429 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 44 to 56,
388, 389, 398, 411, 412, 425 and/or 429 or its herein described
functional homologues and has the herein mentioned activity.
[2218] A protein having an activity conferring an increase in the
amount or level of glutamine preferably has the structure of the
polypeptide described herein, in particular of a polypeptides
comprising a consensus sequence as indicated in Table IV, column 7,
lines 57 to 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 or of a polypeptide
as indicated in Table II, columns 5 or 7, lines 57 to 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 or the functional homologues thereof as
described herein, or is encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 57 to 62, 392 to 395, 397, 402, 404,
407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to
433 or its herein described functional homologues and has the
herein mentioned activity.
[2219] [0050.0.4.4] For the purposes of the present invention, the
term "arginine" and/or "glutamate" and/or "glutamine" and/or
"proline" and "L-arginine" and/or "L-glutamate" and/or
"L-glutamine" and/or "L-proline" also encompass the corresponding
salts, such as, for example, arginine- and/or glutamate- and/or
glutamine- and/or proline-hydrochloride or arginine and/or
glutamate and/or glutamine and/or proline sulfate. Preferably the
term arginine and/or glutamate and/or glutamine and/or proline is
intended to encompass the term L-arginine and/or L-glutamate and/or
L-glutamine and/or L-proline.
[2220] [0051.0.0.4] and [0052.0.0.4]: see [0051.0.0.0] and
[0052.0.0.0]
[2221] [0053.0.4.4] In one embodiment, the process of the present
invention comprises one or more of the following steps [2222] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 34
to 37, 390, 405 and/or 430 for arginine [2223] and/or lines 43,
386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421,
424, 427, 434 and/or 435 for glutamate [2224] and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 for proline [2225]
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 for glutamine resp.,
or its homologs, e.g. as indicated in Table II, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 for arginine and/or lines 43,
386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421,
424, 427, 434 and/or 435 for glutamate and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 for proline and/or lines 62, 392
to 395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423,
426, 428 and/or 431 to 433 for glutamine resp., activity having
herein-mentioned the respective fine chemical-increasing activity;
[2226] b) stabilizing a mRNA conferring the increased expression of
a protein encoded by the nucleic acid molecule of the invention,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434 and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp. or its homologs activity, e.g. as indicated in
Table II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434 and/or 435 and/or lines 54 to 56,
388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp or of a mRNA encoding the polypeptide of
the present invention having herein-mentioned the respective fine
chemical-increasing activity; [2227] c) increasing the specific
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the present invention having herein-mentioned
the respective fine chemical-increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 34 to 37, 390, 405 and/or 430 and/or lines 43,
386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421,
424, 427, 434 and/or 435 and/or lines 54 to 56, 388, 389, 398, 411,
412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407
to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433
respor its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434 and/or 435 and/or lines 54 to 56, 388, 389, 398,
411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402,
404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431
to 433 resp or decreasing the inhibiitory regulation of the
polypeptide of the invention; [2228] d) generating or increasing
the expression of an endogenous or artificial transcription factor
mediating the expression of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptide of the invention having
herein-mentioned the respective fine chemical-increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434 and/or 435 and/or lines 54 to 56,
388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434 and/or 435 and/or lines
54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62,
392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422,
423, 426, 428 and/or 431 to 433 resp; [2229] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned the respective fine chemical-increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434 and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp or its homologs activity, e.g. as indicated in
Table II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434 and/or 435 and/or lines 54 to 56,
388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp, by adding one or more exogenous
inducing factors to the organisms or parts thereof; [2230] f)
expressing a transgenic gene encoding a protein conferring the
increased expression of a polypeptide encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention, having herein-mentioned the respective fine
chemical-increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 34
to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399
to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434 and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 34
to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399
to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434 and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp, and/or [2231]
g) increasing the copy number of a gene conferring the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or the
polypeptide of the invention having herein-mentioned the respective
fine chemical-increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 34
to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399
to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434 and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp or its
homologs, e.g. as indicated in Table II, columns 5 or 7, lines 34
to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399
to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434 and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp, activity.
[2232] h) Increasing the expression of the endogenous gene encoding
the polypeptide of the invention, e.g. a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 34
to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399
to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434 and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 34
to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399
to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434 and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp by adding
positive expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; [2233] i)
Modulating growth conditions of an organism in such a manner, that
the expression or activity of the gene encoding the protein of the
invention or the protein itself is enhanced for example
microorganisms or plants can be grown for example under a higher
temperature regime leading to an enhanced expression of heat shock
proteins, which can lead an enhanced the fine chemical production;
and/or [2234] j) selecting of organisms with expecially high
activity of the proteins of the invention from natural or from
mutagenized resources and breeding them into the target organisms,
eg the elite crops.
[2235] [0054.0.4.4] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of arginine and/or
glutamate and/or proline and/or glutamine after increasing the
expression or activity of the encoded polypeptide or having the
activity of a polypeptide having an activity of a protein according
to Table II, lines 34 to 37, 390, 405 and/or 430 and/or lines 43,
386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421,
424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398,
411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402,
404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431
to 433 resp. or its homologs activity, e.g. as indicated in Table
II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp.
[2236] [0055.0.0.4] to [0071.0.0.4]: see [0055.0.0.0] to
[0071.0.0.0]
[2237] [0072.0.4.4] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to arginine and/or glutamate and/or glutamine and/or
proline Argininosuccinate, Citrulline, Ornithine, Urea,
Pyrroline-5-carboxylate, Hydroxyproline,
Hydroxypyrroline-carboxylate, 3-Hydroxypyrroline-5-carboxylate,
.gamma.-Glutamylcysteine, Glutathione, Hydroxyglutamate,
4-Hydroxyglutamate, Oxoglutarate, 4-Hydroxy-2-oxoglutarate,
Glutamine.
[2238] [0073.0.4.4] Accordingly, in one embodiment, the process
according to the invention relates to a process which comprises:
[2239] (c) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [2240] (d) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table II, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp. or of a polypeptide
being encoded by the nucleic acid molecule of the present invention
and described below, e.g. conferring an increase of the respective
fine chemical in an organism, preferably in a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant, [2241] (e) growing the organism, preferably the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue or the plant under conditions which permit
the production of the fine chemical in the organism, preferably the
microorganism, the plant cell, the plant tissue or the plant; and
if desired, revovering, optionally isolating, the free and/or bound
the fine chemical and, optionally further free and/or bound amino
acids synthetized by the organism, the microorganism, the non-human
animal, the plant or animal cell, the plant or animal tissue or the
plant.
[2242] [0074.0.0.4] to [0084.0.0.4]: see [0074.0.0.0] to
[0084.0.0.0]
[2243] [0085.0.4.4] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [2244] a) the nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp. or a derivative
thereof, or [2245] b) a genetic regulatory element, for example a
promoter, which is functionally linked to the nucleic acid sequence
as indicated in Table I, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp. or a derivative
thereof, or [2246] c) (a) and (b) is/are not present in its/their
natural genetic environment or has/have been modified by means of
genetic manipulation methods, it being possible for the
modification to be, by way of example, a substitution, addition,
deletion, inversion or insertion of one or more nucleotide.
"Natural genetic environment" means the natural chromosomal locus
in the organism of origin or the presence in a genomic library. In
the case of a genomic library, the natural, genetic environment of
the nucleic acid sequence is preferably at least partially still
preserved. The environment flanks the nucleic acid sequence at
least on one side and has a sequence length of at least 50 bp,
preferably at least 500 bp, particularly preferably at least 1000
bp, very particularly preferably at least 5000 bp.
[2247] [0086.0.0.4] and [0088.1.0.4]: see [0086.0.0.0] and
[0088.1.0.0]
[2248] [0089.0.0.4] to [0097.0.0.4]: see [0089.0.0.0] to
[0097.0.0.0]
[2249] [0098.0.4.4] In a preferred embodiment, the fine chemical
(arginine and/or glutamate and/or glutamine and/or proline) is
produced in accordance with the invention and, if desired, is
isolated. The production of further amino acids such as methionine,
lysine and/or threonine mixtures of amino acid by the process
according to the invention is advantageous.
[2250] [0099.0.0.4] to [0102.0.0.4]: see [0099.0.0.0] to
[0102.0.0.0]
[2251] [0103.0.4.4] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [2252]
a) nucleic acid molecule encoding, preferably at least the mature
form, of the polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp. or a fragment thereof, which confers an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [2253] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule having a sequence as indicated in Table I, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. [2254] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [2255] d) nucleic acid molecule encoding a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [2256] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [2257] f) nucleic acid molecule encoding a
polypeptide, the polypeptide being derived by substituting,
deleting and/or adding one or more amino acids of the amino acid
sequence of the polypeptide encoded by the nucleic acid molecules
(a) to (d), preferably to (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[2258] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [2259] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers pairs having a sequence as indicated in Table
III, column 7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43,
386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421,
424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398,
411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402,
404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431
to 433 resp. and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [2260]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [2261] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence having a
sequences as indicated in Table IV, column 7, lines 34 to 37, 390,
405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401,
403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp., and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [2262] k) nucleic acid molecule
comprising one or more of the nucleic acid molecule encoding the
amino acid sequence of a polypeptide encoding a domain of the
polypeptide indicated in Table II, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; and [2263] l) nucleic acid molecule
which is obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably to (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; or which comprises a sequence which is complementary
thereto.
[2264] [00103.1.0.4.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., by one or
more nucleotides. In one embodiment, the nucleic acid molecule used
in the process of the invention does not consist of the sequence
shown in indicated in Table I A, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. In one
embodiment, the nucleic acid molecule used in the process of the
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I A, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. In
another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II A, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2265] [00103.2.0.4.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., by one or
more nucleotides. In one embodiment, the nucleic acid molecule used
in the process of the invention does not consist of the sequence
shown in indicated in Table I B, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.: In one
embodiment, the nucleic acid molecule used in the process of the
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I B, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. In
another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II B, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2266] [0104.0.4.4] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence indicated in
Table I, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp., by one or more nucleotides. In one
embodiment, the nucleic acid molecule used in the process of the
invention does not consist of the sequence indicated in Table I,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp. In one embodiment, the nucleic acid molecule of
the present invention is less than 100%, 99.999%, 99.99%, 99.9% or
99% identical to the sequence indicated in Table I, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. In
another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2267] [0105.0.0.4] to [0107.0.0.4]: see [0105.0.0.0] to
[0107.0.0.0]
[2268] [0108.0.4.4] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp., nucleic acid
molecules which are derived from a amino acid sequences as
indicated in Table II, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp. or from
polypeptides comprising the consensus sequence as indicated in
Table IV, column 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp. or their derivatives or homologues encoding
polypeptides with the enzymatic or biological activity of a
polypeptide as indicated in Table I, column 3, 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. or e.g.
conferring a increase of the fine chemical after increasing its
expression or activity are advantageously increased in the process
according to the invention.
[2269] [0109.0.0.4]: see [0109.0.0.0]
[2270] [0110.0.4.4] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide of the invention or
the polypeptide used in the method of the invention or used in the
process of the invention, e.g. of a protein as indicated in Table
II, column 5, lines 34 to 37, 390, 405 and/or 430 and/or lines 43,
386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421,
424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398,
411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402,
404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431
to 433 respor being encoded by a nucleic acid molecule indicated in
Table I, column 5, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp. or of its homologs, e.g. as indicated in Table II,
column 7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386,
387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424,
427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411,
412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407
to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433
resp., can be determined from generally accessible databases.
[2271] [0111.0.0.4]: see [0111.0.0.0]
[2272] [0112.0.4.4] The nucleic acid molecules used in the process
according to the invention take the form of isolated nucleic acid
sequences, which encode polypeptides with protein activity of
proteins as indicated in Table II, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., and
conferring a arginine and/or glutamate and/or proline and/or
glutamine increase.
[2273] [0113.0.0.4] to [0120.0.0.4]: [0113.0.0.0] to
[0120.0.0.0]
[2274] [0121.0.4.4] However, it is also possible to use artificial
sequences, which differ in one or more bases from the nucleic acid
sequences found in organisms, or in one or more amino d molecules
from polypeptide sequences found in organisms, in particular from
the polypeptide sequences indicated in faUe II, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. or
the functional homologues thereof as described herein, preferably
conferring above-mentioned activity, i.e. conferring a increase of
the respective fine chemical after increasing its activity.
[2275] [0122.0.0.4] to [0127.0.0.4]: see [0122.0.0.0] to
[0127.0.0.0]
[2276] [0128.0.4.4] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, column 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. by
means of polymerase chain reaction can be generated on the basis of
a sequence shown herein, for example the sequence as indicated in
Table I, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp. or the sequences derived from a sequence as
indicated in Table II, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2277] [0129.0.4.4] Moreover, it is possible to identify conserved
regions from various organisms by carrying out protein sequence
alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence indicated in Table IV,
column 7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386,
387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424,
427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411,
412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407
to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433
resp. is derived from said alignments.
[2278] [0130.0.0.4] to [0138.0.0.4]: see [0130.0.0.0] to
[0138.0.0.0]
[2279] [0139.0.4.4] Polypeptides having above-mentioned activity,
i.e. conferring the increase of the respective fine chemical level,
derived from other organisms, can be encoded by other DNA sequences
which hybridize to a sequences indicated in Table I, columns 5 or
7, preferably table I B, lines 34 to 37, 390, 405 and/or 430 for
arginine and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427, 434, and/or 435 for glutamate
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 for
proline and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for
glutamine resp., under relaxed hybridization conditions and which
code on expression for peptides having the respective fine
chemical, in particular, of arginine and/or glutamate and/or
proline and/or glutamine, resp., increasing activity.
[2280] [0140.0.0.4] to [0146.0.0.4]: see [0140.0.0.0] to
[0146.0.0.0]
[2281] [0147.0.4.4] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., is one which is sufficiently complementary to one
of said nucleotide sequences such that it can hybridize to one of
said nucleotide sequences, thereby forming a stable duplex.
Preferably, the hybridisation is performed under stringent
hybrization conditions. However, a complement of one of the herein
disclosed sequences is preferably a sequence complement thereto
according to the base pairing of nucleic acid molecules well known
to the skilled person. For example, the bases A and G undergo base
pairing with the bases T and U or C, resp. and visa versa.
Modifications of the bases can influence the base-pairing
partner.
[2282] [0148.0.4.4] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7,
preferably table I B, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp., preferably table I B or a portion thereof
and preferably has above mentioned activity, in particular, of
arginine and/or glutamate and/or proline and/or glutamine
increasing activity after increasing the activity or an activity of
a product of a gene encoding said sequences or their homologs.
[2283] [0149.0.4.4] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.,
preferably of Table I B, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp. or a portion
thereof and encodes a protein having above-mentioned activity and
as indicated in indicated in Table II.
[2284] [00149.1.4.4] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., preferably of Table I B, columns 5 or 7, lines 34
to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399
to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or
435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. has further
one or more of the activities annotated or known for the a protein
as indicated in Table II, column 3, lines 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2285] [0150.0.4.4] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table I, columns 5 or 7, preferably
table I B, lines 34 to 37, 390, 405 and/or 430 and/or lines 43,
386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421,
424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398,
411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402,
404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431
to 433 resp. for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of arginine and/or glutamate
and/or proline and/or glutamine, resp., if its activity is
increased. The nucleotide sequences determined from the cloning of
the present protein-according-to-the-invention-encoding gene allows
for the generation of probes and primers designed for use in
identifying and/or cloning its homologues in other cell types and
organisms. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, 15 preferably about 20 or 25, more
preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one of the sequences set forth, e.g., as indicated in
Table I, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp. an anti-sense sequence of one of the
sequences, e.g., as indicated in Table I, columns 5 or 7, lines 34
to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399
to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or
435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. or naturally
occurring mutants thereof. Primers based on a nucleotide of
invention can be used in PCR reactions to clone homologues of the
polypeptide of the invention or of the polypeptide used in the
process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primer pairs indicated in Table III, column 7, lines
34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.,
will result in a fragment of a polynucleotide sequence as indicated
in Table I, columns 5 or 7 lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp., or its gene product
[2286] [0151.0.0.4]: see [0151.0.0.0]
[2287] [0152.0.4.4] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., such that the
protein or portion thereof maintains the ability to participate in
the respective fine chemical production, in particular an activity
increasing the level of arginine and/or glutamate and/or proline
and/or glutamine, resp., as mentioned above or as described in the
examples in plants or microorganisms is comprised.
[2288] [0153.0.4.4] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., such that the
protein or portion thereof is able to participate in the increase
of the respective fine chemical production. In one embodiment, a
protein or portion thereof as indicated in Table II, columns 5 or
7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387,
391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427,
434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., has
for example an activity of a polypeptide as indicated in Table II,
column 3, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386,
387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424,
427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411,
412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407
to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433
resp.
[2289] [0154.0.4.4] In one embodiment, the nucleic acid molecule of
the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., and has
above-mentioned activity, e.g. conferring preferably the increase
of the respective fine chemical.
[2290] [0155.0.0.4] and [0156.0.0.4]: see [0155.0.0.0] and
[0156.0.0.0]
[2291] [0157.0.4.4] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp. (and portions
thereof) due to degeneracy of the genetic code and thus encode a
polypeptide of the present invention, in particular a polypeptide
having above mentioned activity, e.g. conferring an increase in the
respective fine chemical in a organism, e.g. as that polypeptides
comprising the consensus sequences as indicated in Table IV, column
7 lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387,
391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427,
434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. or
of the polypeptide as indicated in Table II, columns 5 or 7, lines
34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. or
their functional homologues. Advantageously, the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, a consensus sequences as indicated in Table IV,
column 7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386,
387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424,
427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411,
412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407
to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433
resp. or of the polypeptide as indicated in Table II, columns 5 or
7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387,
391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427,
434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. or
the functional homologues. In a still further embodiment, the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention encodes a full length protein
which is substantially homologous to an amino acid sequence
comprising a consensus sequence as indicated in Table IV, column 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. or
of a polypeptide as indicated in Table II, columns 5 or 7, lines 34
to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399
to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or
435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. or the
functional homologues thereof. However, in a preferred embodiment,
the nucleic acid molecule of the present invention does not consist
of a sequence as indicated in Table I, columns 5 or 7 lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., preferably as
indicated in Table I A, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp. Preferably the
nucleic acid molecule of the invention is a functional homologue or
identical to a nucleic acid molecule indicated in Table I B,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp.
[2292] [0158.0.0.4] to [0160.0.0.4]: see [0158.0.0.0] to
[0160.0.0.0]
[2293] [0161.0.4.4] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. The nucleic
acid molecule is preferably at least 20, 30, 50, 100, 250 or more
nucleotides in length.
[2294] [0162.0.0.4]: see [0162.0.0.0]
[2295] [0163.0.4.4] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp., corresponds to a
naturally-occurring nucleic acid molecule of the invention. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein). Preferably, the nucleic
acid molecule encodes a natural protein having above-mentioned
activity, e.g. conferring the respective fine chemical increase
after increasing the expression or activity thereof or the activity
of a protein of the invention or used in the process of the
invention.
[2296] [0164.0.0.4]: see [0164.0.0.0]
[2297] [0165.0.4.4] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2298] [0166.0.0.4] and [0167.0.0.4]: see [0166.0.0.0] and
[0167.0.0.0]
[2299] [0431.0.0.0] [0168.0.4.4] Accordingly, the invention relates
to nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., preferably of Table II B, column 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. yet retain
said activity described herein. The nucleic acid molecule can
comprise a nucleotide sequence encoding a polypeptide, wherein the
polypeptide comprises an amino acid sequence at least about 50%
identical to an amino acid sequence as indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., preferably of Table II B, column 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. and is capable
of participation in the increase of production of the respective
fine chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., preferably of Table II B, column 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., more
preferably at least about 70% identical to one of the sequences as
indicated in Table II, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp., preferably of
Table II B, column 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp., even more preferably at least about 80%,
90%, or 95% homologous to a sequence as indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., preferably of Table II B, column 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., and most
preferably at least about 96%, 97%, 98%, or 99% identical to the
sequence as indicated in Table II, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., preferably of
Table II B, column 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp.
[2300] [0169.0.0.4] to [0172.0.0.4]: see [0169.0.0.0] to
[0172.0.0.0]
[2301] [0173.0.4.4] For example a sequence which has a 80% homology
with sequence SEQ ID No 1982 at the nucleic acid level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID No 1982 by the above Gap program algorithm with the
above parameter set, has a 80% homology.
[2302] [0174.0.0.4]: see [0174.0.0.0]
[2303] [0175.0.4.4] For example a sequence which has a 80% homology
with sequence SEQ ID No 1983 at the protein level is understood as
meaning a sequence which, upon comparison with the sequence SEQ ID
No 1983 by the above program algorithm with the above parameter
set, has a 80% homology.
[2304] [0176.0.4.4] Functional equivalents derived from one of the
polypeptides as indicated in Table II, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., according to
the invention by substitution, insertion or deletion have at least
30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%
by preference at least 80%, especially preferably at least 85% or
90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%,
97%, 98% or 99% homology with one of the polypeptides as indicated
in Table II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp., according to the invention and are
distinguished by essentially the same properties as a polypeptide
as indicated in Table II, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2305] [0177.0.4.4] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., preferably of
Table I B, column 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp. according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptide as indicated in Table II,
columns 5 or 7 lines 34 to 37, 390, 405 and/or 430 and/or lines 43,
386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421,
424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398,
411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402,
404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431
to 433 resp. according to the invention and encode polypeptides
having essentially the same properties as a polypeptide as
indicated in Table II, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp., preferably of
Table I B, column 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp.
[2306] [0178.0.0.4]: see [0178.0.0.0]
[2307] [0179.0.4.4] A nucleic acid molecule encoding an homologous
to a protein sequence as indicated in Table II, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.,
preferably of Table II B, column 7 lines 34 to 37, 390, 405 and/or
430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp. can be created by introducing one or
more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table I, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.,
such that one or more amino acid substitutions, additions or
deletions are introduced into the encoded protein. Mutations can be
introduced into the encoding sequences of a sequences as indicated
in Table I, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp., by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
[2308] [0180.0.0.4] to [0183.0.0.4]: see [0180.0.0.0] to
[0183.0.0.0]
[2309] [0184.0.4.4] Homologues of the nucleic acid sequences used,
with a sequence as indicated in Table I, preferably table I B,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., or of the nucleic acid sequences derived from a
sequences as indicated in Table II, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. comprise also
allelic variants with at least approximately 30%, 35%, 40% or 45%
homology, by preference at least approximately 50%, 60% or 70%,
more preferably at least approximately 90%, 91%, 92%, 93%, 94% or
95% and even more preferably at least approximately 96%, 97%, 98%,
99% or more homology with one of the nucleotide sequences shown or
the abovementioned derived nucleic acid sequences or their
homologues, derivatives or analogues or parts of these. Allelic
variants encompass in particular functional variants which can be
obtained by deletion, insertion or substitution of nucleotides from
the sequences shown, preferably from a sequence as indicated in
Table I, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp., or from the derived nucleic acid
sequences, the intention being, however, that the enzyme activity
or the biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[2310] [0185.0.4.4] In one embodiment of the present invention, the
nucleic acid molecule of the invention or used in the process of
the invention comprises one or more sequences as indicated in Table
I, preferably table I B, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp. In one embodiment,
it is preferred that the nucleic acid molecule comprises as little
as possible other nucleotides not shown in any one of sequences as
indicated in Table I, preferably table I B, columns 5 or 7, lines
34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. In
one embodiment, the nucleic acid molecule comprises less than 500,
400, 300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides.
In a further embodiment, the nucleic acid molecule comprises less
than 30, 20 or 10 further nucleotides. In one embodiment, a nucleic
acid molecule used in the process of the invention is identical to
a sequences as indicated in Table I, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2311] [0186.0.4.4] Also preferred is that one or more nucleic acid
molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II,
preferably table II B, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp. In one embodiment,
the nucleic acid molecule encodes less than 150, 130, 100, 80, 60,
50, 40 or 30 further amino acids. In a further embodiment, the
encoded polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5
further amino acids. In one embodiment, the encoded polypeptide
used in the process of the invention is identical to the sequences
as indicated in Table II, preferably table II B, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2312] [0187.0.4.4] In one embodiment, a nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.,
comprises less than 100 further nucleotides. In a further
embodiment, said nucleic acid molecule comprises less than 30
further nucleotides. In one embodiment, the nucleic acid molecule
used in the process is identical to a coding sequence as indicated
in Table II, preferably table II B, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. Idealerweise
wurde man in diesen Abschnitten nur column 7 von Table II
bevorzugen, wurde auch teilweise so gemacht, andererseits
erheblicher Aufwand and ggf. nicht unbedingt notwendig??
[2313] [0188.0.4.4] Polypeptides (=proteins), which still have the
essential biological or enzymatic activity of the polypeptide of
the present invention conferring an increase of the respective fine
chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp., and is expressed under identical
conditions.
[2314] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, columns 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp.
[2315] [0189.0.4.4] Homologues of a sequences as indicated in Table
I, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., or of a derived sequences as indicated in Table
II, columns 5 or 7 lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., also mean truncated sequences, cDNA,
single-stranded DNA or RNA of the coding and noncoding DNA
sequence. Homologues of said sequences are also understood as
meaning derivatives, which comprise noncoding regions such as, for
example, UTRs, terminators, enhancers or promoter variants. The
promoters upstream of the nucleotide sequences stated can be
modified by one or more nucleotide substitution(s), insertion(s)
and/or deletion(s) without, however, interfering with the
functionality or activity either of the promoters, the open reading
frame (=ORF) or with the 3'-regulatory region such as terminators
or other 3' regulatory regions, which are far away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[2316] [0190.0.0.4] to [0203.0.0.4]: see [0190.0.0.0] to
[0203.0.0.0]
[2317] [0204.0.4.4] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule which comprises a nucleic acid
molecule selected from the group consisting of:
(haben die 435 automatisch erganzt, daher ggf. Hier etwas
unabersichtlich) [2318] a) nucleic acid molecule encoding,
preferably at least the mature form, of a polypeptide as indicated
in Table II, preferably table II B, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.; or a fragment
thereof conferring an increase in the amount of the respective fine
chemical, in particular, of arginine (lines 30 to 37, 390, 405
and/or 430) and/or glutamate (lines 38 to 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434 and/or
435) and/or proline (lines 44 to 56, 388, 389, 398, 411, 412, 425
and/or 429) and/or glutamine (lines 57 to 62, 392 to 395, 397, 402,
404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431
to 433), resp., in an organism or a part thereof [2319] b) nucleic
acid molecule comprising, preferably at least the mature form, of a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., or
a fragment thereof conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [2320]
c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [2321] d) nucleic acid molecule encoding a
polypeptide whose sequence has at least 50% identity with the amino
acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the fine chemical in an organism or a part thereof; [2322] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under under stringent hybridisation conditions and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [2323] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [2324] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [2325] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table III, column 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp., and conferring an
increase in the amount of the respective fine chemical, in
particular, of arginine (lines 30 to 37, 390, 405 and/or 430)
and/or glutamate (lines 38 to 43, 386, 387, 391, 396, 399 to 401,
403, 406, 413, 414, 417, 418, 421, 424, 427, 434 and/or 435/or
proline (lines 44 to 56, 388, 389, 398, 411, 412, 425 and/or 429)
and/or glutamine (lines 57 to 62, 392 to 395, 397, 402, 404, 407 to
410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433),
resp. in an organism or a part thereof; [2326] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[2327] j) nucleic acid molecule which encodes a polypeptide
comprising a consensus sequence as indicated in Table IV, columns 5
or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386,
387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424,
427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411,
412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407
to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433
resp., and conferring an increase in the amount of the respective
fine chemical, in particular, of arginine (lines 30 to 37, 390, 405
and/or 430) and/or glutamate (lines 38 to 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434
and/or, 435) and/or proline (lines 44 to 56, 388, 389, 398, 411,
412, 425 and/or 429) and/or glutamine (lines 57 to 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433), resp., in an organism or a part thereof; [2328]
k) nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domaine of a polypeptide as indicated in
Table II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp., and conferring an increase in the
amount of the respective fine chemical, in particular, of arginine
(lines 30 to 37, 390, 405 and/or 430) and/or glutamate (lines 38 to
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434 and/or 435) and/or proline (lines 44 to 56, 388,
389, 398, 411, 412, 425 and/or 429) and/or glutamine (lines 57 to
62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422,
423, 426, 428 and/or 431 to 433), in an organism or a part thereof;
and [2329] l) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., or a nucleic acid molecule encoding, preferably
at least the mature form of, a polypeptide as indicated in Table
II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp. and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof; or
which encompasses a sequence which is complementary thereto;
whereby, preferably, the nucleic acid molecule according to (a) to
(l) distinguishes over the sequence indicated in Table IA, columns
5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386,
387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424,
427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411,
412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407
to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433
resp., by one or more nucleotides. In one embodiment, the nucleic
acid molecule does not consist of the sequence shown and indicated
in Table I A or I B, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp.: In one embodiment,
the nucleic acid molecule is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence indicated in Table I A or I B,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp. In another embodiment, the nucleic acid molecule
does not encode a polypeptide of a sequence indicated in Table II A
or II B, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp. In an other embodiment, the nucleic acid
molecule of the present invention is at least 30%, 40%, 50%, or 60%
identical and less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I A or I B, columns 5 or
7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387,
391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427,
434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. In a
further embodiment the nucleic acid molecule does not encode a
polypeptide sequence as indicated in Table II A or II B, columns 5
or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386,
387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424,
427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411,
412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407
to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433
resp. Accordingly, in one embodiment, the nucleic acid molecule of
the differs at least in one or more residues from a nucleic acid
molecule indicated in Table I A or I B, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. Accordingly,
in one embodiment, the nucleic acid molecule of the present
invention encodes a polypeptide, which differs at least in one or
more amino acids from a polypeptide indicated in Table II A or I B,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp. In another embodiment, a nucleic acid molecule
indicated in Table I A or I B, columns 5 or 7, lines 34 to 37, 390,
405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401,
403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp. does not encode a
protein of a sequence indicated in Table II A or II B, columns 5 or
7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387,
391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427,
434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.
Accordingly, in one embodiment, the protein encoded by a sequences
of a nucleic acid according to (a) to (l) does not consist of a
sequence as indicated in Table II A or II B, columns 5 or 7, lines
34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. In a
further embodiment, the protein of the present invention is at
least 30%, 40%, 50%, or 60% identical to a protein sequence
indicated in Table II A or II B, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. and less than
100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 985, 97%, 96% or 95% identical to a
sequence as indicated in Table I A or II B, columns 5 or 7, lines
34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2330] [0205.0.0.4] to [0226.0.0.4]: see [0205.0.0.0] to
[0226.0.0.0]
[2331] [0227.0.4.4] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorgansims.
[2332] In addition to a sequence indicated in Table I, columns 5 or
7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387,
391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427,
434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., or
its derivatives, it is advantageous additionally to express and/or
mutate further genes in the organisms. Especially advantageously,
additionally at least one further gene of the amino acid
biosynthetic pathway such as for L-lysine, L-threonine and/or
L-methionine and/or L-leucine and/or isoleucine and/or valine is
expressed in the organisms such as plants or microorganisms. It is
also possible that the regulation of the natural genes has been
modified advantageously so that the gene and/or its gene product is
no longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine one or more of the sequences indicated in
Table I, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp., with genes which generally support or
enhances to growth or yield of the target organisms, for example
genes which lead to faster growth rate of microorganisms or genes
which produces stress-, pathogen, or herbicide resistant
plants.
[2333] [0228.0.0.4] to [0230.0.0.4]: see [0228.0.0.0] to
[0230.0.0.0]
[2334] [0231.0.4.4] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously an arginine and/or glutamate and/or
glutamine and/or proline degrading protein is attenuated, in
particular by reducing the rate of expression of the corresponding
gene.
[2335] [0232.0.0.4] to [0282.0.0.4]: see [0232.0.0.0] to
[0282.0.0.0]
[2336] [0283.0.4.4] Moreover, a native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table II,
column 3, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386,
387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424,
427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411,
412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407
to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433
resp., or an antibody against a polypeptide as indicated in Table
II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp. which can be produced by standard
techniques utilizing the polypeptid of the present invention or
fragment thereof, i.e., the polypeptide of this invention.
Preferred are monoclonal antibodies.
[2337] [0284.0.0.4]: see [0284.0.0.0].
[2338] [0285.0.4.4] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp., or as encoded by a nucleic acid molecule
as indicated in Table I, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp., or functional
homologues thereof.
[2339] [0286.0.4.4] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp. In another embodiment, the present
invention relates to a polypeptide comprising or consisting of a
consensus sequence as indicated in Table IV, column 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., whereby 20 or
less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred
5 or 4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table IV, column 7, lines lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp [0287.0.0.4] to [0290.0.0.4]: see
[0287.0.0.0] to [0290.0.0.0]
[2340] [0291.0.4.4] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[2341] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table II A or IIB,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp. by one or more amino acids. In one embodiment,
polypeptide distinguishes form a sequence as indicated in Table II
A or IIB, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp. by more than 1, 2, 3, 4, 5, 6, 7, 8 or
9 amino acids, preferably by more than 10, 15, 20, 25 or 30 amino
acids, evenmore preferred are more than 40, 50, or 60 amino acids
and, preferably, the sequence of the polypeptide of the invention
distinguishes from a sequence as indicated in Table II A or II B,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp. by not more than 80% or 70% of the amino acids,
preferably not more than 60% or 50%, more preferred not more than
40% or 30%, even more preferred not more than 20% or 10%. In an
other embodiment, said polypeptide of the invention does not
consist of a sequence as indicated in Table II A or II B, columns 5
or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386,
387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424,
427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398, 411,
412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407
to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433
resp.
[2342] [0292.0.0.4]: see [0292.0.0.0]
[2343] [0293.0.4.4] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, the polypeptide of
the invention has a sequence which distinguishes from a sequence as
indicated in Table II A or II B, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. by one or more
amino acids. In an other embodiment, said polypeptide of the
invention does not consist of the sequence as indicated in Table II
A or II B, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp. In a further embodiment, said
polypeptide of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical. In one embodiment, said polypeptide
does not consist of the sequence encoded by a nucleic acid
molecules as indicated in Table I A or IB, columns 5 or 7, lines 34
to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399
to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or
435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2344] [0294.0.4.4] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 34 to 37, 390, 405 and/or
430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp., which distinguishes over a sequence as
indicated in Table IIA or table II B, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., by one or
more amino acids, preferably by more than 5, 6, 7, 8 or 9 amino
acids, preferably by more than 10, 15, 20, 25 or 30 amino acids,
evenmore preferred are more than 40, 50, or 60 amino acids but even
more preferred by less than 70% of the amino acids, more preferred
by less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[2345] [0295.0.0.4] and [0297.0.0.4]: see [0295.0.0.0] and
[0297.0.0.0]
[2346] [0297.1.4.4] Non polypeptide of the invention-chemicals are
e.g. polypeptides having not the activity of a polypeptide
indicated in Table II, columns 3, 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2347] [0298.0.4.4] A polypeptide of the invention can participate
in the process of the present invention. The polypeptide or a
portion thereof comprises preferably an amino acid sequence which
is sufficiently homologous to an amino acid sequence as indicated
in Table II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp. The portion of the protein is
preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp.
[2348] [0299.0.4.4] Further, the polypeptide can have an amino acid
sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. The
preferred polypeptide of the present invention preferably possesses
at least one of the activities according to the invention and
described herein. A preferred polypeptide of the present invention
includes an amino acid sequence encoded by a nucleotide sequence
which hybridizes, preferably hybridizes under stringent conditions,
to a nucleotide sequence as indicated in Table I, columns 5 or 7,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., or
which is homologous thereto, as defined above.
[2349] [0300.0.4.4] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., in amino acid sequence due to natural variation
or mutagenesis, as described in detail herein. Accordingly, the
polypeptide comprise an amino acid sequence which is at least about
35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about
75%, 80%, 85% or 90, and more preferably at least about 91%, 92%,
93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%,
99% or more homologous to an entire amino acid sequence of a
sequence as indicated in Table II A or II B, columns 5 or 7, lines
34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.
[2350] [0301.0.0.4]: see [0301.0.0.0]
[2351] [0302.0.4.4] Biologically active portions of an polypeptide
of the present invention include peptides comprising amino acid
sequences derived from the amino acid sequence of the polypeptide
of the present invention or used in the process of the present
invention, e.g., an amino acid sequence as indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., or the amino acid sequence of a protein
homologous thereto, which include fewer amino acids than a full
length polypeptide of the present invention or used in the process
of the present invention or the full length protein which is
homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[2352] [0303.0.0.4]: see [0303.0.0.0]
[2353] [0304.0.4.4] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 34 to 37, 390, 405 and/or 430 and/or lines 43,
386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421,
424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389, 398,
411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397, 402,
404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431
to 433 resp., but having differences in the sequence from said
wild-type protein. These proteins may be improved in efficiency or
activity, may be present in greater numbers in the cell than is
usual, or may be decreased in efficiency or activity in relation to
the wild type protein.
[2354] [0305.0.0.4] to [0308.0.0.4]: see [0306.0.0.0] to
[0308.0.0.0]
[2355] [0309.0.4.4] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., refers to a polypeptide having an amino acid
sequence corresponding to the polypeptide of the invention or used
in the process of the invention, whereas a "non-polypeptide of the
invention" or "other polypeptide" not being indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., refers to a polypeptide having an amino acid
sequence corresponding to a protein which is not substantially
homologous a polypeptide of the invention, preferably which is not
substantially homologous to a polypeptide as indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., e.g., a protein which does not confer the
activity described herein or annotated or known for as indicated in
Table II, column 3, lines 34 to 37, 390, 405 and/or 430 and/or
lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417,
418, 421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388,
389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395,
397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428
and/or 431 to 433 resp., and which is derived from the same or a
different organism. In one embodiment, a "non-polypeptide of the
invention" or "other polypeptide" not being indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., does not confer an increase of the respective
fine chemical in an organism or part thereof.
[2356] [0310.0.0.4] to [0334.0.0.4]: see [0310.0.0.0] to
[0334.0.0.0]
[2357] [0335.0.4.4] The dsRNAi method has proved to be particularly
effective and advantageous for reducing the expression of a nucleic
acid sequences as indicated in Table I, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., and/or
homologs thereof. As described inter alia in WO 99/32619, dsRNAi
approaches are clearly superior to traditional antisense
approaches. The invention therefore furthermore relates to
double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived therefrom), bring about altered
metabolic activity by the reduction in the expression of a nucleic
acid sequences as indicated in Table I, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., and/or
homologs thereof. In a double-stranded RNA molecule for reducing
the expression of an protein encoded by a nucleic acid sequence
sequences as indicated in Table I, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., and/or
homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[2358] [0336.0.0.4] to [0342.0.0.4]: see [0336.0.0.0] to
[0342.0.0.0]
[2359] [0343.0.4.4] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., or its
homolog is not necessarily required in order to bring about
effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table I, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp., or homologs
thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[2360] [0344.0.0.4] to [0361.0.0.4]: see [0344.0.0.0] to
[0361.0.0.0]
[2361] [0362.0.4.4] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, columns 5 or 7, lines 34 to 37, 390, 405 and/or 430
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 and/or lines 54 to
56, 388, 389, 398, 411, 412, 425 and/or 429 and/or lines 62, 392 to
395, 397, 402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426,
428 and/or 431 to 433 resp. e.g. encoding a polypeptide having
protein activity, as indicated in Table II, columns 3, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. Due to the
above mentioned activity the respective fine chemical content in a
cell or an organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. Transgenic for a polypeptide having an activity of a
polypeptide as indicated in Table II, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., means herein
that due to modulation or manipulation of the genome, an activity
as annotated for a polypeptide as indicated in Table II, column 3,
lines 34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391,
396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp. e.g.
having a sequence as indicated in Table II, columns 5 or 7, lines
34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., is
increased in a cell or an organism or a part thereof. Examples are
described above in context with the process of the invention
[2362] [0363.0.0.4]: to [0382.0.0.4]: see [0363.0.0.0] to
[0382.0.0.0]
[2363] [0383.0.4.4] For preparing arginine and/or glutamate and/or
glutamine and/or proline compound-containing fine chemicals, in
particular the fine chemical, it is possible to use as arginine
and/or glutamate and/or glutamine and/or proline amino acid source
organic compounds such as, for example, citrulline,
argininosuccinate, ornithine, aspartate, 2-Oxoglutarate, glutamyl,
glutamic-semialdehyde, Pyrroline-5-carboxylate, Glutamine or else
organic arginine and/or glutamate and/or glutamine and/or proline
acid precursor compounds.
[2364] [0384.0.0.4]: see [0384.0.0.0]
[2365] [0385.0.4.4] The fermentation broths obtained in this way,
containing in particular L-arginine and/or L-glutamate and/or
L-proline and/or L-tryptophane, L-methionine, L-threonine and/or
L-lysine, normally have a dry matter content of from 7.5 to 25% by
weight. Sugar-limited fermentation is additionally advantageous, at
least at the end, but especially over at least 30% of the
fermentation time. This means that the concentration of utilizable
sugar in the fermentation medium is kept at, or reduced to, 0 to 3
g/l during this time. The fermentation broth is then processed
further. Depending on requirements, the biomass can be removed
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation or a combination of these
methods, from the fermentation broth or left completely in it. The
fermentation broth can then be thickened or concentrated by known
methods, such as, for example, with the aid of a rotary evaporator,
thin-film evaporator, falling film evaporator, by reverse osmosis
or by nanofiltration. This concentrated fermentation broth can then
be worked up by freeze-drying, spray drying, spray granulation or
by other processes.
[2366] [0386.0.0.4] to [0392.0.0.4]: see [0386.0.0.0] to
[0392.0.0.0]
[2367] [0393.0.4.4] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [2368] d) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [2369] e) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table I, preferably Table I B, columns 5 or 7, lines 34 to 37, 390,
405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401,
403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp., and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[2370] f) introducing the candidate nucleic acid molecules in host
cells, preferably in a plant cell or a microorganism, appropriate
for producing the fine chemical; [2371] g) expressing the
identified nucleic acid molecules in the host cells; [2372] h)
assaying the the fine chemical level in the host cells; and [2373]
i) identifying the nucleic acid molecule and its gene product which
expression confers an increase in the the fine chemical level in
the host cell after expression compared to the wild type.
[2374] [0394.0.0.4] to [0399.0.0.4]: see [0394.0.0.0] to
[0399.0.0.0]
[2375] [00399.1.4.4]: One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 and/or lines
43, 386, 387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418,
421, 424, 427, 434, and/or 435 and/or lines 54 to 56, 388, 389,
398, 411, 412, 425 and/or 429 and/or lines 62, 392 to 395, 397,
402, 404, 407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or
431 to 433 resp., or a homolog thereof, e.g. comparing the phenotyp
of nearly identical organisms with low and high activity of a
protein as indicated in Table II, columns 5 or 7, lines 34 to 37,
390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., after
incubation with the drug.
[2376] [0400.0.0.4] to [0423.0.0.4]: see [0400.0.0.0] to
[0423.0.0.0]
[2377] [0424.0.0.4]: see [0424.0.0.2]
[2378] [0425.0.0.4] to [0460.0.0.4]: see [0425.0.0.0] to
[0460.0.0.0]
[0461.0.4.4] Example 10
Cloning SEQ ID NO: 1982 for the Expression in Plants
[2379] [0462.0.0.4]: see [0462.0.0.0]
[2380] [0463.0.4.4] SEQ ID NO: 1982 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[2381] [0464.0.0.4] to [0466.0.0.4]: see [0464.0.0.0] to
[0466.0.0.0]
[2382] [0467.0.4.4] The following primer sequences were selected
for the gene SEQ ID No:
[2383] 1982:
TABLE-US-00018 i) forward primer (SEQ ID No: 2046) atgaataacg
aacccttacg tccc ii) reverse primer (SEQ ID No: 2047) ttacatatcc
tcatgaaatt cttcaagt
[2384] [0468.0.0.4] to [0479.0.0.4]: see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.4.4] Example 11
Generation of Transgenic Plants which Express SEQ ID No: 1982
[2385] [0481.0.0.4] to [0513.0.0.4]: see [0481.0.0.0] to
[0513.0.0.0]
[2386] [0514.0.4.4] As an alternative, the amino acids can be
detected advantageously via
[2387] HPLC separation in ethanolic extract as described by
Geigenberger et al. (Plant Cell & Environ, 19, 1996:
43-55).
[2388] The results of the different plant analyses can be seen from
the table which follows:
TABLE-US-00019 TABLE 1 ORF Metabolite Method Min Max b0730
Glutamate LC 1.55 2.15 b0730 Proline GC 1.35 3.72 b1829 Glutamine
LC 1.50 1.68 b1829 Arginine LC 1.45 12.41 b2699 Proline GC + LC
1.32 2.41 YBR030W Proline LC 1.51 3.82 YBR204C Glutamate GC 1.55
1.76 YDL106C Proline GC + LC 1.51 1.99 YDR271C Proline GC + LC 1.36
5.82 YDR316W Arginine LC 1.45 2.02 YEL045C Proline GC 1.41 1.89
YER173W Glutamine GC 1.86 3.85 YER173W Proline GC 1.34 2.91 YFL013C
Glutamate GC + LC 1.81 2.34 YFL050C Proline GC 1.44 1.74 YFR042W
Glutamine GC 1.41 1.43 YGR104C Glutamate LC 1.64 1.96 YGR135W
Proline GC 1.32 3.89 YHR130C Arginine LC 1.67 1.85 YIL150C Proline
GC 1.33 4.04 YKR057W Arginine LC 1.57 5.57 YKR057W Glutamine GC
1.41 3.84 YNL090w Proline GC + LC 1.73 6.29 YNL090w Arginine LC
1.54 4.23 YPR024W Glutamate LC 1.26 1.43 YPR133W-A Glutamate GC
1.34 1.68 YPR138C Proline GC 1.54 6.20 b0695 Arginine LC 1.51 4.19
b1284 Arginine LC 1.47 2.83 b1827 Proline GC 1.42 2.26 b1852
Glutamine GC 1.40 1.42 b2095 Arginine LC 1.55 1.59 b4265 Glutamine
GC 1.32 1.47 b0050 Glutamate LC 1.37 1.97 b0057 Glutamate GC + LC
1.35 1.83 b0138 Proline LC 1.50 2.80 b0149 Proline GC 1.33 2.20
b0161 Arginine LC 7.28 9.81 b0161 Glutamate LC 1.35 1.65 b0161
Glutamine GC 1.43 3.56 b0486 Glutamine LC 1.51 2.28 b0849 Glutamine
LC 1.37 1.50 b0970 Glutamine GC + LC 1.59 3.80 b1343 Glutamine LC
1.37 1.39 b1343 Glutamate GC 1.48 1.99 b1360 Proline GC 1.33 1.70
b1693 Glutamate LC 1.39 2.49 b1736 Glutamate LC 1.46 1.97 b1738
Glutamate LC 1.38 2.07 b1886 Glutamine LC 1.36 2.24 b1896 Glutamate
GC 1.67 2.62 b1926 Glutamine LC 1.07 1.27 b2307 Arginine LC 1.95
3.47 b2307 Glutamate LC 1.35 1.89 b2414 Glutamine LC 1.30 1.56
b2426 Glutamine LC 1.31 1.62 b2489 Glutamine LC 1.33 1.44 b2553
Proline LC 1.49 1.68 b2553 Glutamine GC + LC 1.55 1.90 b2664
Proline GC + LC 1.35 9.53 b2710 Glutamate LC 1.35 1.38 b2818
Glutamine GC 1.45 6.19 b2818 Glutamate GC 1.50 2.29 b3064 Glutamine
GC 1.72 2.41 b3074 Glutamate LC 1.34 1.85 b3116 Glutamate GC + LC
1.35 1.98 b3160 Glutamine LC 1.38 1.64 b3160 Glutamine GC 1.51 2.89
b3166 Glutamine LC 1.29 1.40 b3169 Glutamine GC + LC 1.55 2.11
b3169 Glutamate GC + LC 1.42 2.40 b3231 Glutamine GC 1.50 2.64
b3619 Glutamate LC 1.40 2.22 b3644 Proline GC + LC 1.32 3.41 b3680
Glutamine GC 1.50 2.99 b3791 Glutamine LC 1.28 1.57 b3791 Glutamate
LC 1.39 1.57 b3919 Proline GC 1.35 2.18 b3936 Arginine LC 2.20 4.98
b4004 Glutamine LC 1.30 1.36 b4074 Glutamine GC 1.40 1.42 b4133
Glutamine GC 1.59 3.12 b4346 Glutamate GC 1.38 1.44 YFL019C
Glutamate GC + LC 1.81 2.34
[2389] [0515.0.0.4] to [0552.0.0.4]: see [0515.0.0.0] to
[0552.0.0.0]
[0552.1.4.4]: Example 15
Metabolite Profiling Info from Zea mays
[2390] Zea mays plants were engineered, grown and analylzed as
described in Example 14c.
[2391] The results of the different Zea mays plants analysed can be
seen from Table 2 which follows:
TABLE-US-00020 TABLE 2 ORF_NAME Metabolite Min Max YKR057W
Glutamine 2.30 2.48 YIL150C Proline 1.67 2.08 b1284 Arginine 1.37
2.39 b1829 Glutamine 1.41 2.17 b1896 Glutamate 1.48 2.65 b2553
Proline 1.76 6.63 b2553 Glutamine 1.54 9.51 b2664 Proline 1.78 2.38
b3116 Glutamate 1.67 1.91
[2392] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in proline and/or
glutamine and/or arginine and/or glutamate in genetically modified
corn plants expressing the Saccharomyces cerevisiae nucleic acid
sequence YIL150C or YKR057Wor E. coli nucleic acid sequence b1284,
b1829, b1896, b2553, b2664 or b3116 resp.
[2393] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YIL150C or its homologs, e.g. "a chromatin
binding protein, required for S-phase (DNA synthesis) initiation or
completion" or its homologs, is increased in corn plants,
preferably, an increase of the fine chemical proline between 67%
and 108% is conferred.
[2394] In case the activity of the Saccharomyces cerevisiae protein
YKR057W or a ribosomal protein, similar to S21 ribosomal proteins,
involved in ribosome biogenesis and translation or its homolog, is
increased in corn plants, preferably, an increase of the fine
chemical glutamine between 130% and 148% is conferred.
[2395] In one embodiment, in case the activity of the E. coli
protein b1284 or its homologs, e.g. "a transcriptional regulator
for regulation of C-compound and carbohydrate utilization,
transcriptional control, transcriptional repressor, DNA binding",
is increased in corn plants, preferably, an increase of the fine
chemical arginine between 37% and 139% is conferred.
[2396] In one embodiment, in case the activity of the E. coli
protein b1829 or its homologs, e.g. "the activity of a heat shock
protein with protease activity (htpx)", is increased in corn
plants, preferably, an increase of the fine chemical glutamine
between 41% and 117% is conferred.
[2397] In one embodiment, in case the activity of the E. coli
protein b1896 or its homologs, e.g. "a trehalose-6-phosphate
synthase or its homologs", is increased in corn plants, preferably,
an increase of the fine chemical glutamate between 48% and 165% is
conferred.
[2398] In one embodiment, in case the activity of the E. coli
protein b2553 or its homologs, e.g. "the activity of a regulatory
protein P-II for glutamine synthetase", is increased in corn
plants, preferably, an increase of the fine chemical proline
between 76% and 563% is conferred and/or an increase of the fine
chemical glutamine between 54% and 851% is conferred.
[2399] In one embodiment, in case the activity of the E. coli
protein b2664 or its homologs, e.g. "the activity of a hydrogenase
Fe--S subunit", is increased in corn plants, preferably, an
increase of the fine chemical proline between 78% and 138% is
conferred.
[2400] In one embodiment, in case the activity of the E. coli
protein b3116 or its homologs, e.g. "the activity of a
L-threonine/L-serine permease, anaerobically inducible (HAAAP
family)", is increased in corn plants, preferably, an increase of
the fine chemical glutamate between 67% and 91% is conferred.
[2401] [0552.2.0.4]: see [0552.2.0.0]
[2402] [0553.0.4.4] [2403] 1. A process for the production of
arginine and/or glutamate and/or proline and/or glutamine, which
comprises (a) increasing or generating the activity of a protein as
indicated in Table II, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 for arginine and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
for glutamate and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 for proline and/or lines 62, 392 to 395, 397, 402, 404,
407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to
433 for glutamine resp., or a functional equivalent thereof in a
non-human organism, or in one or more parts thereof; and (b)
growing the organism under conditions which permit the production
of arginine and/or glutamate and/or proline and/or glutamine resp.
in said organism. [2404] 2. A process for the production of
arginine and/or glutamate and/or proline and/or glutamine,
comprising the increasing or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [2405] (a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 for arginine [2406] and/or lines 43, 386,
387, 391, 396, 399 to 401, 403, 406, 413, 414, 417, 418, 421, 424,
427, 434, and/or 435 for glutamate [2407] and/or lines 54 to 56,
388, 389, 398, 411, 412, 425 and/or 429 for proline [2408] and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 for glutamine resp., or a
fragment thereof, which confers an increase in the amount of
arginine and/or glutamate and/or proline and/or glutamine in an
organism or a part thereof; [2409] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 for arginine
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 for glutamate [2410]
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 for
proline and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for
glutamine resp.; [2411] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of arginine and/or
glutamate and/or proline and/or glutamine in an organism or a part
thereof; [2412] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of arginine
and/or glutamate and/or proline and/or glutamine in an organism or
a part thereof; [2413] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of arginine and/or glutamate and/or proline and/or glutamine in an
organism or a part thereof; [2414] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table III,
columns 5 or 7, lines 34 to 37, 390, 405 and/or 430 for arginine
and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406, 413,
414, 417, 418, 421, 424, 427, 434, and/or 435 for glutamate [2415]
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 for
proline [2416] and/or lines 62, 392 to 395, 397, 402, 404, 407 to
410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 for
glutamine resp., and conferring an increase in the amount of
arginine and/or glutamate and/or proline and/or glutamine in an
organism or a part thereof; [2417] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
arginine and/or glutamate and/or proline and/or glutamine in an
organism or a part thereof; [2418] h) nucleic acid molecule
encoding a polypeptide comprising a consensus as indicated in Table
IV, column 7, lines 34 to 37, 390, 405 and/or 430 for arginine
[2419] and/or lines 43, 386, 387, 391, 396, 399 to 401, 403, 406,
413, 414, 417, 418, 421, 424, 427, 434, and/or 435 for glutamate
[2420] and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or
429 for proline [2421] and/or lines 62, 392 to 395, 397, 402, 404,
407 to 410, 415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to
433 for glutamine resp., and conferring an increase in the amount
of arginine and/or glutamate and/or proline and/or glutamine in an
organism or a part thereof; and [2422] i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof.
[2423] or comprising a sequence which is complementary thereto.
[2424] 3. The process of claim 1 or 2, comprising recovering of the
free or bound arginine and/or glutamate and/or proline and/or
glutamine. [2425] 4. The process of any one of claims 1 to 3,
comprising the following steps: [2426] (a) selecting an organism or
a part thereof expressing a polypeptide encoded by the nucleic acid
molecule characterized in claim 2; [2427] (b) mutagenizing the
selected organism or the part thereof; [2428] (c) comparing the
activity or the expression level of said polypeptide in the
mutagenized organism or the part thereof with the activity or the
expression of said polypeptide of the selected organisms or the
part thereof; [2429] (d) selecting the mutated organisms or parts
thereof, which comprise an increased activity or expression level
of said polypeptide compared to the selected organism or the part
thereof; [2430] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [2431] (f) recovering, and
optionally isolating, the free or bound arginine and/or glutamate
and/or proline and/or glutamine produced by the selected mutated
organisms or parts thereof. [2432] 5. The process of any one of
claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. [2433] 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [2434] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., or a fragment
thereof, which confers an increase in the amount of arginine and/or
glutamate and/or proline and/or glutamine in an organism or a part
thereof; [2435] b) nucleic acid molecule comprising of a nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines 34 to
37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to
401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435
and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp.; [2436] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of arginine and/or glutamate
and/or proline and/or glutamine in an organism or a part thereof;
[2437] d) nucleic acid molecule which encodes a polypeptide which
has at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of arginine and/or glutamate
and/or proline and/or glutamine in an organism or a part thereof;
[2438] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of arginine
and/or glutamate and/or proline and/or glutamine in an organism or
a part thereof; [2439] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, column 7, lines
34 to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396,
399 to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434,
and/or 435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425
and/or 429 and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410,
415, 416, 419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., and
conferring an increase in the amount of arginine and/or glutamate
and/or proline and/or glutamine in an organism or a part thereof;
[2440] g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of arginine and/or
glutamate and/or proline and/or glutamine in an organism or a part
thereof; [2441] h) nucleic acid molecule encoding a polypeptide
comprising a consensus as indicated in Table IV, column 7, lines 34
to 37, 390, 405 and/or 430 and/or lines 43, 386, 387, 391, 396, 399
to 401, 403, 406, 413, 414, 417, 418, 421, 424, 427, 434, and/or
435 and/or lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429
and/or lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416,
419, 420, 422, 423, 426, 428 and/or 431 to 433 resp., and
conferring an increase in the amount of arginine and/or glutamate
and/or proline and/or glutamine in an organism or a part thereof;
and [2442] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof.
whereby the nucleic acid molecule distinguishes over the sequence
as indicated in Table I A, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp., by one or more
nucleotides. [2443] 7. A nucleic acid construct which confers the
expression of the nucleic acid molecule of claim 6, comprising one
or more regulatory elements. [2444] 8. A vector comprising the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7. [2445] 9. The vector as claimed in claim 8,
wherein the nucleic acid molecule is in operable linkage with
regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic, host. [2446] 10. A
host cell, which has been transformed stably or transiently with
the vector as claimed in claim 9 or 10 or the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 2 to 5. [2447] 11.
The host cell of claim 10, which is a transgenic host cell. [2448]
12. The host cell of claim 10 or 11, which is a plant cell, an
animal cell, a microorganism, or a yeast cell, a fungus cell, a
prokaryotic cell, an eukaryotic cell or an archaebacterium. [2449]
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. [2450] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table II A, columns 5 or 7, lines 34 to 37, 390, 405
and/or 430 and/or lines 43, 386, 387, 391, 396, 399 to 401, 403,
406, 413, 414, 417, 418, 421, 424, 427, 434, and/or 435 and/or
lines 54 to 56, 388, 389, 398, 411, 412, 425 and/or 429 and/or
lines 62, 392 to 395, 397, 402, 404, 407 to 410, 415, 416, 419,
420, 422, 423, 426, 428 and/or 431 to 433 resp., by one or more
amino acids. [2451] 15. An antibody, which binds specifically to
the polypeptide as claimed in claim 14. [2452] 16. A plant tissue,
propagation material, harvested material or a plant comprising the
host cell as claimed in claim 12 which is plant cell or an
Agrobacterium. [2453] 17. A method for screening for agonists and
antagonists of the activity of a polypeptide encoded by the nucleic
acid molecule of claim 6 conferring an increase in the amount of
arginine and/or glutamate and/or proline and/or glutamine in an
organism or a part thereof comprising: (a) contacting cells,
tissues, plants or microorganisms which express the a polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of arginine and/or glutamate and/or proline
and/or glutamine in an organism or a part thereof with a candidate
compound or a sample comprising a plurality of compounds under
conditions which permit the expression the polypeptide; (b)
assaying the arginine and/or glutamate and/or proline and/or
glutamine level or the polypeptide expression level in the cell,
tissue, plant or microorganism or the media the cell, tissue, plant
or microorganisms is cultured or maintained in; and (c) identifying
a agonist or antagonist by comparing the measured arginine and/or
glutamate and/or proline and/or glutamine level or polypeptide
expression level with a standard arginine and/or glutamate and/or
proline and/or glutamine or polypeptide expression level measured
in the absence of said candidate compound or a sample comprising
said plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist.
[2454] 18. A process for the identification of a compound
conferring increased arginine and/or glutamate and/or proline
and/or glutamine production in a plant or microorganism, comprising
the steps: a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of arginine and/or glutamate and/or proline and/or glutamine
in an organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with dais readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 5
conferring an increase in the amount of arginine and/or glutamate
and/or proline and/or glutamine in an organism or a part thereof;
b) identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [2455] 19. A method for the identification of a
gene product conferring an increase in arginine and/or glutamate
and/or proline and/or glutamine production in a cell, comprising
the following steps: (a) contacting the nucleic acid molecules of a
sample, which can contain a candidate gene encoding a gene product
conferring an increase in arginine and/or glutamate and/or proline
and/or glutamine after expression with the nucleic acid molecule of
claim 6; (b) identifying the nucleic acid molecules, which
hybridise under relaxed stringent conditions with the nucleic acid
molecule of claim 6; (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing arginine and/or
glutamate and/or proline and/or glutamine; (d) expressing the
identified nucleic acid molecules in the host cells; (e) assaying
the arginine and/or glutamate and/or proline and/or glutamine level
in the host cells; and (f) identifying nucleic acid molecule and
its gene product which expression confers an increase in the
arginine and/or glutamate and/or proline and/or glutamine level in
the host cell in the host cell after expression compared to the
wild type. [2456] 20. A method for the identification of a gene
product conferring an increase in arginine and/or glutamate and/or
proline and/or glutamine production in a cell, comprising the
following steps: (a) identifiying in a data bank nucleic acid
molecules of an organism; which can contain a candidate gene
encoding a gene product conferring an increase in the arginine
and/or glutamate and/or proline and/or glutamine amount or level in
an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing arginine and/or glutamate and/or proline
and/or glutamine; (c) expressing the identified nucleic acid
molecules in the host cells; (d) assaying the arginine and/or
glutamate and/or proline and/or glutamine level in the host cells;
and (e) identifying nucleic acid molecule and its gene product
which expression confers an increase in the arginine and/or
glutamate and/or proline and/or glutamine level in the host cell
after expression compared to the wild type. [2457] 21. A method for
the production of an agricultural composition comprising the steps
of the method of any one of claims 17 to 20 and formulating the
compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. [2458] 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [2459] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
arginine and/or glutamate and/or proline and/or glutamine after
expression. [2460] 24. Use of the polypeptide of claim 14 or the
nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of arginine and/or glutamate
and/or proline and/or glutamine levels in an organism. [2461] 25.
Food or feed composition comprising the nucleic acid molecule of
claim 6, the polypeptide of claim 14, the nucleic acid construct of
claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 14, the
plant or plant tissue of claim 16, the harvested material of claim
16, the host cell of claims 10 to 12 or the gene product identified
according to the method of claim 19 or 20. [2462] 26. Use of the
nucleic acid molecule of claim 6, the polypeptide of claim 14, the
nucleic acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claims 10 to 12 or
the gene product identified according to the method of claim 19 or
20 for the protection of a plant against a arginine and/or
glutamate and/or proline and/or glutamine synthesis inhibiting
herbicide.
[2463] [0554.0.0.4] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[2464] [0000.0.0.5] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[2465] [0001.0.0.5] for the disclosure of this paragraph see
[0001.0.0.0].
[2466] [0002.0.5.5] Fatty acids are the building blocks of
triglycerides, lipids, oils and fats. Some of the fatty acids such
as linoleic or linolenic acid are "essential" because the human
body is not able to synthesize them but needs them, so humans must
ingest them through the diet. Other fatty acids can be synthesized
by the human body, therefore they are not labeled as "essential".
Nevertheless very often the amount of production of for example
fatty acids such as eicosapentaenoic acid (=EPA,
C.sub.20:5.sup..DELTA.5,8,11,14,17) or docosahexaenoic acid (=DHA,
C.sub.226.sup..DELTA.4,7,10,13,16,19) in the body is not sufficient
for an optimal body function. Polyunsaturated fatty acids (=PUFA)
that means fatty acids, which have more than 1 double bond in the
carbon chain are divided into families depending on where their
end-most double bond is located. There are two main subtypes of
fatty acids: the omega-3 and omega-6 fatty acids. The Omega-3's are
those with their endmost double bond 3 carbons from their methyl
end. The Omega-6's are those with their endmost double bond 6
carbons from their methyl end. Linoleic acid (an omega-6) and
alpha-linolenic acid (an omega-3) are the only true "essential"
fatty acids. Both are used inside the body as starting material to
synthesize others such as EPA or DHA.
[2467] [0003.0.5.5] Fatty acids and triglycerides have numerous
applications in the food and feed industry, in cosmetics and in the
drug sector. Depending on whether they are free saturated or
unsaturated fatty acids or triglycerides with an increased content
of saturated or unsaturated fatty acids, they are suitable for the
most varied applications; thus, for example, polyunsaturated fatty
acids (=PUFAs) are added to infant formula to increase its
nutritional value. The various fatty acids and triglycerides are
mainly obtained from microorganisms such as fungi or from
oil-producing plants including phytoplankton and algae, such as
soybean, oilseed rape, sunflower and others, where they are usually
obtained in the form of their triacylglycerides.
[2468] [0004.0.5.5] Principally microorganisms such as Mortierella
or oil producing plants such as soybean, rapeseed or sunflower or
algae such as Crytocodinium or Phaeodactylum are a common source
for oils containing PUFAs, where they are usually obtained in the
form of their triacyl glycerides. Alternatively, they are obtained
advantageously from animals, such as fish. The free fatty acids are
prepared advantageously by hydrolysis with a strong base such as
potassium or sodium hydroxide. Higher poly unsaturated fatty acids
such as DHA, EPA, ARA, Dihomo-.gamma.-linoleic acid
(C.sub.20.3.sup..DELTA.8,11,14) or Docosapentaenoic acid (=DPA,
C.sub.22:5.sup..DELTA.7,10,13,16,19) are not produced by oil
producing plants such as soybean, rapeseed, safflower or sunflower.
A natural sources for said fatty acids are fish for example
herring, salmon, sardine, redfish, eel, carp, trout, halibut,
mackerel, pike-perch or tuna or algae.
[2469] [0005.0.5.5] Whether oils with unsaturated or with saturated
fatty acids are preferred depends on the intended purpose; thus,
for example, lipids with unsaturated fatty acids, specifically
polyunsaturated fatty acids, are preferred in human nutrition since
they have a positive effect on the cholesterol level in the blood
and thus on the possibility of heart disease. They are used in a
variety of dietetic foodstuffs or medicaments. In addition PUFAs
are commonly used in food, feed and in the cosmetic industry. Poly
unsaturated .omega.-3- and/or .omega.-6-fatty acids are an
important part of animal feed and human food. Because of the common
composition of human food poly unsaturated w-3-fatty acids, which
are an essential component of fish oil, should be added to the food
to increase the nutritional value of the food; thus, for example,
polyunsaturated fatty acids such as DHA or EPA are added as
mentioned above to infant formula to increase its nutritional
value. The true essential fatty acids linoleic and linolenic fatty
acid have a lot of positive effects in the human body such as a
positive effect on healthy heart, arteries and skin. They bring for
example relieve from eczema, diabetic neuropathy or PMS and
cyclical breast pain.
[2470] [0006.0.5.5] Poly unsaturated .omega.-3- and .omega.-6-fatty
acids are for example precursor of a family of paracrine hormones
called eicosanoids such as prostaglandins which are products of the
metabolism of Dihomo-.gamma.-linoleic acid, ARA or EPA. Eicosanoids
are involved in the regulation of lipolysis, the initiation of
inflammatory responses, the regulation of blood circulation and
pressure and other central functions of the body. Eicosanoids
comprise prostaglandins, leukotrienes, thromboxanes, and
prostacyclins. .omega.-3-fatty acids seem to prevent
artherosclerosis and cardiovascular diseases primarily by
regulating the levels of different eicosanoids. Other Eicosanoids
are the thromboxanes and leukotrienes, which are products of the
metabolism of ARA or EPA.
[2471] [0007.0.5.5] On account of their positive properties there
has been no shortage of attempts in the past to make available
genes which participate in the synthesis of fatty acids or
triglycerides for the production of oils in various organisms
having a modified content of unsaturated fatty acids.
[2472] [0008.0.5.5] Methods of recombinant DNA technology have also
been used for some years to improve the oil content in
microorganisms or plants by amplifying individual fatty acid
biosynthesis genes and investigating the effect on fatty acid
production. For example in WO 91/13972 a .DELTA.-9-desaturase is
described, which is involved in the synthesis of polyunsaturated
fatty acids. In WO 93/11245 a .DELTA.-15-desaturase and in WO
94/11516 a .DELTA.-12-desaturase is claimed. Other desaturases are
described, for example, in EP-A-0 550 162, WO 94/18337, WO
97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al.,
J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347,
1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. To date,
however, the various desaturases have been only inadequately
characterized biochemically since the enzymes in the form of
membrane-bound proteins are isolable and characterizable only with
very great difficulty (McKeon et al., Methods in Enzymol. 71, 1981:
12141-12147, Wang et al., Plant Physiol. Biochem., 26, 1988:
777-792). Generally, membrane-bound desaturases are characterized
by introduction into a suitable organism, which is then
investigated for enzyme activity by means of analysis of starting
materials and products. With regard to the effectiveness of the
expression of desaturases and their effect on the formation of
polyunsaturated fatty acids it may be noted that through expression
of a desaturases and elongases as described to date only low
contents of poly-unsaturated fatty acids/lipids have been achieved.
Therefore, an alternative and more effective pathway with higher
product yield is desirable.
[2473] [0009.0.5.5] As described above, the essential fatty acids
are necessary for humans and many mammals, for example for
livestock. In a study of middle-aged men disclosed by Finnish
researchers (International Journal of Cancer, Sep. 1, 2004), high
intake of linoleic acid seemed to lower the risk of prostate and
other cancers. In another publication the positive influence on
stroke is disclosed (Umemura et al., Stroke, 2002, vol. 33, pp.
2086-2093).
[2474] [0010.0.5.5] Therefore improving the quality of foodstuffs
and animal feeds is an important task of the food-and-feed
industry. This is necessary since, for example, as mentioned above
certain fatty acids, which occur in plants are limited with regard
to the supply of mammals. Especially advantageous for the quality
of foodstuffs and animal feeds is as balanced as possible fatty
acid profile in the diet since a great excess of omega-3-fatty
acids above a specific concentration in the food has no positive
effect unless the omega-3-fatty acid content is in balance to the
omega-6-fatty acid content of the diet. A further increase in
quality is only possible via addition of further fatty acids, which
are limiting under these conditions. The targeted addition of the
limiting fatty acid in the form of synthetic products must be
carried out with extreme caution in order to avoid fatty acid
imbalance.
[2475] [0011.0.5.5] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add a plurality of fatty acids
in a balanced manner to suit the organism.
[2476] [0012.0.5.5] Accordingly, there is still a great demand for
new and more suitable genes which encode proteins which participate
in the biosynthesis of fatty acids and make it possible to produce
certain fatty acids specifically on an industrial scale without
unwanted byproducts forming. In the selection of genes for
biosynthesis two characteristics above all are particularly
important. On the one hand, there is as ever a need for improved
processes for obtaining the highest possible contents of
polyunsaturated fatty acids on the other hand as less as possible
byproducts should be produced in the production process.
[2477] [0013.0.0.5] for the disclosure of this paragraph see
[0013.0.0.0] above.
[2478] [0014.0.5.5] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is linoleic acid or
tryglycerides, lipids, oils or fats containing linoleic acid.
Accordingly, in the present invention, the term "the fine chemical"
as used herein relates to "linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid". Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising linoleic acid and/or triglycerides, lipids,
oils and/or fats containing linoleic acid.
[2479] [0015.0.5.5] In one embodiment, the term "the fine chemical"
or "the respective fine chemical" means linoleic acid and/or
tryglycerides, lipids, oils and/or fats containing linoleic acid.
Throughout the specification the term "the fine chemical" or "the
respective fine chemical" means linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, linoleic acid
and its salts, ester, thioester or linoleic acid in free form or
bound to other compounds such as triglycerides, glycolipids,
phospholipids etc. In a preferred embodiment, the term "the fine
chemical" means linoleic acid, in free form or its salts or bound
to triglycerides. Triglycerides, lipids, oils, fats or lipid
mixture thereof shall mean any triglyceride, lipid, oil and/or fat
containing any bound or free linoleic acid for example
sphingolipids, phosphoglycerides, lipids, glycolipids such as
glycosphingolipids, phospholipids such as phosphatidylethanolamine,
phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol or diphosphatidylglycerol, or as
monoacylglyceride, diacylglyceride or triacylglyceride or other
fatty acid esters such as acetyl-Coenzym A thioester, which contain
further saturated or unsaturated fatty acids in the fatty acid
molecule. In one embodiment, the term "the fine chemical" and the
term "the respective fine chemical" mean at least one chemical
compound with an activity of the above mentioned fine chemical.
[2480] [0016.0.5.5] Accordingly, the present invention relates to a
process comprising [2481] (a) increasing or generating the activity
of one or more b0730, b3256, YBR089C-A, YDR447C, YOR024W, b0050,
b0251, b0255, b0577, b0849, b1097, b1693, b2710, b2822, b3064,
b3166, b3457, b3644 and/or b4129 protein(s) or of a protein having
the sequence of a polypeptide encoded by a nucleic acid molecule
indicated in Table I, columns 5 or 7, lines 63 to 67 and 436 to 449
in a non-human organism or in one or more parts thereof and [2482]
(b) growing the organism under conditions which permit the
production of the fine chemical, thus, linoleic acid or fine
chemicals comprising linoleic acid, in said organism.
[2483] Accordingly, the present invention relates to a process for
the production of a fine chemical comprising [2484] (a) increasing
or generating the activity of one or more proteins having the
activity of a protein indicated in Table II, column 3, lines 63 to
67 and 436 to 449 or having the sequence of a polypeptide encoded
by a nucleic acid molecule indicated in Table I, column 5 or 7,
lines 63 to 67 and 436 to 449, in a non-human organism in one or
more parts thereof and [2485] (b) growing the organism under
conditions which permit the production of the fine chemical, in
particular linoleic acid.
[2486] [0017.0.0.5] and [0018.0.0.5] for the disclosure of the
paragraphs [0017.0.0.5] and [0018.0.0.5] see paragraphs
[0017.0.0.0] and [0018.0.0.0] above.
[2487] [0019.0.5.5] Advantageously the process for the production
of the fine chemical leads to an enhanced production of the fine
chemical. The terms "enhanced" or "increase" mean at least a 10%,
20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or
100%, more preferably 150%, 200%, 300%, 400% or 500% higher
production of the fine chemical in comparison to the reference as
defined below, e.g. that means in comparison to an organism without
the aforementioned modification of the activity of a protein having
the activity of a protein indicated in Table II, column 3, lines 63
to 67 and 436 to 449 or encoded by nucleic acid molecule indicated
in Table I, columns 5 or 7, lines 63 to 67 and 436 to 449.
[2488] [0020.0.5.5] Surprisingly it was found, that the transgenic
expression of at least one of the Saccaromyces cerevisiae
protein(s) indicated in Table II, Column 3, lines 65 to 67, and/or
the Escherichia coli K12 protein(s) indicated in Table II, Column
3, lines 63 to 64 and 436 to 449 in Arabidopsis thaliana conferred
an increase in the linoleic acid (or fine chemical) content of the
transformed plants.
[2489] [0021.0.0.5] for the disclosure of this paragraph see
[0021.0.0.0] above.
[2490] [0022.0.5.5] The sequence of b0730 from Escherichia coli K12
has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as
transcriptional regulator of succinylCoA synthetase operon.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a transcriptional regulator of
succinylCoA synthetase operon from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, in particular for increasing the amount
of linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, preferably linoleic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a transcriptional regulator of succinylCoA synthetase operon is
increased or generated, e.g. from E. coli or a homolog thereof.
[2491] The sequence of b3256 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as acetyl CoA carboxylase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a acetyl CoA carboxylase from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, in particular
for increasing the amount of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, preferably
linoleic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a acetyl CoA carboxylase is
increased or generated, e.g. from E. coli or a homolog thereof.
[2492] The sequence of YBR089C-A from Saccharomyces cerevisiae has
been published in Feldmann et al., EMBO J., 13 (24), 5795-5809
(1994) and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its cellular activity has not been characterized yet. It shows
homology to mammalian high mobility group proteins 1 and 2. Its
function may be redundantly with the highly homologous gene NHP6A.
Furthermore it shows homology to the high-mobility group
non-histone chromatin protein Nhp6 bp. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a YBR089C-A activity from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of linoleic acid and/or tryglycerides, lipids,
oils and/or fats containing linoleic acid, in particular for
increasing the amount of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, preferably
linoleic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a YBR089C-A protein is increased
or generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[2493] The sequence of YDR447C from Saccharomyces cerevisiae has
been published in Jacq et al., Nature 387 (6632 Suppl), 75-78,
1997, and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is being defined as ribosomal protein 51 (rp51) of the
small (40 s) subunit; nearly identical to Rps17Ap and has
similarity to rat S17 ribosomal protein; Rpsl7 bp. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a "ribosomal protein 51" or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, in particular for increasing the amount
of linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, preferably linoleic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a ribosomal protein 51 is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[2494] The sequence of YOR024W from Saccharomyces cerevisiae has
been submitted from de Haan to the EMBL Protein Sequence Database,
July 1996 and its cellular activity has not been characterized yet.
It is probably a membrane protein. Accordingly, in one embodiment,
the process of the present invention comprises the use of YOR024W,
from Saccharomyces cerevisiae or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of linoleic acid
and/or tryglycerides, lipids, oils and/or fats containing linoleic
acid, in particular for increasing the amount of linoleic acid
and/or tryglycerides, lipids, oils and/or fats containing linoleic
acid, preferably linoleic acid in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a YOR024W is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[2495] The sequence of b0050 (Accession number NP.sub.--414592)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a conserved protein potentially involved in protein
protein interaction. Accordingly, in one embodiment, the process of
the present invention comprises the use of said protein from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, in particular
for increasing the amount of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, preferably
linoleic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of said protein is increased or
generated, e.g. from E. coli or a homolog thereof.
[2496] The sequence of b0251 (Accession number NP.sub.--414785)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as putative HTH-type transcriptional regulator.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a putative HTH-type transcriptional
regulator protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, in particular for increasing the amount
of linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, preferably linoleic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a putative HTH-type transcriptional regulator protein is increased
or generated, e.g. from E. coli or a homolog thereof.
[2497] The sequence of b0255 (Accession number NP.sub.--414789)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a CP4-6 prophage; IS911 homolog. Accordingly, in one
embodiment, the process of the present invention comprises the use
of said CP4-6 prophage; IS911 homolog protein from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of linoleic acid and/or tryglycerides, lipids,
oils and/or fats containing linoleic acid, in particular for
increasing the amount of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, preferably
linoleic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of said CP4-6 prophage; IS911
homolog protein is increased or generated, e.g. from E. coli or a
homolog thereof.
[2498] The sequence of b0577 (Accession number NP.sub.--415109)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a putative transport protein. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a putative transport protein from E. coli or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
of linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, in particular for increasing the amount
of linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, preferably linoleic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a putative transport protein is increased or generated, e.g. from
E. coli or a homolog thereof.
[2499] The sequence of b0849 (Accession number NP.sub.--415370)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a glutaredoxin 1 redox coenzyme for
glutathione-dependent ribonucleotide reductase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a glutaredoxin 1 redox coenzyme for glutathione-dependent
ribonucleotide reductase protein from E. coli or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
of linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, in particular for increasing the amount
of linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, preferably linoleic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a glutaredoxin 1 redox coenzyme for glutathione-dependent
ribonucleotide reductase protein is increased or generated, e.g.
from E. coli or a homolog thereof.
[2500] The sequence of b1097 (Accession number NP.sub.--415615)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a putative thymidylate kinase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a putative thymidylate kinase protein from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of linoleic acid and/or tryglycerides, lipids,
oils and/or fats containing linoleic acid, in particular for
increasing the amount of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, preferably
linoleic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a putative thymidylate kinase
protein is increased or generated, e.g. from E. coli or a homolog
thereof.
[2501] The sequence of b1693 (Accession number NP.sub.--416208)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a 3-dehydroquinate dehydratase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a 3-dehydroquinate dehydratase protein from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of linoleic acid and/or tryglycerides, lipids,
oils and/or fats containing linoleic acid, in particular for
increasing the amount of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, preferably
linoleic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a 3-dehydroquinate dehydratase
protein is increased or generated, e.g. from E. coli or a homolog
thereof.
[2502] The sequence of b2710 (Accession number NP.sub.--417190)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a flavorubredoxin (FIRd) bifunctional NO and O.sub.2
reductase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a flavorubredoxin (FIRd)
bifunctional NO and O.sub.2 reductase protein from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of linoleic acid and/or tryglycerides, lipids,
oils and/or fats containing linoleic acid, in particular for
increasing the amount of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, preferably
linoleic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a flavorubredoxin (FIRd)
bifunctional NO and O.sub.2 reductase protein is increased or
generated, e.g. from E. coli or a homolog thereof.
[2503] The sequence of b2822 (Accession number NP.sub.--417299)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a DNA helicase, ATP-dependent dsDNA/ssDNA exonuclease V
subunit, ssDNA endonuclease. Accordingly, in one embodiment, the
process of the present invention comprises the use of a DNA
helicase, ATP-dependent dsDNA/ssDNA exonuclease V subunit, ssDNA
endonuclease protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, in particular for increasing the amount
of linoleic acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid, preferably linoleic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a DNA helicase, ATP-dependent dsDNA/ssDNA exonuclease V subunit,
ssDNA endonuclease protein is increased or generated, e.g. from E.
coli or a homolog thereof.
[2504] The sequence of b3064 (Accession number NP.sub.--417536)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a putative O-sialoglycoprotein endopeptidase, with
actin-like ATPase domain. Accordingly, in one embodiment, the
process of the present invention comprises the use of a putative
O-sialoglycoprotein endopeptidase, with actin-like ATPase domain
protein from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of linoleic acid and/or
tryglycerides, lipids, oils and/or fats containing linoleic acid,
in particular for increasing the amount of linoleic acid and/or
tryglycerides, lipids, oils and/or fats containing linoleic acid,
preferably linoleic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a putative
0-sialoglycoprotein endopeptidase, with actin-like ATPase domain
protein is increased or generated, e.g. from E. coli or a homolog
thereof.
[2505] The sequence of b3166 (Accession number NP.sub.--417635)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a tRNA pseudouridine 5S synthase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a tRNA pseudouridine 5S synthase protein from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of linoleic acid and/or tryglycerides, lipids,
oils and/or fats containing linoleic acid, in particular for
increasing the amount of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, preferably
linoleic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a tRNA pseudouridine 5S synthase
protein is increased or generated, e.g. from E. coli or a homolog
thereof.
[2506] The sequence of b3457 (Accession number NP.sub.--417914)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a high-affinity branched-chain amino acid transport
protein (ABC superfamily). Accordingly, in one embodiment, the
process of the present invention comprises the use of a
high-affinity branched-chain amino acid transport protein from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, in particular
for increasing the amount of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, preferably
linoleic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a high-affinity branched-chain
amino acid transport protein is increased or generated, e.g. from
E. coli or a homolog thereof.
[2507] The sequence of b3644 (Accession number NP.sub.--418101)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as an uncharacterized stress-induced protein. Accordingly,
in one embodiment, the process of the present invention comprises
the use of an uncharacterized stress-induced protein from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, in particular
for increasing the amount of linoleic acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid, preferably
linoleic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of an uncharacterized stress-induced
protein is increased or generated, e.g. from E. coli or a homolog
thereof.
[2508] The sequence of b4129 (Accession number NP.sub.--418553)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a lysine tRNA synthetase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a lysine tRNA synthetase protein from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of linoleic acid and/or tryglycerides, lipids, oils and/or
fats containing linoleic acid, in particular for increasing the
amount of linoleic acid and/or tryglycerides, lipids, oils and/or
fats containing linoleic acid, preferably linoleic acid in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a lysine tRNA synthetase protein is increased or generated, e.g.
from E. coli or a homolog thereof.
[2509] [0023.0.5.5] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the fine chemical amount or content. Further, in the
present invention, the term "homologue" relates to the sequence of
an organism having the highest sequence homology to the herein
mentioned or listed sequences of all expressed sequences of said
organism.
[2510] However, the person skilled in the art knows, that,
preferably, the homologue has said fine-chemical increasing
activity and, if known, the same biological function or activity in
the organism as at least one of the protein(s) indicated in Table
II, column 3, lines 63 to 67 and 436 to 449, e.g. having the
sequence of a polypeptide encoded by a nucleic acid molecule
comprising the sequence indicated in Table I, columns 5 or 7, lines
63 to 67 and 436 to 449.
[2511] In one embodiment, the homolog of any one of the
polypeptides indicated in Table II, columns 5 or 7, lines 65 to 67
is a homolog having the same or a similar activity, in particular
an increase of activity confers an increase in the content of the
fine chemical in the organisms and being derived from an Eukaryot.
In one embodiment, the homolog of a polypeptide indicated in Table
II, column 3, lines 63, 64 and 436 to 449 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from bacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 65 to 67 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in an organisms or part
thereof, and being derived from Fungi. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 63,
64 and 436 to 449 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or part
thereof and being derived from Proteobacteria. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
65 to 67 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof and
being derived from Ascomycota. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 63, 64 and 436
to 449 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or part thereof, and
being derived from Gammaproteobacteria. In one embodiment, the
homolog of a polypeptide polypeptide indicated in Table II, column
3, lines 65 to 67 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or part
thereof, and being derived from Saccharomycotina. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 63, 64 and 436 to 449 is a homolog having the same
or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 65 to 67 is a homolog having
the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms or a part thereof, and being derived from
Saccharomycetes. In one embodiment, the homolog of the a
polypeptide indicated in Table II, column 3, lines 63, 64 and 436
to 449 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or part thereof, and
being derived from Enterobacteriaceae. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 65
to 67 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms, and being derived
from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 63, 64 and 436
to 449 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof,
and being derived from Escherichia. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 65 to 67 is
a homolog having the same or a similar activity, in particular an
increase of activity confers an increase in the content of the fine
chemical in the organisms or a part thereof, and being derived from
Saccharomycetaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 65 to 67 is a homolog having
the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms or a part thereof, and being derived from
Saccharomycetes.
[2512] [0023.1.0.5] and [0024.0.0.5] for the disclosure of the
paragraphs [0023.1.0.5] and [0024.0.0.5] see [0023.1.0.0] and
[0024.0.0.0] above.
[2513] [0025.0.5.5] In accordance with the invention, a protein or
polypeptide has the "activity of a protein as indicated in Table
II, column 3, lines 63 to 67 and 436 to 449" if its de novo
activity, or its increased expression directly or indirectly leads
to an increased linoleic acid and/or tryglycerides, lipids, oils
and/or fats containing linoleic acid level in the organism or a
part thereof, preferably in a cell of said organism and the protein
has the above mentioned activities of a protein as indicated in
Table II, column 3, lines 63 to 67 and 436 to 449. Throughout the
specification the activity or preferably the biological activity of
such a protein or polypeptide or an nucleic acid molecule or
sequence encoding such protein or polypeptide is identical or
similar if it still has the biological or enzymatic activity of a
protein as indicated in Table II, column 3, lines 63 to 67 and 436
to 449, or which has at least 10% of the original enzymatic
activity, preferably 20%, particularly preferably 30%, most
particularly preferably 40% in comparison to a protein of
Saccharomyces cerevisiae as indicated in Table II, column 3, lines
65 to 67 and/or a protein of E. coli K12 as indicated in Table II,
column 3, lines 63, 64 and 436 to 449.
[2514] [0025.1.0.5] and [0025.2.0.5] for the disclosure of the
paragraphs [0025.1.0.5] and [0025.2.0.5] see paragraphs
[0025.1.0.0] and [0025.2.0.0] above.
[2515] [0026.0.0.5] to [0033.0.0.5] for the disclosure of the
paragraphs [0026.0.0.5] to [0033.0.0.5] see paragraphs [0026.0.0.0]
and [0033.0.0.0] above.
[2516] [0034.0.5.5] Preferably, the reference, control or wild type
differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. as result of
an increase in the level of the nucleic acid molecule of the
present invention or an increase of the specific activity of the
polypeptide of the invention or the polypeptide used in the method
of the invention. E.g., it differs by or in the expression level or
activity of an protein having the activity of a protein as
indicated in Table II, column 3, lines 63 to 67 and 436 to 449 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 63 to 67 and 436 to 449 or its homologs, e.g. as
indicated in Table I, column 7, lines 63 to 67 and 436 to 449, its
biochemical or genetical causes and therefore shows the increased
amount of the fine chemical.
[2517] [0035.0.0.5] to [0044.0.0.5] for the disclosure of the
paragraphs [0035.0.0.5] to [0044.0.0.5] see paragraphs [0035.0.0.0]
and [0044.0.0.0] above.
[2518] [0045.0.5.5] In one embodiment, in case the activity of the
Escherichia coli K12 protein b0730 or its homologs e.g. a
transcriptional regulator of succinylCoA synthetase operon e.g. a
transcriptional regulator for regulation of C-compound and
carbohydrate utilization, transcriptional control, prokaryotic
nucleoid, transcriptional repressor, DNA binding e.g. as indicated
in Table II, columns 5 or 7, line 63, is increased, preferably, in
one embodiment the increase of the fine chemical, preferably of
linoleic acid between 13% and 34% or more is conferred. In case the
activity of the Escherichia coli K12 protein b3256 or its homologs
e.g. a acetyl CoA carboxylase e.g. as indicated in Table II,
columns 5 or 7, line 64, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
linoleic acid between 13% and 20% or more is conferred.
[2519] In case the activity of the Saccharomyces cerevisiae protein
YBR089C-A or its homologs, e.g. a "uncharacterized protein
YBR089C-A", which shows homology to mammalian high mobility group
proteins 1 and 2, gene NHP6A and to high-mobility group non-histone
chromatin protein Nhp6 bp, e.g. as indicated in Table II, columns 5
or 7, line 65 is increased, preferably, in one embodiment an
increase of the fine chemical, preferably of linoleic acid between
36% and 70% or more is conferred.
[2520] In case the activity of the Saccharomyces cerevisiae protein
YDR447C or its homologs, e.g. a ribosomal protein 51 (rp51) of the
small (40 s) subunit e.g. as indicated in Table II, columns 5 or 7,
line 66 is increased, preferably, in one embodiment an increase of
the fine chemical, preferably of linoleic acid between 18% and 140%
or more is conferred.
[2521] In case the activity of the Saccaromyces cerevisiae protein
YOR024W or its homologs e.g. a "uncharacterized protein YOR024W",
which is probably a membrane protein e.g. as indicated in Table II,
columns 5 or 7, line 67 is increased, preferably, in one embodiment
the increase of the fine chemical, preferably of linoleic acid,
between 16% and 33% or more is conferred.
[2522] In case the activity of the Escherichia coli K12 protein
b0050 or its homologs e.g. a conserved protein potentially involved
in protein protein interaction e.g. as indicated in Table II,
columns 5 or 7, line 436, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
linoleic acid between 15% and 37% or more is conferred.
[2523] In case the activity of the Escherichia coli K12 protein
b0251 or its homologs e.g. a putative HTH-type transcriptional
regulator e.g. as indicated in Table II, columns 5 or 7, line 437,
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of linoleic acid between 14% and 31% or
more is conferred.
[2524] In case the activity of the Escherichia coli K12 protein
b0255 or its homologs e.g. a CP4-6 prophage; IS911 homolog e.g. as
indicated in Table II, columns 5 or 7, line 438, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of linoleic acid between 22% and 31% or more is
conferred.
[2525] In case the activity of the Escherichia coli K12 protein
b0577 or its homologs e.g. a putative transport protein e.g. as
indicated in Table II, columns 5 or 7, line 439, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of linoleic acid between 17% and 40% or more is
conferred.
[2526] In case the activity of the Escherichia coli K12 protein
b0849 or its homologs e.g. a glutaredoxin 1 redox coenzyme for
glutathione-dependent ribonucleotide reductase e.g. as indicated in
Table II, columns 5 or 7, line 440, is increased, preferably, in
one embodiment the increase of the fine chemical, preferably of
linoleic acid between 19% and 34% or more is conferred.
[2527] In case the activity of the Escherichia coli K12 protein
b1097 or its homologs e.g. a putative thymidylate kinase e.g. as
indicated in Table II, columns 5 or 7, line 441, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of linoleic acid between 15% and 30% or more is
conferred.
[2528] In case the activity of the Escherichia coli K12 protein
b1693 or its homologs e.g. a 3-dehydroquinate dehydratase e.g. as
indicated in Table II, columns 5 or 7, line 442, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of linoleic acid between 17% and 32% or more is
conferred.
[2529] In case the activity of the Escherichia coli K12 protein
b2710 or its homologs e.g. a flavorubredoxin (FIRd) bifunctional NO
and O.sub.2 reductase e.g. as indicated in Table II, columns 5 or
7, line 443, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of linoleic acid between
15% and 29% or more is conferred.
[2530] In case the activity of the Escherichia coli K12 protein
b2822 or its homologs e.g. a DNA helicase, ATP-dependent
dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease e.g. as
indicated in Table II, columns 5 or 7, line 444, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of linoleic acid between 15% and 27% or more is
conferred.
[2531] In case the activity of the Escherichia coli K12 protein
b3064 or its homologs e.g. a putative O-sialoglycoprotein
endopeptidase, with actin-like ATPase domain e.g. as indicated in
Table II, columns 5 or 7, line 445, is increased, preferably, in
one embodiment the increase of the fine chemical, preferably of
linoleic acid between 17% and 30% or more is conferred.
[2532] In case the activity of the Escherichia coli K12 protein
b3166 or its homologs e.g. a tRNA pseudouridine 5S synthase e.g. as
indicated in Table II, columns 5 or 7, line 446, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of linoleic acid between 14% and 21% or more is
conferred.
[2533] In case the activity of the Escherichia coli K12 protein
b3457 or its homologs e.g. a high-affinity branched-chain amino
acid transport protein (ABC superfamily) e.g. as indicated in Table
II, columns 5 or 7, line 447, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
linoleic acid between 20% and 44% or more is conferred.
[2534] In case the activity of the Escherichia coli K12 protein
b3644 or its homologs e.g. an uncharacterized stress-induced
protein e.g. as indicated in Table II, columns 5 or 7, line 448, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of linoleic acid between 14% and 35% or more
is conferred.
[2535] In case the activity of the Escherichia coli K12 protein
b4129 or its homologs e.g. a lysine tRNA synthetase e.g. as
indicated in Table II, columns 5 or 7, line 449, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of linoleic acid between 20% and 47% or more is
conferred.
[2536] [0046.0.5.5] In one embodiment, in case the activity of the
Escherichia coli K12 protein b0730 or its homologs e.g. a
transcriptional regulator of succinylCoA synthetase operon is
increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing linoleic acid is
conferred.
[2537] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3256 or its homologs e.g. a acetyl CoA
carboxylase is increased, preferably an increase of the fine
chemical and of tryglycerides, lipids, oils and/or fats containing
linoleic acid is conferred.
[2538] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YBR089C-A or its homologs e.g. a
"uncharacterized protein YBR089C-A" is increased, preferably an
increase of the fine chemical and of tryglycerides, lipids, oils
and/or fats containing linoleic acid is conferred.
[2539] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YDR447C or its homologs, e.g. a ribosomal
protein 51 (rp51) of the small (40 s) subunit is increased,
preferably an increase of the fine chemical and of tryglycerides,
lipids, oils and/or fats containing linoleic acid is conferred.
[2540] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YOR024W or its homologs e.g. a "uncharacterized
protein YOR024W" is increased, preferably an increase of the fine
chemical and of tryglycerides, lipids, oils and/or fats containing
linoleic acid is conferred.
[2541] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0050 or its homologs e.g. a conserved protein
potentially involved in protein protein interaction is increased,
preferably an increase of the fine chemical and of tryglycerides,
lipids, oils and/or fats containing linoleic acid is conferred.
[2542] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0251 or its homologs e.g. a putative HTH-type
transcriptional regulator is increased, preferably an increase of
the fine chemical and of tryglycerides, lipids, oils and/or fats
containing linoleic acid is conferred.
[2543] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0255 or its homologs e.g. a CP4-6 prophage; IS911
homolog is increased, preferably an increase of the fine chemical
and of tryglycerides, lipids, oils and/or fats containing linoleic
acid is conferred.
[2544] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0577 or its homologs e.g. a putative transport
protein is increased, preferably an increase of the fine chemical
and of tryglycerides, lipids, oils and/or fats containing linoleic
acid is conferred.
[2545] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0849 or its homologs e.g. a glutaredoxin 1 redox
coenzyme for glutathione-dependent ribonucleotide reductase is
increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing linoleic acid is
conferred.
[2546] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1097 or its homologs e.g. a putative thymidylate
kinase is increased, preferably an increase of the fine chemical
and of tryglycerides, lipids, oils and/or fats containing linoleic
acid is conferred.
[2547] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1693 or its homologs e.g. a 3-dehydroquinate
dehydratase is increased, preferably an increase of the fine
chemical and of tryglycerides, lipids, oils and/or fats containing
linoleic acid is conferred.
[2548] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2710 or its homologs e.g. a flavorubredoxin
(FIRd) bifunctional NO and O.sub.2 reductase is increased,
preferably an increase of the fine chemical and of tryglycerides,
lipids, oils and/or fats containing linoleic acid is conferred.
[2549] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2822 or its homologs e.g. a DNA helicase,
ATP-dependent dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease
is increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing linoleic acid is
conferred.
[2550] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3064 or its homologs e.g. putative
O-sialoglycoprotein endopeptidase, with actin-like ATPase domain is
increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing linoleic acid is
conferred.
[2551] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3166 or its homologs e.g. a tRNA pseudouridine 5S
synthase is increased, preferably an increase of the fine chemical
and of tryglycerides, lipids, oils and/or fats containing linoleic
acid is conferred.
[2552] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3457 or its homologs e.g. a high-affinity
branched-chain amino acid transport protein (ABC superfamily) is
increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing linoleic acid is
conferred.
[2553] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3644 or its homologs e.g. an uncharacterized
stress-induced protein is increased, preferably an increase of the
fine chemical and of tryglycerides, lipids, oils and/or fats
containing linoleic acid is conferred.
[2554] In one embodiment, in case the activity of the Escherichia
coli K12 protein b4129 or its homologs e.g. a lysine tRNA
synthetase is increased, preferably an increase of the fine
chemical and of tryglycerides, lipids, oils and/or fats containing
linoleic acid is conferred.
[2555] [0047.0.0.5] and [0048.0.0.5] for the disclosure of the
paragraphs [0047.0.0.5] and [0048.0.0.5] see paragraphs
[0047.0.0.0] and [0048.0.0.0] above.
[2556] [0049.0.5.5] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
preferably has the structure of the polypeptide described herein,
in particular a polypeptide comprising a consensus sequence as
indicated in Table IV, column 7, lines 63 to 67 and 436 to 449 or
of a polypeptide as indicated in Table II, columns 5 or 7, lines 63
to 67 and 436 to 449 or the functional homologues thereof as
described herein, or is encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 63 to 67 and 436 to 449 or its
herein described functional homologues and has the herein mentioned
activity.
[2557] [0050.0.5.5] For the purposes of the present invention, the
term "linoleic acid" also encompasses the corresponding salts, such
as, for example, the potassium or sodium salts of linoleic acid or
the salts of linoleic acid with amines such as diethylamine.
[2558] [0051.0.5.5] and [0052.0.0.5] for the disclosure of the
paragraphs [0051.0.5.5] and [0052.0.0.5] see paragraphs
[0051.0.0.0] and [0052.0.0.0] above.
[2559] [0053.0.5.5] In one embodiment, the process of the present
invention comprises one or more of the following steps [2560] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention or the nucleic acid molecule or the
polypeptide used in the method of the invention, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 63 to 67 and 436 to 449 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 63 to 67
and 436 to 449, having herein-mentioned the fine chemical
increasing activity; [2561] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention, e.g. of a polypeptide having an activity
of a protein as indicated in Table II, column 3, lines 63 to 67 and
436 to 449 or its homolog's activity, e.g. as indicated in Table
II, column 7, lines 63 to 67 and 436 to 449 or of a mRNA encoding
the polypeptide of the present invention having herein mentioned
linoleic acid increasing activity; [2562] c) increasing the
specific activity of a protein conferring the increased expression
of a protein encoded by the nucleic acid molecule of the invention
or of the polypeptide of the present invention or the nucleic acid
molecule or the polypeptide used in the method of the invention,
having herein-mentioned linoleic acid increasing activity, e.g. of
a polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 63 to 67 and 436 to 449 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 63 to 67
and 436 to 449, or decreasing the inhibitory regulation of the
polypeptide of the invention or of the polypeptide used in the
method of the invention; [2563] d) generating or increasing the
expression of an endogenous or artificial transcription factor
mediating the expression of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention or of the polypeptide of the invention or the polypeptide
used in the method of the invention having herein-mentioned
linoleic acid increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 63
to 67 and 436 to 449 or its homolog's activity, e.g. as indicated
in Table II, column 7, lines 63 to 67 and 436 to 449; [2564] e)
stimulating activity of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
present invention or a polypeptide of the present invention having
herein-mentioned linoleic acid increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 63 to 67 and 436 to 449 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 63 to 67
and 436 to 449, by adding one or more exogenous inducing factors to
the organism or parts thereof; [2565] f) expressing a transgenic
gene encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned linoleic acid increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 63 to 67 and 436 to 449 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 63 to 67
and 436 to 449, [2566] g) increasing the copy number of a gene
conferring the increased expression of a nucleic acid molecule
encoding a polypeptide encoded by the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention or the polypeptide of the invention or the polypeptide
used in the method of the invention having herein-mentioned
linoleic acid increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 63
to 67 and 436 to 449 or its homolog's activity, e.g. as indicated
in Table II, column 7, lines 63 to 67 and 436 to 449, [2567] h)
increasing the expression of the endogenous gene encoding the
polypeptide of the invention or the polypeptide used in the method
of the invention, e.g. a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 63 to 67 and 436
to 449 or its homolog's activity, e.g. as indicated in Table II,
column 7, lines 63 to 67 and 436 to 449, by adding positive
expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; [2568] i)
Modulating growth conditions of an organism in such a manner, that
the expression or activity of the gene encoding the protein of the
invention or the protein itself is enhanced for example
microorganisms or plants can be grown for example under a higher
temperature regime leading to an enhanced expression of heat shock
proteins, which can lead an enhanced fine chemical production.
[2569] j) selecting of organisms with especially high activity of
the proteins of the invention from natural or from mutagenized
resources and breeding them into the target organisms, eg the elite
crops.
[2570] [0054.0.5.5] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of linoleic acid after
increasing the expression or activity of the encoded polypeptide or
having the activity of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 63 to 67 and 436
to 449 or its homolog's activity, e.g. as indicated in Table II,
columns 5 or 7, lines 63 to 67 and 436 to 449.
[2571] [0055.0.0.5] to [0067.0.0.5] for the disclosure of the
paragraphs [0055.0.0.5] to [0067.0.0.5] see paragraphs [0055.0.0.0]
to [0067.0.0.0] above.
[2572] [0068.0.5.5] and [0069.0.5.5] for the disclosure of the
paragraphs [0068.0.5.5] and [0069.0.5.5] see paragraphs
[0068.0.0.0] and [0069.0.0.0] above.
[2573] [0070.0.5.5] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention or the polypeptide of the invention or the
polypeptide used in the method of the invention as described below,
for example the nucleic acid construct mentioned below into an
organism alone or in combination with other genes, it is possible
not only to increase the biosynthetic flux towards the end product,
but also to increase, modify or create de novo an advantageous,
preferably novel metabolites composition in the organism, e.g. an
advantageous fatty acid composition comprising a higher content of
(from a viewpoint of nutritional physiology limited) fatty acids,
like palmitate, palmitoleate, stearate and/or oleate.
[2574] [0071.0.5.5] for the disclosure of this paragraph see
[0071.0.0.0] above.
[2575] [0072.0.5.5] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to linoleic acid, triglycerides, lipids, oils and/or fats
containing linoleic acid compounds such as palmitate, palmitoleate,
stearate and/or oleate.
[2576] [0073.0.5.5] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[2577] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [2578] b) increasing the activity of a
protein having the activity of a polypeptide of the invention or
the polypeptide used in the method of the invention or a homolog
thereof, e.g. as shown in Table II, columns 5 or 7, lines 63 to 67
and 436 to 449 or of a polypeptide being encoded by the nucleic
acid molecule of the present invention and described below, i.e.
conferring an increase of the respective fine chemical in the
organism, preferably a microorganism, the a non-human animal, a
plant or animal cell, a plant or animal tissue or the plant, [2579]
c) growing the organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or the
plant under conditions which permit the production of the fine
chemical in the organism, preferably a microorganism, a plant cell,
a plant tissue or the plant; and [2580] d) if desired, revovering,
optionally isolating, the free and/or bound the respective fine
chemical and, optionally further free and/or bound fatty acids
synthetized by the organism, the microorganism, the non-human
animal, the plant or animal cell, the plant or animal tissue or the
plant.
[2581] [0074.0.5.5] The organism, in particular the microorganism,
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant is advantageously grown in such a way that it
is not only possible to recover, if desired isolate the free or
bound the fine chemical or the free and bound the fine chemical but
as option it is also possible to produce, recover and, if desired
isolate, other free or/and bound fatty acids, in particular oleic
acid.
[2582] [0075.0.0.5] to [0084.0.0.5] for the disclosure of the
paragraphs [0075.0.0.5] to [0084.0.0.5] see paragraphs [0075.0.0.0]
to [0084.0.0.0] above.
[2583] [0085.0.5.5] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [2584] a) the nucleic acid sequence as
depicted and shown in Table I, columns 5 or 7, lines 63 to 67 and
436 to 449 or a derivative thereof, or [2585] b) a genetic
regulatory element, for example a promoter, which is functionally
linked to the nucleic acid sequence as depicted and shown in Table
I, columns 5 or 7, lines 63 to 67 and 436 to 449 or a derivative
thereof, or [2586] c) (a) and (b) is/are not present in its/their
natural genetic environment or has/have been modified by means of
genetic manipulation methods, it being possible for the
modification to be, by way of example, a substitution, addition,
deletion, inversion or insertion of one or more nucleotide.
"Natural genetic environment" means the natural chromosomal locus
in the organism of origin or the presence in a genomic library. In
the case of a genomic library, the natural, genetic environment of
the nucleic acid sequence is preferably at least partially still
preserved. The environment flanks the nucleic acid sequence at
least on one side and has a sequence length of at least 50 bp,
preferably at least 500 bp, particularly preferably at least 1000
bp, very particularly preferably at least 5000 bp.
[2587] [0086.0.0.5] and [0087.0.0.5] for the disclosure of the
paragraphs [0086.0.0.5] and [0087.0.0.5] see paragraphs
[0086.0.0.0] and [0087.0.0.0] above.
[2588] [0088.0.5.5] In an advantageous embodiment of the invention,
the organism takes the form of a plant whose fatty acid content is
modified advantageously owing to the nucleic acid molecule of the
present invention expressed. This is important for plant breeders
since, for example, the nutritional value of plants for poultry is
dependent on the abovementioned essential fatty acids and the
general amount of fatty acids as energy source in feed. After the
activity of a protein as shown in Table II, columns 5 or 7, lines
63 to 67 and 436 to 449 has been increased or generated, or after
the expression of nucleic acid molecule or polypeptide according to
the invention has been generated or increased, the transgenic plant
generated thus is grown on or in a nutrient medium or else in the
soil and subsequently harvested.
[2589] [0088.1.0.5], [0089.0.0.5], [0090.0.0.5] and [0091.0.5.5]
for the disclosure of the paragraphs [0088.1.0.5], [0089.0.0.5],
[0090.0.0.5] and [0091.0.5.5] see paragraphs [0088.1.0.0],
[0089.0.0.0], [0090.0.0.0] and [0091.0.0.0] above.
[2590] [0092.0.0.5] to [0094.0.0.5] for the disclosure of the
paragraphs [0092.0.0.5] to [0094.0.0.5] see paragraphs [0092.0.0.0]
to [0094.0.0.0] above.
[2591] [0095.0.5.5] It may be advantageous to increase the pool of
free fatty acids in the transgenic organisms by the process
according to the invention in order to isolate high amounts of the
pure fine chemical.
[2592] [0096.0.5.5] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid for example a fatty acid
transporter protein or a compound, which functions as a sink for
the desired fatty acid for example for linoleic or linolenic acid
in the organism is useful to increase the production of the
respective fine chemical (see Bao and Ohlrogge, Plant Physiol. 1999
August; 120 (4): 1057-1062). Such fatty acid transporter protein
may serve as a link between the location of fatty acid synthesis
and the socalled sink tissue, in which fatty acids, triglycerides,
oils and fats are stored.
[2593] [0097.0.5.5] for the disclosure of this paragraph see
[0097.0.0.0].
[2594] [0098.0.5.5] In a preferred embodiment, the fine chemical
(linoleic acid) is produced in accordance with the invention and,
if desired, is isolated. The production of further fatty acids such
as palmitic acid, stearic acid, palmitoleic acid and/or oleic acid
mixtures thereof or mixtures of other fatty acids by the process
according to the invention is advantageous.
[2595] [0099.0.5.5] In the case of the fermentation of
microorganisms, the abovementioned fatty acids may accumulate in
the medium and/or the cells. If microorganisms are used in the
process according to the invention, the fermentation broth can be
processed after the cultivation. Depending on the requirement, all
or some of the biomass can be removed from the fermentation broth
by separation methods such as, for example, centrifugation,
filtration, decanting or a combination of these methods, or else
the biomass can be left in the fermentation broth. The fermentation
broth can subsequently be reduced, or concentrated, with the aid of
known methods such as, for example, rotary evaporator, thin-layer
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. Afterwards advantageously further compounds for
formulation can be added such as corn starch or silicates. This
concentrated fermentation broth advantageously together with
compounds for the formulation can subsequently be processed by
lyophilization, spray drying, spray granulation or by other
methods. Preferably the fatty acids or the fatty acid compositions
are isolated from the organisms, such as the microorganisms or
plants or the culture medium in or on which the organisms have been
grown, or from the organism and the culture medium, in the known
manner, for example via extraction, distillation, crystallization,
chromatography or a combination of these methods. These
purification methods can be used alone or in combination with the
aforementioned methods such as the separation and/or concentration
methods.
[2596] [0100.0.5.5] Transgenic plants which comprise the fatty
acids such as saturated or polyunsaturated fatty acids synthesized
in the process according to the invention can advantageously be
marketed directly without there being any need for the oils, lipids
or fatty acids synthesized to be isolated. Plants for the process
according to the invention are listed as meaning intact plants and
all plant parts, plant organs or plant parts such as leaf, stem,
seeds, root, tubers, anthers, fibers, root hairs, stalks, embryos,
calli, cotelydons, petioles, harvested material, plant tissue,
reproductive tissue and cell cultures which are derived from the
actual transgenic plant and/or can be used for bringing about the
transgenic plant. In this context, the seed comprises all parts of
the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue. However, the fine chemical produced
in the process according to the invention can also be isolated from
the organisms, advantageously plants, in the form of their oils,
fats, lipids and/or free fatty acids. Fatty acids produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts, preferably the plant
seeds. To increase the efficiency of oil extraction it is
beneficial to clean, to temper and if necessary to hull and to
flake the plant material especially the seeds. In this context, the
oils, fats, lipids and/or free fatty acids can be obtained by what
is known as cold beating or cold pressing without applying heat. To
allow for greater ease of disruption of the plant parts,
specifically the seeds, they are previously comminuted, steamed or
roasted. The seeds, which have been pretreated in this manner can
subsequently be pressed or extracted with solvents such as warm
hexane. The solvent is subsequently removed. In the case of
microorganisms, the latter are, after harvesting, for example
extracted directly without further processing steps or else, after
disruption, extracted via various methods with which the skilled
worker is familiar. In this manner, more than 96% of the compounds
produced in the process can be isolated. Thereafter, the resulting
products are processed further, i.e. degummed and/or refined. In
this process, substances such as the plant mucilages and suspended
matter are first removed. What is known as desliming can be
affected enzymatically or, for example, chemico-physically by
addition of acid such as phosphoric acid. Thereafter otionally, the
free fatty acids are removed by treatment with a base like alkali,
for example aqueous KOH or NaOH, or acid hydrolysis, advantageously
in the presence of an alcohol such as methanol or ethanol, or via
enzymatic cleavage, and isolated via, for example, phase separation
and subsequent acidification via, for example, H.sub.2SO.sub.4. The
fatty acids can also be liberated directly without the
above-described processing step. If desired the resulting product
can be washed thoroughly with water to remove traces of soap and
the alkali remaining in the product and then dried. To remove the
pigment remaining in the product, the products can be subjected to
bleaching, for example using filler's earth or active charcoal. At
the end, the product can be deodorized, for example using steam
distillation under vacuum. These chemically pure fatty acids or
fatty acid compositions are advantageous for applications in the
food industry sector, the cosmetic sector and especially the
pharmacological industry sector.
[2597] [0101.0.5.5] for the disclosure of this paragraph see
[0101.0.0.0].
[2598] [0102.0.5.5] Fatty acids can for example be detected
advantageously via GC separation methods. The unambiguous detection
for the presence of fatty acid products can be obtained by
analyzing recombinant organisms using analytical standard methods:
GC, GC-MS or TLC, as described on several occasions by Christie and
the references therein (1997, in: Advances on Lipid Methodology,
Fourth Edition: Christie, Oily Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). One
example is the analysis of fatty acids via FAME and GC-MS or TLC
(abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer chromatography.
The material to be analyzed can be disrupted by sonication,
grinding in a glass mill, liquid nitrogen and grinding or via other
applicable methods. After disruption, the material must be
centrifuged. The sediment is resuspended in distilled water, heated
for 10 minutes at 100.degree. C., cooled on ice and recentrifuged,
followed by extraction for one hour at 90.degree. C. in 0.5 M
sulfuric acid in methanol with 2% dimethoxypropane, which leads to
hydrolyzed oil and lipid compounds, which give transmethylated
lipids. These fatty acid methyl esters are extracted in petroleum
ether and finally subjected to a GC analysis using a capillary
column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 .mu.m, 0.32
mm) at a temperature gradient of between 170.degree. C. and
240.degree. C. for 20 minutes and 5 minutes at 240.degree. C. The
identity of the resulting fatty acid methyl esters must be defined
using standards which are available from commercial sources (i.e.
Sigma).
[2599] [0103.0.5.5] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [2600] a) nucleic acid molecule encoding, preferably
at least the mature form, of a polypeptide having a sequence as
shown in Table II, columns 5 or 7, lines 63 to 67 and 436 to 449 in
or a fragment thereof, which confers an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[2601] b) nucleic acid molecule comprising, preferably at least the
mature form, of the nucleic acid molecule having a sequence as
shown in Table I, columns 5 or 7, lines 63 to 67 and 436 to 449,
[2602] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [2603] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[2604] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [2605]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [2606] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably of (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [2607] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primer pairs
having a sequence as shown in Table III, column 7, lines 63 to 67
and 436 to 449 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [2608]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably of (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [2609] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequence as shown in Table IV, column 7, lines 63
to 67 and 436 to 449 and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[2610] k) nucleic acid molecule comprising one or more of the
nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of a polypeptide as shown in Table
II, columns 5 or 7, lines 63 to 67 and 436 to 449 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; and [2611] l) nucleic acid molecule
which is obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably of (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; or which comprises a sequence which is complementary
thereto.
[2612] [0103.1.0.5] and [0103.2.0.5] for the disclosure of the
paragraphs [0103.1.0.5] and [0103.2.0.5] see paragraphs
[0103.1.0.0] and [0103.2.0.0] above.
[2613] [0104.0.5.5] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence as shown in
Table I, columns 5 or 7, lines 63 to 67 and 436 to 449, preferably
over the sequences as shown in Table IA, columns 5 or 7, lines 63
to 67 and 436 to 449. In one embodiment, the nucleic acid molecule
of the present invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to the sequences shown in Table I, columns 5 or 7,
lines 63 to 67 and 436 to 449, preferably over the sequences as
shown in Table IA, columns 5 or 7, lines 63 to 67 and 436 to 449.
In another embodiment, the nucleic acid molecule does not encode a
polypeptide of the sequence shown in Table II, columns 5 or 7,
lines 63 to 67 and 436 to 449, preferably over the sequences as
shown in Table IA, columns 5 or 7, lines 63 to 67 and 436 to
449.
[2614] [0105.0.0.5] to [0107.0.0.5] for the disclosure of the
paragraphs [0105.0.0.5] to [0107.0.0.5] see paragraphs [0105.0.0.0]
and [0107.0.0.0] above.
[2615] [0108.0.5.5] Nucleic acid molecules with the sequence shown
in Table I, columns 5 or 7, lines 63 to 67 and 436 to 449, nucleic
acid molecules which are derived from the amino acid sequences
shown in Table II, columns 5 or 7, lines 63 to 67 and 436 to 449 or
from polypeptides comprising the consensus sequence shown in Table
IV, column 7, lines 63 to 67 and 436 to 449, or their derivatives
or homologues encoding polypeptides with the enzymatic or
biological activity of a protein as shown in Table II, columns 5 or
7, lines 63 to 67 and 436 to 449 or e.g. conferring a linoleic acid
increase after increasing its expression or activity are
advantageously increased in the process according to the
invention.
[2616] [0109.0.5.5] for the disclosure of this paragraph see
[0109.0.0.0] above.
[2617] [0110.0.5.5] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide used in the method
of the invention or used in the process of the invention, e.g. of a
protein as shown in Table II, columns 5 or 7, lines 63 to 67 and
436 to 449 or being encoded by a nucleic acid molecule indicated in
Table I, columns 5 or 7, lines 63 to 67 and 436 to 449 or of its
homologs, e.g. as indicated in Table II, columns 5 or 7, lines 63
to 67 and 436 to 449 can be determined from generally accessible
databases.
[2618] [0111.0.0.5] for the disclosure of this paragraph see
[0111.0.0.0] above.
[2619] [0112.0.5.5] The nucleic acid molecules used in the process
according to the invention take the form of isolated nucleic acid
sequences, which encode polypeptides with an activity of a
polypeptide as indicated in Table II, column 3, lines lines 63 to
67 and 436 to 449 or having the sequence of a polypeptide as
indicated in Table II, columns 5 and 7, lines 63 to 67 and 436 to
449 and conferring an increase of the respective fine chemical.
[2620] [0113.0.0.5] to [0120.0.0.5] for the disclosure of the
paragraphs [0113.0.0.5] to [0120.0.0.5] see paragraphs [0113.0.0.0]
and [0120.0.0.0] above.
[2621] [0121.0.5.5] However, it is also possible to use artificial
sequences, which differ in one or more bases from the nucleic acid
sequences found in organisms, or in one or more amino acid
molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences shown in Table II,
columns 5 or 7, lines 63 to 67 and 436 to 449 or the functional
homologues thereof as described herein, preferably conferring
above-mentioned activity, i.e. conferring an increase of the
respective fine chemical after increasing its activity.
[2622] [0122.0.0.5] to [0127.0.0.5] for the disclosure of the
paragraphs [0122.0.0.5] to [0127.0.0.5] see paragraphs [0122.0.0.0]
and [0127.0.0.0] above.
[2623] [0128.0.5.5] Synthetic oligonucleotide primers for the
amplification, e.g. as shown in Table III, column 7, lines 63 to 67
and 436 to 449, by means of polymerase chain reaction can be
generated on the basis of a sequence shown herein, for example the
sequence shown in Table I, columns 5 or 7, lines 63 to 67 and 436
to 449 or the sequences as shown in Table II, columns 5 or 7, lines
63 to 67 and 436 to 449.
[2624] [0129.0.5.5] Moreover, it is possible to identify conserved
regions from various organisms by carrying out protein sequence
alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention or the polypeptide used in the method of the invention,
from which conserved regions, and in turn, degenerate primers can
be derived. Conserved regions are those, which show a very little
variation in the amino acid in one particular position of several
homologs from different origin. The consensus sequence shown in
Table IV, column 7, lines 63 to 67 and 436 to 449 is derived from
said alignments and represent such conserved regions.
[2625] [0130.0.5.5] for the disclosure of this paragraph see
[0130.0.0.0].
[2626] [0131.0.0.5] to [0138.0.0.5] for the disclosure of the
paragraphs [0131.0.0.5] to [0138.0.0.5] see paragraphs [0131.0.0.0]
to [0138.0.0.0] above.
[2627] [0139.0.5.5] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical increase, derived from
other organisms, can be encoded by other DNA sequences which
hybridize to a sequences indicated in Table I, columns 5 or 7,
lines 63 to 67 and 436 to 449, preferably of Table I B, columns 5
or 7, lines 63 to 67 and 436 to 449 under relaxed hybridization
conditions and which code on expression for peptides having the
linoleic asid increasing activity.
[2628] [0140.0.0.5] to [0146.0.0.5] for the disclosure of the
paragraphs [0140.0.0.5] to [0146.0.0.5] see paragraphs [0140.0.0.0]
and [0146.0.0.0] above.
[2629] [0147.0.5.5] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 63 to 67 and 436 to 449, preferably in
Table I B, columns 5 or 7, lines 63 to 67 and 436 to 449 is one
which is sufficiently complementary to one of said nucleotide
sequences such that it can hybridize to one of said nucleotide
sequences thereby forming a stable duplex. Preferably, the
hybridisation is performed under stringent hybridization
conditions. However, a complement of one of the herein disclosed
sequences is preferably a sequence complement thereto according to
the base pairing of nucleic acid molecules well known to the
skilled person. For example, the bases A and G undergo base pairing
with the bases T and U or C, resp. and visa versa. Modifications of
the bases can influence the base-pairing partner.
[2630] [0148.0.5.5] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines
63 to 67 and 436 to 449, preferably of Table I B, columns 5 or 7,
lines 63 to 67 and 436 to 449, or a functional portion thereof and
preferably has above mentioned activity, in particular has
the--fine-chemical-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homologs.
[2631] [0149.0.5.5] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 63 to 67 and 436 to 449, preferably of Table I B, columns 5
or 7, lines 63 to 67 and 436 to 449 or a portion thereof and
encodes a protein having above-mentioned activity and as indicated
in indicated in Table II, columns 5 or 7, lines 63 to 67 and 436 to
449, preferably of Table II B, columns 5 or 7, lines 63 to 67 and
436 to 449, e.g. conferring an increase of the respective fine
chemical.
[2632] [00149.1.0.5] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 63 to 67 and 436 to 449, preferably of Table
I B, columns 5 or 7, lines 63 to 67 and 436 to 449 has further one
or more of the activities annotated or known for the a protein as
indicated in Table II, column 3, lines 63 to 67 and 436 to 449,
preferably of Table II B, columns 3, lines 63 to 67 and 436 to
449.
[2633] [0150.0.5.5] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table I, columns 5 or 7, lines 63 to 67 and 436 to 449, preferably
of Table I B, columns 5 or 7, lines 63 to 67 and 436 to 449, for
example a fragment which can be used as a probe or primer or a
fragment encoding a biologically active portion of the polypeptide
of the present invention or of a polypeptide used in the process of
the present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of linoleic acid if its activity is
increased. The nucleotide sequences determined from the cloning of
the present protein-according-to-the-invention-encoding gene allows
for the generation of probes and primers designed for use in
identifying and/or cloning its homologues in other cell types and
organisms. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, 15 preferably about 20 or 25, more
preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one of the sequences indicated in Table I, columns 5 or
7, lines 63 to 67 and 436 to 449, an anti-sense sequence of one of
the sequences indicated in Table I, columns 5 or 7, lines 63 to 67
and 436 to 449, or naturally occurring mutants thereof. Primers
based on a nucleotide sequence of the invention can be used in PCR
reactions to clone homologues of the polypeptide of the invention
or of the polypeptide used in the process of the invention, e.g. as
the primers described in the examples of the present invention,
e.g. as shown in the examples. A PCR with the primer pairs
indicated in Table III, column 7, lines 63 to 67 and 436 to 449
will result in a fragment of a polynucleotide sequence as indicated
in Table I, columns 5 or 7, lines 63 to 67 and 436 to 449.
Preferred is Table I B, column 7, lines 63 to 67 and 436 to
449.
[2634] [0151.0.0.5] for the disclosure of this paragraph see
[0151.0.0.0] above.
[2635] [0152.0.5.5] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to the amino acid
sequence as shown in Table II, columns 5 or 7, lines 63 to 67 and
436 to 449 such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular a linoleic acid increasing activity as mentioned
above or as described in the examples in plants or microorganisms
is comprised.
[2636] [0153.0.5.5] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence shown in Table II, columns 5 or 7, lines 63 to 67 and
436 to 449 such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
shown in Table II, columns 5 or 7, lines 63 to 67 and 436 to 449
has for example an activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 63 to 67 and 436 to 449 are described
herein.
[2637] [0154.0.5.5] In one embodiment, the nucleic acid molecule of
the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as shown in Table II, columns 5 or 7, lines 63 to 67 and
436 to 449 and having above-mentioned activity, e.g. conferring
preferably the increase of the respective fine chemical.
[2638] [0155.0.0.5] and [0156.0.0.5] for the disclosure of the
paragraphs [0155.0.0.5] and [0156.0.0.5] see paragraphs
[0155.0.0.0] and [0156.0.0.0] above.
[2639] [0157.0.5.5] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 63 to 67 and 436 to 449
(and portions thereof) due to degeneracy of the genetic code and
thus encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides comprising the consensus sequences as indicated
in Table IV, column 7, lines 63 to 67 and 436 to 449 or of the
polypeptide as indicated in Table II, columns 5 or 7, lines 63 to
67 and 436 to 449 or their functional homologues. Advantageously,
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention comprises, or in an
other embodiment has, a nucleotide sequence encoding a protein
comprising, or in an other embodiment having, a consensus sequences
as indicated in Table IV, column 7, lines 63 to 67 and 436 to 449
or of the polypeptide as indicated in Table II, columns 5 or 7,
lines 63 to 67 and 436 to 449 or the functional homologues. In a
still further embodiment, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention encodes a full length protein which is substantially
homologous to an amino acid sequence comprising a consensus
sequence as indicated in Table IV, columns 5 or 7, lines 63 to 67
and 436 to 449 or of a polypeptide as indicated in Table II,
columns 5 or 7, lines 63 to 67 and 436 to 449 or the functional
homologues thereof. However, in a preferred embodiment, the nucleic
acid molecule of the present invention does not consist of a
sequence as indicated in Table I, columns 5 or 7, lines 63 to 67
and 436 to 449, preferably as indicated in Table I A, columns 5 or
7, lines 63 to 67 and 436 to 449. Preferably the nucleic acid
molecule of the invention is a functional homologue or identical to
a nucleic acid molecule indicated in Table I B, columns 5 or 7,
lines 63 to 67 and 436 to 449.
[2640] [0158.0.0.5] to [0160.0.0.5] for the disclosure of the
paragraphs [0158.0.0.5] to [0160.0.0.5] see paragraphs [0158.0.0.0]
to [0160.0.0.0] above.
[2641] [0161.0.5.5] Accordingly, in another embodiment, a nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising the
sequence shown in Table I, columns 5 or 7, lines 63 to 67 and 436
to 449. The nucleic acid molecule is preferably at least 20, 30,
50, 100, 250 or more nucleotides in length.
[2642] [0162.0.0.5] for the disclosure of this paragraph see
paragraph [0162.0.0.0] above.
[2643] [0163.0.5.5] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 63 to 67 and 436 to
449 corresponds to a naturally-occurring nucleic acid molecule of
the invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[2644] [0164.0.0.5] for the disclosure of this paragraph see
paragraph [0164.0.0.0] above.
[2645] [0165.0.5.5] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as shown in
Table I, columns 5 or 7, lines 63 to 67 and 436 to 449.
[2646] [0166.0.0.5] and [0167.0.0.5] for the disclosure of the
paragraphs [0166.0.0.5] and [0167.0.0.5] see paragraphs
[0166.0.0.0] and [0167.0.0.0] above.
[2647] [0168.0.5.5] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organism or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 63 to 67 and 436 to 449 yet retain said activity described
herein. The nucleic acid molecule can comprise a nucleotide
sequence encoding a polypeptide, wherein the polypeptide comprises
an amino acid sequence at least about 50% identical to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 63 to
67 and 436 to 449 and is capable of participation in the increase
of production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to the
sequence as indicated in Table II, columns 5 or 7, lines 63 to 67
and 436 to 449, more preferably at least about 70% identical to one
of the sequences as indicated in Table II, columns 5 or 7, lines 63
to 67 and 436 to 449, even more preferably at least about 80%, 90%,
95% homologous to the sequence as indicated in Table II, columns 5
or 7, lines 63 to 67 and 436 to 449, and most preferably at least
about 96%, 97%, 98%, or 99% identical to the sequence as indicated
in Table II, columns 5 or 7, lines 63 to 67 and 436 to 449.
[2648] Accordingly, the invention relates to nucleic acid molecules
encoding a polypeptide having above-mentioned activity, e.g.
conferring an increase in the the respective fine chemical in an
organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 63 to 67 and 436 to 449, preferably of Table II B, column 7,
lines 63 to 67 and 436 to 449 yet retain said activity described
herein. The nucleic acid molecule can comprise a nucleotide
sequence encoding a polypeptide, wherein the polypeptide comprises
an amino acid sequence at least about 50% identical to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 63 to
67 and 436 to 449, preferably of Table II B, column 7, lines 63 to
67 and 436 to 449 and is capable of participation in the increase
of production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table II, columns 5 or 7, lines 63 to 67
and 436 to 449, preferably of Table II B, column 7, lines 63 to 67
and 436 to 449, more preferably at least about 70% identical to one
of the sequences as indicated in Table II, columns 5 or 7, lines 63
to 67 and 436 to 449, preferably of Table II B, column 7, lines 63
to 67 and 436 to 449, even more preferably at least about 80%, 90%,
or 95% homologous to a sequence as indicated in Table II, columns 5
or 7, lines 63 to 67 and 436 to 449, preferably of Table II B,
column 7, lines 63 to 67 and 436 to 449, and most preferably at
least about 96%, 97%, 98%, or 99% identical to the sequence as
indicated in Table II, columns 5 or 7, lines 63 to 67 and 436 to
449, preferably of Table II B, column 7, lines 63 to 67 and 436 to
449.
[2649] [0169.0.0.5] to [0175.0.5.5] for the disclosure of the
paragraphs [0169.0.0.5] to [0175.0.5.5] see paragraphs [0169.0.0.0]
to [0175.0.0.0] above.
[2650] [0176.0.5.5] Functional equivalents derived from one of the
polypeptides as shown in Table II, columns 5 or 7, lines 63 to 67
and 436 to 449 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as shown in Table II, columns
5 or 7, lines 63 to 67 and 436 to 449 according to the invention
and are distinguished by essentially the same properties as the
polypeptide as shown in Table II, columns 5 or 7, lines 63 to 67
and 436 to 449.
[2651] [0177.0.5.5] Functional equivalents derived from the nucleic
acid sequence as shown in Table I, columns 5 or 7, lines 63 to 67
and 436 to 449 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as shown in Table II, columns
5 or 7, lines 63 to 67 and 436 to 449 according to the invention
and encode polypeptides having essentially the same properties as
the polypeptide as shown in Table II, columns 5 or 7, lines 63 to
67 and 436 to 449.
[2652] [0178.0.0.5] for the disclosure of this paragraph see
[0178.0.0.0] above.
[2653] [0179.0.5.5] A nucleic acid molecule encoding a homologous
protein to a protein sequence of as indicated in Table II, columns
5 or 7, lines 63 to 67 and 436 to 449, preferably of Table II B,
column 7, lines 63 to 67 and 436 to 449 can be created by
introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of the nucleic acid molecule
of the present invention, in particular as indicated in Table I,
columns 5 or 7, lines 63 to 67 and 436 to 449 such that one or more
amino acid substitutions, additions or deletions are introduced
into the encoded protein. Mutations can be introduced into the
encoding sequences for example into a sequences as indicated in
Table I, columns 5 or 7, lines 63 to 67 and 436 to 449 by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[2654] [0180.0.0.5] to [0183.0.0.5] for the disclosure of the
paragraphs [0180.0.0.5] to [0183.0.0.5] see paragraphs [0180.0.0.0]
to [0183.0.0.0] above.
[2655] [0184.0.5.5] Homologues of the nucleic acid sequences used,
with a sequence as indicated in Table I, columns 5 or 7, lines 63
to 67 and 436 to 449, preferably of Table I B, column 7, lines 63
to 67 and 436 to 449, or of the nucleic acid sequences derived from
a sequences as indicated in Table II, columns 5 or 7, lines 63 to
67 and 436 to 449, preferably of Table II B, column 7, lines 63 to
67 and 436 to 449, comprise also allelic variants with at least
approximately 30%, 35%, 40% or 45% homology, by preference at least
approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
I, columns 5 or 7, lines 63 to 67 and 436 to 449, or from the
derived nucleic acid sequences, the intention being, however, that
the enzyme activity or the biological activity of the resulting
proteins synthesized is advantageously retained or increased.
[2656] [0185.0.5.5] In one embodiment of the present invention, the
nucleic acid molecule of the invention or used in the process of
the invention comprises one or more sequences as indicated in Table
I, columns 5 or 7, lines 63 to 67 and 436 to 449, preferably of
Table I B, column 7, lines 63 to 67 and 436 to 449. In one
embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotide sequences not
shown in any one of sequences as indicated in Table I, columns 5 or
7, lines 63 to 67 and 436 to 449, preferably of Table I B, column
7, lines 63 to 67 and 436 to 449. In one embodiment, the nucleic
acid molecule comprises less than 500, 400, 300, 200, 100, 90, 80,
70, 60, 50 or 40 further nucleotides. In a further embodiment, the
nucleic acid molecule comprises less than 30, 20 or 10 further
nucleotides. In one embodiment, a nucleic acid molecule used in the
process of the invention is identical to a sequences as indicated
in Table I, columns 5 or 7, lines 63 to 67 and 436 to 449,
preferably of Table I B, column 7, lines 63 to 67 and 436 to
449.
[2657] [0186.0.5.5] Also preferred is that one or more nucleic acid
molecule(s) used in the process of the invention encode a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 63 to 67 and 436 to 449, preferably of Table II B,
column 7, lines 63 to 67 and 436 to 449. In one embodiment, the
nucleic acid molecule encodes less than 150, 130, 100, 80, 60, 50,
40 or 30 further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table II, columns 5 or 7, lines 63 to 67 and 436 to 449,
preferably of Table II B, column 7, lines 63 to 67 and 436 to
449.
[2658] [0187.0.5.5] In one embodiment, the nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table II, columns 5 or 7,
lines 63 to 67 and 436 to 449, preferably of Table II B, column 7,
lines 63 to 67 and 436 to 449 and comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a sequences as indicated in Table II, columns 5
or 7, lines 63 to 67 and 436 to 449, preferably of Table II B,
column 7, lines 63 to 67 and 436 to 449.
[2659] [0188.0.5.5] Polypeptides (=proteins), which still have the
essential biological activity of the polypeptide of the present
invention conferring an increase of the fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide shown in Table II, columns 5 or 7, lines 63 to 67
and 436 to 449 expressed under identical conditions.
[2660] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, columns 7, lines 63 to 67 and 436 to 449.
[2661] [0189.0.5.5] Homologues of sequences as indicated in Table
I, columns 5 or 7, lines 63 to 67 and 436 to 449 or of the derived
sequences shown in Table II, columns 5 or 7, lines 63 to 67 and 436
to 449 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[2662] [0190.0.0.5], [0191.0.5.5], [00191.1.0.5] and [0192.0.0.5]
to [0203.0.0.5] for the disclosure of the paragraphs [0190.0.0.5],
[0191.0.5.5], [0191.1.0.5] and [0192.0.0.5] to [0203.0.0.5] see
paragraphs [0190.0.0.0], [0191.0.0.0], [0191.1.0.0] and
[0192.0.0.0] to [0203.0.0.0] above.
[2663] [0204.0.5.5] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [2664] a) nucleic
acid molecule encoding, preferably at least the mature form, of the
polypeptide shown in Table II, columns 5 or 7, lines 63 to 67 and
436 to 449; preferably of Table II B, column 7, lines 63 to 67 and
436 to 449; or a fragment thereof conferring an increase in the
amount of the fine chemical in an organism or a part thereof [2665]
b) nucleic acid molecule comprising, preferably at least the mature
form, of the nucleic acid molecule shown in Table I, columns 5 or
7, lines 63 to 67 and 436 to 449 preferably of Table I B, column 7,
lines 63 to 67 and 436 to 449; or a fragment thereof conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [2666] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [2667] d) nucleic
acid molecule encoding a polypeptide whose sequence has at least
50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [2668] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [2669] f) nucleic acid molecule encoding a polypeptide,
the polypeptide being derived by substituting, deleting and/or
adding one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[2670] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to [2671] (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [2672] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying a cDNA library or a genomic library using the primers or
primer pairs as indicated in Table III, column 7, lines 63 to 67
and 436 to 449 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [2673]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from a expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (g), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [2674] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence shown in
Table IV, column 7, lines 63 to 67 and 436 to 449 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [2675] k) nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a
domaine of the polypeptide shown in Table II, columns 5 or 7, lines
63 to 67 and 436 to 449, preferably of Table II B, column 7, lines
63 to 67 and 436 to 449; and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
and [2676] l) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of the nucleic acid molecule shown in Table I, columns 5
or 7, lines 63 to 67 and 436 to 449 or a nucleic acid molecule
encoding, preferably at least the mature form of, the polypeptide
shown in Table II, columns 5 or 7, lines 63 to 67 and 436 to 449,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which encompasses a
sequence which is complementary thereto; whereby, preferably, the
nucleic acid molecule according to (a) to (l) distinguishes over
the sequence as depicted in Table IA or IB, columns 5 or 7, lines
63 to 67 and 436 to 449 by one or more nucleotides. In one
embodiment, the nucleic acid molecule of the invention does not
consist of the sequence shown in Table IA or IB, columns 5 or 7,
lines 63 to 67 and 436 to 449. In an other embodiment, the nucleic
acid molecule of the present invention is at least 30% identical
and less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to the
sequence shown in Table IA or IB, columns 5 or 7, lines 63 to 67
and 436 to 449. In a further embodiment the nucleic acid molecule
does not encode the polypeptide sequence shown in Table IIA or IIB,
columns 5 or 7, lines 63 to 67 and 436 to 449. Accordingly, in one
embodiment, the nucleic acid molecule of the present invention
encodes in one embodiment a polypeptide which differs at least in
one or more amino acids from the polypeptide as depicted in Table
IIA or IIB, columns 5 or 7, lines 63 to 67 and 436 to 449 and
therefore does not encode a protein of the sequence shown in Table
IIA or IIB, columns 5 or 7, lines 63 to 67 and 436 to 449.
Accordingly, in one embodiment, the protein encoded by a sequence
of a nucleic acid according to (a) to (l) does not consist of the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 63 to 67
and 436 to 449. In a further embodiment, the protein of the present
invention is at least 30% identical to protein sequence depicted in
Table IIA or IIB, columns 5 or 7, lines 63 to 67 and 436 to 449 and
less than 100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 985, 97%, 96% or 95% identical to the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 63 to 67
and 436 to 449.
[2677] [0205.0.0.5] to [0226.0.0.5] for the disclosure of the
paragraphs [0205.0.0.5] to [0226.0.0.5] see paragraphs [0205.0.0.0]
to [0226.0.0.0] above.
[2678] [0227.0.5.5] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[2679] In addition to the sequence mentioned in Table I, columns 5
or 7, lines 63 to 67 and 436 to 449 or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the fatty acid biosynthetic pathway such as for
palmitate, palmitoleate, stearate and/or oleate is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the respective
desired fine chemical since, for example, feedback regulations no
longer exist to the same extent or not at all. In addition it might
be advantageously to combine the sequences shown in Table I,
columns 5 or 7, lines 63 to 67 and 436 to 449 with genes which
generally support or enhances to growth or yield of the target
organisms, for example genes which lead to faster growth rate of
microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[2680] [0228.0.5.5] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the fatty acid
metabolism, in particular in fatty acid synthesis.
[2681] [0229.0.5.5] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the saturated, poly unsaturated
fatty acid biosynthesis such as desaturases like
.DELTA.-4-desaturases, .DELTA.-5-desaturases,
.DELTA.-6-desaturases, .DELTA.-8-desaturases,
.DELTA.-9-desaturases, .DELTA.-12-desaturases,
.DELTA.-17-desaturases, .omega.-3-desaturases, elongases like
.DELTA.-5-elongases, .DELTA.-6-elongases, 9-elongases,
acyl-CoA-dehydrogenases, acyl-ACP-desaturases,
acyl-ACP-thioesterases, fatty acid acyl-transferases, acyl-CoA
lysophospholipid-acyltransferases, acyl-CoA carboxylases, fatty
acid synthases, fatty acid hydroxylases, acyl-CoA oxydases,
acetylenases, lipoxygenases, triacyl-lipases etc. as described in
WO 98/46765, WO 98/46763, WO 98/46764, WO 99/64616, WO 00/20603, WO
00/20602, WO 00/40705, US 20040172682, US 20020156254, U.S. Pat.
No. 6,677,145 US 20040053379 or US 20030101486. These genes lead to
an increased synthesis of the essential fatty acids.
[2682] [0230.0.5.5] for the disclosure of this paragraph see
paragraph [0230.0.0.0] above.
[2683] [0231.0.5.5] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a linoleic acid degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[2684] [0232.0.0.5] to [0276.0.0.5] for the disclosure of the
paragraphs [0232.0.0.5] to [0276.0.0.5] see paragraphs [0232.0.0.0]
to [0276.0.0.0] above.
[2685] [0277.0.5.5] The fatty acids produced can be isolated from
the organism by methods with which the skilled worker is familiar.
For example via extraction, salt precipitation and/or different
chromatography methods. The process according to the invention can
be conducted batchwise, semibatchwise or continuously. The fine
chemical produced in the process according to the invention can be
isolated as mentioned above from the organisms, advantageously
plants, in the form of their oils, fats, lipids and/or free fatty
acids. Fatty acids produced by this process can be obtained by
harvesting the organisms, either from the crop in which they grow,
or from the field. This can be done via pressing or extraction of
the plant parts, preferably the plant seeds. Hexane is preferably
used as solvent in the process, in which more than 96% of the
compounds produced in the process can be isolated. Thereafter, the
resulting products are processed further, i.e. degummed, refined,
bleached and/or deodorized.
[2686] [0278.0.0.5] to [0282.0.0.5] for the disclosure of the
paragraphs [0278.0.0.5] to [0282.0.0.5] see paragraphs [0278.0.0.0]
to [0282.0.0.0] above.
[2687] [0283.0.5.5] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an antibody against polypeptides as shown in
Table II, columns 5 or 7, lines 63 to 67 and 436 to 449 or an
antigenic part thereof, which can be produced by standard
techniques utilizing polypeptides comprising or consisting of
abovementioned sequences, e.g. the polypeptide of the present
invention or fragment thereof. Preferred are monoclonal antibodies
specifically binding to polypeptides as indicated in Table II,
columns 5 or 7, lines 63 to 67 and 436 to 449.
[2688] [0284.0.0.5] for the disclosure of this paragraph see
[0284.0.0.0] above.
[2689] [0285.0.5.5] In one embodiment, the present invention
relates to a polypeptide having the sequence shown in Table II,
columns 5 or 7, lines 63 to 67 and 436 to 449 or as coded by the
nucleic acid molecule shown in Table I, columns 5 or 7, lines 63 to
67 and 436 to 449 or functional homologues thereof.
[2690] [0286.0.5.5] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 63 to 67 and 436 to 449 and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table IV, column 7, lines 63 to 67 and 436 to 449, whereby 20 or
less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred
5 or 4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a poylpeptide or to a polypeptide comprising more than
one consensus sequences as indicated in Table IV, column 7, lines
63 to 67 and 436 to 449.
[2691] [0287.0.0.5] to [0290.0.0.5] for the disclosure of the
paragraphs [0287.0.0.5] to [0290.0.0.5] see paragraphs [0287.0.0.0]
to [0290.0.0.0] above.
[2692] [0291.0.5.5] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of a
plant or microorganism specific consensus sequences.
[2693] In one embodiment, said polypeptide of the invention
distinguishes over the sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 63 to 67 and 436 to 449 by one or more amino
acids. In one embodiment, polypeptide distinguishes form the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 63 to 67
and 436 to 449 by more than 5, 6, 7, 8 or 9 amino acids, preferably
by more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred
are more than 40, 50, or 60 amino acids and, preferably, the
sequence of the polypeptide of the invention distinguishes from the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 63 to 67
and 436 to 449 by not more than 80% or 70% of the amino acids,
preferably not more than 60% or 50%, more preferred not more than
40% or 30%, even more preferred not more than 20% or 10%. In
another embodiment, said polypeptide of the invention does not
consist of the sequence shown in Table IIA or IIB, columns 5 or 7,
lines 63 to 67 and 436 to 449.
[2694] [0292.0.0.5] for the disclosure of this paragraph see
[0292.0.0.0] above.
[2695] [0293.0.5.5] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the
process.
[2696] In one embodiment, the polypeptide of the invention has a
sequence, which distinguishes from the sequence as shown in Table
IIA or IIB, columns 5 or 7, lines 63 to 67 and 436 to 449 by one or
more amino acids. In another embodiment, said polypeptide of the
invention does not consist of the sequence shown in Table IIA or
IIB, columns 5 or 7, lines 63 to 67 and 436 to 449. In a further
embodiment, said polypeptide of the present invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical. In one embodiment,
said polypeptide does not consist of the sequence encoded by the
nucleic acid molecules shown in Table IA or IB, columns 5 or 7,
lines 63 to 67 and 436 to 449.
[2697] [0294.0.5.5] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table IIA or IIB, column 3, lines 63 to 67 and 436 to
449, which distinguishes over the sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 63 to 67 and 436 to 449 by one or
more amino acids, preferably by more than 5, 6, 7, 8 or 9 amino
acids, preferably by more than 10, 15, 20, 25 or 30 amino acids,
evenmore preferred are more than 40, 50, or 60 amino acids but even
more preferred by less than 70% of the amino acids, more preferred
by less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[2698] [0295.0.0.5], [0296.0.0.5] and [0297.0.5.5] for the
disclosure of the paragraphs [0295.0.0.5], [0296.0.0.5] and
[0297.0.5.5] see paragraphs [0295.0.0.0] to [0297.0.0.0] above.
[2699] [00297.1.0.5] Non-polypeptide of the invention-chemicals are
e.g. polypeptides having not the activity and/or the amino acid
sequence of a polypeptide indicated in Table II, columns 3, 5 or 7,
lines 63 to 67 and 436 to 449.
[2700] [0298.0.5.5] A polypeptide of the invention can participate
in the process of the present invention. The polypeptide or a
portion thereof comprises preferably an amino acid sequence which
is sufficiently homologous to an amino acid sequence as indicated
in Table II, columns 5 or 7, lines 63 to 67 and 436 to 449 such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table II, columns 5 or 7, lines 63 to 67 and 436 to 449.
[2701] [0299.0.5.5] Further, the polypeptide can have an amino acid
sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences of Table II, columns 5 or 7, lines 63 to 67
and 436 to 449. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence of Table I, columns
5 or 7, lines 63 to 67 and 436 to 449 or which is homologous
thereto, as defined above.
[2702] [0300.0.5.5] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 63 to 67 and 436 to 449 in amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 63 to 67 and 436 to 449.
[2703] [0301.0.0.5] for the disclosure of this paragraph see
[0301.0.0.0] above.
[2704] [0302.0.5.5] Biologically active portions of an polypeptide
of the present invention include peptides comprising amino acid
sequences derived from the amino acid sequence of the polypeptide
of the present invention or used in the process of the present
invention, e.g., an amino acid sequence shown in Table II, columns
5 or 7, lines 63 to 67 and 436 to 449 or the amino acid sequence of
a protein homologous thereto, which include fewer amino acids than
a full length polypeptide of the present invention or used in the
process of the present invention or the full length protein which
is homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[2705] [0303.0.0.5] for the disclosure of this paragraph see
[0303.0.0.0] above.
[2706] [0304.0.5.5] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, columns 5 or 7, lines 63 to 67 and 436 to 449 but having
differences in the sequence from said wild-type protein. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity in relation to the wild type protein.
[2707] [0305.0.5.5], [0306.0.5.5] and [0306.1.0.5] for the
disclosure of the paragraphs [0305.0.5.5], [0306.0.5.5] and
[0306.1.0.5] see paragraphs [0305.0.0.0], [0306.0.0.0] and
[0306.1.0.0] above.
[2708] [0307.0.0.5] and [0308.0.0.5] for the disclosure of the
paragraphs [0307.0.0.5] and [0308.0.0.5] see paragraphs [0307.0.0.0
and [0308.0.0.0] above.
[2709] [0309.0.0.5] In one embodiment, a reference to protein
(=polypeptide)" of the invention e.g. as indicated in Table II,
columns 5 or 7, lines 63 to 67 and 436 to 449 refers to a
polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas a "non-polypeptide" or "other polypeptide" e.g.
not indicated in Table II, columns 5 or 7, lines 63 to 67 and 436
to 449 refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to
a polypeptide of the invention, preferably which is not
substantially homologous to a polypeptide having a protein
activity, e.g., a protein which does not confer the activity
described herein and which is derived from the same or a different
organism.
[2710] [0310.0.0.5] to [0334.0.0.5] for the disclosure of the
paragraphs [0310.0.0.5] to [0334.0.0.5] see paragraphs [0310.0.0.0]
to [0334.0.0.0] above.
[2711] [0335.0.5.5] The dsRNAi method has proved to be particularly
effective and advantageous for reducing the expression of a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 63 to
67 and 436 to 449 and/or homologs thereof. As described inter alia
in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table I,
columns 5 or 7, lines 63 to 67 and 436 to 449 and/or homologs
thereof. In a double-stranded RNA molecule for reducing the
expression of an protein encoded by a nucleic acid sequence of one
of the sequences as indicated in Table I, columns 5 or 7, lines 63
to 67 and 436 to 449 and/or homologs thereof, one of the two RNA
strands is essentially identical to at least part of a nucleic acid
sequence, and the respective other RNA strand is essentially
identical to at least part of the complementary strand of a nucleic
acid sequence.
[2712] [0336.0.0.5] to [0342.0.0.5] for the disclosure of the
paragraphs [0336.0.0.5] to [0342.0.0.5] see paragraphs [0336.0.0.0]
to [0342.0.0.0] above.
[2713] [0343.0.5.5] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 63 to 67
and 436 to 449 or its homolog is not necessarily required in order
to bring about effective reduction in the expression. The advantage
is, accordingly, that the method is tolerant with regard to
sequence deviations as may be present as a consequence of genetic
mutations, polymorphisms or evolutionary divergences. Thus, for
example, using the dsRNA, which has been generated starting from a
sequence of one of the sequences as indicated in Table I, columns 5
or 7, lines 63 to 67 and 436 to 449 or homologs thereof of the one
organism, may be used to suppress the corresponding expression in
another organism.
[2714] [0344.0.0.5] to [0350.0.0.5], [0351.0.5.5] and [0352.0.0.5]
to [0361.0.0.5] for the disclosure of the paragraphs [0344.0.0.5]
to [0350.0.0.5], [0351.0.5.5] and [0352.0.0.5] to [0361.0.0.5] see
paragraphs [0344.0.0.0] to [0361.0.0.0] above.
[2715] [0362.0.5.5] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, the nucleic acid construct of
the invention, the antisense molecule of the invention, the vector
of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. encoding a polypeptide having an
activity of a protein such as the polypeptides as indicated in
Table II, column 3, lines 63 to 67 and 436 to 449. Due to the above
mentioned activity the fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a protein such
as the polypeptides as indicated in Table II, column 3, lines 63 to
67 and 436 to 449. Activity means herein that due to modulation or
manipulation of the genome, the activity of polypeptide having an
activity of a protein such as the polypeptides as indicated in
Table II, column 3, lines 63 to 67 and 436 to 449 or a similar
activity, which is increased in the cell or organism or part
thereof. Examples are described above in context with the process
of the invention.
[2716] [0363.0.0.5], [0364.0.5.5] and [0365.0.0.5] to [0379.0.5.5]
for the disclosure of the paragraphs [0363.0.0.5], [0364.0.5.5] and
[0365.0.0.5] to [0379.0.5.5] see paragraphs [0363.0.0.0] to
[0379.0.0.0] above.
[2717] [0380.0.5.5] The fatty acids obtained in the process are
suitable as starting material for the synthesis of further products
of value. For example, they can be used in combination with each
other or alone for the production of pharmaceuticals, foodstuffs,
animal feeds or cosmetics. Accordingly, the present invention
relates a method for the production of a pharmaceuticals, food
stuff, animal feeds, nutrients or cosmetics comprising the steps of
the process according to the invention, including the isolation of
the fatty acid composition produced or the fine chemical produced
if desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the fatty acids produced in the
process or of the transgenic organisms in animal feeds, foodstuffs,
medicines, food supplements, cosmetics or pharmaceuticals.
[2718] [0381.0.0.5] and [0382.0.0.5] for the disclosure of the
paragraphs [0381.0.0.5] and [0382.0.0.5] see paragraphs
[0381.0.0.0] and [0382.0.0.0] above.
[2719] [0383.0.5.5] For preparing fatty acid compound-containing
fine chemicals, in particular the fine chemical, it is possible to
use as fatty acid source organic compounds such as, for example,
oils, fats and/or lipids comprising fatty acids such as fatty acids
having a carbon back bone between C.sub.10- to C.sub.16-carbon
atoms and/or small organic acids such acetic acid, propionic acid
or butanoic acid as precursor compounds.
[2720] [0384.0.0.5] and [0385.0.5.5] for the disclosure of the
paragraphs [0384.0.0.5] and [0385.0.5.5] see paragraphs
[0384.0.0.0] and [0385.0.0.0] above.
[2721] [0386.0.5.5] However, it is also possible to purify the
fatty acid produced further. For this purpose, the
product-containing composition is subjected for example to a thin
layer chromatography on silica gel plates or to a chromatography
such as a Florisil column (Bouhours J. F., J. Chromatrogr. 1979,
169, 462), in which case the desired product or the impurities are
retained wholly or partly on the chromatography resin. These
chromatography steps can be repeated if necessary, using the same
or different chromatography resins. The skilled worker is familiar
with the choice of suitable chromatography resins and their most
effective use. An alternative method to purify the fatty acids is
for example a crystallization in the presence of urea. These
methods can be combined with each other.
[2722] [0387.0.0.5] to [0392.0.0.5] for the disclosure of the
paragraphs [0387.0.0.5] to [0392.0.0.5] see paragraphs [0387.0.0.0]
to [0392.0.0.0] above.
[2723] [0393.0.5.5] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [2724] (a) contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [2725] (b) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence shown in Table I, columns 5 or 7, lines 63 to 67
and 436 to 449, preferably in Table I B, columns 5 or 7, lines 63
to 67 and 436 to 449 and, optionally, isolating the full length
cDNA clone or complete genomic clone; [2726] (c) introducing the
candidate nucleic acid molecules in host cells, preferably in a
plant cell or a microorganism, appropriate for producing the
respective fine chemical; [2727] (d) expressing the identified
nucleic acid molecules in the host cells; [2728] (e) assaying the
respective fine chemical level in the host cells; and [2729] (f)
identifying the nucleic acid molecule and its gene product which
expression confers an increase in the respective fine chemical
level in the host cell after expression compared to the wild
type.
[2730] [0394.0.0.5] to [0415.0.0.5] and [0416.0.5.5] for the
disclosure of the paragraphs [0394.0.0.5] to [0415.0.0.5] and
[0416.0.5.5] see paragraphs [0394.0.0.0] to [0416.0.0.0] above.
[2731] [0417.0.5.5] The nucleic acid molecule of the invention, the
vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the fatty acid production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect an organism such as a
microorganism or a plant against inhibitors, which block the fatty
acid, in particular the fine chemical, synthesis in said organism.
Examples of inhibitors or herbicides blocking the fatty acid
synthesis in organism such as microorganism or plants are for
example cerulenin, Thiolactomycin, Diazoborines or Triclosan, which
inhibit the fatty acids (beta-ketoacyl thioester synthetase
inhibitors) and sterol biosynthesis inhibitors,
aryloxyphenoxypropionates such as diclofop, fenoxaprop, haloxyfop,
fluazifop or quizalofop or cyclohexanediones such as clethodim or
sethoxydim
[(2-[1-{ethoxyimino}butyl]-542-{ethylthio}propyl]-3-hydroxy-2-cyclohexen--
1-one], which inhibit the plant acetyl-coenzyme A carboxylase or
thiocarbamates such as butylate, EPTC [=S-ethyl
dipropylcarbamothioat] or vernolate.
[2732] [0418.0.0.5] to [0430.0.0.5] for the disclosure of the
paragraphs [0418.0.0.5] to
[2733] [0430.0.0.5] see paragraphs [0418.0.0.0] to [0430.0.0.0]
above.
[2734] [0431.0.5.5], [0432.0.5.5], [0433.0.0.5] and [0434.0.0.5]
for the disclosure of the paragraphs [0431.0.5.5], [0432.0.5.5],
[0433.0.0.5] and [0434.0.0.5] see paragraphs [0431.0.0.0] to
[0434.0.0.0] above.
[0435.0.5.5] Example 3
In-Vivo and In-Vitro Mutagenesis
[2735] [0436.0.5.5] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of fatty acids can be
carried out by passing a plasmid DNA (or another vector DNA)
containing the desired nucleic acid sequence or nucleic acid
sequences through E. coli and other microorganisms (for example
Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are
not capable of maintaining the integrity of its genetic
information. Usual mutator strains have mutations in the genes for
the DNA repair system [for example mutHLS, mutD, mutT and the like;
for comparison, see Rupp, W. D. (1996) DNA repair mechanisms in
Escherichia coli and Salmonella, pp. 2277-2294, ASM: Washington].
The skilled worker knows these strains. The use of these strains is
illustrated for example in Greener, A. and Callahan, M. (1994)
Strategies 7; 32-34.
[2736] In-vitro mutation methods such as increasing the spontaneous
mutation rates by chemical or physical treatment are well known to
the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below. Site-directed mutagensis method such as the
introduction of desired mutations with an M13 or phagemid vector
and short oligonucleotides primers is a well-known approach for
site-directed mutagensis. The clou of this method involves cloning
of the nucleic acid sequence of the invention into an M13 or
phagemid vector, which permits recovery of single-stranded
recombinant nucleic acid sequence. A mutagenic oligonucleotide
primer is then designed whose sequence is perfectly complementary
to nucleic acid sequence in the region to be mutated, but with a
single difference: at the intended mutation site it bears a base
that is complementary to the desired mutant nucleotide rather than
the original. The mutagenic oligonucleotide is then allowed to
prime new DNA synthesis to create a complementary full-length
sequence containing the desired mutation. Another site-directed
mutagensis method is the PCR mismatch primer mutagensis method also
known to the skilled person. Dpnl site-directed mutagensis is a
further known method as described for example in the Stratagene
Quickchange.TM. site-directed mutagenesis kit protocol. A huge
number of other methods are also known and used in common
practice.
[2737] Positive mutation events can be selected by screening the
organisms for the production of the desired fine chemical.
[0437.0.5.5] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpina
[2738] [0438.0.5.5] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology, VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[2739] [0439.0.5.5] Using standard methods, it is possible to clone
a gene of interest into one of the above-described shuttle vectors
and to introduce such hybrid vectors into the microorganism strains
used in the inventive process. The transformation of Saccharomyces
can be achieved for example by LiCI or sheroplast transformation
(Bishop et al., Mol. Cell. Biol., 6, 1986: 3401-3409; Sherman et
al., Methods in Yeasts in Genetics, [Cold Spring Harbor Lab. Cold
Spring Harbor, N. Y.] 1982, Agatep et al., Technical Tips Online
1998, 1:51: P01525 or Gietz et al., Methods Mol. Cell. Biol. 5,
1995: 255-269) or electroporation (Delorme E., Appl. Environ.
Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[2740] [0440.0.5.5] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002,) teaches a homolog recombination system dependent
on rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this
system for homologous recombination are vectors derived from the
Ylp series. Plasmid vectors derived for example from the 2p-Vector
are known by the skilled worker and used for the expression in
yeasts. Other preferred vectors are for example pART1, pCHY21 or
pEVP11 as they have been described by McLeod et al. (EMBO J. 1987,
6:729-736) and Hoffman et al. (Genes Dev. 5, 1991: :561-571.) or
Russell et al. (J. Biol. Chem. 258, 1983: 143-149.). Other
beneficial yeast vectors are plasmids of the REP, REP-X, pYZ or RIP
series.
[2741] [0441.0.0.5] for the disclosure of this paragraph see
[0441.0.0.0] above.
[2742] [0442.0.5.5] The observations of the acivity of a mutated,
or transgenic, protein in a transformed host cell are based on the
fact that the protein is expressed in a similar manner and in a
similar quantity as the wild-type protein. A suitable method for
determining the transcription quantity of the mutant, or
transgenic, gene (a sign for the amount of mRNA which is available
for the translation of the gene product) is to carry out a Northern
blot (see, for example, Ausubel et al., (1988) Current Protocols in
Molecular Biology, Wiley: New York), where a primer which is
designed in such a way that it binds to the gene of interest is
provided with a detectable marker (usually a radioactive or
chemiluminescent marker) so that, when the total RNA of a culture
of the organism is extracted, separated on a gel, applied to a
stable matrix and incubated with this probe, the binding and
quantity of the binding of the probe indicates the presence and
also the amount of mRNA for this gene. Another method is a
quantitative PCR. This information detects the extent to which the
gene has been transcribed. Total cell RNA can be isolated for
example from yeasts or E. coli by a variety of methods, which are
known in the art, for example with the Ambion kit according to the
instructions of the manufacturer or as described in Edgington et
al., Promega Notes Magazine Number 41, 1993, p. 14.
[2743] [0443.0.0.5] for the disclosure of this paragraph see
[0443.0.0.0] above.
[0444.0.5.5] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[2744] [0445.0.5.5] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[2745] [0446.0.0.5] to [0453.0.0.5] for the disclosure of the
paragraphs [0446.0.0.5] to [0453.0.0.5] see paragraphs [0446.0.0.0]
to [0453.0.0.0] above.
[0454.0.5.5] Example 8
Analysis of the Effect of the Nucleic Acid Molecule on the
Production of the Fatty Acid
[2746] [0455.0.5.5] The effect of the genetic modification in
plants, fungi, algae or ciliates on the production of a desired
compound (such as a fatty acid) can be determined by growing the
modified microorganisms or the modified plant under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the elevated production of
desired product (i.e. of the lipids or a fatty acid). These
analytical techniques are known to the skilled worker and comprise
spectroscopy, thin-layer chromatography, various types of staining
methods, enzymatic and microbiological methods and analytical
chromatography such as high-performance liquid chromatography (see,
for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2,
p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.,
(1987) "Applications of HPLC in Biochemistry" in: Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et
al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery
and purification", p. 469-714, VCH: Weinheim; Belter, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[2747] In addition to the abovementioned processes, plant lipids
are extracted from plant material as described by Cahoon et al.
(1999) Proc. Natl. Acad. Sci. USA 96 (22): 12935-12940 and Browse
et al. (1986) Analytic Biochemistry 152:141-145. The qualitative
and quantitative analysis of lipids or fatty acids is described by
Christie, William W., Advances in Lipid Methodology, Ayr/Scotland:
Oily Press (Oily Press Lipid Library; 2); Christie, William W., Gas
Chromatography and Lipids. A Practical Guide--Ayr, Scotland: Oily
Press, 1989, Repr. 1992, IX, 307 pp. (Oily Press Lipid Library; 1);
"Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952)--16
(1977) under the title: Progress in the Chemistry of Fats and Other
Lipids CODEN.
[2748] [0456.0.0.5] for the disclosure of this paragraph see
[0456.0.0.0] above.
[0457.0.5.5] Example 9
Purification of the Fatty Acid
[2749] [0458.0.5.5] One example is the analysis of fatty acids
(abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid
chromatography/mass spectrometry; TAG, triacylglycerol; TLC,
thin-layer chromatography).
[2750] The unambiguous detection for the presence of fatty acid
products can be obtained by analyzing recombinant organisms using
analytical standard methods: GC, GC-MS or TLC, as described on
several occasions by Christie and the references therein (1997, in:
Advances on Lipid Methodology, Fourth Edition: Christie, Oily
Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). The
total fatty acids produced in the organism for example in yeasts
used in the inventive process can be analysed for example according
to the following procedure: The material such as yeasts, E. coli or
plants to be analyzed can be disrupted by sonication, grinding in a
glass mill, liquid nitrogen and grinding or via other applicable
methods. After disruption, the material must be centrifuged
(1000.times.g, 10 min., 4.degree. C.) and washed once with 100 mM
NaHCO.sub.3, pH 8.0 to remove residual medium and fatty acids. For
preparation of the fatty acid methyl esters (FAMES) the sediment is
resuspended in distilled water, heated for 10 minutes at
100.degree. C., cooled on ice and recentrifuged, followed by
extraction for one hour at 90.degree. C. in 0.5 M sulfuric acid in
methanol with 2% dimethoxypropane, which leads to hydrolyzed oil
and lipid compounds, which give transmethylated lipids.
[2751] The FAMES are then extracted twice with 2 ml petrolether,
washed once with 100 mM NaHCO.sub.3, pH 8.0 and once with distilled
water and dried with Na.sub.2SO.sub.4. The organic solvent can be
evaporated under a stream of Argon and the FAMES were dissolved in
50 .mu.l of petrolether. The samples can be separated on a ZEBRON
ZB-Wax capillary column (30 m, 0.32 mm, 0.25 .mu.m; Phenomenex) in
a Hewlett Packard 6850 gas chromatograph with a flame ionisation
detector. The oven temperature is programmed from 70.degree. C. (1
min. hold) to 200.degree. C. at a rate of 20.degree. C./min., then
to 250.degree. C. (5 min. hold) at a rate of 5.degree. C./min and
finally to 260.degree. C. at a rate of 5.degree. C./min. Nitrogen
is used as carrier gas (4.5 ml/min. at 70.degree. C.). The identity
of the resulting fatty acid methyl esters can be identified by
comparison with retention times of FAME standards, which are
available from commercial sources (i.e. Sigma).
[2752] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[2753] This is followed by heating at 100.degree. C. for 10 minutes
and, after cooling on ice, by resedimentation. The cell sediment is
hydrolyzed for one hour at 90.degree. C. with 1 M methanolic
sulfuric acid and 2% dimethoxypropane, and the lipids are
transmethylated. The resulting fatty acid methyl esters (FAMEs) are
extracted in petroleum ether. The extracted FAMEs are analyzed by
gas liquid chromatography using a capillary column (Chrompack, WCOT
Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and a temperature
gradient of from 170.degree. C. to 240.degree. C. in 20 minutes and
5 minutes at 240.degree. C. The identity of the fatty acid methyl
esters is confirmed by comparison with corresponding FAME standards
(Sigma). The identity and position of the double bond can be
analyzed further by suitable chemical derivatization of the FAME
mixtures, for example to give 4,4-dimethoxyoxazoline derivatives
(Christie, 1998) by means of GC-MS.
[2754] The methodology is described for example in Napier and
Michaelson, 2001, Lipids. 36(8):761-766; Sayanova et al., 2001,
Journal of Experimental Botany. 52(360):1581-1585, Sperling et al.,
2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al.,
1998, FEBS Letters. 439(3):215-218.
[2755] [0459.0.5.5] If required and desired, further chromatography
steps with a suitable resin may follow. Advantageously the fatty
acids can be further purified with a so-called RTHPLC. As eluent
different an acetonitrile/water or chloroform/acetonitrile mixtures
are advantageously is used. For example canola oil can be separated
said HPLC method using an RP-18-column (ET 250/3 Nucleosil 120-5
C.sub.18 Macherey and Nagel, Duren, Germany). A
chloroform/acetonitrile mixture (v/v 30:70) is used as eluent. The
flow rate is beneficial 0.8 ml/min. For the analysis of the fatty
acids an ELSD detector (evaporative light-scattering detector) is
used. MPLC, dry-flash chromatography or thin layer chromatography
are other beneficial chromatography methods for the purification of
fatty acids. If necessary, these chromatography steps may be
repeated, using identical or other chromatography resins. The
skilled worker is familiar with the selection of suitable
chromatography resin and the most effective use for a particular
molecule to be purified.
[2756] [0460.0.5.5] In addition depending on the produced fine
chemical purification is also possible with cristalisation or
destilation. Both methods are well known to a person skilled in the
art.
[0461.0.5.5] Example 10
Cloning SEQ ID NO: 4464 for the Expression in Plants
[2757] [0462.0.0.5] for the disclosure of this paragraph see
[0462.0.0.0] above.
[2758] [0463.0.5.5] SEQ ID NO: 4464 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[2759] [0464.0.5.5] The composition for the protocol of the Pfu
Turbo DNA polymerase was as follows: 1.times.PCR buffer
(Stratagene), 0.2 mM of each dNTP, 100 ng genomic DNA of
Saccharomyces cerevisiae (strain S288C; Research Genetics, Inc.,
now Invitrogen) or Escherichia coli (strain MG1655; E. coli Genetic
Stock Center), 50 .mu.mol forward primer, 50 .mu.mol reverse
primer, 2.5 u Pfu Turbo DNA polymerase. The amplification cycles
were as follows:
[2760] [0465.0.5.5] 1 cycle of 3 minutes at 94-95.degree. C.,
followed by 25-36 cycles of in each case 1 minute at 95.degree. C.
or 30 seconds at 94.degree. C., 45 seconds at 50.degree. C., 30
seconds at 50.degree. C. or 30 seconds at 55.degree. C. and 210-480
seconds at 72.degree. C., followed by 1 cycle of 8 minutes at
72.degree. C., then 4.degree. C.
[2761] [0466.0.0.5] for the disclosure of this paragraph see
[0466.0.0.0] above.
[2762] [0467.0.5.5] The following primer sequences were selected
for the gene SEQ ID NO: 4464:
TABLE-US-00021 i) forward primer (SEQ ID NO: 4556) atgggacaca
agcccttata ccg ii) reverse primer (SEQ ID NO: 4557) ttatcgcgat
gattttcgct gcg
[2763] [0468.0.0.5] to [0479.0.0.5] for the disclosure of the
paragraphs [0468.0.0.5] to [0479.0.0.5] see paragraphs [0468.0.0.0]
to [0479.0.0.0] above.
[0480.0.5.5] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 4464
[2764] [0481.0.0.5] for the disclosure of this paragraph see
[0481.0.0.0] above.
[2765] [0482.0.0.5] to [0513.0.0.5] for the disclosure of the
paragraphs [0482.0.0.5] to [0513.0.0.5] see paragraphs [0482.0.0.0]
to [0513.0.0.0] above.
[2766] [0514.0.5.5] As an alternative, the fatty acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55). [2767] The results of the different plant analyses
can be seen from the table which follows:
TABLE-US-00022 [2767] TABLE 1 ORF Metabolite Method Min Max b0730
Linoleic Acid (C18:2 (c9,c12)) GC 1.13 1.34 b3256 Linoleic Acid
(C18:2 (c9,c12)) GC 1.13 1.20 YBR089C-A Linoleic Acid (C18:2
(c9,c12)) GC 1.36 1.70 YDR447C Linoleic Acid (C18:2 (c9,c12)) GC
1.18 2.40 YOR024W Linoleic Acid (C18:2 (c9,c12)) GC 1.16 1.33 b0050
Linoleic acid (C18:cis[9,12]2) GC 1.15 1.37 b0251 Linoleic acid
(C18:cis[9,12]2) GC 1.14 1.31 b0255 Linoleic acid (C18:cis[9,12]2)
GC 1.22 1.31 b0577 Linoleic acid (C18:cis[9,12]2) GC 1.17 1.40
b0849 Linoleic acid (C18:cis[9,12]2) GC 1.19 1.34 b1097 Linoleic
acid (C18:cis[9,12]2) GC 1.15 1.30 b1693 Linoleic acid
(C18:cis[9,12]2) GC 1.17 1.32 b2710 Linoleic acid (C18:cis[9,12]2)
GC 1.15 1.29 b2822 Linoleic acid (C18:cis[9,12]2) GC 1.15 1.27
b3064 Linoleic acid (C18:cis[9,12]2) GC 1.17 1.30 b3166 Linoleic
acid (C18:cis[9,12]2) GC 1.14 1.21 b3457 Linoleic acid
(C18:cis[9,12]2) GC 1.20 1.44 b3644 Linoleic acid (C18:cis[9,12]2)
GC 1.14 1.35 b4129 Linoleic acid (C18:cis[9,12]2) GC 1.20 1.47
[2768] [0515.0.5.5] Column 2 shows the fatty acid analyzed. Columns
4 and 5 shows the ratio of the analyzed fatty acid between the
transgenic plants and the wild type; Increase of the metabolites:
Max: maximal x-fold (normalised to wild type)-Min: minimal x-fold
(normalised to wild type). Decrease of the metabolites: Max:
maximal x-fold (normalised to wild type) (minimal decrease), Min:
minimal x-fold (normalised to wild type) (maximal decrease). Column
3 indicates the analytical method.
[2769] [0516.0.0.5] and [0517.0.5.5] for the disclosure of the
paragraphs [0516.0.0.5] and [0517.0.5.5] see paragraphs
[0516.0.0.0] and [0517.0.0.0] above.
[2770] [0518.0.0.5] to [0529.0.0.5] and [0530.0.5.5] for the
disclosure of the paragraphs [0518.0.0.5] to [0529.0.0.5] and
[0530.0.5.5] see paragraphs [0518.0.0.0] to [0530.0.0.0] above.
[2771] [0530.1.0.5] to [0530.6.0.5] for the disclosure of the
paragraphs [0530.1.0.5] to [0530.6.0.5] see paragraphs [0530.1.0.0]
to [0530.6.0.0] above.
[2772] [0531.0.0.5] to [0533.0.0.5] and [0534.0.5.5] for the
disclosure of the paragraphs [0531.0.0.5] to [0533.0.0.5] and
[0534.0.5.5] see paragraphs [0531.0.0.0] to [0534.0.0.0] above.
[2773] [0535.0.0.5] to [0537.0.0.5] and [0538.0.5.5] for the
disclosure of the paragraphs [0535.0.0.5] to [0537.0.0.5] and
[0538.0.5.5] see paragraphs [0535.0.0.0] to [0538.0.0.0] above.
[2774] [0539.0.0.5] to [0542.0.0.5] and [0543.0.5.5] for the
disclosure of the paragraphs [0539.0.0.5] to [0542.0.0.5] and
[0543.0.5.5] see paragraphs [0539.0.0.0] to [0543.0.0.0] above.
[2775] [0544.0.0.5] to [0547.0.0.5] and [0548.0.5.5] to
[0552.0.0.5] for the disclosure of the paragraphs [0544.0.0.5] to
[0547.0.0.5] and [0548.0.5.5] to [0552.0.0.5] see paragraphs
[0544.0.0.0] to [0552.0.0.0] above.
[2776] [0552.2.0.5] for the disclosure of this paragraph see
[0552.2.0.0] above.
[2777] [0553.0.5.5] [2778] 1. A process for the production of
linoleic acid, which comprises [2779] (a) increasing or generating
the activity of a protein as indicated in Table II, columns 5 or 7,
lines 63 to 67 and 436 to 449 or a functional equivalent thereof in
a non-human organism, or in one or more parts thereof; and [2780]
(b) growing the organism under conditions which permit the
production of linoleic acid in said organism. [2781] 2. A process
for the production of linoleic acid, comprising the increasing or
generating in an organism or a part thereof the expression of at
least one nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [2782] a) nucleic acid
molecule encoding of a polypeptide as indicated in Table II,
columns 5 or 7, lines 63 to 67 and 436 to 449 or a fragment
thereof, which confers an increase in the amount of linoleic acid
in an organism or a part thereof; [2783] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 63 to 67 and 436 to 449; [2784] c) nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as a
result of the degeneracy of the genetic code and conferring an
increase in the amount of linoleic acid in an organism or a part
thereof; [2785] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of linoleic
acid in an organism or a part thereof; [2786] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under stringent hybridisation conditions and conferring an
increase in the amount of linoleic acid in an organism or a part
thereof; [2787] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, column 7, lines
63 to 67 and 436 to 449 and conferring an increase in the amount of
linoleic acid in an organism or a part thereof; [2788] g) nucleic
acid molecule encoding a polypeptide which is isolated with the aid
of monoclonal antibodies against a polypeptide encoded by one of
the nucleic acid molecules of (a) to (f) and conferring an increase
in the amount of linoleic acid in an organism or a part thereof;
[2789] h) nucleic acid molecule encoding a polypeptide comprising a
consensus sequence as indicated in Table IV, column 7, lines 63 to
67 and 436 to 449 and conferring an increase in the amount of
linoleic acid in an organism or a part thereof; and [2790] i)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment thereof having at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (k) and
conferring an increase in the amount of linoleic acid in an
organism or a part thereof. [2791] or comprising a sequence which
is complementary thereto. [2792] 3. The process of claim 1 or 2,
comprising recovering of the free or bound linoleic acid. [2793] 4.
The process of any one of claims 1 to 3, comprising the following
steps: [2794] (a) selecting an organism or a part thereof
expressing a polypeptide encoded by the nucleic acid molecule
characterized in claim 2; [2795] (b) mutagenizing the selected
organism or the part thereof; [2796] (c) comparing the activity or
the expression level of said polypeptide in the mutagenized
organism or the part thereof with the activity or the expression of
said polypeptide of the selected organisms or the part thereof;
[2797] (d) selecting the mutated organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
[2798] (e) optionally, growing and cultivating the organisms or the
parts thereof; and [2799] (f) recovering, and optionally isolating,
the free or bound linoleic acid produced by the selected mutated
organisms or parts thereof. [2800] 5. The process of any one of
claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. [2801] 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [2802] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 63 to
67 and 436 to 449 or a fragment thereof, which confers an increase
in the amount of linoleic acid in an organism or a part thereof;
[2803] b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 63 to 67
and 436 to 449; [2804] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of linoleic acid in
an organism or a part thereof; [2805] d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of linoleic acid in an organism or a part thereof; [2806] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of linoleic acid in an
organism or a part thereof; [2807] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table III,
columns 5 or 7, lines 63 to 67 and 436 to 449 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [2808] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
linoleic acid in an organism or a part thereof; [2809] h) nucleic
acid molecule encoding a polypeptide comprising a consensus
sequence as indicated in Table IV, column 7, lines 63 to 67 and 436
to 449 and conferring an increase in the amount of linoleic acid in
an organism or a part thereof; and [2810] i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of linoleic acid in an organism or a part thereof. [2811]
whereby the nucleic acid molecule distinguishes over the sequence
as indicated in Table I A, columns 5 or 7, lines 63 to 67 and 436
to 449 by one or more nucleotides. [2812] 7. A nucleic acid
construct which confers the expression of the nucleic acid molecule
of claim 6, comprising one or more regulatory elements. [2813] 8. A
vector comprising the nucleic acid molecule as claimed in claim 6
or the nucleic acid construct of claim 7. [2814] 9. The vector as
claimed in claim 8, wherein the nucleic acid molecule is in
operable linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [2815] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [2816] 11. The host cell of claim 10, which is a
transgenic host cell. [2817] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [2818] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [2819] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table II A, columns 5
or 7, lines 63 to 67 and 436 to 449 by one or more amino acids
[2820] 15. An antibody, which binds specifically to the polypeptide
as claimed in claim 14. [2821] 16. A plant tissue, propagation
material, harvested material or a plant comprising the host cell as
claimed in claim 12 which is plant cell or an Agrobacterium. [2822]
17. A method for screening for agonists and antagonists of the
activity of a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of linoleic acid in an
organism or a part thereof comprising: [2823] (a) contacting cells,
tissues, plants or microorganisms which express the a polypeptide
encoded by the nucleic acid molecule of claim 5 conferring an
increase in the amount of linoleic acid in an organism or a part
thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; [2824] (b) assaying the linoleic acid level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [2825] (c)
identifying a agonist or antagonist by comparing the measured
linoleic acid level or polypeptide expression level with a standard
of linoleic acid or polypeptide expression level measured in the
absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. [2826] 18. A process for
the identification of a compound conferring increased linoleic acid
production in a plant or microorganism, comprising the steps:
[2827] (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of linoleic acid in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with dais readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
linoleic acid in an organism or a part thereof; [2828] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [2829] 19. A method for the identification of a
gene product conferring an increase in linoleic acid production in
a cell, comprising the following steps: [2830] (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in linoleic
acid after expression with the nucleic acid molecule of claim 6;
[2831] (b) identifying the nucleic acid molecules, which hybridise
under relaxed stringent conditions with the nucleic acid molecule
of claim 6; [2832] (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing linoleic acid;
[2833] (d) expressing the identified nucleic acid molecules in the
host cells; [2834] (e) assaying the linoleic acid level in the host
cells; and [2835] (f) identifying nucleic acid molecule and its
gene product which expression confers an increase in the linoleic
acid level in the host cell in the host cell after expression
compared to the wild type. [2836] 20. A method for the
identification of a gene product conferring an increase in linoleic
acid production in a cell, comprising the following steps: [2837]
(a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the linoleic acid amount or level
in an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; [2838]
(b) introducing the candidate nucleic acid molecules in host cells
appropriate for producing linoleic acid; [2839] (c) expressing the
identified nucleic acid molecules in the host cells; [2840] (d)
assaying the linoleic acid level in the host cells; and [2841] (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the linoleic acid level in the
host cell after expression compared to the wild type. [2842] 21. A
method for the production of an agricultural composition comprising
the steps of the method of any one of claims 17 to 20 and
formulating the compound identified in any one of claims 17 to 20
in a form acceptable for an application in agriculture. [2843] 22.
A composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [2844] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
linoleic acid after expression. [2845] 24. Use of the polypeptide
of claim 14 or the nucleic acid construct claim 7 or the gene
product identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of
linoleic acid levels in an organism. [2846] 25. Food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20.
[2847] 26. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the host cell of claim 10 to 12 or the gene
product identified according to the method of claim 19 or 20 for
the protection of a plant against a linoleic acid synthesis
inhibiting herbicide.
[2848] [0554.0.0.5] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[2849] [0000.0.0.6] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[2850] [0001.0.0.6] for the disclosure of this paragraph see
[0001.0.0.0].
[2851] [0002.0.5.6] to [0008.0.5.6] for the disclosure of the
paragraphs [0002.0.5.6] to [0008.0.5.6] see [0002.0.0.0] and
[0008.0.0.0] above.
[2852] [0009.0.6.6] As described above, the essential fatty acids
are necessary for humans and many mammals, for example for
livestock. Essential fatty acids, such as alpha-linolenic acid, are
extremely important for healing and maintaining good health.
Compounds made from alpha-linolenic acid have been shown to
decrease blood clotting and decrease inflammatory processes in the
body.
[2853] [0010.0.6.6] to [0012.0.6.6] for the disclosure of the
paragraphs [0010.0.6.6] to [0012.0.6.6] see [0010.0.0.0] and
[0012.0.0.0] above.
[2854] [0013.0.0.6] for the disclosure of this paragraph see
[0013.0.0.0] above.
[2855] [0014.0.6.6] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is .alpha.-linolenic acid or
tryglycerides, lipids, oils or fats containing .alpha.-linolenic
acid. Accordingly, in the present invention, the term "the fine
chemical" as used herein relates to ".alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid". Further, the term "the fine chemicals" as
used herein also relates to fine chemicals comprising
.alpha.-linolenic acid and/or triglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid.
[2856] [0015.0.6.6] In one embodiment, the term "the fine chemical"
means .alpha.-linolenic acid and/or tryglycerides, lipids, oils
and/or fats containing .alpha.-linolenic acid. Throughout the
specification the term "the fine chemical" means .alpha.-linolenic
acid and/or tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, .alpha.-linolenic acid and its salts,
ester, thioester or .alpha.-linolenic acid in free form or bound to
other compounds such as triglycerides, glycolipids, phospholipids
etc. In a preferred embodiment, the term "the fine chemical" means
.alpha.-linolenic acid, in free form or its salts or bound to
triglycerides. Triglycerides, lipids, oils, fats or lipid mixture
thereof shall mean any triglyceride, lipid, oil and/or fat
containing any bound or free .alpha.-linolenic acid for example
sphingolipids, phosphoglycerides, lipids, glycolipids such as
glycosphingolipids, phospholipids such as phosphatidylethanolamine,
phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol or diphosphatidylglycerol, or as
monoacylglyceride, diacylglyceride or triacylglyceride or other
fatty acid esters such as acetyl-Coenzym A thioester, which contain
further saturated or unsaturated fatty acids in the fatty acid
molecule.
[2857] [0016.0.6.6] Accordingly, the present invention relates to a
process comprising [2858] (a) increasing or generating the activity
of a b2699, YER173W, YGL205W, YIL150C, b0050, b0251, b0376, b0577,
b0849, b2822, b3129, b3457, b3462 and/or b3644 protein(s) or of a
protein having the sequence of a polypeptide encoded by a nucleic
acid molecule indicated in Table I, columns 5 or 7, lines 68 to 71
and 450 to 459 in a non-human organism in one or more parts thereof
and [2859] (b) growing the organism under conditions which permit
the production of the fine chemical, thus, .alpha.-linolenic acid
or fine chemicals comprising .alpha.-linolenic acid, in said
organism.
[2860] Accordingly, the present invention relates to a process for
the production of a fine chemical comprising [2861] (a) increasing
or generating the activity of one or more proteins having the
activity of a protein indicated in Table II, column 3, lines 68 to
71 and 450 to 459 or having the sequence of a polypeptide encoded
by a nucleic acid molecule indicated in Table I, column 5 or 7,
lines 68 to 71 and 450 to 459, in a non-human organism in one or
more parts thereof and [2862] (b) growing the organism under
conditions which permit the production of the fine chemical, in
particular .alpha.-linolenic acid.
[2863] [0017.0.0.6] and [0018.0.0.6] for the disclosure of the
paragraphs [0017.0.0.6] and [0018.0.0.6] see paragraphs
[0017.0.0.0] and [0018.0.0.0] above.
[2864] [0019.0.6.6] Advantageously the process for the production
of the fine chemical leads to an enhanced production of the fine
chemical. The terms "enhanced" or "increase" mean at least a 10%,
20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or
100%, more preferably 150%, 200%, 300%, 400% or 500% higher
production of the fine chemical in comparison to the reference as
defined below, e.g. that means in comparison to an organism without
the aforementioned modification of the activity of a protein having
the activity of a protein indicated in Table II, column 3, lines 68
to 71 and 450 to 459 or encoded by nucleic acid molecule indicated
in Table I, columns 5 or 7, lines 68 to 71 and 450 to 459.
[2865] [0020.0.6.6] Surprisingly it was found, that the transgenic
expression of at least one of the Saccaromyces cerevisiae
protein(s) indicated in Table II, Column 3, lines 69 to 71, and/or
the Escherichia coli K12 protein(s) indicated in Table II, Column
3, lines 68 and 450 to 459 in Arabidopsis thaliana conferred an
increase in the .alpha.-linolenic acid (or fine chemical) content
of the transformed plants.
[2866] [0021.0.0.6] for the disclosure of this paragraph see
[0021.0.0.0] above.
[2867] [0022.0.6.6] The sequence of b2699 from Escherichia coli K12
has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as DNA-dependent
ATPase or DNA- and ATP-dependent coprotease. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a DNA-dependent ATPase or DNA- and ATP-dependent coprotease from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, in particular for increasing the amount of
.alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, preferably
.alpha.-linolenic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a DNA-dependent ATPase or
DNA- and ATP-dependent coprotease is increased or generated, e.g.
from E. coli or a homolog thereof.
[2868] The sequence of YER173W from Saccharomyces cerevisiae has
been published in Dietrich et al., Nature 387 (6632 Suppl), 78-81
(1997) and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its cellular activity has not been characterized yet. It seams to
be a "checkpoint protein, involved in the activation of the DNA
damage and meiotic pachytene checkpoints; subunit of a clamp loader
that loads Rad17p-Mec3p-Ddc1p onto DNA. Its properly a homolog of
human and S. pombe Rad17 protein". Accordingly, in one embodiment,
the process of the present invention comprises the use of a YER173W
activity from Saccharomyces cerevisiae or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
.alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, in particular for
increasing the amount of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, preferably .alpha.-linolenic acid in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a YER173W protein is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[2869] The sequence of YGL205W from Saccharomyces cerevisiae has
been published in Tettelin et al., Nature 387 (6632 Suppl), 81-84
(1997), and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is being defined as a "fatty-acyl coenzyme A oxidase,
which is involved in the fatty acid beta-oxidation pathway;
localized to the peroxisomal matrix". Accordingly, in one
embodiment, the process of the present invention comprises the use
of a "fatty-acyl coenzyme A oxidase" or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
.alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, in particular for
increasing the amount of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, preferably .alpha.-linolenic acid in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a fatty-acyl coenzyme A oxidase is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[2870] The sequence of YIL150C from Saccharomyces cerevisiae has
been published in Churcher et al., Nature 387 (6632 Suppl), 84-87
(1997) and in Goffeau et al., Science 274 (5287), 546-547, 1996 and
its cellular activity is being defined as "chromatin binding
protein, required for S-phase (DNA synthesis) initation or
completion". Accordingly, in one embodiment, the process of the
present invention comprises the use of a chromatin binding protein,
from Saccharomyces cerevisiae or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of
.alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, in particular for
increasing the amount of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, preferably .alpha.-linolenic acid in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a chromatin binding protein is increased or generated,
e.g. from Saccharomyces cerevisiae or a homolog thereof.
[2871] The sequence of b0050 (Accession number NP.sub.--414592)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a conserved protein potentially involved in protein
protein interaction. Accordingly, in one embodiment, the process of
the present invention comprises the use of said protein from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, in particular for increasing the amount of
.alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, preferably
.alpha.-linolenic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of said protein is increased or
generated, e.g. from E. coli or a homolog thereof.
[2872] The sequence of b0251 (Accession number NP.sub.--414785)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as putative HTH-type transcriptional regulator.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a putative HTH-type transcriptional
regulator protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
.alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, in particular for
increasing the amount of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, preferably .alpha.-linolenic acid in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a putative HTH-type transcriptional regulator protein
is increased or generated, e.g. from E. coli or a homolog
thereof.
[2873] The sequence of b0376 (Accession number NP.sub.--414910)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a beta-lactamase/D-ala carboxypeptidase, penicillin
binding protein. Accordingly, in one embodiment, the process of the
present invention comprises the use of a beta-lactamase/D-ala
carboxypeptidase, penicillin binding protein from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of .alpha.-linolenic acid and/or tryglycerides,
lipids, oils and/or fats containing .alpha.-linolenic acid, in
particular for increasing the amount of .alpha.-linolenic acid
and/or tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, preferably .alpha.-linolenic acid in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a beta-lactamase/D-ala carboxypeptidase, penicillin
binding protein is increased or generated, e.g. from E. coli or a
homolog thereof.
[2874] The sequence of b0577 (Accession number NP.sub.--415109)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a putative transport protein. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a putative transport protein from E. coli or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
of .alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, in particular for
increasing the amount of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, preferably .alpha.-linolenic acid in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a putative transport protein is increased or generated,
e.g. from E. coli or a homolog thereof.
[2875] The sequence of b0849 (Accession number NP.sub.--415370)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a glutaredoxin 1 redox coenzyme for
glutathione-dependent ribonucleotide reductase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a glutaredoxin 1 redox coenzyme for glutathione-dependent
ribonucleotide reductase protein from E. coli or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
of .alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, in particular for
increasing the amount of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, preferably .alpha.-linolenic acid in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a glutaredoxin 1 redox coenzyme for
glutathione-dependent ribonucleotide reductase protein is increased
or generated, e.g. from E. coli or a homolog thereof.
[2876] The sequence of b2822 (Accession number NP.sub.--417299)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a DNA helicase, ATP-dependent dsDNA/ssDNA exonuclease V
subunit, ssDNA endonuclease. Accordingly, in one embodiment, the
process of the present invention comprises the use of a DNA
helicase, ATP-dependent dsDNA/ssDNA exonuclease V subunit, ssDNA
endonuclease protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
.alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, in particular for
increasing the amount of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, preferably .alpha.-linolenic acid in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a DNA helicase, ATP-dependent dsDNA/ssDNA exonuclease V
subunit, ssDNA endonuclease protein is increased or generated, e.g.
from E. coli or a homolog thereof.
[2877] The sequence of b3129 (Accession number NP.sub.--417598)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a putative protease; htrA suppressor protein.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a putative protease; htrA suppressor
protein from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of .alpha.-linolenic acid
and/or tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, in particular for increasing the amount of
.alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, preferably
.alpha.-linolenic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a putative protease; htrA
suppressor protein is increased or generated, e.g. from E. coli or
a homolog thereof.
[2878] The sequence of b3457 (Accession number NP.sub.--417914)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a high-affinity branched-chain amino acid transport
protein (ABC superfamily). Accordingly, in one embodiment, the
process of the present invention comprises the use of a
high-affinity branched-chain amino acid transport protein from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, in particular for increasing the amount of
.alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, preferably
.alpha.-linolenic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a high-affinity
branched-chain amino acid transport protein is increased or
generated, e.g. from E. coli or a homolog thereof.
[2879] The sequence of b3462 (Accession number NP.sub.--417919)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a integral membrane cell division protein. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a integral membrane cell division protein from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, in particular for increasing the amount of
.alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, preferably
.alpha.-linolenic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a integral membrane cell
division protein is increased or generated, e.g. from E. coli or a
homolog thereof.
[2880] The sequence of b3644 (Accession number NP.sub.--418101)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as an uncharacterized stress-induced protein. Accordingly,
in one embodiment, the process of the present invention comprises
the use of an uncharacterized stress-induced protein from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of .alpha.-linolenic acid and/or
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid, in particular for increasing the amount of
.alpha.-linolenic acid and/or tryglycerides, lipids, oils and/or
fats containing .alpha.-linolenic acid, preferably
.alpha.-linolenic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of an uncharacterized
stress-induced protein is increased or generated, e.g. from E. coli
or a homolog thereof.
[2881] [0023.0.6.6] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the fine chemical amount or content. Further, in the
present invention, the term "homologue" relates to the sequence of
an organism having the highest sequence homology to the herein
mentioned or listed sequences of all expressed sequences of said
organism.
[2882] However, the person skilled in the art knows, that,
preferably, the homologue has said fine-chemical increasing
activity and, if known, the same biological function or activity in
the organism as at least one of the protein(s) indicated in Table
II, Column 3, lines 68 to 71 and 450 to 459, e.g. having the
sequence of a polypeptide encoded by a nucleic acid molecule
comprising the sequence indicated in indicated in Table I, Column 5
or 7, lines 68 to 71 and 450 to 459.
[2883] In one embodiment, the homolog of any one of the
polypeptides indicated in Table II, lines 69 to 71 is a homolog
having the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms and being derived from an Eukaryot. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 68 and 450 to 459 is a homolog having the same or a
similar activity, in particular an increase of activity confers an
increase in the content of the fine chemical in the organisms or
part thereof, and being derived from bacteria. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
69 to 71 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in an organisms or part thereof, and
being derived from Fungi. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 68 and 450 to
459 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or part thereof and
being derived from Proteobacteria. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 69 to 71 is
a homolog having the same or a similar activity, in particular an
increase of activity confers an increase in the content of the fine
chemical in the organisms or a part thereof and being derived from
Ascomycota. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 68 and 450 to 459 is a
homolog having the same or a similar activity, in particular an
increase of activity confers an increase in the content of the fine
chemical in the organisms or part thereof, and being derived from
Gammaproteobacteria. In one embodiment, the homolog of a
polypeptide polypeptide indicated in Table II, column 3, lines 69
to 71 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or part thereof, and
being derived from Saccharomycotina. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 68 and 450
to 459 is a homolog having the samie or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or part thereof, and
being derived from Enterobacteriales. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 69
to 71 is a homolog having the samie or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof,
and being derived from Saccharomycetes. In one embodiment, the
homolog of the a polypeptide indicated in Table II, column 3, lines
68 and 450 to 459 is a homolog having the samie or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or part
thereof, and being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 69 to 71 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms, and being
derived from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 68 and 450 to
459 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof,
and being derived from Escherichia. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 69 to 71 is
a homolog having the same or a similar activity, in particular an
increase of activity confers an increase in the content of the fine
chemical in the organisms or a part thereof, and being derived from
Saccharomycetaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 69 to 71 is a homolog having
the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms or a part thereof, and being derived from
Saccharomycetes.
[2884] [0023.1.0.6] and [0024.0.0.6] for the disclosure of the
paragraphs [0023.1.0.6] and [0024.0.0.6] see [0023.1.0.0] and
[0024.0.0.0] above.
[2885] [0025.0.6.6] In accordance with the invention, a protein or
polypeptide has the "activity of a protein as indicated in Table
II, column 3, lines 68 to 71 and 450 to 459" if its de novo
activity, or its increased expression directly or indirectly leads
to an increased .alpha.-linolenic acid and/or tryglycerides,
lipids, oils and/or fats containing .alpha.-linolenic acid level in
the organism or a part thereof, preferably in a cell of said
organism and the protein has the above mentioned activities of a
protein as indicated in Table II, column 3, lines 68 to 71 and 450
to 459. Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as indicated in Table II, column 3, lines 68
to 71 and 450 to 459, or which has at least 10% of the original
enzymatic activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein of
Saccharomyces cerevisiae as indicated in Table II, column 3, lines
69 to 71 and/or a protein of E. coli K12 as indicated in Table II,
column 3, lines 68 and 450 to 459.
[2886] [0025.1.0.6] and [0025.2.0.6] for the disclosure of the
paragraphs [0025.1.0.6] and [0025.2.0.6] see paragraphs
[0025.1.0.0] and [0025.2.0.0] above.
[2887] [0026.0.0.6] to [0033.0.0.6] for the disclosure of the
paragraphs [0026.0.0.6] to [0033.0.0.6] see paragraphs [0026.0.0.0]
to [0033.0.0.0] above.
[2888] [0034.0.6.6] Preferably, the reference, control or wild type
differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. as result of
an increase in the level of the nucleic acid molecule of the
present invention or an increase of the specific activity of the
polypeptide of the invention or the polypeptide used in the method
of the invention. E.g., it differs by or in the expression level or
activity of an protein having the activity of a protein as
indicated in Table II, column 3, lines 68 to 71 and 450 to 459 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 68 to 71 and 450 to 459 or its homologs, e.g. as
indicated in Table I, column 7, lines 68 to 71 and 450 to 459, its
biochemical or genetical causes and therefore shows the increased
amount of the fine chemical.
[2889] [0035.0.0.6] to [0038.0.0.6] and [0039.0.5.6] for the
disclosure of the paragraphs [0035.0.0.6] to [0038.0.0.6] and
[0039.0.5.6] see paragraphs [0035.0.0.0] to [0039.0.0.0] above.
[2890] [0040.0.0.6] to [0044.0.0.6] for the disclosure of the
paragraphs [0040.0.0.6] to [0044.0.0.6] see paragraphs [0035.0.0.0]
and [0044.0.0.0] above.
[2891] [0045.0.6.6] In one embodiment, in case the activity of the
Escherichia coli K12 protein b2699 or its homologs e.g. a
DNA-dependent ATPase or DNA- and ATP-dependent coprotease protein
e.g. as indicated in Table II, columns 5 or 7, line 68, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of .alpha.-linolenic acid between 13% and 32%
or more is conferred.
[2892] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YER173W or its homologs, e.g. a "uncharacterized
protein YER173W", which seams to be a "checkpoint protein, involved
in the activation of the DNA damage and meiotic pachytene
checkpoints; subunit of a clamp loader that loads
Rad17p-Mec3p-Ddc1p onto DNA. Its properly a homolog of human and S.
pombe Rad17 protein" e.g. as indicated in Table II, columns 5 or 7,
line 69, is increased, preferably, in one embodiment an increase of
the fine chemical, preferably of .alpha.-linolenic acid between 15%
and 54% or more is conferred.
[2893] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YGL205W or its homologs, e.g. a fatty-acyl
coenzyme A oxidase protein e.g. as indicated in Table II, columns 5
or 7, line 70, is increased, preferably, in one embodiment an
increase of the fine chemical, preferably of .alpha.-linolenic acid
between 13% and 24% or more is conferred.
[2894] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YIL150C or its homologs e.g. a "chromatin
binding protein" e.g. as indicated in Table II, columns 5 or 7,
line 71, is increased, preferably, in one embodiment the increase
of the fine chemical, preferably of .alpha.-linolenic acid, by at
least 243%.
[2895] In case the activity of the Escherichia coli K12 protein
b0050 or its homologs e.g. a conserved protein potentially involved
in protein protein interaction e.g. as indicated in Table II,
columns 5 or 7, line 450, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
.alpha.-linolenic acid between 11% and 19% or more is
conferred.
[2896] In case the activity of the Escherichia coli K12 protein
b0251 or its homologs e.g. a putative HTH-type transcriptional
regulator e.g. as indicated in Table II, columns 5 or 7, line 451,
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of .alpha.-linolenic acid between 14% and
29% or more is conferred.
[2897] In case the activity of the Escherichia coli K12 protein
b0376 or its homologs e.g. a beta-lactamase/D-ala carboxypeptidase,
penicillin binding protein e.g. as indicated in Table II, columns 5
or 7, line 452, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of .alpha.-linolenic acid
between 15% and 34% or more is conferred.
[2898] In case the activity of the Escherichia coli K12 protein
b0577 or its homologs e.g. a putative transport protein e.g. as
indicated in Table II, columns 5 or 7, line 453, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of .alpha.-linolenic acid between 12% and 96% or more is
conferred.
[2899] In case the activity of the Escherichia coli K12 protein
b0849 or its homologs e.g. a glutaredoxin 1 redox coenzyme for
glutathione-dependent ribonucleotide reductase e.g. as indicated in
Table II, columns 5 or 7, line 454, is increased, preferably, in
one embodiment the increase of the fine chemical, preferably of
.alpha.-linolenic acid between 13% and 31% or more is
conferred.
[2900] In case the activity of the Escherichia coli K12 protein
b2822 or its homologs e.g. a DNA helicase, ATP-dependent
dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease e.g. as
indicated in Table II, columns 5 or 7, line 455, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of .alpha.-linolenic acid between 15% and 20% or more is
conferred.
[2901] In case the activity of the Escherichia coli K12 protein
b3129 or its homologs e.g. a putative protease; htrA suppressor
protein e.g. as indicated in Table II, columns 5 or 7, line 456, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of .alpha.-linolenic acid between 12% and 26%
or more is conferred.
[2902] In case the activity of the Escherichia coli K12 protein
b3457 or its homologs e.g. a high-affinity branched-chain amino
acid transport protein (ABC superfamily) e.g. as indicated in Table
II, columns 5 or 7, line 457, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
.alpha.-linolenic acid between 15% and 28% or more is
conferred.
[2903] In case the activity of the Escherichia coli K12 protein
b3462 or its homologs e.g. a integral membrane cell division
protein e.g. as indicated in Table II, columns 5 or 7, line 458, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of .alpha.-linolenic acid between 13% and 17%
or more is conferred.
[2904] In case the activity of the Escherichia coli K12 protein
b3644 or its homologs e.g. an uncharacterized stress-induced
protein e.g. as indicated in Table II, columns 5 or 7, line 459, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of .alpha.-linolenic acid between 13% and 36%
or more is conferred.
[2905] [0046.0.6.6] In case the activity of the Escherichia coli
K12 protein b2699 or its homologs e.g. a DNA-dependent ATPase or
DNA- and ATP-dependent coprotease protein is increased, preferably
an increase of the fine chemical and of tryglycerides, lipids, oils
and/or fats containing .alpha.-linolenic acid is conferred.
[2906] In case the activity of the Saccaromyces cerevisiae protein
YER173W or its homologs e.g. a "uncharacterized protein YER173W" is
increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid is conferred.
[2907] In case the activity of the Saccharomyces cerevisiae protein
YGL205W or its homologs, e.g. a fatty-acyl coenzyme A oxidase
protein is increased, preferably an increase of the fine chemical
and of tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid is conferred.
[2908] In case the activity of the Saccharomyces cerevisiae protein
YIL150C or its homologs e.g. a "chromatin binding protein, required
for S-phase (DNA synthesis) initation or completion" is increased,
preferably an increase of the fine chemical and of tryglycerides,
lipids, oils and/or fats containing .alpha.-linolenic acid is
conferred.
[2909] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0050 or its homologs e.g. a conserved protein
potentially involved in protein protein interaction is increased,
preferably an increase of the fine chemical and of tryglycerides,
lipids, oils and/or fats containing .alpha.-linolenic acid is
conferred.
[2910] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0251 or its homologs e.g. a putative HTH-type
transcriptional regulator is increased, preferably an increase of
the fine chemical and of tryglycerides, lipids, oils and/or fats
containing .alpha.-linolenic acid is conferred.
[2911] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0376 or its homologs e.g. a beta-lactamase/D-ala
carboxypeptidase, penicillin binding protein is increased,
preferably an increase of the fine chemical and of tryglycerides,
lipids, oils and/or fats containing .alpha.-linolenic acid is
conferred.
[2912] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0577 or its homologs e.g. a putative transport
protein is increased, preferably an increase of the fine chemical
and of tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid is conferred.
[2913] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0849 or its homologs e.g. a glutaredoxin 1 redox
coenzyme for glutathione-dependent ribonucleotide reductase is
increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid is conferred.
[2914] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2822 or its homologs e.g. a DNA helicase,
ATP-dependent dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease
is increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid is conferred.
[2915] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3129 or its homologs e.g. a putative protease;
htrA suppressor protein is increased, preferably an increase of the
fine chemical and of tryglycerides, lipids, oils and/or fats
containing .alpha.-linolenic acid is conferred.
[2916] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3457 or its homologs e.g. a high-affinity
branched-chain amino acid transport protein (ABC superfamily) is
increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing
.alpha.-linolenic acid is conferred.
[2917] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3462 or its homologs e.g. a integral membrane
cell division protein is increased, preferably an increase of the
fine chemical and of tryglycerides, lipids, oils and/or fats
containing .alpha.-linolenic acid is conferred.
[2918] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3644 or its homologs e.g. an uncharacterized
stress-induced protein is increased, preferably an increase of the
fine chemical and of tryglycerides, lipids, oils and/or fats
containing .alpha.-linolenic acid is conferred.
[2919] [0047.0.0.6] to [0048.0.0.6] for the disclosure of the
paragraphs [0047.0.0.6] and [0048.0.0.6] see paragraphs
[0047.0.0.0] and [0048.0.0.0] above.
[2920] [0049.0.6.6] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
preferably has the structure of the polypeptide described herein,
in particular a polypeptide comprising a consensus sequence as
indicated in Table IV, column 7, lines 68 to 71 and 450 to 459 or
of the polypeptide as shown in the amino acid sequences as
disclosed in table II, columns 5 and 7, lines 68 to 71 and 450 to
459 or the functional homologues thereof as described herein, or is
encoded by the nucleic acid molecule characterized herein or the
nucleic acid molecule according to the invention, for example by
the nucleic acid molecule as shown in table I, columns 5 and 7,
lines 68 to 71 and 450 to 459 or its herein described functional
homologues and has the herein mentioned activity.
[2921] [0050.0.6.6] For the purposes of the present invention, the
term ".alpha.-linolenic acid" also encompasses the corresponding
salts, such as, for example, the potassium or sodium salts of
.alpha.-linolenic acid or the salts of .alpha.-linolenic acid with
amines such as diethylamine.
[2922] [0051.0.5.6] and [0052.0.0.6] for the disclosure of the
paragraphs [0051.0.5.6] and [0052.0.0.6] see paragraphs
[0051.0.0.0] and [0052.0.0.0] above.
[2923] [0053.0.6.6] In one embodiment, the process of the present
invention comprises one or more of the following steps [2924] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention or the nucleic acid molecule or the
polypeptide used in the method of the invention, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 68 to 71 and 450 to 459 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 68 to 71
and 450 to 459, having herein-mentioned the fine chemical
increasing activity; [2925] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention, e.g. of a polypeptide having an activity
of a protein as indicated in Table II, column 3, lines 68 to 71 and
450 to 459 or its homologs activity, e.g. as indicated in Table II,
column 7, lines 68 to 71 and 450 to 459 or of a mRNA encoding the
polypeptide of the present invention having herein-mentioned
.alpha.-linolenic acid increasing activity; [2926] c) increasing
the specific activity of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptide of the present invention or the
nucleic acid molecule or the polypeptide used in the method of the
invention, having herein-mentioned .alpha.-linolenic acid
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 68 to 71 and 450
to 459 or its homologs activity, e.g. as indicated in Table II,
column 7, lines 68 to 71 and 450 to 459, or decreasing the
inhibiitory regulation of the polypeptide of the invention or of
the polypeptide used in the method of the invention; [2927] d)
generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention or of the polypeptide
of the invention or the polypeptide used in the method of the
invention having herein-mentioned .alpha.-linolenic acid increasing
activity, e.g. of a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 68 to 71 and 450 to 459 or
its homolog's activity, e.g. as indicated in Table II, column 7,
lines 68 to 71 and 450 to 459; [2928] e) stimulating activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the present invention or a polypeptide
of the present invention having herein-mentioned .alpha.-linolenic
acid increasing activity, e.g. of a polypeptide having an activity
of a protein as indicated in Table II, column 3, lines 68 to 71 and
450 to 459 or its homolog's activity, e.g. as indicated in Table
II, column 7, lines 68 to 71 and 450 to 459, by adding one or more
exogenous inducing factors to the organisms or parts thereof;
[2929] f) expressing a transgenic gene encoding a protein
conferring the increased expression of a polypeptide encoded by the
nucleic acid molecule of the present invention or a polypeptide of
the present invention, having herein-mentioned .alpha.-linolenic
acid increasing activity, e.g. of a polypeptide having an activity
of a protein as indicated in Table II, column 3, lines 68 to 71 and
450 to 459 or its homolog's activity, e.g. as indicated in Table
II, column 7, lines 68 to 71 and 450 to 459; [2930] g) increasing
the copy number of a gene conferring the increased expression of a
nucleic acid molecule encoding a polypeptide encoded by the nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention or the polypeptide of the invention or
the polypeptide used in the method of the invention having
herein-mentioned .alpha.-linolenic acid increasing activity, e.g.
of a polypeptide having an activity of a protein as indicated in
Table II, column 3, lines 68 to 71 and 450 to 459 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 68 to 71
and 450 to 459; [2931] h) increasing the expression of the
endogenous gene encoding the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 68 to 71 and 450 to 459 or its homolog's activity, e.g. as
indicated in Table II, column 7, lines 68 to 71 and 450 to 459, by
adding positive expression or removing negative expression
elements, e.g. homologous recombination can be used to either
introduce positive regulatory elements like for plants the 35S
enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; [2932]
i) Modulating growth conditions of an organism in such a manner,
that the expression or activity of the gene encoding the protein of
the invention or the protein itself is enhanced for example
microorganisms or plants can be grown for example under a higher
temperature regime leading to an enhanced expression of heat shock
proteins, which can lead an enhanced fine chemical production.
[2933] j) selecting of organisms with especially high activity of
the proteins of the invention from natural or from mutagenized
resources and breeding them into the target organisms, eg the elite
crops.
[2934] [0054.0.6.6] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of .alpha.-linolenic acid
after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 68
to 71 and 450 to 459 or its homolog's activity, e.g. as indicated
in Table II, columns 5 or 7, lines 68 to 71 and 450 to 459.
[2935] [0055.0.0.6] to [0067.0.0.6] for the disclosure of the
paragraphs [0055.0.0.6] to [0067.0.0.6] see paragraphs [0055.0.0.0]
to [0067.0.0.0] above.
[2936] [0068.0.5.6] and [0069.0.5.6] for the disclosure of the
paragraphs [0068.0.5.6] and [0069.0.5.6] see paragraphs
[0068.0.0.0] and [0069.0.0.0] above.
[2937] [0070.0.6.6] and [0071.0.5.6] for the disclosure of the
paragraphs [0070.0.6.6] and [0071.0.5.6] see paragraphs
[0070.0.5.5] and [0071.0.0.0] above.
[2938] [0072.0.6.6] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to .alpha.-linolenic acid, triglycerides, lipids, oils
and/or fats containing .alpha.-linolenic acid compounds such as
palmitate, palmitoleate, stearate, oleate and/or linoleic acid.
[2939] [0073.0.6.6] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[2940] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [2941] b) increasing the activity of a
protein having the activity of a polypeptide of the invention or
the polypeptide used in the method of the invention or a homolog
thereof, e.g. as shown in Table II, columns 5 or 7, lines 68 to 71
and 450 to 459 or of a polypeptide being encoded by the nucleic
acid molecule of the present invention and described below, i.e.
conferring an increase of the respective fine chemical in the
organism, preferably a microorganism, the a non-human animal, a
plant or animal cell, a plant or animal tissue or the plant, [2942]
c) growing the organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or the
plant under conditions which permit the production of the fine
chemical in the organism, preferably a microorganism, a plant cell,
a plant tissue or the plant; and [2943] d) if desired, recovering,
optionally isolating, the free and/or bound the respective fine
chemical and, optionally further free and/or bound fatty acids
synthetized by the organism, the microorganism, the non-human
animal, the plant or animal cell, the plant or animal tissue or the
plant.
[2944] [0074.0.5.6] for the disclosure of this paragraph see
[0074.0.5.5] above.
[2945] [0075.0.0.6] to [0084.0.0.6] for the disclosure of the
paragraphs [0075.0.0.6] to [0084.0.0.6] see paragraphs [0075.0.0.0]
to [0084.0.0.0] above.
[2946] [0085.0.6.6] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [2947] a) the nucleic acid sequence as
shown in table I, lines 68 to 71 and 450 to 459, columns 5 and 7 or
a derivative thereof, or [2948] b) a genetic regulatory element,
for example a promoter, which is functionally linked to the nucleic
acid sequence as shown table I, lines 68 to 71 and 450 to 459,
columns 5 and 7 or a derivative thereof, or [2949] c) (a) and (b)
is/are not present in its/their natural genetic environment or
has/have been modified by means of genetic manipulation methods, it
being possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[2950] [0086.0.0.6] and [0087.0.0.6] for the disclosure of the
paragraphs [0086.0.0.6] and [0087.0.0.6] see paragraphs
[0086.0.0.0] and [0087.0.0.0] above.
[2951] [0088.0.6.6] In an advantageous embodiment of the invention,
the organism takes the form of a plant whose fatty acid content is
modified advantageously owing to the nucleic acid molecule of the
present invention expressed. This is important for plant breeders
since, for example, the nutritional value of plants for poultry is
dependent on the abovementioned essential fatty acids and the
general amount of fatty acids as energy source in feed. After the
activity of a protein as shown in Table II, columns 5 or 7, lines
68 to 71 and 450 to 459 has been increased or generated, or after
the expression of nucleic acid molecule or polypeptide according to
the invention has been generated or increased, the transgenic plant
generated thus is grown on or in a nutrient medium or else in the
soil and subsequently harvested.
[2952] [0088.1.0.6], [0089.0.0.6], [0090.0.0.6] and [0091.0.5.6]
for the disclosure of the paragraphs [0088.1.0.6], [0089.0.0.6],
[0090.0.0.6] and [0091.0.5.6] see paragraphs [0088.1.0.0],
[0089.0.0.0], [0090.0.0.0] and [0091.0.0.0] above.
[2953] [0092.0.0.6] to [0094.0.0.6] for the disclosure of the
paragraphs [0092.0.0.6] to [0094.0.0.6] see paragraphs [0092.0.0.0]
to [0094.0.0.0] above.
[2954] [0095.0.5.6], [0096.0.5.6] and [0097.0.5.6] for the
disclosure of the paragraphs [0095.0.5.6], [0096.0.5.6] and
[0097.0.5.6] see paragraphs [0095.0.5.5], [0096.0.5.5] and
[0097.0.0.0] above.
[2955] [0098.0.6.6] In a preferred embodiment, the fine chemical
(.alpha.-linolenic acid) is produced in accordance with the
invention and, if desired, is isolated. The production of further
fatty acids such as palmitic acid, stearic acid, palmitoleic acid,
oleic acid and/or linoleic acid mixtures thereof or mixtures of
other fatty acids by the process according to the invention is
advantageous.
[2956] [0099.0.5.6] and [0100.0.5.6] for the disclosure of the
paragraphs [0099.0.5.6] and [0100.0.5.6] see paragraphs
[0099.0.5.5] and [0100.0.5.5] above.
[2957] [0101.0.5.6] and [0102.0.5.6] for the disclosure of the
paragraphs [0101.0.5.6] and [0102.0.5.6] see [0101.0.0.0] and
[0102.0.5.5] above.
[2958] [0103.0.6.6] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [2959]
a) nucleic acid molecule encoding, preferably at least the mature
form, of a polypeptide having a sequence as shown in Table II,
columns 5 or 7, lines 68 to 71 and 450 to 459 in or a fragment
thereof, which confers an increase in the amount of the respective
fine chemical in an organism or a part thereof; [2960] b) nucleic
acid molecule comprising, preferably at least the mature form, of
the nucleic acid molecule having a sequence as shown in Table I,
columns 5 or 7, lines 68 to 71 and 450 to 459, [2961] c) nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as result
of the degeneracy of the genetic code and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[2962] d) nucleic acid molecule encoding a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [2963] e) nucleic acid molecule which hybridizes with
a nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [2964] f)
nucleic acid molecule encoding a polypeptide, the polypeptide being
derived by substituting, deleting and/or adding one or more amino
acids of the amino acid sequence of the polypeptide encoded by the
nucleic acid molecules (a) to (d), preferably to (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [2965] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [2966] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primer pairs
having a sequence as shown in Table III, column 7, lines 68 to 71
and 450 to 459 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [2967]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [2968] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequence as shown in Table IV, column 7, lines 68
to 71 and 450 to 459 and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[2969] k) nucleic acid molecule comprising one or more of the
nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of a polypeptide as shown in Table
II, columns 5 or 7, lines 68 to 71 and 450 to 459 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; and [2970] l) nucleic acid molecule
which is obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably to (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; or which comprises a sequence which is complementary
thereto.
[2971] [0103.1.0.6] and [0103.2.0.6] for the disclosure of the
paragraphs [0103.1.0.6] and [0103.2.0.6] see paragraphs
[0103.1.0.0] and [0103.2.0.0] above.
[2972] [0104.0.6.6] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence shown in Table
I, columns 5 or 7, lines 68 to 71 and 450 to 459, preferably over
the sequences as shown in Table IA, columns 5 or 7, lines 68 to 71
and 450 to 459 by one or more nucleotides or does not consist of
the sequence shown in Table I, columns 5 or 7, lines 68 to 71 and
450 to 459, preferably does not consist of the sequences as shown
in Table IA, columns 5 or 7, lines 68 to 71 and 450 to 459. In one
embodiment, the nucleic acid molecule of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to the
sequence shown in Table I, columns 5 or 7, lines 68 to 71 and 450
to 459, preferably to the sequences as shown in Table IA, columns 5
or 7, lines 68 to 71 and 450 to 459. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of the sequence
shown in Table II, columns 5 or 7, lines 68 to 71 and 450 to 459,
preferably of the sequences as shown in Table IA, columns 5 or 7,
lines 68 to 71 and 450 to 459.
[2973] [0105.0.0.6] to [0107.0.0.6] for the disclosure of the
paragraphs [0105.0.0.6] to [0107.0.0.6] see paragraphs [0105.0.0.0]
and [0107.0.0.0] above.
[2974] [0108.0.6.6] Nucleic acid molecules with the sequence shown
in Table I, columns 5 or 7, lines 68 to 71 and 450 to 459, nucleic
acid molecules which are derived from the amino acid sequences
shown in Table II, columns 5 or 7, lines 68 to 71 and 450 to 459 or
from polypeptides comprising the consensus sequence shown in Table
IV, column 7, lines 68 to 71 and 450 to 459, or their derivatives
or homologues encoding polypeptides with the enzymatic or
biological activity of a protein as shown in Table II, columns 5 or
7, lines 68 to 71 and 450 to 459 or e.g. conferring a
.alpha.-linolenic acid increase after increasing its expression or
activity are advantageously increased in the process according to
the invention.
[2975] [0109.0.5.6] for the disclosure of this paragraph see
[0109.0.0.0] above.
[2976] [0110.0.6.6] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide used in the method
of the invention or used in the process of the invention, e.g. of a
protein as shown in Table II, columns 5 or 7, lines 68 to 71 and
450 to 459 or being encoded by a nucleic acid molecule indicated in
Table I, columns 5 or 7, lines 68 to 71 and 450 to 459 or of its
homologs, e.g. as indicated in Table II, columns 5 or 7, lines 68
to 71 and 450 to 459 can be determined from generally accessible
databases.
[2977] [0111.0.0.6] for the disclosure of this paragraph see
[0111.0.0.0] above.
[2978] [0112.0.6.6] The nucleic acid molecules used in the process
according to the invention take the form of isolated nucleic acid
sequences, which encode polypeptides with an activity of a
polypeptide as indicated in Table II, column 3, lines lines 68 to
71 and 450 to 459 or having the sequence of a polypeptide as
indicated in Table II, columns 5 and 7, lines 68 to 71 and 450 to
459 and conferring an increase of the respective fine chemical.
[2979] [0113.0.0.6] to [0120.0.0.6] for the disclosure of the
paragraphs [0113.0.0.6] to [0120.0.0.6] see paragraphs [0113.0.0.0]
and [0120.0.0.0] above.
[2980] [0121.0.6.6] However, it is also possible to use artificial
sequences, which differ in one or more bases from the nucleic acid
sequences found in organisms, or in one or more amino acid
molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences shown in Table II,
columns 5 or 7, lines 68 to 71 and 450 to 459 or the functional
homologues thereof as described herein, preferably conferring
above-mentioned activity, i.e. conferring an increase of the
respective fine chemical after increasing its activity.
[2981] [0122.0.0.6] to [0127.0.0.6] for the disclosure of the
paragraphs [0122.0.0.6] to [0127.0.0.6] see paragraphs [0122.0.0.0]
and [0127.0.0.0] above.
[2982] [0128.0.6.6] Synthetic oligonucleotide primers for the
amplification, e.g. as shown in Table III, column 7, lines 68 to 71
and 450 to 459, by means of polymerase chain reaction can be
generated on the basis of a sequence shown herein, for example the
sequence shown in Table I, columns 5 or 7, lines 68 to 71 and 450
to 459 or the sequences as shown in Table II, columns 5 or 7, lines
68 to 71 and 450 to 459.
[2983] [0129.0.6.6] Moreover, it is possible to identify conserved
regions from various organisms by carrying out protein sequence
alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence shown in Table IV, column
7, lines 68 to 71 and 450 to 459 is derived from said
alignments.
[2984] [0130.0.6.6] for the disclosure of this paragraph see
[0130.0.0.0].
[2985] [0131.0.0.6] to [0138.0.0.6] for the disclosure of the
paragraphs [0131.0.0.6] to [0138.0.0.6] see paragraphs [0131.0.0.0]
to [0138.0.0.0] above.
[2986] [0139.0.6.6] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical increase, derived from
other organisms, can be encoded by other DNA sequences which
hybridize to the sequences shown in Table I, columns 5 or 7, lines
68 to 71 and 450 to 459, preferably of Table I B, columns 5 or 7,
lines 68 to 71 and 450 to 459 under relaxed hybridization
conditions and which code on expression for peptides having the
.alpha.-linolenic acid increasing activity.
[2987] [0140.0.0.6] to [0146.0.0.6] for the disclosure of the
paragraphs [0140.0.0.6] to [0146.0.0.6] see paragraphs [0140.0.0.0]
and [0146.0.0.0] above.
[2988] [0147.0.6.6] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 68 to 71 and 450 to 459, preferably in
Table I B, columns 5 or 7, lines 68 to 71 and 450 to 459 is one
which is sufficiently complementary to one of said nucleotide
sequences such that it can hybridize to one of said nucleotide
sequences thereby forming a stable duplex. Preferably, the
hybridisation is performed under stringent hybridization
conditions. However, a complement of one of the herein disclosed
sequences is preferably a sequence complement thereto according to
the base pairing of nucleic acid molecules well known to the
skilled person. For example, the bases A and G undergo base pairing
with the bases T and U or C, resp. and visa versa. Modifications of
the bases can influence the base-pairing partner.
[2989] [0148.0.6.6] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines
68 to 71 and 450 to 459, preferably of Table I B, columns 5 or 7,
lines 68 to 71 and 450 to 459, or a functional portion thereof and
preferably has above mentioned activity, in particular has
the--fine-chemical-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homologs.
[2990] [0149.0.6.6] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 68 to 71 and 450 to 459, preferably of Table I B, columns 5
or 7, lines 68 to 71 and 450 to 459 or a portion thereof and
encodes a protein having above-mentioned activity as indicated in
Table II, columns 5 or 7, lines 68 to 71 and 450 to 459, preferably
of Table II B, columns 5 or 7, lines 68 to 71 and 450 to 459, e.g.
conferring an increase of the respective fine chemical.
[2991] [00149.1.0.5] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 68 to 71 and 450 to 459, preferably of Table
I B, columns 5 or 7, lines 68 to 71 and 450 to 459 has further one
or more of the activities annotated or known for the a protein as
indicated in Table II, column 3, lines 68 to 71 and 450 to 459.
[2992] [0150.0.6.6] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table I, columns 5 or 7, lines 68 to 71 and 450 to 459, preferably
of Table I B, columns 5 or 7, lines 68 to 71 and 450 to 459, for
example a fragment which can be used as a probe or primer or a
fragment encoding a biologically active portion of the polypeptide
of the present invention or of a polypeptide used in the process of
the present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of methionine if its activity is increased.
The nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences indicated in Table I, columns 5 or 7, lines 68 to
71 and 450 to 459, an anti-sense sequence of one of the sequences
indicated in Table I, columns 5 or 7, lines 68 to 71 and 450 to
459, or naturally occurring mutants thereof. Primers based on a
nucleotide sequence of the invention can be used in PCR reactions
to clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table III, column 7, lines 68 to 71 and 450 to 459 will result in a
fragment of a polynucleotide sequence as indicated in Table I,
columns 5 or 7, lines 68 to 71 and 450 to 459. Preferred is Table I
B, column 7, lines 68 to 71 and 450 to 459.
[2993] [0151.0.0.6] for the disclosure of this paragraph see
[0151.0.0.0] above.
[2994] [0152.0.6.6] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to the amino acid
sequence shown in Table II, columns 5 or 7, lines 68 to 71 and 450
to 459 such that the protein or portion thereof maintains the
ability to participate in the fine chemical production, in
particular a .alpha.-linolenic acid increasing the activity as
mentioned above or as described in the examples in plants or
microorganisms is comprised.
[2995] [0153.0.6.6] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence shown in Table II, columns 5 or 7, lines 68 to 71 and
450 to 459 such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
shown in Table II, columns 5 or 7, lines 68 to 71 and 450 to 459
has for example an activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 68 to 71 and 450 to 459.
[2996] [0154.0.6.6] In one embodiment, the nucleic acid molecule of
the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as shown in Table II, columns 5 or 7, lines 68 to 71 and
450 to 459 and having above-mentioned activity, e.g. conferring
preferably the increase of the respective fine chemical.
[2997] [0155.0.0.6] and [0156.0.0.6] for the disclosure of the
paragraphs [0155.0.0.6] and [0156.0.0.6] see paragraphs
[0155.0.0.0] and [0156.0.0.0] above.
[2998] [0157.0.6.6] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 68 to 71 and 450 to 459
(and portions thereof) due to degeneracy of the genetic code and
thus encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides comprising the consensus sequences as indicated
in Table IV, column 7, lines 68 to 71 and 450 to 459 or of the
polypeptide as indicated in Table II, columns 5 or 7, lines 68 to
71 and 450 to 459 or their functional homologues. Advantageously,
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention comprises, or in an
other embodiment has, a nucleotide sequence encoding a protein
comprising, or in an other embodiment having, a consensus sequences
as indicated in Table IV, column 7, lines 68 to 71 and 450 to 459
or of the polypeptide as indicated in Table II, columns 5 or 7,
lines 68 to 71 and 450 to 459 or the functional homologues. In a
still further embodiment, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention encodes a full length protein which is substantially
homologous to an amino acid sequence comprising a consensus
sequence as indicated in Table IV, column 7, lines 68 to 71 and 450
to 459 or of a polypeptide as indicated in Table II, columns 5 or
7, lines 68 to 71 and 450 to 459 or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table I, columns 5 or 7, lines 68 to 71 and 450 to
459, preferably as indicated in Table I A, columns 5 or 7, lines 68
to 71 and 450 to 459. Preferably the nucleic acid molecule of the
invention is a functional homologue or identical to a nucleic acid
molecule indicated in Table I B, columns 5 or 7, lines 68 to 71 and
450 to 459.
[2999] [0158.0.0.6] to [0160.0.0.6] for the disclosure of the
paragraphs [0158.0.0.6] to [0160.0.0.6] see paragraphs [0158.0.0.0]
to [0160.0.0.0] above.
[3000] [0161.0.6.6] Accordingly, in another embodiment, a nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising the
sequence shown in Table I, columns 5 or 7, lines 68 to 71 and 450
to 459. The nucleic acid molecule is preferably at least 20, 30,
50, 100, 250 or more nucleotides in length.
[3001] [0162.0.0.6] for the disclosure of this paragraph see
paragraph [0162.0.0.0] above.
[3002] [0163.0.6.6] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 68 to 71 and 450 to
459 corresponds to a naturally-occurring nucleic acid molecule of
the invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[3003] [0164.0.0.6] for the disclosure of this paragraph see
paragraph [0164.0.0.0] above.
[3004] [0165.0.6.6] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as shown in
Table I, columns 5 or 7, lines 68 to 71 and 450 to 459.
[3005] [0166.0.0.6] and [0167.0.0.6] for the disclosure of the
paragraphs [0166.0.0.6] and [0167.0.0.6] see paragraphs
[0166.0.0.0] and [0167.0.0.0] above.
[3006] [0168.0.6.6] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organism or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 68 to 71 and 450 to 459 yet retain said activity described
herein. The nucleic acid molecule can comprise a nucleotide
sequence encoding a polypeptide, wherein the polypeptide comprises
an amino acid sequence at least about 50% identical to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 68 to
71 and 450 to 459 and is capable of participation in the increase
of production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to the
sequence as indicated in Table II, columns 5 or 7, lines 68 to 71
and 450 to 459, more preferably at least about 70% identical to one
of the sequences as indicated in Table II, columns 5 or 7, lines 68
to 71 and 450 to 459, even more preferably at least about 80%, 90%,
95% homologous to the sequence as indicated in Table II, columns 5
or 7, lines 68 to 71 and 450 to 459, and most preferably at least
about 96%, 97%, 98%, or 99% identical to the sequence as indicated
in Table II, columns 5 or 7, lines 68 to 71 and 450 to 459.
[3007] Accordingly, the invention relates to nucleic acid molecules
encoding a polypeptide having above-mentioned activity, e.g.
conferring an increase in the the respective fine chemical in an
organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 68 to 71 and 450 to 459, preferably of Table II B, column 7,
lines 68 to 71 and 450 to 459 yet retain said activity described
herein. The nucleic acid molecule can comprise a nucleotide
sequence encoding a polypeptide, wherein the polypeptide comprises
an amino acid sequence at least about 50% identical to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 68 to
71 and 450 to 459, preferably of Table II B, column 7, lines 68 to
71 and 450 to 459 and is capable of participation in the increase
of production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table II, columns 5 or 7, lines 68 to 71
and 450 to 459, preferably of Table II B, column 7, lines 68 to 71
and 450 to 459, more preferably at least about 70% identical to one
of the sequences as indicated in Table II, columns 5 or 7, lines 68
to 71 and 450 to 459, preferably of Table II B, column 7, lines 68
to 71 and 450 to 459, even more preferably at least about 80%, 90%,
or 95% homologous to a sequence as indicated in Table II, columns 5
or 7, lines 68 to 71 and 450 to 459, preferably of Table II B,
column 7, lines 68 to 71 and 450 to 459, and most preferably at
least about 96%, 97%, 98%, or 99% identical to the sequence as
indicated in Table II, columns 5 or 7, lines 68 to 71 and 450 to
459, preferably of Table II B, column 7, lines 68 to 71 and 450 to
459.
[3008] [0169.0.0.6] to [0175.0.5.6] for the disclosure of the
paragraphs [0169.0.0.6] to [0175.0.5.6] see paragraphs [0169.0.0.0]
to [0175.0.0.0] above.
[3009] [0176.0.6.6] Functional equivalents derived from one of the
polypeptides as shown in Table II, columns 5 or 7, lines 68 to 71
and 450 to 459 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as shown in Table II, columns
5 or 7, lines 68 to 71 and 450 to 459 according to the invention
and are distinguished by essentially the same properties as the
polypeptide as shown in Table II, columns 5 or 7, lines 68 to 71
and 450 to 459.
[3010] [0177.0.6.6] Functional equivalents derived from the nucleic
acid sequence as shown in Table I, columns 5 or 7, lines 68 to 71
and 450 to 459 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as shown in Table II, columns
5 or 7, lines 68 to 71 and 450 to 459 according to the invention
and encode polypeptides having essentially the same properties as
the polypeptide as shown in Table II, columns 5 or 7, lines 68 to
71 and 450 to 459.
[3011] [0178.0.0.6] for the disclosure of this paragraph see
[0178.0.0.0] above.
[3012] [0179.0.6.6] A nucleic acid molecule encoding a homologous
to a protein sequence of as indicated in Table II, columns 5 or 7,
lines 68 to 71 and 450 to 459, preferably of Table II B, column 7,
lines 68 to 71 and 450 to 459 can be created by introducing one or
more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table I, columns 5 or 7,
lines 68 to 71 and 450 to 459 such that one or more amino acid
substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced into the encoding
sequences for example into a sequences as indicated in Table I,
columns 5 or 7, lines 68 to 71 and 450 to 459 by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[3013] [0180.0.0.6] to [0183.0.0.6] for the disclosure of the
paragraphs [0180.0.0.6] to [0183.0.0.6] see paragraphs [0180.0.0.0]
to [0183.0.0.0] above.
[3014] [0184.0.6.6] Homologues of the nucleic acid sequences used,
with a sequence as indicated in Table I, columns 5 or 7, lines 68
to 71 and 450 to 459, preferably of Table I B, column 7, lines 68
to 71 and 450 to 459, or of the nucleic acid sequences derived from
a sequences as indicated in Table II, columns 5 or 7, lines 68 to
71 and 450 to 459, preferably of Table II B, column 7, lines 68 to
71 and 450 to 459, comprise also allelic variants with at least
approximately 30%, 35%, 40% or 45% homology, by preference at least
approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
I, columns 5 or 7, lines 68 to 71 and 450 to 459, or from the
derived nucleic acid sequences, the intention being, however, that
the enzyme activity or the biological activity of the resulting
proteins synthesized is advantageously retained or increased.
[3015] [0185.0.6.6] In one embodiment of the present invention, the
nucleic acid molecule of the invention or used in the process of
the invention comprises one or more sequences as indicated in Table
I, columns 5 or 7, lines 68 to 71 and 450 to 459, preferably of
Table I B, column 7, lines 68 to 71 and 450 to 459. In one
embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotide sequences not
shown in any one of sequences as indicated in Table I, columns 5 or
7, lines 68 to 71 and 450 to 459, preferably of Table I B, column
7, lines 68 to 71 and 450 to 459. In one embodiment, the nucleic
acid molecule comprises less than 500, 400, 300, 200, 100, 90, 80,
70, 60, 50 or 40 further nucleotides. In a further embodiment, the
nucleic acid molecule comprises less than 30, 20 or 10 further
nucleotides. In one embodiment, a nucleic acid molecule used in the
process of the invention is identical to a sequences as indicated
in Table I, columns 5 or 7, lines 68 to 71 and 450 to 459,
preferably of Table I B, column 7, lines 68 to 71 and 450 to
459.
[3016] [0186.0.6.6] Also preferred is that one or more nucleic acid
molecule(s) used in the process of the invention encode a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 68 to 71 and 450 to 459, preferably of Table II B,
column 7, lines 68 to 71 and 450 to 459. In one embodiment, the
nucleic acid molecule encodes less than 150, 130, 100, 80, 60, 50,
40 or 30 further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table II, columns 5 or 7, lines 68 to 71 and 450 to 459,
preferably of Table II B, column 7, lines 68 to 71 and 450 to
459.
[3017] [0187.0.6.6] In one embodiment, the nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table II, columns 5 or 7,
lines 68 to 71 and 450 to 459, preferably of Table II B, column 7,
lines 68 to 71 and 450 to 459 comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a sequences as indicated in Table II, columns 5
or 7, lines 68 to 71 and 450 to 459, preferably of Table II B,
column 7, lines 68 to 71 and 450 to 459.
[3018] [0188.0.6.6] Polypeptides (=proteins), which still have the
essential biological activity of the polypeptide of the present
invention conferring an increase of the fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide shown in Table II, columns 5 or 7, lines 68 to 71
and 450 to 459 expressed under identical conditions.
[3019] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, columns 7, lines 68 to 71 and 450 to 459.
[3020] [0189.0.6.6] Homologues of sequences as indicated in Table
I, columns 5 or 7, lines 68 to 71 and 450 to 459 or of the derived
sequences shown in Table II, columns 5 or 7, lines 68 to 71 and 450
to 459 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[3021] [0190.0.0.6], [0191.0.5.6], [00191.1.0.6] and [0192.0.0.6]
to [0203.0.0.6] for the disclosure of the paragraphs [0190.0.0.6],
[0191.0.5.6], [0191.1.0.6] and [0192.0.0.6] to [0203.0.0.6] see
paragraphs [0190.0.0.0], [0191.0.0.0], [0191.1.0.0] and
[0192.0.0.0] to [0203.0.0.0] above.
[3022] [0204.0.6.6] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [3023] a) nucleic
acid molecule encoding, preferably at least the mature form, of the
polypeptide shown in Table II, columns 5 or 7, lines 68 to 71 and
450 to 459; preferably of Table II B, column 7, lines 68 to 71 and
450 to 459; or a fragment thereof conferring an increase in the
amount of the fine chemical in an organism or a part thereof [3024]
b) nucleic acid molecule comprising, preferably at least the mature
form, of the nucleic acid molecule shown in Table I, columns 5 or
7, lines 68 to 71 and 450 to 459 preferably of Table I B, column 7,
lines 68 to 71 and 450 to 459; or a fragment thereof conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [3025] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [3026] d) nucleic
acid molecule encoding a polypeptide whose sequence has at least
50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [3027] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [3028] f) nucleic acid molecule encoding a polypeptide,
the polypeptide being derived by substituting, deleting and/or
adding one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[3029] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to [3030] (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [3031] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying a cDNA library or a genomic library using the primers or
primer pairs as indicated in Table III, column 7, lines 68 to 71
and 450 to 459 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [3032]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from a expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (g), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [3033] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence shown in
Table IV, column 7, lines 68 to 71 and 450 to 459 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [3034] k) nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of the polypeptide shown in Table II, columns 5 or 7, lines 68 to
71 and 450 to 459, preferably of Table II B, column 7, lines 68 to
71 and 450 to 459; and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[3035] l) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment of at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (h) or of the
nucleic acid molecule shown in Table I, columns 5 or 7, lines 68 to
71 and 450 to 459 or a nucleic acid molecule encoding, preferably
at least the mature form of, the polypeptide shown in Table II,
columns 5 or 7, lines 68 to 71 and 450 to 459, and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; or which encompasses a sequence which
is complementary thereto; whereby, preferably, the nucleic acid
molecule according to (a) to (l) distinguishes over the sequence as
depicted in Table IA, columns 5 or 7, lines 68 to 71 and 450 to 459
by one or more nucleotides. In one embodiment, the nucleic acid
molecule of the invention does not consist of the sequence shown in
Table IA or IB, columns 5 or 7, lines 68 to 71 and 450 to 459. In
an other embodiment, the nucleic acid molecule of the present
invention is at least 30% identical and less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to the sequence shown in Table IA or
IB, columns 5 or 7, lines 68 to 71 and 450 to 459. In a further
embodiment the nucleic acid molecule does not encode the
polypeptide sequence shown in Table IIA or IIB, columns 5 or 7,
lines 68 to 71 and 450 to 459. Accordingly, in one embodiment, the
nucleic acid molecule of the present invention encodes in one
embodiment a polypeptide which differs at least in one or more
amino acids from the polypeptide as depicted in Table IIA or IIB,
columns 5 or 7, lines 68 to 71 and 450 to 459 and therefore does
not encode a protein of the sequence shown in Table IIA or IIB,
columns 5 or 7, lines 68 to 71 and 450 to 459. Accordingly, in one
embodiment, the protein encoded by a sequence of a nucleic acid
according to (a) to (l) does not consist of the sequence shown in
Table IIA or IIB, columns 5 or 7, lines 68 to 71 and 450 to 459. In
a further embodiment, the protein of the present invention is at
least 30% identical to protein sequence depicted in Table IIA or
IIB, columns 5 or 7, lines 68 to 71 and 450 to 459 and less than
100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 985, 97%, 96% or 95% identical to the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 68 to 71
and 450 to 459.
[3036] [0205.0.0.6] to [0226.0.0.6] for the disclosure of the
paragraphs [0205.0.0.6] to [0226.0.0.6] see paragraphs [0205.0.0.0]
to [0226.0.0.0] above.
[3037] [0227.0.6.6] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[3038] In addition to the sequence mentioned in Table I, columns 5
or 7, lines 68 to 71 and 450 to 459 or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the fatty acid biosynthetic pathway such as for
palmitate, palmitoleate, stearate and/or oleate is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine the sequences shown in Table I, columns 5
or 7, lines 68 to 71 and 450 to 459 with genes which generally
support or enhances to growth or yield of the target organisms, for
example genes which lead to faster growth rate of microorganisms or
genes which produces stress-, pathogen, or herbicide resistant
plants.
[3039] [0228.0.5.6] to [0230.0.5.6] for the disclosure of the
paragraphs [0228.0.5.6] to [0230.0.5.6] see paragraphs [0228.0.0.0]
to [0230.0.0.0] above.
[3040] [0231.0.6.6] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a .alpha.-linolenic acid degrading
protein is attenuated, in particular by reducing the rate of
expression of the corresponding gene.
[3041] [0232.0.0.6] to [0276.0.0.6] for the disclosure of the
paragraphs [0232.0.0.6] to [0276.0.0.6] see paragraphs [0232.0.0.0]
to [0276.0.0.0] above.
[3042] [0277.0.5.6] for the disclosure of this paragraph see
paragraph [0277.0.5.5] above.
[3043] [0278.0.0.6] to [0282.0.0.6] for the disclosure of the
paragraphs [0278.0.0.6] to [0282.0.0.6] see paragraphs [0278.0.0.0]
to [0282.0.0.0] above.
[3044] [0283.0.6.6] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an antibody against polypeptides as shown in
Table II, columns 5 or 7, lines 68 to 71 and 450 to 459 or an
antigenic part thereof, which can be produced by standard
techniques utilizing polypeptides comprising or consisting of
abovementioned sequences, e.g. the polypeptid of the present
invention or fragment thereof. Preferred are monoclonal antibodies
specifically binding to polypeptides as indicated in Table II,
columns 5 or 7, lines 68 to 71 and 450 to 459.
[3045] [0284.0.0.6] for the disclosure of this paragraph see
[0284.0.0.0] above.
[3046] [0285.0.6.6] In one embodiment, the present invention
relates to a polypeptide having the sequence shown in Table II,
columns 5 or 7, lines 68 to 71 and 450 to 459 or as coded by the
nucleic acid molecule shown in Table I, columns 5 or 7, lines 68 to
71 and 450 to 459 or functional homologues thereof.
[3047] [0286.0.6.6] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 68 to 71 and 450 to 459 and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table IV, column 7, lines 68 to 71 and 450 to 459, whereby 20 or
less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred
5 or 4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a poylpeptide or to a polypeptide comprising more than
one consensus sequences as indicated in Table IV, column 7, lines
68 to 71 and 450 to 459.
[3048] [0287.0.0.6] to [0290.0.0.6] for the disclosure of the
paragraphs [0287.0.0.6] to [0290.0.0.6] see paragraphs [0287.0.0.0]
to [0290.0.0.0] above.
[3049] [0291.0.6.6] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of a
plant or microorganism specific consensus sequences.
[3050] In one embodiment, said polypeptide of the invention
distinguishes over the sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 68 to 71 and 450 to 459 by one or more amino
acids. In one embodiment, polypeptide distinguishes form the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 68 to 71
and 450 to 459 by more than 5, 6, 7, 8 or 9 amino acids, preferably
by more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred
are more than 40, 50, or 60 amino acids and, preferably, the
sequence of the polypeptide of the invention distinguishes from the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 68 to 71
and 450 to 459 by not more than 80% or 70% of the amino acids,
preferably not more than 60% or 50%, more preferred not more than
40% or 30%, even more preferred not more than 20% or 10%. In
another embodiment, said polypeptide of the invention does not
consist of the sequence shown in Table IIA or IIB, columns 5 or 7,
lines 68 to 71 and 450 to 459.
[3051] [0292.0.0.6] for the disclosure of this paragraph see
[0292.0.0.0] above.
[3052] [0293.0.6.6] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the
process.
[3053] In one embodiment, the polypeptide of the invention has a
sequence, which distinguishes from the sequence as shown in Table
IIA or IIB, columns 5 or 7, lines 68 to 71 and 450 to 459 by one or
more amino acids. In another embodiment, said polypeptide of the
invention does not consist of the sequence shown in Table IIA or
IIB, columns 5 or 7, lines 68 to 71 and 450 to 459. In a further
embodiment, said polypeptide of the present invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical. In one embodiment,
said polypeptide does not consist of the sequence encoded by the
nucleic acid molecules shown in Table IA or IB, columns 5 or 7,
lines 68 to 71 and 450 to 459.
[3054] [0294.0.6.6] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 68 to 71 and 450 to 459,
which distinguishes over the sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 68 to 71 and 450 to 459 by one or more
amino acids, preferably by more than 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, evenmore
preferred are more than 40, 50, or 60 amino acids but even more
preferred by less than 70% of the amino acids, more preferred by
less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[3055] [0295.0.0.6], [0296.0.0.6] and [0297.0.5.6] for the
disclosure of the paragraphs [0295.0.0.6], [0296.0.0.6] and
[0297.0.5.6] see paragraphs [0295.0.0.0] to [0297.0.0.0] above.
[3056] [00297.1.0.6] Non-polypeptide of the invention-chemicals are
e.g. polypeptides having not the activity and/or the amino acid
sequence of a polypeptide indicated in Table II, columns 3, 5 or 7,
lines 68 to 71 and 450 to 459.
[3057] [0298.0.6.6] A polypeptide of the invention can participate
in the process of the present invention. The polypeptide or a
portion thereof comprises preferably an amino acid sequence which
is sufficiently homologous to an amino acid sequence as indicated
in Table II, columns 5 or 7, lines 68 to 71 and 450 to 459 such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table II, columns 5 or 7, lines 68 to 71 and 450 to 459.
[3058] [0299.0.6.6] Further, the polypeptide can have an amino acid
sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences of Table II, columns 5 or 7, lines 68 to 71
and 450 to 459. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence of Table I, columns
5 or 7, lines 68 to 71 and 450 to 459 or which is homologous
thereto, as defined above.
[3059] [0300.0.6.6] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 68 to 71 and 450 to 459 in amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 68 to 71 and 450 to 459.
[3060] [0301.0.0.6] for the disclosure of this paragraph see
[0301.0.0.0] above.
[3061] [0302.0.6.6] Biologically active portions of an polypeptide
of the present invention include peptides comprising amino acid
sequences derived from the amino acid sequence of the polypeptide
of the present invention or used in the process of the present
invention, e.g., an amino acid sequence shown in Table II, columns
5 or 7, lines 68 to 71 and 450 to 459 or the amino acid sequence of
a protein homologous thereto, which include fewer amino acids than
a full length polypeptide of the present invention or used in the
process of the present invention or the full length protein which
is homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[3062] [0303.0.0.6] for the disclosure of this paragraph see
[0303.0.0.0] above.
[3063] [0304.0.6.6] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, columns 5 or 7, lines 68 to 71 and 450 to 459 but having
differences in the sequence from said wild-type protein. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity in relation to the wild type protein.
[3064] [0305.0.5.6], [0306.0.5.6] and [0306.1.0.6] for the
disclosure of the paragraphs [0305.0.5.6], [0306.0.5.6] and
[0306.1.0.6] see paragraphs [0305.0.0.0], [0306.0.0.0] and
[0306.1.0.0] above.
[3065] [0307.0.0.6] and [0308.0.0.6] for the disclosure of the
paragraphs [0307.0.0.6] and [0308.0.0.6] see paragraphs [0307.0.0.0
and [0308.0.0.0] above.
[3066] [0309.0.6.6] In one embodiment, a reference to protein
(=polypeptide)" of the invention e.g. as indicated in Table II,
columns 5 or 7, lines 68 to 71 and 450 to 459 refers to a
polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas a "non-polypeptide" or "other polypeptide" e.g.
not indicated in Table II, columns 5 or 7, lines 68 to 71 and 450
to 459 refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to
a polypeptide of the invention, preferably which is not
substantially homologous to a polypeptide having a protein
activity, e.g., a protein which does not confer the activity
described herein and which is derived from the same or a different
organism.
[3067] [0310.0.0.6] to [0334.0.0.6] for the disclosure of the
paragraphs [0310.0.0.6] to [0334.0.0.6] see paragraphs [0310.0.0.0]
to [0334.0.0.0] above.
[3068] [0335.0.6.6] The dsRNAi method has proved to be particularly
effective and advantageous for reducing the expression of a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 68 to
71 and 450 to 459 and/or homologs thereof. As described inter alia
in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table I,
columns 5 or 7, lines 68 to 71 and 450 to 459 and/or homologs
thereof. In a double-stranded RNA molecule for reducing the
expression of an protein encoded by a nucleic acid sequence of one
of the sequences as indicated in Table I, columns 5 or 7, lines 68
to 71 and 450 to 459 and/or homologs thereof, one of the two RNA
strands is essentially identical to at least part of a nucleic acid
sequence, and the respective other RNA strand is essentially
identical to at least part of the complementary strand of a nucleic
acid sequence.
[3069] [0336.0.0.6] to [0342.0.0.6] for the disclosure of the
paragraphs [0336.0.0.6] to [0342.0.0.6] see paragraphs [0336.0.0.0]
to [0342.0.0.0] above.
[3070] [0343.0.6.6] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 68 to 71
and 450 to 459 or its homolog is not necessarily required in order
to bring about effective reduction in the expression. The advantage
is, accordingly, that the method is tolerant with regard to
sequence deviations as may be present as a consequence of genetic
mutations, polymorphisms or evolutionary divergences. Thus, for
example, using the dsRNA, which has been generated starting from a
sequence of one of the sequences as indicated in Table I, columns 5
or 7, lines 68 to 71 and 450 to 459 or homologs thereof of the one
organism, may be used to suppress the corresponding expression in
another organism.
[3071] [0344.0.0.6] to [0350.0.0.6], [0351.0.5.6] and [0352.0.0.6]
to [0361.0.0.6] for the disclosure of the paragraphs [0344.0.0.5]
to [0350.0.0.5], [0351.0.5.5] and [0352.0.0.5] to [0361.0.0.5] see
paragraphs [0344.0.0.0] to [0361.0.0.0] above.
[3072] [0362.0.6.6] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, the nucleic acid construct of
the invention, the antisense molecule of the invention, the vector
of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. encoding a polypeptide having an
activity of a protein such as the polypeptides as indicated in
Table II, column 3, lines 68 to 71 and 450 to 459. Due to the above
mentioned activity the fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a protein such
as the polypeptides as indicated in Table II, column 3, lines 68 to
71 and 450 to 459. Activity means herein that due to modulation or
manipulation of the genome, the activity of polypeptide having an
activity of a protein such as the polypeptides as indicated in
Table II, column 3, lines 68 to 71 and 450 to 459 or a similar
activity, which is increased in the cell or organism or part
thereof. Examples are described above in context with the process
of the invention.
[3073] [0363.0.0.6], [0364.0.5.6] and [0365.0.0.6] to [0379.0.5.6]
for the disclosure of the paragraphs [0363.0.0.6], [0364.0.5.6] and
[0365.0.0.6] to [0379.0.5.6] see paragraphs [0363.0.0.0] to
[0379.0.0.0] above.
[3074] [0380.0.5.6], [0381.0.0.6] and [0382.0.0.6] for the
disclosure of the paragraphs [0380.0.5.6], [0381.0.0.6] and
[0382.0.0.6] see paragraphs [0380.0.5.5], [0381.0.0.0] and
[0382.0.0.0] above.
[3075] [0383.0.5.6], [0384.0.0.6], [0385.0.5.6] and [0386.0.5.6]
for the disclosure of the paragraphs [0383.0.5.6], [0384.0.0.6],
[0385.0.5.6] and [0386.0.5.6] see paragraphs [0383.0.5.5],
[0384.0.0.0], [0385.0.5.5] and [0386.0.5.5] above.
[3076] [0387.0.0.6] to [0392.0.0.6] for the disclosure of the
paragraphs [0387.0.0.6] to [0392.0.0.6] see paragraphs [0387.0.0.0]
to [0392.0.0.0] above.
[3077] [0393.0.6.6] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [3078] (a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the respective fine chemical after expression, with the
nucleic acid molecule of the present invention; [3079] (b)
identifying the nucleic acid molecules, which hybridize under
relaxed stringent conditions with the nucleic acid molecule of the
present invention in particular to the nucleic acid molecule
sequence shown in Table I, columns 5 or 7, lines 68 to 71 and 450
to 459, preferably in Table I B, columns 5 or 7, lines 68 to 71 and
450 to 459 and, optionally, isolating the full length cDNA clone or
complete genomic clone; [3080] (c) introducing the candidate
nucleic acid molecules in host cells, preferably in a plant cell or
a microorganism, appropriate for producing the respective fine
chemical; [3081] (d) expressing the identified nucleic acid
molecules in the host cells; [3082] (e) assaying the respective
fine chemical level in the host cells; and [3083] (f) identifying
the nucleic acid molecule and its gene product which expression
confers an increase in the respective fine chemical level in the
host cell after expression compared to the wild type.
[3084] [0394.0.0.6] to [0415.0.0.6] and [0416.0.5.6] for the
disclosure of the paragraphs [0394.0.0.6] to [0415.0.0.6] and
[0416.0.5.6] see paragraphs [0394.0.0.0] to [0416.0.0.0] above.
[3085] [0417.0.5.6] and [0418.0.0.6] to [0430.0.0.6] for the
disclosure of the paragraphs [0417.0.5.6] and [0418.0.0.6] to
[0430.0.0.6] see paragraphs [0417.0.5.5] and [0418.0.0.0] to
[0430.0.0.0] above.
[3086] [0431.0.5.6], [0432.0.5.6], [0433.0.0.6] and [0434.0.0.6]
for the disclosure of the paragraphs [0431.0.5.6], [0432.0.5.6],
[0433.0.0.6] and [0434.0.0.6] see paragraphs [0431.0.0.0] to
[0434.0.0.0] above.
[3087] [0435.0.5.6] to [0440.0.5.6] for the disclosure of the
paragraphs [0435.0.5.6] to [0440.0.5.6] see paragraphs [0435.0.5.5]
to [0440.0.5.5] above.
[3088] [0441.0.0.6] and [0442.0.5.6] for the disclosure of the
paragraphs [0441.0.0.6] and [0442.0.5.6] see [0441.0.0.0] and
[0442.0.5.5] above.
[3089] [0443.0.0.6] for the disclosure of this paragraph see
[0443.0.0.0] above.
[3090] [0444.0.5.6] and [0445.0.5.6] for the disclosure of the
paragraphs [0444.0.5.6] and [0445.0.5.6] see [0444.0.5.5] and
[0445.0.5.5] above.
[3091] [0446.0.0.6] to [0453.0.0.6] for the disclosure of the
paragraphs [0446.0.0.6] to [0453.0.0.6] see paragraphs [0446.0.0.0]
to [0453.0.0.0] above.
[3092] [0454.0.5.6] and [0455.0.5.6] for the disclosure of the
paragraphs [0454.0.5.6] and [0455.0.5.6] see [0454.0.5.5] and
[0455.0.5.5] above.
[3093] [0456.0.0.6] for the disclosure of this paragraph see
[0456.0.0.0] above.
[3094] [0457.0.5.6] to [0460.0.5.6] for the disclosure of the
paragraphs [0457.0.5.6] to [0460.0.6.6] see paragraphs [0457.0.5.5]
to [0460.0.5.5] above.
[0461.0.6.6] Example 10
Cloning SEQ ID NO: 5304 for the Expression in Plants
[3095] [0462.0.0.6] for the disclosure of this paragraph see
[0462.0.0.0] above.
[3096] [0463.0.6.6] SEQ ID NO: 5304 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[3097] [0464.0.5.6], [0465.0.5.6] and [0466.0.0.6] for the
disclosure of the paragraphs [0464.0.5.6], [0465.0.5.6] and
[0466.0.0.6] see paragraphs [0464.0.5.5], [0465.0.5.5] and
[0466.0.0.0] above.
[3098] [0467.0.6.6] The following primer sequences were selected
for the gene SEQ ID NO: 5304:
TABLE-US-00023 i) forward primer (SEQ ID NO: 5640) atggctatcg
acgaaaacaa acag ii) reverse primer (SEQ ID NO: 5641) ttaaaaatct
tcgttagttt ctgctac
[3099] [0468.0.0.6] to [0479.0.0.6] for the disclosure of the
paragraphs [0468.0.0.6] to [0479.0.0.6] see paragraphs [0468.0.0.0]
to [0479.0.0.0] above.
[0480.0.6.6] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 5304
[3100] [0481.0.0.6] for the disclosure of this paragraph see
[0481.0.0.0] above.
[3101] [0482.0.0.6] to [0513.0.0.6] for the disclosure of the
paragraphs [0482.0.0.6] to [0513.0.0.6] see paragraphs [0482.0.0.0]
to [0513.0.0.0] above.
[3102] [0514.0.6.6] As an alternative, the fatty acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55).
[3103] The results of the different plant analyses can be seen from
the table which follows:
TABLE-US-00024 TABLE 1 ORF Metabolite Method Min Max b2699 alpha
Linolenic Acid GC 1.13 1.32 (C18:3 (c9,c12,c15)) YER173W alpha
Linolenic Acid GC 1.15 1.54 (C18:3 (c9,c12,c15)) YGL205W alpha
Linolenic Acid GC 1.13 1.24 (C18:3 (c9,c12,c15)) YIL150C alpha
Linolenic Acid GC 3.43 3.43 (C18:3 (c9,c12,c15)) b0050 alpha
Linolenic Acid GC 1.11 1.19 (C18:3 (c9,c12,c15)) b0251 alpha
Linolenic Acid GC 1.14 1.29 (C18:3 (c9,c12,c15)) b0376 alpha
Linolenic Acid GC 1.15 1.34 (C18:3 (c9,c12,c15)) b0577 alpha
Linolenic Acid GC 1.12 1.96 (C18:3 (c9,c12,c15)) b0849 alpha
Linolenic Acid GC 1.13 1.31 (C18:3 (c9,c12,c15)) b2822 alpha
Linolenic Acid GC 1.15 1.20 (C18:3 (c9,c12,c15)) b3129 alpha
Linolenic Acid GC 1.12 1.26 (C18:3 (c9.c12.c151) b3457 alpha
Linolenic Acid GC 1.15 1.28 (C18:3 (c9,c12,c15)) b3462 alpha
Linolenic Acid GC 1.13 1.17 (C18:3 (c9,c12,c15)) b3644 alpha
Linolenic Acid GC 1.13 1.36 (C18:3 (c9,c12,c15))
[3104] [0515.0.5.6] Column 2 shows the fatty acid analyzed. Columns
4 and 5 shows the ratio of the analyzed fatty acid between the
transgenic plants and the wild type; Increase of the metabolites:
Max: maximal x-fold (normalised to wild type)-Min: minimal x-fold
(normalised to wild type). Decrease of the metabolites: Max:
maximal x-fold (normalised to wild type) (minimal decrease), Min:
minimal x-fold (normalised to wild type) (maximal decrease). Column
3 indicates the analytical method.
[3105] [0516.0.0.6] and [0517.0.5.6] for the disclosure of the
paragraphs [0516.0.0.6] and [0517.0.5.6] see paragraphs
[0516.0.0.0] and [0517.0.0.0] above.
[3106] [0518.0.0.6] to [0529.0.0.6] and [0530.0.5.6] for the
disclosure of the paragraphs [0518.0.0.6] to [0529.0.0.6] and
[0530.0.5.6] see paragraphs [0518.0.0.0] to [0530.0.0.0] above.
[3107] [0530.1.0.6] to [0530.6.0.6] for the disclosure of the
paragraphs [0530.1.0.6] to [0530.6.0.6] see paragraphs [0530.1.0.0]
to [0530.6.0.0] above.
[3108] [0531.0.0.6] to [0533.0.0.6] and [0534.0.5.6] for the
disclosure of the paragraphs [0531.0.0.6] to [0533.0.0.6] and
[0534.0.5.6] see paragraphs [0531.0.0.0] to [0534.0.0.0] above.
[3109] [0535.0.0.6] to [0537.0.0.6] and [0538.0.5.6] for the
disclosure of the paragraphs [0535.0.0.6] to [0537.0.0.6] and
[0538.0.5.6] see paragraphs [0535.0.0.0] to [0538.0.0.0] above.
[3110] [0539.0.0.6] to [0542.0.0.6] and [0543.0.5.6] for the
disclosure of the paragraphs [0539.0.0.6] to [0542.0.0.6] and
[0543.0.5.6] see paragraphs [0539.0.0.0] to [0543.0.0.0] above.
[3111] [0544.0.0.6] to [0547.0.0.6] and [0548.0.5.6] to
[0552.0.0.6] for the disclosure of the paragraphs [0544.0.0.6] to
[0547.0.0.6] and [0548.0.5.6] to [0552.0.0.6] see paragraphs
[0544.0.0.0] to [0552.0.0.0] above.
[0552.1.6.6]: Example 15
Metabolite Profiling Info from Zea mays
[3112] Zea mays plants were engineered, grown and transformed as
described in Example 14c.
[3113] The results of the different Zea mays plants analysed can be
seen from Table 2, which follows:
TABLE-US-00025 TABLE 2 ORF_NAME Metabolite Min Max YIL150C alpha
Linolenic Acid (C18:3 (c9,c12,c15)) 1.47 2.24
[3114] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in .alpha.-linolenic acid
in genetically modified corn plants expressing the Saccharomyces
cerevisiae nucleic acid sequence YIL150C.
[3115] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YIL150C or its homologs, e.g. a "chromatin
binding protein, required for S-phase (DNA synthesis) initiation or
completion" or its homologs, is increased in corn plants,
preferably, an increase of the fine chemical .alpha.-linolenic acid
between 47% and 124% is conferred.
[3116] [0552.2.0.6] for the disclosure of this paragraph see
[0552.2.0.0] above.
[3117] [0553.0.6.6] [3118] 1. A process for the production of
.alpha.-linolenic acid, which comprises [3119] (a) increasing or
generating the activity of a protein as indicated in Table II,
columns 5 or 7, lines 68 to 71 and 450 to 459 or a functional
equivalent thereof in a non-human organism, or in one or more parts
thereof; and [3120] (b) growing the organism under conditions which
permit the production of .alpha.-linolenic acid in said organism.
[3121] 2. A process for the production of .alpha.-linolenic acid,
comprising the increasing or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [3122] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 68 to
71 and 450 to 459 or a fragment thereof, which confers an increase
in the amount of .alpha.-linolenic acid in an organism or a part
thereof; [3123] b) nucleic acid molecule comprising of a nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines 68 to
71 and 450 to 459; [3124] c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as a result of the degeneracy of the
genetic code and conferring an increase in the amount of
.alpha.-linolenic acid in an organism or a part thereof; [3125] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of .alpha.-linolenic acid in an organism
or a part thereof; [3126] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under stringent
hybridisation conditions and conferring an increase in the amount
of .alpha.-linolenic acid in an organism or a part thereof; [3127]
f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table III, column 7, lines 68 to 71 and 450 to 459 and
conferring an increase in the amount of .alpha.-linolenic acid in
an organism or a part thereof; [3128] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
.alpha.-linolenic acid in an organism or a part thereof; [3129] h)
nucleic acid molecule encoding a polypeptide comprising a consensus
sequence as indicated in Table IV, column 7, lines 68 to 71 and 450
to 459 and conferring an increase in the amount of
.alpha.-linolenic acid in an organism or a part thereof; and [3130]
i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of .alpha.-linolenic
acid in an organism or a part thereof. [3131] or comprising a
sequence which is complementary thereto. [3132] 3. The process of
claim 1 or 2, comprising recovering of the free or bound
.alpha.-linolenic acid. [3133] 4. The process of any one of claims
1 to 3, comprising the following steps: [3134] (a) selecting an
organism or a part thereof expressing a polypeptide encoded by the
nucleic acid molecule characterized in claim 2; [3135] (b)
mutagenizing the selected organism or the part thereof; [3136] (c)
comparing the activity or the expression level of said polypeptide
in the mutagenized organism or the part thereof with the activity
or the expression of said polypeptide of the selected organisms or
the part thereof; [3137] (d) selecting the mutated organisms or
parts thereof, which comprise an increased activity or expression
level of said polypeptide compared to the selected organism or the
part thereof; [3138] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [3139] (f) recovering, and
optionally isolating, the free or bound .alpha.-linolenic acid
produced by the selected mutated organisms or parts thereof. [3140]
5. The process of any one of claims 1 to 4, wherein the activity of
said protein or the expression of said nucleic acid molecule is
increased or generated transiently or stably. [3141] 6. An isolated
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: [3142] a) nucleic acid molecule
encoding of a polypeptide as indicated in Table II, columns 5 or 7,
lines 68 to 71 and 450 to 459 or a fragment thereof, which confers
an increase in the amount of .alpha.-linolenic acid in an organism
or a part thereof; [3143] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in
[3144] Table I, columns 5 or 7, lines 68 to 71 and 450 to 459;
[3145] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of .alpha.-linolenic acid in
an organism or a part thereof; [3146] d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of .alpha.-linolenic acid in an organism or a part thereof;
[3147] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of
.alpha.-linolenic acid in an organism or a part thereof; [3148] f)
nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table III, column 7, lines 68 to 71 and 450 to 459 and
conferring an increase in the amount of .alpha.-linolenic acid in
an organism or a part thereof; [3149] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
.alpha.-linolenic acid in an organism or a part thereof; [3150] h)
nucleic acid molecule encoding a polypeptide comprising a consensus
sequence as indicated in Table IV, column 7, lines 68 to 71 and 450
to 459 and conferring an increase in the amount of
.alpha.-linolenic acid in an organism or a part thereof; and [3151]
i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of .alpha.-linolenic
acid in an organism or a part thereof. [3152] whereby the nucleic
acid molecule distinguishes over the sequence as indicated in Table
I A, columns 5 or 7, lines 68 to 71 and 450 to 459 by one or more
nucleotides. [3153] 7. A nucleic acid construct which confers the
expression of the nucleic acid molecule of claim 6, comprising one
or more regulatory elements. [3154] 8. A vector comprising the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7. [3155] 9. The vector as claimed in claim 8,
wherein the nucleic acid molecule is in operable linkage with
regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic, host. [3156] 10. A
host cell, which has been transformed stably or transiently with
the vector as claimed in claim 8 or 9 or the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 2 to 5. [3157] 11.
The host cell of claim 10, which is a transgenic host cell. [3158]
12. The host cell of claim 10 or 11, which is a plant cell, an
animal cell, a microorganism, or a yeast cell, a fungus cell, a
prokaryotic cell, an eukaryotic cell or an archaebacterium. [3159]
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. [3160] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table II A, columns 5 or 7, lines 68 to 71 and 450 to
459 by one or more amino acids 15. An antibody, which binds
specifically to the polypeptide as claimed in claim 14. [3161] 16.
A plant tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [3162] 17. A method for screening for agonists
and antagonists of the activity of a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of .alpha.-linolenic acid in an organism or a part thereof
comprising: [3163] (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of .alpha.-linolenic acid in an organism or a part thereof
with a candidate compound or a sample comprising a plurality of
compounds under conditions which permit the expression the
polypeptide; [3164] (b) assaying the .alpha.-linolenic acid level
or the polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [3165] (c)
identifying a agonist or antagonist by comparing the measured
.alpha.-linolenic acid level or polypeptide expression level with a
standard of .alpha.-linolenic acid or polypeptide expression level
measured in the absence of said candidate compound or a sample
comprising said plurality of compounds, whereby an increased level
over the standard indicates that the compound or the sample
comprising said plurality of compounds is an agonist and a
decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[3166] 18. A process for the identification of a compound
conferring increased .alpha.-linolenic acid production in a plant
or microorganism, comprising the steps: [3167] (a) culturing a
plant cell or tissue or microorganism or maintaining a plant
expressing the polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of .alpha.-linolenic
acid in an organism or a part thereof and a readout system capable
of interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with dais readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of .alpha.-linolenic acid in
an organism or a part thereof; [3168] (b) identifying if the
compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout system.
[3169] 19. A method for the identification of a gene product
conferring an increase in .alpha.-linolenic acid production in a
cell, comprising the following steps: [3170] (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in
.alpha.-linolenic acid after expression with the nucleic acid
molecule of claim 6; [3171] (b) identifying the nucleic acid
molecules, which hybridise under relaxed stringent conditions with
the nucleic acid molecule of claim 6; [3172] (c) introducing the
candidate nucleic acid molecules in host cells appropriate for
producing .alpha.-linolenic acid; [3173] (d) expressing the
identified nucleic acid molecules in the host cells; [3174] (e)
assaying the .alpha.-linolenic acid level in the host cells; and
[3175] (f) identifying nucleic acid molecule and its gene product
which expression confers an increase in the .alpha.-linolenic acid
level in the host cell in the host cell after expression compared
to the wild type. [3176] 20. A method for the identification of a
gene product conferring an increase in .alpha.-linolenic acid
production in a cell, comprising the following steps: [3177] (a)
identifying in a data bank nucleic acid molecules of an organism;
which can contain a candidate gene encoding a gene product
conferring an increase in the .alpha.-linolenic acid amount or
level in an organism or a part thereof after expression, and which
are at least 20% homolog to the nucleic acid molecule of claim 6;
[3178] (b) introducing the candidate nucleic acid molecules in host
cells appropriate for producing .alpha.-linolenic acid; [3179] (c)
expressing the identified nucleic acid molecules in the host cells;
[3180] (d) assaying the .alpha.-linolenic acid level in the host
cells; and [3181] (e) identifying nucleic acid molecule and its
gene product which expression confers an increase in the
.alpha.-linolenic acid level in the host cell after expression
compared to the wild type. [3182] 21. A method for the production
of an agricultural composition comprising the steps of the method
of any one of claims 17 to 20 and formulating the compound
identified in any one of claims 17 to 20 in a form acceptable for
an application in agriculture. [3183] 22. A composition comprising
the nucleic acid molecule of claim 6, the polypeptide of claim 14,
the nucleic acid construct of claim 7, the vector of any one of
claim 8 or 9, an antagonist or agonist identified according to
claim 17, the compound of claim 18, the gene product of claim 19 or
20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. [3184] 23. Use of the nucleic acid molecule as
claimed in claim 6 for the identification of a nucleic acid
molecule conferring an increase of .alpha.-linolenic acid after
expression. [3185] 24. Use of the polypeptide of claim 14 or the
nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of .alpha.-linolenic acid levels
in an organism. [3186] 25. Food or feed composition comprising the
nucleic acid molecule of claim 6, the polypeptide of claim 14, the
nucleic acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20. [3187] 26. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the host cell of claim 10 to 12 or the gene
product identified according to the method of claim 19 or 20 for
the protection of a plant against a .alpha.-linolenic acid
synthesis inhibiting herbicide.
[3188] [0554.0.0.6] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[3189] [0000.0.0.7] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[3190] [0001.0.0.7] for the disclosure of this paragraph see
[0001.0.0.0].
[3191] [0002.0.7.7] Fatty acids and triglycerides have numerous
applications in the food and feed industry, in cosmetics and in the
drug sector. Depending on whether they are free saturated or
unsaturated fatty acids or triglycerides with an increased content
of saturated or unsaturated fatty acids, they are suitable for the
most varied applications; thus, for example, polyunsaturated fatty
acids (=PUFAs) are added to infant formula to increase its
nutritional value. The various fatty acids and triglycerides are
mainly obtained from microorganisms such as fungi or from
oil-producing plants including phytoplankton and algae, such as
soybean, oilseed rape, sunflower and others, where they are usually
obtained in the form of their triacylglycerides.
[3192] [0003.0.7.7] Stearic acid (=octadecanoic acid) is one of the
many useful types of saturated fatty acids that comes from many
animal and vegetable fats and oils. It is a waxy solid that melts
at around 70.degree. C. Commenly stearic acid is either prepared by
treating animal fat with water at a high pressure and temperature
or starting with vegetable oils by hydrogenation of said oils. It
is useful as an ingredient in making candles, soaps, and cosmetics
and for softening rubber.
[3193] [0004.0.7.7] Principally microorganisms such as Mortierella
or oil producing plants such as soybean, rapeseed or sunflower or
algae such as Crytocodinium or Phaeodactylum are a common source
for oils containing fatty acids, where they are usually obtained in
the form of their triacyl glycerides. Alternatively, they are
obtained advantageously from animals, such as fish. The free fatty
acids are prepared advantageously by hydrolysis with a strong base
such as potassium or sodium hydroxide.
[3194] [0005.0.5.7] for the disclosure of this paragraph see
[0005.0.5.5] above.
[3195] [0006.0.7.7] Unlike most saturated fats, stearic acid does
not seem to increase cholesterol levels in the blood, because liver
enzymes convert it to an unsaturated fat during digestion.
[3196] [0007.0.7.7] Stearic acid is the most common one of the
long-chain fatty acids. It is found in many foods, such as beef
fat, and cocoa butter. It is widely used as mentioned above as a
lubricant, in soaps, cosmetics, food packaging, deodorant sticks,
toothpastes, and as a softener in rubber.
[3197] [0008.0.7.7] Encouraging research shows that stearic acid,
one of the components of the fat found in the cocoa butter of
chocolate, may have some positive effects on platelets. The
mechanism believed to be responsible for the potential platelet
activation by stearic acid involves Arachidonic metabolism, which
includes thromboxane A2, a potent aggregating compound, and
prostaglandin 12, a potent anti-aggregating compound.
[3198] [0009.0.7.7] As described above, fatty acids are used in a
lot of different applications, for example in cosmetics,
pharmaceuticals and in feed and food.
[3199] [0010.0.7.7] Therefore improving the productivity of such
fatty acids and improving the quality of foodstuffs and animal
feeds is an important task of the different industries.
[3200] [0011.0.7.7] To ensure a high productivity of certain fatty
acids in plants or microorganism, it is necessary to manipulate the
natural biosynthesis of fatty acids in said organism.
[3201] [0012.0.7.7] Accordingly, there is still a great demand for
new and more suitable genes which encode enzymes which participate
in the biosynthesis of fatty acids and make it possible to produce
certain fatty acids specifically on an industrial scale without
unwanted byproducts forming. In the selection of genes for
biosynthesis two characteristics above all are particularly
important. On the one hand, there is as ever a need for improved
processes for obtaining the highest possible contents of fatty
acids on the other hand as less as possible byproducts should be
produced in the production process.
[3202] [0013.0.0.7] for the disclosure of this paragraph see
[0013.0.0.0] above.
[3203] [0014.0.7.7] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is stearic acid or
tryglycerides, lipids, oils or fats containing stearic acid.
Accordingly, in the present invention, the term "the fine chemical"
as used herein relates to "stearic acid and/or tryglycerides,
lipids, oils and/or fats containing stearic acid". Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising stearic acid and/or triglycerides, lipids,
oils and/or fats containing stearic acid.
[3204] [0015.0.7.7] In one embodiment, the term "the fine chemical"
means stearic acid and/or tryglycerides, lipids, oils and/or fats
containing stearic acid. Throughout the specification the term "the
fine chemical" means stearic acid and/or tryglycerides, lipids,
oils and/or fats containing stearic acid, stearic acid and its
salts, ester, thioester or stearic acid in free form or bound to
other compounds such as triglycerides, glycolipids, phospholipids
etc. In a preferred embodiment, the term "the fine chemical" means
stearic acid, in free form or its salts or bound to triglycerides.
Triglycerides, lipids, oils, fats or lipid mixture thereof shall
mean any triglyceride, lipid, oil and/or fat containing any bound
or free stearic acid for example sphingolipids, phosphoglycerides,
lipids, glycolipids such as glycosphingolipids, phospholipids such
as phosphatidylethanolamine, phosphatidylcholine,
phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or
diphosphatidylglycerol, or as monoacylglyceride, diacylglyceride or
triacylglyceride or other fatty acid esters such as acetyl-Coenzym
A thioester, which contain further saturated or unsaturated fatty
acids in the fatty acid molecule.
[3205] In one embodiment, the term "the fine chemical" and the term
"the respective fine chemical" mean at least one chemical compound
with an activity of the above mentioned fine chemical.
[3206] [0016.0.7.7] Accordingly, the present invention relates to a
process comprising [3207] (a) increasing or generating the activity
of a b2699, b2095, b3256, b2699, b1093,
[3208] YOR024W, YBR089C, YFR042W, YIL150C, YDR513W, YLR010C, b0161,
b1896 and/or b3457 protein(s) or of a protein having the sequence
of a polypeptide encoded by a nucleic acid molecule indicated in
Table I, columns 5 or 7, lines 72 to 81 and 460 to 462 in a
non-human organism in one or more parts thereof and [3209] (b)
growing the organism under conditions which permit the production
of the fine chemical, thus, stearic acid or fine chemicals
comprising stearic acid, in said organism.
[3210] Accordingly, the present invention relates to a process for
the production of a fine chemical comprising [3211] (a) increasing
or generating the activity of one or more proteins having the
activity of a protein indicated in Table II, column 3, lines 72 to
81 and 460 to 462 or having the sequence of a polypeptide encoded
by a nucleic acid molecule indicated in Table I, column 5 or 7,
lines 72 to 81 and 460 to 462, in a non-human organism in one or
more parts thereof and [3212] (b) growing the organism under
conditions which permit the production of the fine chemical, in
particular stearic acid.
[3213] [0017.0.0.7] and [0018.0.0.7] for the disclosure of the
paragraphs [0017.0.0.7] and [0018.0.0.7] see paragraphs
[0017.0.0.0] and [0018.0.0.0] above.
[3214] [0019.0.7.7] Advantageously the process for the production
of the fine chemical leads to an enhanced production of the fine
chemical. The terms "enhanced" or "increase" mean at least a 10%,
20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or
100%, more preferably 150%, 200%, 300%, 400% or 500% higher
production of the fine chemical in comparison to the reference as
defined below, e.g. that means in comparison to an organism without
the aforementioned modification of the activity of a protein having
the activity of a protein indicated in Table II, column 3, lines 72
to 81 and 460 to 462 or encoded by nucleic acid molecule indicated
in Table I, columns 5 or 7, lines 72 to 81 and 460 to 462.
[3215] [0020.0.7.7] Surprisingly it was found, that the transgenic
expression of at least one of the Saccaromyces cerevisiae
protein(s) indicated in Table II, Column 3, lines 75 to 80 and/or
the Escherichia coli K12 protein(s) indicated in Table II, Column
3, lines 72 to 74, 81 and 460 to 462 in Arabidopsis thaliana
conferred an increase in the stearic acid (or fine chemical)
content of the transformed plants.
[3216] [0021.0.0.7] for the disclosure of this paragraph see
[0021.0.0.0] above.
[3217] [0022.0.7.7] The sequence of b3256 from Escherichia coli K12
has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as acetyl CoA
carboxylase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a acetyl CoA carboxylase
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of stearic acid and/or
tryglycerides, lipids, oils and/or fats containing stearic acid, in
particular for increasing the amount of stearic acid and/or
tryglycerides, lipids, oils and/or fats containing stearic acid,
preferably stearic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a acetyl CoA carboxylase is
increased or generated, e.g. from E. coli or a homolog thereof.
[3218] The sequence of b2699 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as DNA-dependent ATPase or DNA-
and ATP-dependent coprotease. Accordingly, in one embodiment, the
process of the present invention comprises the use of a
DNA-dependent ATPase or DNA- and ATP-dependent coprotease from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of stearic acid and/or tryglycerides,
lipids, oils and/or fats containing stearic acid, in particular for
increasing the amount of stearic and/or tryglycerides, lipids, oils
and/or fats containing stearic acid, preferably stearic acid in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of a DNA-dependent ATPase or DNA- and ATP-dependent
coprotease is increased or generated, e.g. from E. coli or a
homolog thereof. The sequence of b2095 from Escherichia coli K12
has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as putative
tagatose-6-phosphate kinase 1. Accordingly, in one embodiment, the
process of the present invention comprises the use of a putative
tagatose-6-phosphate kinase 1 from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
stearic acid and/or tryglycerides, lipids, oils and/or fats
containing stearic acid, in particular for increasing the amount of
stearic acid and/or tryglycerides, lipids, oils and/or fats
containing stearic acid, preferably stearic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a putative tagatose-6-phosphate kinase 1 is increased or generated,
e.g. from E. coli or a homolog thereof.
[3219] The sequence of b1093 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as
3-oxoacyl-[acyl-carrier-protein] reductase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a 3-oxoacyl-[acyl-carrier-protein] reductase from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of stearic acid and/or tryglycerides, lipids,
oils and/or fats containing stearic acid, in particular for
increasing the amount of stearic acid and/or tryglycerides, lipids,
oils and/or fats containing stearic acid, preferably stearic acid
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a 3-oxoacyl-[acyl-carrier-protein]
reductase is increased or generated, e.g. from E. coli or a homolog
thereof. The sequence of YOR024W from Saccharomyces cerevisiae has
been submitted from de Haan to the EMBL Protein Sequence Database,
July 1996 and its cellular activity has not been characterized yet.
It is probably a membrane protein. Accordingly, in one embodiment,
the process of the present invention comprises the use of YOR024W,
from Saccharomyces cerevisiae or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of stearic acid
and/or tryglycerides, lipids, oils and/or fats containing stearic
acid, in particular for increasing the amount of stearic acid
and/or tryglycerides, lipids, oils and/or fats containing stearic
acid, preferably stearic acid in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a YOR024W is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog thereof.
The sequence of YBR089C-A from Saccharomyces cerevisiae has been
published in Feldmann et al., EMBO J., 13 (24), 5795-5809 (1994)
and Goffeau et al., Science 274 (5287), 546-547, 1996, and its
cellular activity has not been characterized yet. It shows homology
to mammalian high mobility group proteins 1 and 2. Its function may
be redundantly with the highly homologous gene NHP6A. Furthermore
it shows homology to the high-mobility group non-histone chromatin
protein Nhp6 bp. Accordingly, in one embodiment, the process of the
present invention comprises the use of a YBR089C-A activity from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of stearic acid and/or
tryglycerides, lipids, oils and/or fats containing stearic acid, in
particular for increasing the amount of stearic acid and/or
tryglycerides, lipids, oils and/or fats containing stearic acid,
preferably stearic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a YBR089C-A protein is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[3220] The sequence of YFR042W from Saccharomyces cerevisiae has
been published in Murakami et al., Nat. Genet. 10 (3), 261-268
(1995) and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its cellular activity has not been clearly characterized yet. It
seams to be a "protein, which is required for cell viability".
Accordingly, in one embodiment, the process of the present
invention comprises the use of a YFR042W activity from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of stearic acid and/or
tryglycerides, lipids, oils and/or fats containing stearic acid, in
particular for increasing the amount of stearic acid and/or
tryglycerides, lipids, oils and/or fats containing stearic acid,
preferably stearic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a YFR042W protein is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof. The sequence of YIL150C from Saccharomyces
cerevisiae has been published in Churcher et al., Nature 387 (6632
Suppl), 84-87 (1997) and in Goffeau et al., Science 274 (5287),
546-547, 1996 and its cellular activity has not been characterized
yet. It seams to be a "protein required for S-phase (DNA synthesis)
initiation or completion". Accordingly, in one embodiment, the
process of the present invention comprises the use of YIL150C, from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of stearic acid and/or
tryglycerides, lipids, oils and/or fats containing stearic acid, in
particular for increasing the amount of stearic acid and/or
tryglycerides, lipids, oils and/or fats containing stearic acid,
preferably stearic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a YIL150C protein is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[3221] The sequence of YDR513w from Saccharomyces cerevisiae has
been published in Jacq et al., Nature 387 (6632 Suppl), 75-78
(1997) and in Goffeau et al., Science 274 (5287), 546-547, 1996 and
its cellular activity has characterized as glutaredoxin
(thioltransferase, glutathione reductase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of YDR513w, from Saccharomyces cerevisiae or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
stearic acid and/or tryglycerides, lipids, oils and/or fats
containing stearic acid, in particular for increasing the amount of
stearic acid and/or tryglycerides, lipids, oils and/or fats
containing stearic acid, preferably stearic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a glutaredoxin is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof.
[3222] The sequence of YLR010c from Saccharomyces cerevisiae has
been published in Johnston et al., Nature 387 (6632 Suppl), 87-90
(1997) and in Goffeau et al., Science 274 (5287), 546-547, 1996 and
its cellular activity has not been characterized yet. It seams to
be a "protein involved in telomeric pathways in association with
Stn1". Accordingly, in one embodiment, the process of the present
invention comprises the use of YLR010C, from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the fine chemical, meaning of stearic acid and/or tryglycerides,
lipids, oils and/or fats containing stearic acid, in particular for
increasing the amount of stearic acid and/or tryglycerides, lipids,
oils and/or fats containing stearic acid, preferably stearic acid
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a YLR010C protein is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[3223] The sequence of b0161 (Accession number NP.sub.--414703)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a periplasmic serine protease (heat shock protein).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a periplasmic serine protease from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of stearic acid and/or tryglycerides,
lipids, oils and/or fats containing stearic acid, in particular for
increasing the amount of stearic acid and/or tryglycerides, lipids,
oils and/or fats containing stearic acid, preferably stearic acid
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a periplasmic serine protease is
increased or generated, e.g. from E. coli or a homolog thereof.
[3224] The sequence of b1896 (Accession number NP.sub.--416410)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a trehalose-6-phosphate synthase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a trehalose-6-phosphate synthase from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of stearic acid and/or tryglycerides, lipids, oils and/or
fats containing stearic acid, in particular for increasing the
amount of stearic acid and/or tryglycerides, lipids, oils and/or
fats containing stearic acid, preferably stearic acid in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a trehalose-6-phosphate synthase is increased or generated, e.g.
from E. coli or a homolog thereof. The sequence of b3457 (Accession
number NP.sub.--417914) from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a high-affinity branched-chain
amino acid transport protein (ABC superfamily). Accordingly, in one
embodiment, the process of the present invention comprises the use
of a high-affinity branched-chain amino acid transport protein from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of stearic acid and/or tryglycerides,
lipids, oils and/or fats containing stearic acid, in particular for
increasing the amount of stearic acid and/or tryglycerides, lipids,
oils and/or fats containing stearic acid, preferably stearic acid
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a high-affinity branched-chain amino acid
transport protein is increased or generated, e.g. from E. coli or a
homolog thereof.
[3225] [0023.0.7.7] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the fine chemical amount or content. Further, in the
present invention, the term "homologue" relates to the sequence of
an organism having the highest sequence homology to the herein
mentioned or listed sequences of all expressed sequences of said
organism.
[3226] However, the person skilled in the art knows, that,
preferably, the homologue has said fine-chemical increasing
activity and, if known, the same biological function or activity in
the organism as at least one of the protein(s) indicated in Table
II, Column 3, lines 72 to 81 and 460 to 462, e.g. having the
sequence of a polypeptide encoded by a nucleic acid molecule
comprising the sequence indicated in Table I, Column 5 or 7, lines
72 to 81 and 460 to 462.
[3227] In one embodiment, the homolog of any one of the
polypeptides indicated in Table II, lines 75 to 80 is a homolog
having the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms and being derived from an Eukaryot. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 72 to 74, 81 and 460 to 462 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from bacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 75 to 80 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in an organisms or part
thereof, and being derived from Fungi. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines
lines 72 to 74, 81 and 460 to 462 is a homolog having the same or a
similar activity, in particular an increase of activity confers an
increase in the content of the fine chemical in the organisms or
part thereof and being derived from Proteobacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 75 to 80 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or a part
thereof and being derived from Ascomycota. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 72
to 74, 81 and 460 to 462 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or part
thereof, and being derived from Gammaproteobacteria. In one
embodiment, the homolog of a polypeptide polypeptide indicated in
Table II, column 3, lines 75 to 80 is a homolog having the same or
a similar activity, in particular an increase of activity confers
an increase in the content of the fine chemical in the organisms or
part thereof, and being derived from Saccharomycotina. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 72 to 74, 81 and 460 to 462 is a homolog having the
samie or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 75 to 80 is a homolog having
the samie or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms or a part thereof, and being derived from
Saccharomycetes. In one embodiment, the homolog of the a
polypeptide indicated in Table II, column 3, lines lines 72 to 74,
81 and 460 to 462 is a homolog having the samie or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or part
thereof, and being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 75 to 80 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms, and being
derived from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 72 to 74, 81 and
460 to 462 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof,
and being derived from Escherichia. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 75 to 80 is
a homolog having the same or a similar activity, in particular an
increase of activity confers an increase in the content of the fine
chemical in the organisms or a part thereof, and being derived from
Saccharomycetaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 75 to 80 is a homolog having
the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms or a part thereof, and being derived from
Saccharomycetes.
[3228] [0023.1.0.7] and [0024.0.0.7] for the disclosure of the
paragraphs [0023.1.0.7] and [0024.0.0.7] see [0023.1.0.0] and
[0024.0.0.0] above.
[3229] [0025.0.7.7] In accordance with the invention, a protein or
polypeptide has the "activity of a protein as indicated in Table
II, column 3, lines 72 to 81 and 460 to 462" if its de novo
activity, or its increased expression directly or indirectly leads
to an increased stearic acid and/or tryglycerides, lipids, oils
and/or fats containing stearic acid level in the organism or a part
thereof, preferably in a cell of said organism and the protein has
the above mentioned activities of a protein as indicated in Table
II, column 3, lines 72 to 81 and 460 to 462. Throughout the
specification the activity or preferably the biological activity of
such a protein or polypeptide or an nucleic acid molecule or
sequence encoding such protein or polypeptide is identical or
similar if it still has the biological or enzymatic activity of a
protein as indicated in Table II, column 3, lines 72 to 81 and 460
to 462, or which has at least 10% of the original enzymatic
activity, preferably 20%, particularly preferably 30%, most
particularly preferably 40% in comparison to a protein of
Saccharomyces cerevisiae as indicated in Table II, column 3, lines
75 to 80 and/or a protein of E. coli K12 as indicated in Table II,
column 3, lines 72 to 74, 81 and 460 to 462.
[3230] [0025.1.0.7] and [0025.2.0.7] for the disclosure of the
paragraphs [0025.1.0.7] and [0025.2.0.7] see paragraphs
[0025.1.0.0] and [0025.2.0.0] above.
[3231] [0026.0.0.7] to [0033.0.0.7] for the disclosure of the
paragraphs [0026.0.0.7] to [0033.0.0.7] see paragraphs [0026.0.0.0]
to [0033.0.0.0] above.
[3232] [0034.0.7.7] Preferably, the reference, control or wild type
differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. as result of
an increase in the level of the nucleic acid molecule of the
present invention or an increase of the specific activity of the
polypeptide of the invention or the polypeptide used in the method
of the invention. E.g., it differs by or in the expression level or
activity of an protein having the activity of a protein as
indicated in Table II, column 3, lines 72 to 81 and 460 to 462 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 72 to 81 and 460 to 462 or its homologs, e.g. as
indicated in Table I, column 7, lines 72 to 81 and 460 to 462, its
biochemical or genetical causes and therefore shows the increased
amount of the fine chemical.
[3233] [0035.0.0.7] to [0038.0.0.7] and [0039.0.5.7] for the
disclosure of the paragraphs [0035.0.0.7] to [0038.0.0.7] and
[0039.0.5.7] see paragraphs [0035.0.0.0] to [0039.0.0.0] above.
[3234] [0040.0.0.7] to [0044.0.0.7] for the disclosure of the
paragraphs [0040.0.0.7] to [0044.0.0.7] see paragraphs [0035.0.0.0]
and [0044.0.0.0] above.
[3235] [0045.0.7.7] In one embodiment, in case the activity of the
Escherichia coli K12 protein b3256 or its homologs e.g. a acetyl
CoA carboxylase protein e.g. as indicated in Table II, columns 5 or
7, line 72, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of stearic acid between
17% and 25% or more is conferred.
[3236] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2699 or its homologs e.g. a DNA-dependent ATPase
or DNA- and ATP-dependent coprotease protein e.g. as indicated in
Table II, columns 5 or 7, line 73, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of stearic
acid between 22% and 83% or more is conferred.
[3237] In case the activity of the Escherichia coli K12 protein
b2095 or its homologs e.g. a putative tagatose-6-phosphate kinase 1
e.g. as indicated in Table II, columns 5 or 7, line 74, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of stearic acid between 17% and 26% or more is
conferred.
[3238] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1093 or its homologs e.g. a
3-oxoacyl-[acyl-carrier-protein] reductase e.g. as indicated in
Table II, columns 5 or 7, line 81, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of stearic
acid between 16% and 31% or more is conferred.
[3239] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YOR024W or its homologs, e.g. a "uncharacterized
protein YOR024W", which seams to be "probably a membrane protein"
e.g. as indicated in Table II, columns 5 or 7, line 75 is
increased, preferably, in one embodiment an increase of the fine
chemical, preferably of stearic acid between 16% and 40% or more is
conferred.
[3240] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YBR089C-A or its homologs, e.g. a
"uncharacterized protein YBR089C-A", which seams to have "homology
to mammalian high mobility group proteins 1 and 2" e.g. as
indicated in Table II, columns 5 or 7, line 76 is increased,
preferably, in one embodiment an increase of the fine chemical,
preferably of stearic acid between 36% and 134% or more is
conferred.
[3241] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YFR042W or its homologs, e.g. a "uncharacterized
protein YFR042W", which seams to be a "protein, which is required
for cell viability" e.g. as indicated in Table II, columns 5 or 7,
line 77 is increased, preferably, in one embodiment an increase of
the fine chemical, preferably of stearic acid between 19% and 26%
or more is conferred.
[3242] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YIL150C or its homologs e.g. a "uncharacterized
protein YIL150C", which is probably a "protein required for S-phase
(DNA synthesis) initiation or completion" e.g. as indicated in
Table II, columns 5 or 7, line 78 is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of stearic
acid between 34% and 220% or more is conferred.
[3243] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YDR513W or its homologs, e.g. a "glutaredoxin
(thioltransferase, glutathione reductase)" e.g. as indicated in
Table II, columns 5 or 7, line 79 is increased, preferably, in one
embodiment an increase of the fine chemical, preferably of stearic
acid between 16% and 51% or more is conferred.
[3244] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YLR010C or its homologs, e.g. a "uncharacterized
protein YLR010C", which is probably a "protein involved in
telomeric pathways in association with Stn1" e.g. as indicated in
Table II, columns 5 or 7, line 80 is increased, preferably, in one
embodiment an increase of the fine chemical, preferably of stearic
acid between 16% and 76% or more is conferred.
[3245] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0161 or its homologs e.g. a periplasmic serine
protease e.g. as indicated in Table II, columns 5 or 7, line 460,
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of stearic acid between 16% and 129% or
more is conferred.
[3246] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs e.g. a trehalose-6-phosphate
synthase e.g. as indicated in Table II, columns 5 or 7, line 461,
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of stearic acid between 23% and 30% or
more is conferred.
[3247] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3457 or its homologs e.g. a high-affinity
branched-chain amino acid transport protein (ABC superfamily) e.g.
as indicated in Table II, columns 5 or 7, line 462, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of stearic acid between 18% and 34% or more is
conferred.
[3248] [0046.0.7.7] In case the activity of the Escherichia coli
K12 protein b3256 or its homologs e.g. a acetyl CoA carboxylase
protein, is increased, preferably an increase of the fine chemical
and of tryglycerides, lipids, oils and/or fats containing stearic
acid is conferred.
[3249] In case the activity of the Escherichia coli K12 protein
b2699 or its homologs e.g. a DNA-dependent ATPase or DNA- and
ATP-dependent coprotease protein, is increased, preferably an
increase of the fine chemical and of tryglycerides, lipids, oils
and/or fats containing stearic acid is conferred.
[3250] In case the activity of the Escherichia coli K12 protein
b2095 or its homologs e.g. a putative tagatose-6-phosphate kinase
1, is increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing stearic acid is
conferred.
[3251] In case the activity of the Escherichia coli K12 protein
b1093 or its homologs e.g. a 3-oxoacyl-[acyl-carrier-protein]
reductase, is increased, preferably, in one embodiment the increase
of the fine chemical, preferably an increase of the fine chemical
and of tryglycerides, lipids, oils and/or fats containing stearic
acid is conferred.
[3252] In case the activity of the Saccharomyces cerevisiae protein
YOR024W or its homologs, e.g. a "uncharacterized protein YOR024W",
which seams to be "probably a membrane protein" is increased,
preferably, in one embodiment an increase of the fine chemical,
preferably an increase of the fine chemical and of tryglycerides,
lipids, oils and/or fats containing stearic acid is conferred.
[3253] In case the activity of the Saccharomyces cerevisiae protein
YBR089C-A or its homologs, e.g. a "uncharacterized protein
YBR089C-A", which seams to have "homology to mammalian high
mobility group proteins 1 and 2" is increased, preferably, an
increase of the fine chemical and of tryglycerides, lipids, oils
and/or fats containing stearic acid is conferred.
[3254] In case the activity of the Saccharomyces cerevisiae protein
YFR042W or its homologs, e.g. a "uncharacterized protein YFR042W",
which seams to be a "protein, which is required for cell viability"
is increased, preferably, in one embodiment an increase of the fine
chemical, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing stearic acid is
conferred.
[3255] In case the activity of the Saccaromyces cerevisiae protein
YIL150C or its homologs e.g. a "uncharacterized protein YIL150C",
which is probably a "protein required for S-phase (DNA synthesis)
initiation or completion" is increased, preferably an increase of
the fine chemical and of tryglycerides, lipids, oils and/or fats
containing stearic acid is conferred.
[3256] In case the activity of the Saccharomyces cerevisiae protein
YDR513W or its homologs, e.g. a "glutaredoxin (thioltransferase,
glutathione reductase)" is increased, preferably, in one embodiment
an increase of the fine chemical, preferably an increase of the
fine chemical and of tryglycerides, lipids, oils and/or fats
containing stearic acid is conferred.
[3257] In case the activity of the Saccharomyces cerevisiae protein
YLR010C or its homologs, e.g. a "uncharacterized protein YLR010C",
which is probably a "protein involved in telomeric pathways in
association with Stn1" is increased, preferably, in one embodiment
an increase of the fine chemical, preferably an increase of the
fine chemical and of tryglycerides, lipids, oils and/or fats
containing stearic acid is conferred.
[3258] In case the activity of the Escherichia coli K12 protein
b0161 or its homologs e.g. a periplasmic serine protease is
increased, preferably, in one embodiment an increase of the fine
chemical, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing stearic acid is
conferred.
[3259] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs e.g. a trehalose-6-phosphate
synthase is increased, preferably, in one embodiment an increase of
the fine chemical, preferably an increase of the fine chemical and
of tryglycerides, lipids, oils and/or fats containing stearic acid
is conferred.
[3260] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3457 or its homologs e.g. a high-affinity
branched-chain amino acid transport protein (ABC superfamily) is
increased, preferably, in one embodiment an increase of the fine
chemical, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing stearic acid is
conferred.
[3261] [0047.0.0.7] to [0048.0.0.7] for the disclosure of the
paragraphs [0047.0.0.7] and [0048.0.0.7] see paragraphs
[0047.0.0.0] and [0048.0.0.0] above.
[3262] [0049.0.7.7] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
preferably has the structure of the polypeptide described herein,
in particular a polypeptide comprising a consensus sequence as
indicated in Table IV, column 7, lines 72 to 81 and 460 to 462 or
of the polypeptide as shown in the amino acid sequences as
disclosed in table II, columns 5 and 7, lines 72 to 81 and 460 to
462 or the functional homologues thereof as described herein, or is
encoded by the nucleic acid molecule characterized herein or the
nucleic acid molecule according to the invention, for example by
the nucleic acid molecule as shown in table I, columns 5 and 7,
lines 72 to 81 and 460 to 462 or its herein described functional
homologues and has the herein mentioned activity.
[3263] [0050.0.7.7] For the purposes of the present invention, the
term "stearic acid" also encompasses the corresponding salts, such
as, for example, the potassium or sodium salts of stearic acid or
the salts of stearic acid with amines such as diethylamine.
[3264] [0051.0.5.7] and [0052.0.0.7] for the disclosure of the
paragraphs [0051.0.5.7] and [0052.0.0.7] see paragraphs
[0051.0.0.0] and [0052.0.0.0] above.
[3265] [0053.0.7.7] In one embodiment, the process of the present
invention comprises one or more of the following steps [3266] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention or the nucleic acid molecule or the
polypeptide used in the method of the invention, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 72 to 81 and 460 to 462 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 72 to 81
and 460 to 462, having herein-mentioned stearic acid increasing
activity; [3267] b) stabilizing a mRNA conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention, e.g. of a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 72 to 81 and 460 to 462 or
its homologs activity, e.g. as indicated in Table II, column 7,
lines 72 to 81 and 460 to 462 or of a mRNA encoding the polypeptide
of the present invention having herein-mentioned stearic acid
increasing activity; [3268] c) increasing the specific activity of
a protein conferring the increased expression of a protein encoded
by the nucleic acid molecule of the invention or of the polypeptide
of the present invention or the nucleic acid molecule or the
polypeptide used in the method of the invention, having
herein-mentioned stearic acid increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 72 to 81 and 460 to 462 or its homologs
activity, e.g. as indicated in Table II, column 7, lines 72 to 81
and 460 to 462, or decreasing the inhibiitory regulation of the
polypeptide of the invention or of the polypeptide used in the
method of the invention; [3269] d) generating or increasing the
expression of an endogenous or artificial transcription factor
mediating the expression of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention or of the polypeptide of the invention or the polypeptide
used in the method of the invention having herein-mentioned stearic
acid increasing activity, e.g. of a polypeptide having an activity
of a protein as indicated in Table II, column 3, lines 72 to 81 and
460 to 462 or its homolog's activity, e.g. as indicated in Table
II, column 7, lines 72 to 81 and 460 to 462; [3270] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned stearic acid increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 72 to 81 and 460 to 462 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 72 to 81
and 460 to 462, by adding one or more exogenous inducing factors to
the organisms or parts thereof; [3271] f) expressing a transgenic
gene encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned stearic acid increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 72 to 81 and 460 to 462 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 72 to 81
and 460 to 462; [3272] g) increasing the copy number of a gene
conferring the increased expression of a nucleic acid molecule
encoding a polypeptide encoded by the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention or the polypeptide of the invention or the polypeptide
used in the method of the invention having herein-mentioned stearic
acid increasing activity, e.g. of a polypeptide having an activity
of a protein as indicated in Table II, column 3, lines 72 to 81 and
460 to 462 or its homolog's activity, e.g. as indicated in Table
II, column 7, lines 72 to 81 and 460 to 462; [3273] h) increasing
the expression of the endogenous gene encoding the polypeptide of
the invention or the polypeptide used in the method of the
invention, e.g. a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 72 to 81 and 460 to 462 or
its homolog's activity, e.g. as indicated in Table II, column 7,
lines 72 to 81 and 460 to 462, by adding positive expression or
removing negative expression elements, e.g. homologous
recombination can be used to either introduce positive regulatory
elements like for plants the 35S enhancer into the promoter or to
remove repressor elements form regulatory regions. Further gene
conversion methods can be used to disrupt repressor elements or to
enhance to activity of positive elements. Positive elements can be
randomly introduced in plants by T-DNA or transposon mutagenesis
and lines can be identified in which the positive elements have be
integrated near to a gene of the invention, the expression of which
is thereby enhanced; [3274] i) Modulating growth conditions of an
organism in such a manner, that the expression or activity of the
gene encoding the protein of the invention or the protein itself is
enhanced for example microorganisms or plants can be grown for
example under a higher temperature regime leading to an enhanced
expression of heat shock proteins, which can lead an enhanced fine
chemical production. [3275] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[3276] [0054.0.7.7] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of stearic acid after
increasing the expression or activity of the encoded polypeptide or
having the activity of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 72 to 81 and 460
to 462 or its homolog's activity, e.g. as indicated in Table II,
columns 5 or 7, lines 72 to 81 and 460 to 462.
[3277] [0055.0.0.7] to [0067.0.0.7] for the disclosure of the
paragraphs [0055.0.0.7] to [0067.0.0.7] see paragraphs [0055.0.0.0]
to [0067.0.0.0] above.
[3278] [0068.0.5.7] and [0069.0.5.7] for the disclosure of the
paragraphs [0068.0.5.7] and [0069.0.5.7] see paragraphs
[0068.0.0.0] and [0069.0.0.0] above.
[3279] [0070.0.6.7] and [0071.0.5.7] for the disclosure of the
paragraphs [0070.0.6.7] and [0071.0.5.7] see paragraphs
[0070.0.5.5] and [0071.0.0.0] above.
[3280] [0072.0.7.7] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to stearic acid, triglycerides, lipids, oils and/or fats
containing stearic acid compounds such as palmitate, palmitoleate,
stearate, oleate, .alpha.-linolenic acid and/or linoleic acid.
[3281] [0073.0.7.7] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
a) providing a non-human organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant; b) increasing the activity of a protein having the
activity of a polypeptide of the invention or the polypeptide used
in the method of the invention or a homolog thereof, e.g. as shown
in Table II, columns 5 or 7, lines 72 to 81 and 460 to 462 or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, i.e. conferring an increase
of the respective fine chemical in the organism, preferably a
microorganism, the a non-human animal, a plant or animal cell, a
plant or animal tissue or the plant, c) growing the organism,
preferably a microorganism, a non-human animal, a plant or animal
cell, a plant or animal tissue or the plant under conditions which
permit the production of the fine chemical in the organism,
preferably a microorganism, a plant cell, a plant tissue or the
plant; and d) if desired, revovering, optionally isolating, the
free and/or bound the respective fine chemical and, optionally
further free and/or bound fatty acids synthetized by the organism,
the microorganism, the non-human animal, the plant or animal cell,
the plant or animal tissue or the plant.
[3282] [0074.0.5.7] for the disclosure of this paragraph see
[0074.0.5.5] above.
[3283] [0075.0.0.7] to [0084.0.0.7] for the disclosure of the
paragraphs [0075.0.0.7] to [0084.0.0.7] see paragraphs [0075.0.0.0]
to [0084.0.0.0] above.
[3284] [0085.0.7.7] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [3285] a) the nucleic acid sequence as
shown in table I, lines 72 to 81 and 460 to 462, columns 5 and 7 or
a derivative thereof, or [3286] b) a genetic regulatory element,
for example a promoter, which is functionally linked to the nucleic
acid sequence as shown table I, lines 72 to 81 and 460 to 462,
columns 5 and 7 or a derivative thereof, or [3287] c) (a) and (b)
is/are not present in its/their natural genetic environment or
has/have been modified by means of genetic manipulation methods, it
being possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[3288] [0086.0.0.7] and [0087.0.0.7] for the disclosure of the
paragraphs [0086.0.0.7] and [0087.0.0.7] see paragraphs
[0086.0.0.0] and [0087.0.0.0] above.
[3289] [0088.0.7.7] In an advantageous embodiment of the invention,
the organism takes the form of a plant whose fatty acid content is
modified advantageously owing to the nucleic acid molecule of the
present invention expressed. This is important for plant breeders
since, for example, the nutritional value of plants for poultry is
dependent on the abovementioned essential fatty acids and the
general amount of fatty acids as energy source in feed. After the
activity of a protein as shown in Table II, columns 5 or 7, lines
72 to 81 and 460 to 462 has been increased or generated, or after
the expression of nucleic acid molecule or polypeptide according to
the invention has been generated or increased, the transgenic plant
generated thus is grown on or in a nutrient medium or else in the
soil and subsequently harvested.
[3290] [0088.1.0.7], [0089.0.0.7], [0090.0.0.7] and [0091.0.5.7]
for the disclosure of the paragraphs [0088.1.0.7], [0089.0.0.7],
[0090.0.0.7] and [0091.0.5.7] see paragraphs [0088.1.0.0],
[0089.0.0.0], [0090.0.0.0] and [0091.0.0.0] above.
[3291] [0092.0.0.7] to [0094.0.0.7] for the disclosure of the
paragraphs [0092.0.0.7] to [0094.0.0.7] see paragraphs [0092.0.0.0]
to [0094.0.0.0] above.
[3292] [0095.0.5.7], [0096.0.5.7] and [0097.0.5.7] for the
disclosure of the paragraphs [0095.0.5.7], [0096.0.5.7] and
[0097.0.5.7] see paragraphs [0095.0.5.5], [0096.0.5.5] and
[0097.0.0.0] above.
[3293] [0098.0.7.7] In a preferred embodiment, the fine chemical
(stearic acid) is produced in accordance with the invention and, if
desired, is isolated. The production of further fatty acids such as
palmitic acid, stearic acid, palmitoleic acid, oleic acid and/or
linoleic acid mixtures thereof or mixtures of other fatty acids by
the process according to the invention is advantageous.
[3294] [0099.0.5.7] and [0100.0.5.7] for the disclosure of the
paragraphs [0099.0.5.7] and [0100.0.5.7] see paragraphs
[0099.0.5.5] and [0100.0.5.5] above.
[3295] [0101.0.5.7] and [0102.0.5.7] for the disclosure of the
paragraphs [0101.0.5.7] and [0102.0.5.7] see [0101.0.0.0] and
[0102.0.5.5] above.
[3296] [0103.0.7.7] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [3297]
a) nucleic acid molecule encoding, preferably at least the mature
form, of a polypeptide having a sequence as shown in Table II,
columns 5 or 7, lines 72 to 81 and 460 to 462 in or a fragment
thereof, which confers an increase in the amount of the respective
fine chemical in an organism or a part thereof; [3298] b) nucleic
acid molecule comprising, preferably at least the mature form, of
the nucleic acid molecule having a sequence as shown in Table I,
columns 5 or 7, lines 72 to 81 and 460 to 462, [3299] c) nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as result
of the degeneracy of the genetic code and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[3300] d) nucleic acid molecule encoding a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [3301] e) nucleic acid molecule which hybridizes with
a nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [3302] f)
nucleic acid molecule encoding a polypeptide, the polypeptide being
derived by substituting, deleting and/or adding one or more amino
acids of the amino acid sequence of the polypeptide encoded by the
nucleic acid molecules (a) to (d), preferably to (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [3303] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [3304] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primer pairs
having a sequence as shown in Table III, column 7, lines 72 to 81
and 460 to 462 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [3305]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [3306] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequence as shown in Table IV, column 7, lines 72
to 81 and 460 to 462 and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[3307] k) nucleic acid molecule comprising one or more of the
nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of a polypeptide as shown in Table
II, columns 5 or 7, lines 72 to 81 and 460 to 462 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; and [3308] l) nucleic acid molecule
which is obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably to (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; or which comprises a sequence which is complementary
thereto.
[3309] [0103.1.0.7] and [0103.2.0.7] for the disclosure of the
paragraphs [0103.1.0.7] and [0103.2.0.7] see paragraphs
[0103.1.0.0] and [0103.2.0.0] above.
[3310] [0104.0.7.7] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence shown in Table
I, columns 5 or 7, lines 72 to 81 and 460 to 462, preferably over
the sequences as shown in Table IA, columns 5 or 7, lines 72 to 81
and 460 to 462 by one or more nucleotides or does not consist of
the sequence shown in Table I, columns 5 or 7, lines 72 to 81 and
460 to 462, preferably over the sequences as shown in Table IA,
columns 5 or 7, lines 72 to 81 and 460 to 462. In one embodiment,
the nucleic acid molecule of the present invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to the sequence shown
in Table I, columns 5 or 7, lines 72 to 81 and 460 to 462,
preferably over the sequences as shown in Table IA, columns 5 or 7,
lines 72 to 81 and 460 to 462. In another embodiment, the nucleic
acid molecule does not encode a polypeptide of the sequence shown
in Table II, columns 5 or 7, lines 72 to 81 and 460 to 462,
preferably of the sequences as shown in Table IA, columns 5 or 7,
lines 72 to 81 and 460 to 462.
[3311] [0105.0.0.7] to [0107.0.0.7] for the disclosure of the
paragraphs [0105.0.0.7] to [0107.0.0.7] see paragraphs [0105.0.0.0]
and [0107.0.0.0] above.
[3312] [0108.0.7.7] Nucleic acid molecules with the sequence shown
in Table I, columns 5 or 7, lines 72 to 81 and 460 to 462, nucleic
acid molecules which are derived from the amino acid sequences
shown in Table II, columns 5 or 7, lines 72 to 81 and 460 to 462 or
from polypeptides comprising the consensus sequence shown in Table
IV, column 7, lines 72 to 81 and 460 to 462, or their derivatives
or homologues encoding polypeptides with the enzymatic or
biological activity of a protein as shown in Table II, columns 5 or
7, lines 72 to 81 and 460 to 462 or e.g. conferring a linoleic acid
increase after increasing its expression or activity are
advantageously increased in the process according to the
invention.
[3313] [0109.0.5.7] for the disclosure of this paragraph see
[0109.0.0.0] above.
[3314] [0110.0.7.7] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide used in the method
of the invention or used in the process of the invention, e.g. of a
protein as shown in Table II, columns 5 or 7, lines 72 to 81 and
460 to 462 or being encoded by a nucleic acid molecule indicated in
Table I, columns 5 or 7, lines 72 to 81 and 460 to 462 or of its
homologs, e.g. as indicated in Table II, columns 5 or 7, lines 72
to 81 and 460 to 462 can be determined from generally accessible
databases.
[3315] [0111.0.0.7] for the disclosure of this paragraph see
[0111.0.0.0] above.
[3316] [0112.0.7.7] The nucleic acid molecules used in the process
according to the invention take the form of isolated nucleic acid
sequences, which encode polypeptides with an activity of a
polypeptide as indicated in Table II, column 3, lines lines 72 to
81 and 460 to 462 or having the sequence of a polypeptide as
indicated in Table II, columns 5 and 7, lines 72 to 81 and 460 to
462 and conferring an increase of the respective fine chemical.
[3317] [0113.0.0.7] to [0120.0.0.7] for the disclosure of the
paragraphs [0113.0.0.7] to [0120.0.0.7] see paragraphs [0113.0.0.0]
and [0120.0.0.0] above.
[3318] [0121.0.7.7] However, it is also possible to use artificial
sequences, which differ in one or more bases from the nucleic acid
sequences found in organisms, or in one or more amino acid
molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences shown in Table II,
columns 5 or 7, lines 72 to 81 and 460 to 462 or the functional
homologues thereof as described herein, preferably conferring
above-mentioned activity, i.e. conferring an increase of the
respective fine chemical after increasing its activity.
[3319] [0122.0.0.7] to [0127.0.0.7] for the disclosure of the
paragraphs [0122.0.0.7] to [0127.0.0.7] see paragraphs [0122.0.0.0]
and [0127.0.0.0] above.
[3320] [0128.0.7.7] Synthetic oligonucleotide primers for the
amplification, e.g. as shown in Table III, column 7, lines 72 to 81
and 460 to 462, by means of polymerase chain reaction can be
generated on the basis of a sequence shown herein, for example the
sequence shown in Table I, columns 5 or 7, lines 72 to 81 and 460
to 462 or the sequences as shown in Table II, columns 5 or 7, lines
72 to 81 and 460 to 462.
[3321] [0129.0.7.7] Moreover, it is possible to identify conserved
regions from various organisms by carrying out protein sequence
alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence shown in Table IV, column
7, lines 72 to 81 and 460 to 462 is derived from said
alignments.
[3322] [0130.0.7.7] for the disclosure of this paragraph see
[0130.0.0.0].
[3323] [0131.0.0.7] to [0138.0.0.7] for the disclosure of the
paragraphs [0131.0.0.7] to [0138.0.0.7] see paragraphs [0131.0.0.0]
to [0138.0.0.0] above.
[3324] [0139.0.7.7] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical increase, derived from
other organisms, can be encoded by other DNA sequences which
hybridize to the sequences shown in Table I, columns 5 or 7, lines
72 to 81 and 460 to 462, preferably of Table I B, columns 5 or 7,
lines 72 to 81 and 460 to 462 under relaxed hybridization
conditions and which code on expression for peptides having the
stearic acid increasing activity.
[3325] [0140.0.0.7] to [0146.0.0.7] for the disclosure of the
paragraphs [0140.0.0.7] to [0146.0.0.7] see paragraphs [0140.0.0.0]
and [0146.0.0.0] above.
[3326] [0147.0.7.7] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 72 to 81 and 460 to 462, preferably in
Table I B, columns 5 or 7, lines 72 to 81 and 460 to 462 is one
which is sufficiently complementary to one of said nucleotide
sequences such that it can hybridize to one of said nucleotide
sequences thereby forming a stable duplex. Preferably, the
hybridisation is performed under stringent hybridization
conditions. However, a complement of one of the herein disclosed
sequences is preferably a sequence complement thereto according to
the base pairing of nucleic acid molecules well known to the
skilled person. For example, the bases A and G undergo base pairing
with the bases T and U or C, resp. and visa versa. Modifications of
the bases can influence the base-pairing partner.
[3327] [0148.0.7.7] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines
72 to 81 and 460 to 462, preferably of Table I B, columns 5 or 7,
lines 72 to 81 and 460 to 462, or a functional portion thereof and
preferably has above mentioned activity, in particular has
the--fine-chemical-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homologs.
[3328] [0149.0.7.7] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 72 to 81 and 460 to 462, preferably of Table I B, columns 5
or 7, lines 72 to 81 and 460 to 462 or a portion thereof and
encodes a protein having above-mentioned activity as indicated in
Table II, columns 5 or 7, lines 72 to 81 and 460 to 462, preferably
of Table II B, columns 5 or 7, lines 72 to 81 and 460 to 462, e.g.
conferring an increase of the respective fine chemical.
[3329] [00149.1.0.7] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 72 to 81 and 460 to 462, preferably of Table
I B, columns 5 or 7, lines 72 to 81 and 460 to 462 has further one
or more of the activities annotated or known for the a protein as
indicated in Table II, column 3, lines 72 to 81 and 460 to 462.
[3330] [0150.0.7.7] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table I, columns 5 or 7, lines 72 to 81 and 460 to 462, preferably
of Table I B, columns 5 or 7, lines 72 to 81 and 460 to 462, for
example a fragment which can be used as a probe or primer or a
fragment encoding a biologically active portion of the polypeptide
of the present invention or of a polypeptide used in the process of
the present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of methionine if its activity is increased.
The nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences indicated in Table I, columns 5 or 7, lines 72 to
81 and 460 to 462, an anti-sense sequence of one of the sequences
indicated in Table I, columns 5 or 7, lines 72 to 81 and 460 to
462, or naturally occurring mutants thereof. Primers based on a
nucleotide sequence of the invention can be used in PCR reactions
to clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table III, column 7, lines 72 to 81 and 460 to 462 will result in a
fragment of a polynucleotide sequence as indicated in Table I,
columns 5 or 7, lines 72 to 81 and 460 to 462. Preferred is Table I
B, column 7, lines 72 to 81 and 460 to 462.
[3331] [0151.0.0.7] for the disclosure of this paragraph see
[0151.0.0.0] above.
[3332] [0152.0.7.7] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to the amino acid
sequence shown in Table II, columns 5 or 7, lines 72 to 81 and 460
to 462 such that the protein or portion thereof maintains the
ability to participate in the fine chemical production, in
particular a stearic acid increasing the activity as mentioned
above or as described in the examples in plants or microorganisms
is comprised.
[3333] [0153.0.7.7] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence shown in Table II, columns 5 or 7, lines 72 to 81 and
460 to 462 such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or protion thereof as
shown in Table II, columns 5 or 7, lines 72 to 81 and 460 to 462
has for example an activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 72 to 81 and 460 to 462 are described
herein.
[3334] [0154.0.7.7] In one embodiment, the nucleic acid molecule of
the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as shown in Table II, columns 5 or 7, lines 72 to 81 and
460 to 462 and having above-mentioned activity, e.g. conferring
preferably the increase of the respective fine chemical.
[3335] [0155.0.0.7] and [0156.0.0.7] for the disclosure of the
paragraphs [0155.0.0.7] and [0156.0.0.7] see paragraphs
[0155.0.0.0] and [0156.0.0.0] above.
[3336] [0157.0.7.7] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 72 to 81 and 460 to 462
(and portions thereof) due to degeneracy of the genetic code and
thus encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides comprising the consensus sequences as indicated
in Table IV, column 7, lines 72 to 81 and 460 to 462 or of the
polypeptide as indicated in Table II, columns 5 or 7, lines 72 to
81 and 460 to 462 or their functional homologues. Advantageously,
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention comprises, or in an
other embodiment has, a nucleotide sequence encoding a protein
comprising, or in an other embodiment having, a consensus sequences
as indicated in Table IV, column 7, lines 72 to 81 and 460 to 462
or of the polypeptide as indicated in Table II, columns 5 or 7,
lines 72 to 81 and 460 to 462 or the functional homologues. In a
still further embodiment, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention encodes a full length protein which is substantially
homologous to an amino acid sequence comprising a consensus
sequence as indicated in Table IV, column 7, lines 72 to 81 and 460
to 462 or of a polypeptide as indicated in Table II, columns 5 or
7, lines 72 to 81 and 460 to 462 or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table I, columns 5 or 7, lines 72 to 81 and 460 to
462, preferably as indicated in Table I A, columns 5 or 7, lines 72
to 81 and 460 to 462. Preferably the nucleic acid molecule of the
invention is a functional homologue or identical to a nucleic acid
molecule indicated in Table I B, columns 5 or 7, lines 72 to 81 and
460 to 462.
[3337] [0158.0.0.7] to [0160.0.0.7] for the disclosure of the
paragraphs [0158.0.0.7] to [0160.0.0.7] see paragraphs [0158.0.0.0]
to [0160.0.0.0] above.
[3338] [0161.0.7.7] Accordingly, in another embodiment, a nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising the
sequence shown in Table I, columns 5 or 7, lines 72 to 81 and 460
to 462. The nucleic acid molecule is preferably at least 20, 30,
50, 100, 250 or more nucleotides in length.
[3339] [0162.0.0.7] for the disclosure of this paragraph see
paragraph [0162.0.0.0] above.
[3340] [0163.0.7.7] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 72 to 81 and 460 to
462 corresponds to a naturally-occurring nucleic acid molecule of
the invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[3341] [0164.0.0.7] for the disclosure of this paragraph see
paragraph [0164.0.0.0] above.
[3342] [0165.0.7.7] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as shown in
Table I, columns 5 or 7, lines 72 to 81 and 460 to 462.
[3343] [0166.0.0.7] and [0167.0.0.7] for the disclosure of the
paragraphs [0166.0.0.7] and [0167.0.0.7] see paragraphs
[0166.0.0.0] and [0167.0.0.0] above.
[3344] [0168.0.7.7] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organism or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 72 to 81 and 460 to 462 yet retain said activity described
herein. The nucleic acid molecule can comprise a nucleotide
sequence encoding a polypeptide, wherein the polypeptide comprises
an amino acid sequence at least about 50% identical to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 72 to
81 and 460 to 462 and is capable of participation in the increase
of production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to the
sequence as indicated in Table II, columns 5 or 7, lines 72 to 81
and 460 to 462, more preferably at least about 70% identical to one
of the sequences as indicated in Table II, columns 5 or 7, lines 72
to 81 and 460 to 462, even more preferably at least about 80%, 90%,
95% homologous to the sequence as indicated in Table II, columns 5
or 7, lines 72 to 81 and 460 to 462, and most preferably at least
about 96%, 97%, 98%, or 99% identical to the sequence as indicated
in Table II, columns 5 or 7, lines 72 to 81 and 460 to 462.
[3345] Accordingly, the invention relates to nucleic acid molecules
encoding a polypeptide having above-mentioned activity, e.g.
conferring an increase in the the respective fine chemical in an
organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 72 to 81 and 460 to 462, preferably of Table II B, column 7,
lines 72 to 81 and 460 to 462 yet retain said activity described
herein. The nucleic acid molecule can comprise a nucleotide
sequence encoding a polypeptide, wherein the polypeptide comprises
an amino acid sequence at least about 50% identical to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 72 to
81 and 460 to 462, preferably of Table II B, column 7, lines 72 to
81 and 460 to 462 and is capable of participation in the increase
of production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table II, columns 5 or 7, lines 72 to 81
and 460 to 462, preferably of Table II B, column 7, lines 72 to 81
and 460 to 462, more preferably at least about 70% identical to one
of the sequences as indicated in Table II, columns 5 or 7, lines 72
to 81 and 460 to 462, preferably of Table II B, column 7, lines 72
to 81 and 460 to 462, even more preferably at least about 80%, 90%,
or 95% homologous to a sequence as indicated in Table II, columns 5
or 7, lines 72 to 81 and 460 to 462, preferably of Table II B,
column 7, lines 72 to 81 and 460 to 462, and indicated in Table II,
columns 5 or 7, lines 72 to 81 and 460 to 462, preferably of Table
II B, column 7, lines 72 to 81 and 460 to 462.
[3346] [0169.0.0.7] to [0175.0.5.7] for the disclosure of the
paragraphs [0169.0.0.7] to [0175.0.5.7] see paragraphs [0169.0.0.0]
to [0175.0.0.0] above.
[3347] [0176.0.7.7] Functional equivalents derived from one of the
polypeptides as shown in Table II, columns 5 or 7, lines 72 to 81
and 460 to 462 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as shown in Table II, columns
5 or 7, lines 72 to 81 and 460 to 462 according to the invention
and are distinguished by essentially the same properties as the
polypeptide as shown in Table II, columns 5 or 7, lines 72 to 81
and 460 to 462.
[3348] [0177.0.7.7] Functional equivalents derived from the nucleic
acid sequence as shown in Table I, columns 5 or 7, lines 72 to 81
and 460 to 462 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as shown in Table II, columns
5 or 7, lines 72 to 81 and 460 to 462 according to the invention
and encode polypeptides having essentially the same properties as
the polypeptide as shown in Table II, columns 5 or 7, lines 72 to
81 and 460 to 462.
[3349] [0178.0.0.7] for the disclosure of this paragraph see
[0178.0.0.0] above.
[3350] [0179.0.7.7] A nucleic acid molecule encoding a homologous
to a protein sequence of as indicated in Table II, columns 5 or 7,
lines 72 to 81 and 460 to 462, preferably of Table II B, column 7,
lines 72 to 81 and 460 to 462 can be created by introducing one or
more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table I, columns 5 or 7,
lines 72 to 81 and 460 to 462 such that one or more amino acid
substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced into the encoding
sequences for example into a sequences as indicated in Table I,
columns 5 or 7, lines 72 to 81 and 460 to 462 by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[3351] [0180.0.0.7] to [0183.0.0.7] for the disclosure of the
paragraphs [0180.0.0.7] to [0183.0.0.7] see paragraphs [0180.0.0.0]
to [0183.0.0.0] above.
[3352] [0184.0.7.7] Homologues of the nucleic acid sequences used,
with a sequence as indicated in Table I, columns 5 or 7, lines 72
to 81 and 460 to 462, preferably of Table I B, column 7, lines 72
to 81 and 460 to 462, or of the nucleic acid sequences derived from
a sequences as indicated in Table II, columns 5 or 7, lines 72 to
81 and 460 to 462, preferably of Table II B, column 7, lines 72 to
81 and 460 to 462, comprise also allelic variants with at least
approximately 30%, 35%, 40% or 45% homology, by preference at least
approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
I, columns 5 or 7, lines 72 to 81 and 460 to 462, or from the
derived nucleic acid sequences, the intention being, however, that
the enzyme activity or the biological activity of the resulting
proteins synthesized is advantageously retained or increased.
[3353] [0185.0.7.7] In one embodiment of the present invention, the
nucleic acid molecule of the invention or used in the process of
the invention comprises one or more sequences as indicated in Table
I, columns 5 or 7, lines 72 to 81 and 460 to 462, preferably of
Table I B, column 7, lines 72 to 81 and 460 to 462. In one
embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotide sequences not
shown in any one of sequences as indicated in Table I, columns 5 or
7, lines 72 to 81 and 460 to 462, preferably of Table I B, column
7, lines 72 to 81 and 460 to 462. In one embodiment, the nucleic
acid molecule comprises less than 500, 400, 300, 200, 100, 90, 80,
70, 60, 50 or 40 further nucleotides. In a further embodiment, the
nucleic acid molecule comprises less than 30, 20 or 10 further
nucleotides. In one embodiment, a nucleic acid molecule used in the
process of the invention is identical to a sequences as indicated
in Table I, columns 5 or 7, lines 72 to 81 and 460 to 462,
preferably of Table I B, column 7, lines 72 to 81 and 460 to
462.
[3354] [0186.0.7.7] Also preferred is that one or more nucleic acid
molecule(s) used in the process of the invention encode a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 72 to 81 and 460 to 462, preferably of Table II B,
column 7, lines 72 to 81 and 460 to 462. In one embodiment, the
nucleic acid molecule encodes less than 150, 130, 100, 80, 60, 50,
40 or 30 further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table II, columns 5 or 7, lines 72 to 81 and 460 to 462,
preferably of Table II B, column 7, lines 72 to 81 and 460 to
462.
[3355] [0187.0.7.7] In one embodiment, the nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table II, columns 5 or 7,
lines 72 to 81 and 460 to 462, preferably of Table II B, column 7,
lines 72 to 81 and 460 to 462 comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a sequences as indicated in Table II, columns 5
or 7, lines 72 to 81 and 460 to 462, preferably of Table II B,
column 7, lines 72 to 81 and 460 to 462.
[3356] [0188.0.7.7] Polypeptides (=proteins), which still have the
essential biological activity of the polypeptide of the present
invention conferring an increase of the fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide shown in Table II, columns 5 or 7, lines 72 to 81
and 460 to 462 expressed under identical conditions.
[3357] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, columns 7, lines 72 to 81 and 460 to 462.
[3358] [0189.0.7.7] Homologues of sequences as indicated in Table
I, columns 5 or 7, lines 72 to 81 and 460 to 462 or of the derived
sequences shown in Table II, columns 5 or 7, lines 72 to 81 and 460
to 462 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[3359] [0190.0.0.7], [0191.0.5.7], [00191.1.0.7] and [0192.0.0.7]
to [0203.0.0.7] for the disclosure of the paragraphs [0190.0.0.7],
[0191.0.5.7], [0191.1.0.7] and [0192.0.0.7] to [0203.0.0.7] see
paragraphs [0190.0.0.0], [0191.0.0.0], [0191.1.0.0] and
[0192.0.0.0] to [0203.0.0.0] above.
[3360] [0204.0.7.7] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [3361] a) nucleic
acid molecule encoding, preferably at least the mature form, of the
polypeptide shown in Table II, columns 5 or 7, lines 72 to 81 and
460 to 462; preferably of Table II B, column 7, lines 72 to 81 and
460 to 462; or a fragment thereof conferring an increase in the
amount of the fine chemical in an organism or a part thereof [3362]
b) nucleic acid molecule comprising, preferably at least the mature
form, of the nucleic acid molecule shown in Table I, columns 5 or
7, lines 72 to 81 and 460 to 462 preferably of Table I B, column 7,
lines 72 to 81 and 460 to 462; or a fragment thereof conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [3363] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [3364] d) nucleic
acid molecule encoding a polypeptide whose sequence has at least
50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [3365] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [3366] f) nucleic acid molecule encoding a polypeptide,
the polypeptide being derived by substituting, deleting and/or
adding one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[3367] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to [3368] (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [3369] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying a cDNA library or a genomic library using the primers or
primer pairs as indicated in Table III, column 7, lines 72 to 81
and 460 to 462 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [3370]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from a expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (g), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [3371] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence shown in
Table IV, column 7, lines 72 to 81 and 460 to 462 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [3372] k) nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a
domaine of the polypeptide shown in Table II, columns 5 or 7, lines
72 to 81 and 460 to 462, preferably of Table II B, column 7, lines
72 to 81 and 460 to 462; and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
and [3373] l) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of the nucleic acid molecule shown in Table I, columns 5
or 7, lines 72 to 81 and 460 to 462 or a nucleic acid molecule
encoding, preferably at least the mature form of, the polypeptide
shown in Table II, columns 5 or 7, lines 72 to 81 and 460 to 462,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which encompasses a
sequence which is complementary thereto; whereby, preferably, the
nucleic acid molecule according to (a) to (l) distinguishes over
the sequence as depicted in Table IA, columns 5 or 7, lines 72 to
81 and 460 to 462 by one or more nucleotides. In one embodiment,
the nucleic acid molecule of the invention does not consist of the
sequence shown in Table IA or IB, columns 5 or 7, lines 72 to 81
and 460 to 462. In an other embodiment, the nucleic acid molecule
of the present invention is at least 30% identical and less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to the sequence shown
in Table IA or IB, columns 5 or 7, lines 72 to 81 and 460 to 462.
In a further embodiment the nucleic acid molecule does not encode
the polypeptide sequence shown in Table IIA or IIB, columns 5 or 7,
lines 72 to 81 and 460 to 462. Accordingly, in one embodiment, the
nucleic acid molecule of the present invention encodes in one
embodiment a polypeptide which differs at least in one or more
amino acids from the polypeptide as depicted in Table IIA or IIB,
columns 5 or 7, lines 72 to 81 and 460 to 462 and therefore does
not encode a protein of the sequence shown in Table IIA or IIB,
columns 5 or 7, lines 72 to 81 and 460 to 462. Accordingly, in one
embodiment, the protein encoded by a sequence of a nucleic acid
according to (a) to (l) does not consist of the sequence shown in
Table IIA or IIB, columns 5 or 7, lines 72 to 81 and 460 to 462. In
a further embodiment, the protein of the present invention is at
least 30% identical to protein sequence depicted in Table IIA or
IIB, columns 5 or 7, lines 72 to 81 and 460 to 462 and less than
100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 985, 97%, 96% or 95% identical to the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 72 to 81
and 460 to 462.
[3374] [0205.0.0.7] to [0226.0.0.7] for the disclosure of the
paragraphs [0205.0.0.7] to [0226.0.0.7] see paragraphs [0205.0.0.0]
to [0226.0.0.0] above.
[3375] [0227.0.7.7] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[3376] In addition to the sequence mentioned in Table I, columns 5
or 7, lines 72 to 81 and 460 to 462 or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the fatty acid biosynthetic pathway such as for
palmitate, palmitoleate, stearate and/or oleate is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine the sequences shown in Table I, columns 5
or 7, lines 72 to 81 and 460 to 462 with genes which generally
support or enhances to growth or yield of the target organisms, for
example genes which lead to faster growth rate of microorganisms or
genes which produces stress-, pathogen, or herbicide resistant
plants.
[3377] [0228.0.5.7] to [0230.0.5.7] for the disclosure of the
paragraphs [0228.0.5.7] to [0230.0.5.7] see paragraphs [0228.0.0.0]
to [0230.0.0.0] above.
[3378] [0231.0.7.7] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a stearic acid degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[3379] [0232.0.0.7] to [0276.0.0.7] for the disclosure of the
paragraphs [0232.0.0.7] to [0276.0.0.7] see paragraphs [0232.0.0.0]
to [0276.0.0.0] above.
[3380] [0277.0.5.7] for the disclosure of this paragraph see
paragraph [0277.0.5.5] above.
[3381] [0278.0.0.7] to [0282.0.0.7] for the disclosure of the
paragraphs [0278.0.0.7] to [0282.0.0.7] see paragraphs [0278.0.0.0]
to [0282.0.0.0] above.
[3382] [0283.0.7.7] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an antibody against polypeptides as shown in
Table II, columns 5 or 7, lines 72 to 81 and 460 to 462 or an
antigenic part thereof, which can be produced by standard
techniques utilizing polypeptides comprising or consisting of
abovementioned sequences, e.g. the polypeptid of the present
invention or fragment thereof. Preferred are monoclonal antibodies
specifically binding to polypeptides as indicated in Table II,
columns 5 or 7, lines 72 to 81 and 460 to 462.
[3383] [0284.0.0.7] for the disclosure of this paragraph see
[0284.0.0.0] above.
[3384] [0285.0.7.7] In one embodiment, the present invention
relates to a polypeptide having the sequence shown in Table II,
columns 5 or 7, lines 72 to 81 and 460 to 462 or as coded by the
nucleic acid molecule shown in Table I, columns 5 or 7, lines 72 to
81 and 460 to 462 or functional homologues thereof.
[3385] [0286.0.7.7] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 72 to 81 and 460 to 462 and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table IV, column 7, lines 72 to 81 and 460 to 462, whereby 20 or
less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred
5 or 4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a poylpeptide or to a polypeptide comprising more than
one consensus sequences as indicated in Table IV, column 7, lines
72 to 81 and 460 to 462.
[3386] [0287.0.0.7] to [0290.0.0.7] for the disclosure of the
paragraphs [0287.0.0.7] to [0290.0.0.7] see paragraphs [0287.0.0.0]
to [0290.0.0.0] above.
[3387] [0291.0.7.7] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of a
plant or microorganism specific consensus sequences.
[3388] In one embodiment, said polypeptide of the invention
distinguishes over the sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 72 to 81 and 460 to 462 by one or more amino
acids. In one embodiment, polypeptide distinguishes form the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 72 to 81
and 460 to 462 by more than 5, 6, 7, 8 or 9 amino acids, preferably
by more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred
are more than 40, 50, or 60 amino acids and, preferably, the
sequence of the polypeptide of the invention distinguishes from the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 72 to 81
and 460 to 462 by not more than 80% or 70% of the amino acids,
preferably not more than 60% or 50%, more preferred not more than
40% or 30%, even more preferred not more than 20% or 10%. In
another embodiment, said polypeptide of the invention does not
consist of the sequence shown in Table IIA or IIB, columns 5 or 7,
lines 72 to 81 and 460 to 462.
[3389] [0292.0.0.7] for the disclosure of this paragraph see
[0292.0.0.0] above.
[3390] [0293.0.7.7] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the
process.
[3391] In one embodiment, the polypeptide of the invention has a
sequence, which distinguishes from the sequence as shown in Table
IIA or IIB, columns 5 or 7, lines 72 to 81 and 460 to 462 by one or
more amino acids. In another embodiment, said polypeptide of the
invention does not consist of the sequence shown in Table IIA or
IIB, columns 5 or 7, lines 72 to 81 and 460 to 462. In a further
embodiment, said polypeptide of the present invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical. In one embodiment,
said polypeptide does not consist of the sequence encoded by the
nucleic acid molecules shown in Table IA or IB, columns 5 or 7,
lines 72 to 81 and 460 to 462.
[3392] [0294.0.7.7] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 72 to 81 and 460 to 462,
which distinguishes over the sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 72 to 81 and 460 to 462 by one or more
amino acids, preferably by more than 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, evenmore
preferred are more than 40, 50, or 60 amino acids but even more
preferred by less than 70% of the amino acids, more preferred by
less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[3393] [0295.0.0.7], [0296.0.0.7] and [0297.0.5.7] for the
disclosure of the paragraphs [0295.0.0.7], [0296.0.0.7] and
[0297.0.5.7] see paragraphs [0295.0.0.0] to [0297.0.0.0] above.
[3394] [00297.1.0.7] Non-polypeptide of the invention-chemicals are
e.g. polypeptides having not the activity and/or the amino acid
sequence of a polypeptide indicated in Table II, columns 3, 5 or 7,
lines 72 to 81 and 460 to 462.
[3395] [0298.0.7.7] A polypeptide of the invention can participate
in the process of the present invention. The polypeptide or a
portion thereof comprises preferably an amino acid sequence which
is sufficiently homologous to an amino acid sequence as indicated
in Table II, columns 5 or 7, lines 72 to 81 and 460 to 462 such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table II, columns 5 or 7, lines 72 to 81 and 460 to 462.
[3396] [0299.0.7.7] Further, the polypeptide can have an amino acid
sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences of Table II, columns 5 or 7, lines 72 to 81
and 460 to 462. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence of Table I, columns
5 or 7, lines 72 to 81 and 460 to 462 or which is homologous
thereto, as defined above.
[3397] [0300.0.7.7] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 72 to 81 and 460 to 462 in amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 72 to 81 and 460 to 462.
[3398] [0301.0.0.7] for the disclosure of this paragraph see
[0301.0.0.0] above.
[3399] [0302.0.7.7] Biologically active portions of an polypeptide
of the present invention include peptides comprising amino acid
sequences derived from the amino acid sequence of the polypeptide
of the present invention or used in the process of the present
invention, e.g., an amino acid sequence shown in Table II, columns
5 or 7, lines 72 to 81 and 460 to 462 or the amino acid sequence of
a protein homologous thereto, which include fewer amino acids than
a full length polypeptide of the present invention or used in the
process of the present invention or the full length protein which
is homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[3400] [0303.0.0.7] for the disclosure of this paragraph see
[0303.0.0.0] above.
[3401] [0304.0.7.7] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, columns 5 or 7, lines 72 to 81 and 460 to 462 but having
differences in the sequence from said wild-type protein. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity in relation to the wild type protein.
[3402] [0305.0.5.7], [0306.0.5.7] and [0306.1.0.7] for the
disclosure of the paragraphs [0305.0.5.7], [0306.0.5.7] and
[0306.1.0.7] see paragraphs [0305.0.0.0], [0306.0.0.0] and
[0306.1.0.0] above.
[3403] [0307.0.0.7] and [0308.0.0.7] for the disclosure of the
paragraphs [0307.0.0.7] and [0308.0.0.7] see paragraphs [0307.0.0.0
and [0308.0.0.0] above.
[3404] [0309.0.7.7] In one embodiment, a reference to protein
(=polypeptide)" of the invention e.g. as indicated in Table II,
columns 5 or 7, lines 72 to 81 and 460 to 462 refers to a
polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas a "non-polypeptide" or "other polypeptide" e.g.
not indicated in Table II, columns 5 or 7, lines 72 to 81 and 460
to 462 refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to
a polypeptide of the invention, preferably which is not
substantially homologous to a polypeptide having a protein
activity, e.g., a protein which does not confer the activity
described herein and which is derived from the same or a different
organism.
[3405] [0310.0.0.7] to [0334.0.0.7] for the disclosure of the
paragraphs [0310.0.0.7] to [0334.0.0.7] see paragraphs [0310.0.0.0]
to [0334.0.0.0] above.
[3406] [0335.0.7.7] The dsRNAi method has proved to be particularly
effective and advantageous for reducing the expression of a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 72 to
81 and 460 to 462 and/or homologs thereof. As described inter alia
in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table I,
columns 5 or 7, lines 72 to 81 and 460 to 462 and/or homologs
thereof. In a double-stranded RNA molecule for reducing the
expression of an protein encoded by a nucleic acid sequence of one
of the sequences as indicated in Table I, columns 5 or 7, lines 72
to 81 and 460 to 462 and/or homologs thereof, one of the two RNA
strands is essentially identical to at least part of a nucleic acid
sequence, and the respective other RNA strand is essentially
identical to at least part of the complementary strand of a nucleic
acid sequence.
[3407] [0336.0.0.7] to [0342.0.0.7] for the disclosure of the
paragraphs [0336.0.0.7] to [0342.0.0.7] see paragraphs [0336.0.0.0]
to [0342.0.0.0] above.
[3408] [0343.0.7.7] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 72 to 81
and 460 to 462 or its homolog is not necessarily required in order
to bring about effective reduction in the expression. The advantage
is, accordingly, that the method is tolerant with regard to
sequence deviations as may be present as a consequence of genetic
mutations, polymorphisms or evolutionary divergences. Thus, for
example, using the dsRNA, which has been generated starting from a
sequence of one of the sequences as indicated in Table I, columns 5
or 7, lines 72 to 81 and 460 to 462 or homologs thereof of the one
organism, may be used to suppress the corresponding expression in
another organism.
[3409] [0344.0.0.7] to [0350.0.0.7], [0351.0.5.7] and [0352.0.0.7]
to [0361.0.0.7] for the disclosure of the paragraphs [0344.0.0.7]
to [0350.0.0.7], [0351.0.5.7] and [0352.0.0.7] to [0361.0.0.7] see
paragraphs [0344.0.0.0] to [0361.0.0.0] above.
[3410] [0362.0.7.7] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, the nucleic acid construct of
the invention, the antisense molecule of the invention, the vector
of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. encoding a polypeptide having an
activity of a protein such as the polypeptides as indicated in
Table II, column 3, lines 72 to 81 and 460 to 462. Due to the above
mentioned activity the fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a protein such
as the polypeptides as indicated in Table II, column 3, lines 72 to
81 and 460 to 462. Activity means herein that due to modulation or
manipulation of the genome, the activity of polypeptide having an
activity of a protein such as the polypeptides as indicated in
Table II, column 3, lines 72 to 81 and 460 to 462 or a similar
activity, which is increased in the cell or organism or part
thereof. Examples are described above in context with the process
of the invention.
[3411] [0363.0.0.7], [0364.0.5.7] and [0365.0.0.7] to [0379.0.5.7
for the disclosure of the paragraphs [0363.0.0.7], [0364.0.5.7] and
[0365.0.0.7] to [0379.0.5.7] see paragraphs [0363.0.0.0] to
[0379.0.0.0] above.
[3412] [0380.0.5.7], [0381.0.0.7] and [0382.0.0.7] for the
disclosure of the paragraphs [0380.0.5.7], [0381.0.0.7] and
[0382.0.0.7] see paragraphs [0380.0.5.5], [0381.0.0.0] and
[0382.0.0.0] above.
[3413] [0383.0.5.7], [0384.0.0.7], [0385.0.5.7] and [0386.0.5.7]
for the disclosure of the paragraphs [0383.0.5.7], [0384.0.0.7],
[0385.0.5.7] and [0386.0.5.7] see paragraphs [0383.0.5.5],
[0384.0.0.0], [0385.0.5.5] and [0386.0.5.5] above.
[3414] [0387.0.0.7] to [0392.0.0.7] for the disclosure of the
paragraphs [0387.0.0.7] to [0392.0.0.7] see paragraphs [0387.0.0.0]
to [0392.0.0.0] above.
[3415] [0393.0.7.7] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps:
(a) contacting e.g. hybridising, the nucleic acid molecules of a
sample, e.g. cells, tissues, plants or microorganisms or a nucleic
acid library, which can contain a candidate gene encoding a gene
product conferring an increase in the respective fine chemical
after expression, with the nucleic acid molecule of the present
invention; (b) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence shown in Table I, columns 5 or 7, lines 72 to 81
and 460 to 462, preferably in Table I B, columns 5 or 7, lines 72
to 81 and 460 to 462 and, optionally, isolating the full length
cDNA clone or complete genomic clone; (c) introducing the candidate
nucleic acid molecules in host cells, preferably in a plant cell or
a microorganism, appropriate for producing the respective fine
chemical; (d) expressing the identified nucleic acid molecules in
the host cells; (e) assaying the respective fine chemical level in
the host cells; and (f) identifying the nucleic acid molecule and
its gene product which expression confers an increase in the
respective fine chemical level in the host cell after expression
compared to the wild type.
[3416] [0394.0.0.7] to [0415.0.0.7] and [0416.0.5.7] for the
disclosure of the paragraphs [0394.0.0.7] to [0415.0.0.7] and
[0416.0.5.7] see paragraphs [0394.0.0.0] to [0416.0.0.0] above.
[3417] [0417.0.5.7] and [0418.0.0.7] to [0430.0.0.7] for the
disclosure of the paragraphs [0417.0.5.7] and [0418.0.0.7] to
[0430.0.0.7] see paragraphs [0417.0.5.5] and [0418.0.0.0] to
[0430.0.0.0] above.
[3418] [0431.0.5.7], [0432.0.5.7], [0433.0.0.7] and [0434.0.0.7]
for the disclosure of the paragraphs [0431.0.5.7], [0432.0.5.7],
[0433.0.0.7] and [0434.0.0.7] see paragraphs [0431.0.0.0] to
[0434.0.0.0] above.
[3419] [0435.0.5.7] to [0440.0.5.7] for the disclosure of the
paragraphs [0435.0.5.7] to [0440.0.5.7] see paragraphs [0435.0.5.5]
to [0440.0.5.5] above.
[3420] [0441.0.0.7] and [0442.0.5.7] for the disclosure of the
paragraphs [0441.0.0.7] and [0442.0.5.7] see [0441.0.0.0] and
[0442.0.5.5] above.
[3421] [0443.0.0.7] for the disclosure of this paragraph see
[0443.0.0.0] above.
[3422] [0444.0.5.7] and [0445.0.5.7] for the disclosure of the
paragraphs [0444.0.5.7] and [0445.0.5.7] see [0444.0.5.5] and
[0445.0.5.5] above.
[3423] [0446.0.0.7] to [0453.0.0.7] for the disclosure of the
paragraphs [0446.0.0.7] to [0453.0.0.7] see paragraphs [0446.0.0.0]
to [0453.0.0.0] above.
[3424] [0454.0.5.7] and [0455.0.5.7] for the disclosure of the
paragraphs [0454.0.5.7] and [0455.0.5.7] see [0454.0.5.5] and
[0455.0.5.5] above.
[3425] [0456.0.0.7] for the disclosure of this paragraph see
[0456.0.0.0] above.
[3426] [0457.0.5.7] to [0460.0.5.7] for the disclosure of the
paragraphs [0457.0.5.7] to [0460.0.6.7] see paragraphs [0457.0.5.5]
to [0460.0.5.5] above.
[0461.0.7.7] Example 10
Cloning SEQ ID NO: 5818 for the Expression in Plants
[3427] [0462.0.0.7] for the disclosure of this paragraph see
[0462.0.0.0] above.
[3428] [0463.0.7.7] SEQ ID NO: 5818 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[3429] [0464.0.5.7], [0465.0.5.7] and [0466.0.0.7] for the
disclosure of the paragraphs [0464.0.5.7], [0465.0.5.7] and
[0466.0.0.7] see paragraphs [0464.0.5.5], [0465.0.5.5] and
[0466.0.0.0] above.
[3430] [0467.0.7.7] The following primer sequences were selected
for the gene SEQ ID NO: 5818:
TABLE-US-00026 i) forward primer (SEQ ID NO: 6426) ttatttttcc
tgaagaccga gttttt ii) reverse primer (SEQ ID NO: 6427) atgctggata
aaattgttat tgcc
[3431] [0468.0.0.7] to [0479.0.0.7] for the disclosure of the
paragraphs [0468.0.0.7] to [0479.0.0.7] see paragraphs [0468.0.0.0]
to [0479.0.0.0] above.
[0480.0.7.7] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 5818
[3432] [0481.0.0.7] for the disclosure of this paragraph see
[0481.0.0.0] above.
[3433] [0482.0.0.7] to [0513.0.0.7] for the disclosure of the
paragraphs [0482.0.0.7] to [0513.0.0.7] see paragraphs [0482.0.0.0]
to [0513.0.0.0] above.
[3434] [0514.0.7.7] As an alternative, the amino acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55).
[3435] The results of the different plant analyses can be seen from
the table, which follows:
TABLE-US-00027 TABLE 1 ORF Metabolite Method Min Max b3256 Stearic
Acid (C18:0) GC 1.17 1.25 b2699 Stearic Acid (C18:0) GC 1.22 1.83
b2095 Stearic Acid (C18:0) GC 1.17 1.26 YOR024W Stearic Acid
(C18:0) GC 1.16 1.40 YBR089C-A Stearic Acid (C18:0) GC 1.36 2.34
YFR042W Stearic Acid (C18:0) GC 1.19 1.26 YIL150C Stearic Acid
(C18:0) GC 1.34 3.20 YDR513W Stearic Acid (C18:0) GC 1.16 1.51
YLR010C Stearic Acid (C18:0) GC 1.16 1.76 b1093 Stearic Acid
(C18:0) GC 1.16 1.31 b0161 Stearic acid (C18:0) GC 1.16 2.29 b1896
Stearic acid (C18:0) GC 1.23 1.30 b3457 Stearic acid (C18:0) GC
1.18 1.34
[3436] [0515.0.5.7] Column 2 shows the fatty acid analyzed. Columns
4 and 5 shows the ratio of the analyzed fatty acid between the
transgenic plants and the wild type; Increase of the metabolites:
Max: maximal x-fold (normalised to wild type)-Min: minimal x-fold
(normalised to wild type). Decrease of the metabolites: Max:
maximal x-fold (normalised to wild type) (minimal decrease), Min:
minimal x-fold (normalised to wild type) (maximal decrease). Column
3 indicates the analytical method.
[3437] [0516.0.0.7] and [0517.0.5.7] for the disclosure of the
paragraphs [0516.0.0.7] and [0517.0.5.7] see paragraphs
[0516.0.0.0] and [0517.0.0.0] above.
[3438] [0518.0.0.7] to [0529.0.0.7] and [0530.0.5.7] for the
disclosure of the paragraphs [0518.0.0.7] to [0529.0.0.7] and
[0530.0.5.7] see paragraphs [0518.0.0.0] to [0530.0.0.0] above.
[3439] [0530.1.0.5] to [0530.6.0.5] for the disclosure of the
paragraphs [0530.1.0.5] to [0530.6.0.5] see paragraphs [0530.1.0.0]
to [0530.6.0.0] above.
[3440] [0531.0.0.7] to [0533.0.0.7] and [0534.0.5.7] for the
disclosure of the paragraphs [0531.0.0.7] to [0533.0.0.7] and
[0534.0.5.7] see paragraphs [0531.0.0.0] to [0534.0.0.0] above.
[3441] [0535.0.0.7] to [0537.0.0.7] and [0538.0.5.7] for the
disclosure of the paragraphs [0535.0.0.7] to [0537.0.0.7] and
[0538.0.5.7] see paragraphs [0535.0.0.0] to [0538.0.0.0] above.
[3442] [0539.0.0.7] to [0542.0.0.7] and [0543.0.5.7] for the
disclosure of the paragraphs [0539.0.0.7] to [0542.0.0.7] and
[0543.0.5.7] see paragraphs [0539.0.0.0] to [0543.0.0.0] above.
[3443] [0544.0.0.7] to [0547.0.0.7] and [0548.0.5.7] to
[0552.0.0.7] for the disclosure of the paragraphs [0544.0.0.7] to
[0547.0.0.7] and [0548.0.5.7] to [0552.0.0.7] see paragraphs
[0544.0.0.0] to [0552.0.0.0] above.
[0552.1.7.7]: Example 15
Metabolite Profiling Info from Zea mays
[3444] Zea mays plants were engineered, grown and analyzed as
described in Example 14c.
[3445] The results of the different Zea mays plants analysed can be
seen from Table 2, which follows:
TABLE-US-00028 TABLE 2 ORF_NAME Metabolite Min Max YIL150C Stearic
Acid (C18:0) 1.67 2.24 b1896 Stearic Acid (C18:0) 1.41 1.74 b3457
Stearic Acid (C18:0) 1.35 1.64
[3446] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in stearic acid in
genetically modified corn plants expressing the Saccharomyces
cerevisiae nucleic acid sequence YIL150Cor E. coli nucleic acid
sequence b1896 or b3457 resp.
[3447] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YIL150C or its homologs, e.g. a "chromatin
binding protein, required for S-phase (DNA synthesis) initiation or
completion", is increased in corn plants, preferably, an increase
of the fine chemical stearic acid between 67% and 124% is
conferred.
[3448] In one embodiment, in case the activity of the E. coli
protein b1896 or its homologs, e.g. "a trehalose-6-phosphate
synthase", is increased in corn plants, preferably, an increase of
the fine chemical stearic acid between 41% and 74% is
conferred.
[3449] In one embodiment, in case the activity of the E. coli
protein b3457 or its homologs, e.g. "a high-affinity branched-chain
amino acid transport protein (ABC superfamily)", is increased in
corn plants, preferably, an increase of the fine chemical stearic
acid between 35% and 64% is conferred.
[3450] [0552.2.0.7] for the disclosure of this paragraph see
[0552.2.0.0] above.
[3451] [0553.0.7.7] [3452] 1. A process for the production of
stearic acid, which comprises [3453] (a) increasing or generating
the activity of a protein as indicated in Table II, columns 5 or 7,
lines 72 to 81 and 460 to 462 or a functional equivalent thereof in
a non-human organism, or in one or more parts thereof; and [3454]
(b) growing the organism under conditions which permit the
production of stearic acid in said organism.
[3455] 2. A process for the production of stearic acid, comprising
the increasing or generating in an organism or a part thereof the
expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [3456]
a) nucleic acid molecule encoding of a polypeptide as indicated in
Table II, columns 5 or 7, lines 72 to 81 and 460 to 462 or a
fragment thereof, which confers an increase in the amount of
stearic acid in an organism or a part thereof; [3457] b) nucleic
acid molecule comprising of a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 72 to 81 and 460 to 462; [3458] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of stearic acid in an organism
or a part thereof; [3459] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of stearic acid
in an organism or a part thereof; [3460] e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of stearic acid in an organism or a part thereof; [3461]
f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table III, column 7, lines 72 to 81 and 460 to 462 and
conferring an increase in the amount of stearic acid in an organism
or a part thereof; [3462] g) nucleic acid molecule encoding a
polypeptide which is isolated with the aid of monoclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (f) and conferring an increase in the amount of stearic
acid in an organism or a part thereof; [3463] h) nucleic acid
molecule encoding a polypeptide comprising a consensus sequence as
indicated in Table IV, column 7, lines 72 to 81 and 460 to 462 and
conferring an increase in the amount of stearic acid in an organism
or a part thereof; and [3464] i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of stearic acid in an organism or a part thereof. [3465] or
comprising a sequence which is complementary thereto. [3466] 3. The
process of claim 1 or 2, comprising recovering of the free or bound
stearic acid. [3467] 4. The process of any one of claims 1 to 3,
comprising the following steps: [3468] (a) selecting an organism or
a part thereof expressing a polypeptide encoded by the nucleic acid
molecule characterized in claim 2; [3469] (b) mutagenizing the
selected organism or the part thereof; [3470] (c) comparing the
activity or the expression level of said polypeptide in the
mutagenized organism or the part thereof with the activity or the
expression of said polypeptide of the selected organisms or the
part thereof; [3471] (d) selecting the mutated organisms or parts
thereof, which comprise an increased activity or expression level
of said polypeptide compared to the selected organism or the part
thereof; [3472] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [3473] (f) recovering, and
optionally isolating, the free or bound stearic acid produced by
the selected mutated organisms or parts thereof. [3474] 5. The
process of any one of claims 1 to 4, wherein the activity of said
protein or the expression of said nucleic acid molecule is
increased or generated transiently or stably. [3475] 6. An isolated
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: [3476] a) nucleic acid molecule
encoding of a polypeptide as indicated in Table II, columns 5 or 7,
lines 72 to 81 and 460 to 462 or a fragment thereof, which confers
an increase in the amount of stearic acid in an organism or a part
thereof; [3477] b) nucleic acid molecule comprising of a nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines 72 to
81 and 460 to 462; [3478] c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as a result of the degeneracy of the
genetic code and conferring an increase in the amount of stearic
acid in an organism or a part thereof; [3479] d) nucleic acid
molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of stearic acid in an organism or a part thereof;
[3480] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of stearic acid
in an organism or a part thereof; [3481] f) nucleic acid molecule
which encompasses a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers or primer pairs as indicated in Table
III, column 7, lines 72 to 81 and 460 to 462 and conferring an
increase in the amount of stearic acid in an organism or a part
thereof; [3482] g) nucleic acid molecule encoding a polypeptide
which is isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of stearic acid in an
organism or a part thereof; [3483] h) nucleic acid molecule
encoding a polypeptide comprising a consensus sequence as indicated
in Table IV, column 7, lines 72 to 81 and 460 to 462 and conferring
an increase in the amount of stearic acid in an organism or a part
thereof; and [3484] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of stearic acid in an organism or a part thereof. [3485]
whereby the nucleic acid molecule distinguishes over the sequence
as indicated in Table I A, columns 5 or 7, lines 72 to 81 and 460
to 462 by one or more nucleotides. [3486] 7. A nucleic acid
construct which confers the expression of the nucleic acid molecule
of claim 6, comprising one or more regulatory elements. [3487] 8. A
vector comprising the nucleic acid molecule as claimed in claim 6
or the nucleic acid construct of claim 7. [3488] 9. The vector as
claimed in claim 8, wherein the nucleic acid molecule is in
operable linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [3489] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [3490] 11. The host cell of claim 10, which is a
transgenic host cell. [3491] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [3492] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [3493] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table II A, columns 5
or 7, lines 72 to 81 and 460 to 462 by one or more amino acids 15.
An antibody, which binds specifically to the polypeptide as claimed
in claim 14. [3494] 16. A plant tissue, propagation material,
harvested material or a plant comprising the host cell as claimed
in claim 12 which is plant cell or an Agrobacterium. [3495] 17. A
method for screening for agonists and antagonists of the activity
of a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of stearic acid in an organism
or a part thereof comprising: [3496] (a) contacting cells, tissues,
plants or microorganisms which express the a polypeptide encoded by
the nucleic acid molecule of claim 6 conferring an increase in the
amount of stearic acid in an organism or a part thereof with a
candidate compound or a sample comprising a plurality of compounds
under conditions which permit the expression the polypeptide;
[3497] (b) assaying the stearic acid level or the polypeptide
expression level in the cell, tissue, plant or microorganism or the
media the cell, tissue, plant or microorganisms is cultured or
maintained in; and [3498] (c) identifying a agonist or antagonist
by comparing the measured stearic acid level or polypeptide
expression level with a standard of stearic acid or polypeptide
expression level measured in the absence of said candidate compound
or a sample comprising said plurality of compounds, whereby an
increased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an agonist and
a decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[3499] 18. A process for the identification of a compound
conferring increased stearic acid production in a plant or
microorganism, comprising the steps: [3500] (a) culturing a plant
cell or tissue or microorganism or maintaining a plant expressing
the polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of stearic acid in an organism
or a part thereof and a readout system capable of interacting with
the polypeptide under suitable conditions which permit the
interaction of the polypeptide with dais readout system in the
presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of stearic acid in an organism
or a part thereof; [3501] (b) identifying if the compound is an
effective agonist by detecting the presence or absence or increase
of a signal produced by said readout system. [3502] 19. A method
for the identification of a gene product conferring an increase in
stearic acid production in a cell, comprising the following steps:
[3503] (a) contacting the nucleic acid molecules of a sample, which
can contain a candidate gene encoding a gene product conferring an
increase in stearic acid after expression with the nucleic acid
molecule of claim 6; [3504] (b) identifying the nucleic acid
molecules, which hybridise under relaxed stringent conditions with
the nucleic acid molecule of claim 6; [3505] (c) introducing the
candidate nucleic acid molecules in host cells appropriate for
producing stearic acid; [3506] (d) expressing the identified
nucleic acid molecules in the host cells; [3507] (e) assaying the
stearic acid level in the host cells; and [3508] (f) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the stearic acid level in the host cell in the host
cell after expression compared to the wild type. [3509] 20. A
method for the identification of a gene product conferring an
increase in stearic acid production in a cell, comprising the
following steps: [3510] (a) identifying in a data bank nucleic acid
molecules of an organism; which can contain a candidate gene
encoding a gene product conferring an increase in the stearic acid
amount or level in an organism or a part thereof after expression,
and which are at least 20% homolog to the nucleic acid molecule of
claim 6; [3511] (b) introducing the candidate nucleic acid
molecules in host cells appropriate for producing stearic acid;
[3512] (c) expressing the identified nucleic acid molecules in the
host cells; [3513] (d) assaying the stearic acid level in the host
cells; and [3514] (e) identifying nucleic acid molecule and its
gene product which expression confers an increase in the stearic
acid level in the host cell after expression compared to the wild
type. [3515] 21. A method for the production of an agricultural
composition comprising the steps of the method of any one of claims
17 to 20 and formulating the compound identified in any one of
claims 17 to 20 in a form acceptable for an application in
agriculture. [3516] 22. A composition comprising the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of any one of claim 8 or 9, an
antagonist or agonist identified according to claim 17, the
compound of claim 18, the gene product of claim 19 or 20, the
antibody of claim 15, and optionally an agricultural acceptable
carrier. [3517] 23. Use of the nucleic acid molecule as claimed in
claim 6 for the identification of a nucleic acid molecule
conferring an increase of stearic acid after expression. [3518] 24.
Use of the polypeptide of claim 14 or the nucleic acid construct
claim 7 or the gene product identified according to the method of
claim 19 or 20 for identifying compounds capable of conferring a
modulation of stearic acid levels in an organism. [3519] 25. Food
or feed composition comprising the nucleic acid molecule of claim
6, the polypeptide of claim 14, the nucleic acid construct of claim
7, the vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. [3520] 26. Use of the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the host cell of claim 10 to
12 or the gene product identified according to the method of claim
19 or 20 for the protection of a plant against a stearic acid
synthesis inhibiting herbicide.
[3521] [0554.0.0.7] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[3522] [0000.0.0.8] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[3523] [0001.0.0.8] and [0002.0.7.8] for the disclosure of the
paragraphs [0001.0.0.8] and [0002.0.7.8] see paragraphs
[0001.0.0.0] and [0002.0.7.7] above.
[3524] [0003.0.8.8] Palmitic acid is a major component for
manufacturing of soaps, lubricating oils and waterproofing
materials. Furhermore it is used for the synthesis of metallic
palmitates. Additional applications are as food additive and in the
synthesis of food-grade additives; as a constituent of cosmetic
formulations. Palmitic acid is a major component of many natural
fats and oils in the form of a glyceryl ester, e.g. palm oil, and
in most commercial-grade stearic acid products.
[3525] [0004.0.7.8] and [0005.0.5.8] for the disclosure of the
paragraphs [0004.0.7.8] and [0005.0.5.8] see paragraphs
[0004.0.7.7] and [0005.0.5.5] above.
[3526] [0006.0.8.8] Palmitic acid is as mentioned above the major
fat in meat and dairy products.
[3527] [0007.0.8.8] Further uses or palmitic acid are as food
ingredients raw material for emulsifiers or personal care
emulsifier for facial creams and lotions. Palmitic acid is also
used in shaving cream formulations, waxes or fruit wax
formulations.
[3528] [0008.0.8.8] Palmitic acid is also used in shaving cream
formulations, waxes or fruit wax formulations.
[3529] [0009.0.7.8] to [0012.0.7.8] for the disclosure of the
paragraphs [0009.0.7.8] to [0012.0.7.8] see paragraphs [0009.0.7.7]
and [0012.0.7.7] above.
[3530] [0013.0.0.8] for the disclosure of this paragraph see
[0013.0.0.0] above.
[3531] [0014.0.8.8] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is palmitic acid or
tryglycerides, lipids, oils or fats containing palmitic acid.
Accordingly, in the present invention, the term "the fine chemical"
as used herein relates to "palmitic acid and/or tryglycerides,
lipids, oils and/or fats containing palmitic acid". Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising palmitic acid and/or triglycerides, lipids,
oils and/or fats containing palmitic acid.
[3532] [0015.0.8.8] In one embodiment, the term "the fine chemical"
means palmitic acid and/or tryglycerides, lipids, oils and/or fats
containing palmitic acid. Throughout the specification the term
"the fine chemical" means palmitic acid and/or tryglycerides,
lipids, oils and/or fats containing palmitic acid, palmitic acid
and its salts, ester, thioester or palmitic acid in free form or
bound to other compounds such as triglycerides, glycolipids,
phospholipids etc. In a preferred embodiment, the term "the fine
chemical" means palmitic acid, in free form or its salts or bound
to triglycerides. Triglycerides, lipids, oils, fats or lipid
mixture thereof shall mean any triglyceride, lipid, oil and/or fat
containing any bound or free palmitic acid for example
sphingolipids, phosphoglycerides, lipids, glycolipids such as
glycosphingolipids, phospholipids such as phosphatidylethanolamine,
phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol or diphosphatidylglycerol, or as
monoacylglyceride, diacylglyceride or triacylglyceride or other
fatty acid esters such as acetyl-Coenzym A thioester, which contain
further saturated or unsaturated fatty acids in the fatty acid
molecule.
[3533] In one embodiment, the term "the fine chemical" and the term
"the respective fine chemical" mean at least one chemical compound
with an activity of the above mentioned fine chemical.
[3534] [0016.0.8.8] Accordingly, the present invention relates to a
process comprising [3535] (a) increasing or generating the activity
of a YDR513W, YDR447C, YBR089C-A, b3256, YGR126W, YPL099C, b0399,
b0849, b3457, b3578, b3644 and/or b4129 protein(s) or of a protein
having the sequence of a polypeptide encoded by a nucleic acid
molecule indicated in Table I, columns 5 or 7, lines 82 to 88 and
463 to 467 in a non-human organism in one or more parts thereof and
[3536] (b) growing the organism under conditions which permit the
production of the fine chemical, thus, palmitic acid or fine
chemicals comprising palmitic acid, in said organism.
[3537] Accordingly, the present invention relates to a process for
the production of a fine chemical comprising [3538] (a) increasing
or generating the activity of one or more proteins having the
activity of a protein indicated in Table II, column 3, lines 82 to
88 and 463 to 467 or having the sequence of a polypeptide encoded
by a nucleic acid molecule indicated in Table I, column 5 or 7,
lines 82 to 88 and 463 to 467, in a non-human organism in one or
more parts thereof and [3539] (b) growing the organism under
conditions which permit the production of the fine chemical, in
particular palmitic acid.
[3540] [0017.0.0.8] and [0018.0.0.8] for the disclosure of the
paragraphs [0017.0.0.8] and [0018.0.0.8] see paragraphs
[0017.0.0.0] and [0018.0.0.0] above.
[3541] [0019.0.8.8] Advantageously the process for the production
of the fine chemical leads to an enhanced production of the fine
chemical. The terms "enhanced" or "increase" mean at least a 10%,
20%, 30%, 40% or 50%, preferably at least 60%, 70%, 80%, 90% or
100%, more preferably 150%, 200%, 300%, 400% or 500% higher
production of the fine chemical in comparison to the reference as
defined below, e.g. that means in comparison to an organism without
the aforementioned modification of the activity of a protein having
the activity of a protein indicated in Table II, column 3, lines 82
to 88 and 463 to 467 or encoded by nucleic acid molecule indicated
in Table I, columns 5 or 7, lines 82 to 88 and 463 to 467.
[3542] [0020.0.8.8] Surprisingly it was found, that the transgenic
expression of at least one of the Saccaromyces cerevisiae
protein(s) indicated in Table II, Column 3, lines 82 to 84 and 86
and 87, and/or the Escherichia coli K12 protein(s) indicated in
Table II, Column 3, lines 85 and 88 and 463 to 467 in Arabidopsis
thaliana conferred an increase in the palmitic acid (or fine
chemical) content of the transformed plants.
[3543] [0021.0.0.8] for the disclosure of this paragraph see
[0021.0.0.0] above.
[3544] [0022.0.8.8] The sequence of b3256 from Escherichia coli K12
has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as acetyl CoA
carboxylase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a acetyl CoA carboxylase
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of palmitic acid and/or
tryglycerides, lipids, oils and/or fats containing palmitic acid,
in particular for increasing the amount of palmitic acid and/or
tryglycerides, lipids, oils and/or fats containing palmitic acid,
preferably palmitic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a acetyl CoA carboxylase is
increased or generated, e.g. from E. coli or a homolog thereof.
[3545] The sequence of b0399 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a positive response regulator
for the pho regulon. Accordingly, in one embodiment, the process of
the present invention comprises the use of a positive response
regulator for the pho regulon from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
palmitic acid and/or tryglycerides, lipids, oils and/or fats
containing palmitic acid, in particular for increasing the amount
of palmitic and/or tryglycerides, lipids, oils and/or fats
containing palmitic acid, preferably palmitic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a positive response regulator for the pho regulon is increased or
generated, e.g. from E. coli or a homolog thereof. The sequence of
YDR513W from Saccharomyces cerevisiae has been published in Jacq et
al., Nature 387 (6632 Suppl), 75-78 (1997) and in Goffeau et al.,
Science 274 (5287), 546-547, 1996 and its cellular activity has
characterized as glutaredoxin (thioltransferase, glutathione
reductase). Accordingly, in one embodiment, the process of the
present invention comprises the use of a glutaredoxin, from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of palmitic acid
and/or tryglycerides, lipids, oils and/or fats containing palmitic
acid, in particular for increasing the amount of palmitic acid
and/or tryglycerides, lipids, oils and/or fats containing palmitic
acid, preferably palmitic acid in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a glutaredoxin is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[3546] The sequence of YDR447C from Saccharomyces cerevisiae has
been published in
[3547] Jacq et al., Nature 387 (6632 Suppl), 75-78 (1997) and in
Goffeau et al., Science 274 (5287), 546-547, 1996 and its cellular
activity has been characterized as a ribosomal protein 51 (rp51) of
the small (40 s) subunit; nearly identical to rps17Ap and has
similarity to rat S17 ribosomal protein; rps17 bp. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a ribosomal protein 51 (rp51) of the small (40 s) subunit,
from Saccharomyces cerevisiae or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of palmitic acid
and/or tryglycerides, lipids, oils and/or fats containing palmitic
acid, in particular for increasing the amount of palmitic acid
and/or tryglycerides, lipids, oils and/or fats containing palmitic
acid, preferably palmitic acid in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a ribosomal protein 51
(rp51) of the small (40 s) subunit is increased or generated, e.g.
from Saccharomyces cerevisiae or a homolog thereof.
[3548] The sequence of YBR089C-A from Saccharomyces cerevisiae has
been published in Feldmann et al., EMBO J., 13 (24), 5795-5809
(1994) and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its cellular activity has not been characterized yet. It shows
homology to mammalian high mobility group proteins 1 and 2. Its
function may be redundantly with the highly homologous gene NHP6A.
Furthermore it shows homology to the high-mobility group
non-histone chromatin protein Nhp6 bp. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a YBR089C-A activity from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of palmitic acid and/or tryglycerides, lipids,
oils and/or fats containing palmitic acid, in particular for
increasing the amount of palmitic acid and/or tryglycerides,
lipids, oils and/or fats containing palmitic acid, preferably
palmitic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a YBR089C-A protein is increased
or generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[3549] The sequence of YGR126W from Saccharomyces cerevisiae has
been published in Tettelin et al., Nature 387 (6632 Suppl), 81-84
(1997) and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its cellular activity has not been characterized yet. It seams to
be a "putative open reading frame". Accordingly, in one embodiment,
the process of the present invention comprises the use of a YGR126W
activity from Saccharomyces cerevisiae or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
palmitic acid and/or tryglycerides, lipids, oils and/or fats
containing palmitic acid, in particular for increasing the amount
of palmitic acid and/or tryglycerides, lipids, oils and/or fats
containing palmitic acid, preferably palmitic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a YGR126W protein is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[3550] The sequence of YPL099C from Saccharomyces cerevisiae has
been published in Bussey et al., Nature 387 (6632 Suppl), 103-105
(1997) and in Goffeau et al., Science 274 (5287), 546-547, 1996 and
its cellular activity has not been characterized yet. "The
authentic, non-tagged protein was localized to the mitochondria".
Accordingly, in one embodiment, the process of the present
invention comprises the use of YPL099C, from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the fine chemical, meaning of palmitic acid and/or
tryglycerides, lipids, oils and/or fats containing palmitic acid,
in particular for increasing the amount of palmitic acid and/or
tryglycerides, lipids, oils and/or fats containing palmitic acid,
preferably palmitic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a YPL099C protein is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[3551] The sequence of b0849 (Accession number NP.sub.--415370)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a glutaredoxin 1 redox coenzyme for
glutathione-dependent ribonucleotide reductase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a glutaredoxin 1 redox coenzyme for glutathione-dependent
ribonucleotide reductase protein from E. coli or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
of palmitic acid and/or tryglycerides, lipids, oils and/or fats
containing palmitic acid, in particular for increasing the amount
of palmitic acid and/or tryglycerides, lipids, oils and/or fats
containing palmitic acid, preferably palmitic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a glutaredoxin 1 redox coenzyme for glutathione-dependent
ribonucleotide reductase protein is increased or generated, e.g.
from E. coli or a homolog thereof. The sequence of b3457 (Accession
number NP.sub.--417914) from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a high-affinity branched-chain
amino acid transport protein (ABC superfamily). Accordingly, in one
embodiment, the process of the present invention comprises the use
of a high-affinity branched-chain amino acid transport protein from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of palmitic acid and/or tryglycerides,
lipids, oils and/or fats containing palmitic acid, in particular
for increasing the amount of palmitic acid and/or tryglycerides,
lipids, oils and/or fats containing palmitic acid, preferably
palmitic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a high-affinity branched-chain
amino acid transport protein is increased or generated, e.g. from
E. coli or a homolog thereof.
[3552] The sequence of b3578 (Accession number YP 026232) from
Escherichia coli K12 has been published in Blattner et al., Science
277(5331), 1453-1474, 1997, and its activity is being defined as
putative component of transport system. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a putative component of transport system from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of palmitic acid and/or tryglycerides, lipids,
oils and/or fats containing palmitic acid, in particular for
increasing the amount of palmitic acid and/or tryglycerides,
lipids, oils and/or fats containing palmitic acid, preferably
palmitic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a putative component of transport
system is increased or generated, e.g. from E. coli or a homolog
thereof.
[3553] The sequence of b3644 (Accession number NP.sub.--418101)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as an uncharacterized stress-induced protein. Accordingly,
in one embodiment, the process of the present invention comprises
the use of an uncharacterized stress-induced protein from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of palmitic acid and/or tryglycerides,
lipids, oils and/or fats containing palmitic acid, in particular
for increasing the amount of palmitic acid and/or tryglycerides,
lipids, oils and/or fats containing palmitic acid, preferably
palmitic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of an uncharacterized stress-induced
protein is increased or generated, e.g. from E. coli or a homolog
thereof.
[3554] The sequence of b4129 (Accession number NP.sub.--418553)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a lysine tRNA synthetase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a lysine tRNA synthetase protein from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of palmitic acid and/or tryglycerides, lipids, oils and/or
fats containing palmitic acid, in particular for increasing the
amount of palmitic acid and/or tryglycerides, lipids, oils and/or
fats containing palmitic acid, preferably palmitic acid in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a lysine tRNA synthetase protein is increased or generated, e.g.
from E. coli or a homolog thereof.
[3555] [0023.0.8.8] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the fine chemical amount or content.
[3556] Further, in the present invention, the term "homologue"
relates to the sequence of an organism having the highest sequence
homology to the herein mentioned or listed sequences of all
expressed sequences of said organism.
[3557] However, the person skilled in the art knows, that,
preferably, the homologue has said fine-chemical increasing
activity and, if known, the same biological function or activity in
the organism as at least one of the protein(s) indicated in Table
II, Column 3, lines 82 to 88 and 463 to 467, e.g. having the
sequence of a polypeptide encoded by a nucleic acid molecule
comprising the sequence indicated in indicated in Table I, Column 5
or 7, lines 82 to 88 and 463 to 467.
[3558] In one embodiment, the homolog of any one of the
polypeptides indicated in Table II, lines 82 to 84 and 86 and 87 is
a homolog having the same or a similar activity, in particular an
increase of activity confers an increase in the content of the fine
chemical in the organisms and being derived from an Eukaryot. In
one embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 85 and 88 and 463 to 467 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from bacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 82 to 84 and 86 and 87 is a homolog having the same
or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in an
organisms or part thereof, and being derived from Fungi. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 85 and 88 and 463 to 467 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof and being derived from Proteobacteria. In
one embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 82 to 84 and 86 and 87 is a homolog having the same
or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or a part thereof and being derived from Ascomycota. In
one embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 85 and 88 and 463 to 467 is a homolog having the
same or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from
Gammaproteobacteria. In one embodiment, the homolog of a
polypeptide polypeptide indicated in Table II, column 3, lines 82
to 84 and 86 and 87 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or part
thereof, and being derived from Saccharomycotina. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 85 and 88 and 463 to 467 is a homolog having the
samie or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 82 to 84 and 86 and 87 is a
homolog having the samie or a similar activity, in particular an
increase of activity confers an increase in the content of the fine
chemical in the organisms or a part thereof, and being derived from
Saccharomycetes. In one embodiment, the homolog of the a
polypeptide indicated in Table II, column 3, lines 85 and 88 and
463 to 467 is a homolog having the samie or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or part thereof, and
being derived from Enterobacteriaceae. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 82
to 84 and 86 and 87 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms, and being
derived from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 85 and 88 and
463 to 467 is a homolog having the same or a similar activity, in
particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof,
and being derived from Escherichia. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 82 to 84
and 86 and 87 is a homolog having the same or a similar activity,
in particular an increase of activity confers an increase in the
content of the fine chemical in the organisms or a part thereof,
and being derived from Saccharomycetaceae. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 82
to 84 and 86 and 87 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the fine chemical in the organisms or a part
thereof, and being derived from Saccharomycetes.
[3559] [0023.1.0.8] and [0024.0.0.8] for the disclosure of the
paragraphs [0023.1.0.8] and [0024.0.0.8] see [0023.1.0.0] and
[0024.0.0.0] above.
[3560] [0025.0.8.8] In accordance with the invention, a protein or
polypeptide has the "activity of a protein as indicated in Table
II, column 3, lines 82 to 88 and 463 to 467" if its de novo
activity, or its increased expression directly or indirectly leads
to an increased palmitic acid and/or tryglycerides, lipids, oils
and/or fats containing palmitic acid level in the organism or a
part thereof, preferably in a cell of said organism and the protein
has the above mentioned activities of a protein as indicated in
Table II, column 3, lines 82 to 88 and 463 to 467. Throughout the
specification the activity or preferably the biological activity of
such a protein or polypeptide or an nucleic acid molecule or
sequence encoding such protein or polypeptide is identical or
similar if it still has the biological or enzymatic activity of a
protein as indicated in Table II, column 3, lines 82 to 88 and 463
to 467, or which has at least 10% of the original enzymatic
activity, preferably 20%, particularly preferably 30%, most
particularly preferably 40% in comparison to a protein of
Saccharomyces cerevisiae as indicated in Table II, column 3, lines
82 to 84 and 86 and 87 and/or a protein of E. coli K12 as indicated
in Table II, column 3, lines 85 and 88 and 463 to 467.
[3561] [0025.1.0.8] and [0025.2.0.8] for the disclosure of the
paragraphs [0025.1.0.8] and [0025.2.0.8] see paragraphs
[0025.1.0.0] and [0025.2.0.0] above.
[3562] [0026.0.0.8] to [0033.0.0.8] for the disclosure of the
paragraphs [0026.0.0.8] to [0033.0.0.8] see paragraphs [0026.0.0.0]
to [0033.0.0.0] above.
[3563] [0034.0.8.8] Preferably, the reference, control or wild type
differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. as result of
an increase in the level of the nucleic acid molecule of the
present invention or an increase of the specific activity of the
polypeptide of the invention or the polypeptide used in the method
of the invention. E.g., it differs by or in the expression level or
activity of an protein having the activity of a protein as
indicated in Table II, column 3, lines 82 to 88 and 463 to 467 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 82 to 88 and 463 to 467 or its homologs, e.g. as
indicated in Table I, column 7, lines 82 to 88 and 463 to 467, its
biochemical or genetical causes and therefore shows the increased
amount of the fine chemical.
[3564] [0035.0.0.8] to [0038.0.0.8] and [0039.0.5.8] for the
disclosure of the paragraphs [0035.0.0.8] to [0038.0.0.8] and
[0039.0.5.8] see paragraphs [0035.0.0.0] to [0039.0.0.0] above.
[3565] [0040.0.0.8] to [0044.0.0.8] for the disclosure of the
paragraphs [0040.0.0.8] to [0044.0.0.8] see paragraphs [0035.0.0.0]
and [0044.0.0.0] above.
[3566] [0045.0.8.8] In one embodiment, in case the activity of the
Escherichia coli K12 protein b3256 or its homologs e.g. a acetyl
CoA carboxylase protein, e.g. as indicated in Table II, columns 5
or 7, line 85, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of palmitic acid between
15% and 17% or more is conferred.
[3567] In case the activity of the Escherichia coli K12 protein
b0399 or its homologs e.g. a positive response regulator for the
pho regulon, e.g. as indicated in Table II, columns 5 or 7, line
88, is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of palmitic acid between 20% and 27% or
more is conferred.
[3568] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YDR513W or its homologs, e.g. a "glutaredoxin",
e.g. as indicated in Table II, columns 5 or 7, line 82, is
increased, preferably, in one embodiment an increase of the fine
chemical, preferably of palmitic acid between 17% and 74% or more
is conferred.
[3569] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YDR447C or its homologs, e.g. a "ribosomal
protein 51 (rp51) of the small (40 s) subunit", e.g. as indicated
in Table II, columns 5 or 7, line 83, is increased, preferably, in
one embodiment an increase of the fine chemical, preferably of
palmitic acid between 19% and 181% or more is conferred.
[3570] In case the activity of the Saccharomyces cerevisiae protein
YBR089C-A or its homologs, e.g. a "uncharacterized protein
YBR089C-A", which seams to have "homology to mammalian high
mobility group proteins 1 and 2", e.g. as indicated in Table II,
columns 5 or 7, line 84, is increased, preferably, in one
embodiment an increase of the fine chemical, preferably of palmitic
acid between 55% and 95% or more is conferred.
[3571] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YGR126W or its homologs, e.g. a "uncharacterized
protein YGR126W", e.g. as indicated in Table II, columns 5 or 7,
line 86, is increased, preferably, in one embodiment an increase of
the fine chemical, preferably of palmitic acid between 34% and 167%
or more is conferred.
[3572] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YPL099C or its homologs e.g. a "uncharacterized
protein YPL099C", e.g. as indicated in Table II, columns 5 or 7,
line 87, is increased, preferably, in one embodiment the increase
of the fine chemical, preferably of of palmitic acid between 21%
and 55% or more is conferred.
[3573] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0849 or its homologs e.g. a glutaredoxin 1 redox
coenzyme for glutathione-dependent ribonucleotide reductase
protein, e.g. as indicated in Table II, columns 5 or 7, line 463,
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of palmitic acid between 17% and 30% or
more is conferred.
[3574] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3457 or its homologs e.g. a high-affinity
branched-chain amino acid transport protein (ABC superfamily), e.g.
as indicated in Table II, columns 5 or 7, line 464, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of palmitic acid between 16% and 36% or more is
conferred.
[3575] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3578 or its homologs e.g. a putative component of
transport system, e.g. as indicated in Table II, columns 5 or 7,
line 465, is increased, preferably, in one embodiment the increase
of the fine chemical, preferably of palmitic acid between 16% and
27% or more is conferred.
[3576] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3644 or its homologs e.g. an uncharacterized
stress-induced protein, e.g. as indicated in Table II, columns 5 or
7, line 466, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of palmitic acid between
16% and 42% or more is conferred.
[3577] In one embodiment, in case the activity of the Escherichia
coli K12 protein b4129 or its homologs e.g. a lysine tRNA
synthetase, e.g. as indicated in Table II, columns 5 or 7, line
467, is increased, preferably, in one embodiment the increase of
the fine chemical, preferably of palmitic acid between 33% and 36%
or more is conferred.
[3578] [0046.0.8.8] In case the activity of the Escherichia coli
K12 protein b3256 or its homologs e.g. a acetyl CoA carboxylase
protein, is increased, preferably an increase of the fine chemical
and of tryglycerides, lipids, oils and/or fats containing palmitic
acid is conferred.
[3579] In case the activity of the Escherichia coli K12 protein
b0399 or its homologs e.g. a positive response regulator for the
pho regulon protein, is increased, preferably an increase of the
fine chemical and of tryglycerides, lipids, oils and/or fats
containing palmitic acid is conferred.
[3580] In case the activity of the Saccharomyces cerevisiae protein
YDR513W or its homologs, e.g. a "glutaredoxin protein" is
increased, preferably, in one embodiment an increase of the fine
chemical, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing palmitic acid is
conferred.
[3581] In case the activity of the Saccharomyces cerevisiae protein
YDR447C or its homologs, e.g. a "ribosomal protein 51 (rp51) of the
small (40 s) subunit protein", which is required for cell
viability" is increased, preferably, in one embodiment an increase
of the fine chemical, preferably an increase of the fine chemical
and of tryglycerides, lipids, oils and/or fats containing palmitic
acid is conferred.
[3582] In case the activity of the Saccharomyces cerevisiae protein
YBR089C-A or its homologs, e.g. a "uncharacterized protein
YBR089C-A", which seams to have "homology to mammalian high
mobility group proteins 1 and 2" is increased, preferably, an
increase of the fine chemical and of tryglycerides, lipids, oils
and/or fats containing palmitic acid is conferred.
[3583] In case the activity of the Saccharomyces cerevisiae protein
YGR126W or its homologs, e.g. a "uncharacterized protein YGR126W"
is increased, preferably, in one embodiment an increase of the fine
chemical, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing palmitic acid is
conferred.
[3584] In case the activity of the Saccaromyces cerevisiae protein
YPL099C or its homologs e.g. a "uncharacterized protein YPL099C
protein" is increased, preferably an increase of the fine chemical
and of tryglycerides, lipids, oils and/or fats containing palmitic
acid is conferred.
[3585] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0849 or its homologs e.g. a glutaredoxin 1 redox
coenzyme for glutathione-dependent ribonucleotide reductase protein
is increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing palmitic acid is
conferred.
[3586] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3457 or its homologs e.g. a high-affinity
branched-chain amino acid transport protein (ABC superfamily) is
increased, preferably an increase of the fine chemical and of
tryglycerides, lipids, oils and/or fats containing palmitic acid is
conferred.
[3587] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3578 or its homologs e.g. a putative component of
transport system is increased, preferably an increase of the fine
chemical and of tryglycerides, lipids, oils and/or fats containing
palmitic acid is conferred.
[3588] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3644 or its homologs e.g. an uncharacterized
stress-induced protein is increased, preferably an increase of the
fine chemical and of tryglycerides, lipids, oils and/or fats
containing palmitic acid is conferred.
[3589] In one embodiment, in case the activity of the Escherichia
coli K12 protein b4129 or its homologs e.g. a lysine tRNA
synthetase is increased, preferably an increase of the fine
chemical and of tryglycerides, lipids, oils and/or fats containing
palmitic acid is conferred.
[3590] [0047.0.0.8] to [0048.0.0.8] for the disclosure of the
paragraphs [0047.0.0.8] and [0048.0.0.8] see paragraphs
[0047.0.0.0] and [0048.0.0.0] above.
[3591] [0049.0.8.8] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
preferably has the structure of the polypeptide described herein,
in particular a polypeptide comprising a consensus sequence as
indicated in Table IV, column 7, lines 82 to 88 and 463 to 467 or
of the polypeptide as shown in the amino acid sequences as
disclosed in table II, columns 5 and 7, lines 82 to 88 and 463 to
467 or the functional homologues thereof as described herein, or is
encoded by the nucleic acid molecule characterized herein or the
nucleic acid molecule according to the invention, for example by
the nucleic acid molecule as shown in table I, columns 5 and 7,
lines 82 to 88 and 463 to 467 or its herein described functional
homologues and has the herein mentioned activity.
[3592] [0050.0.8.8] For the purposes of the present invention, the
term "palmitic acid" also encompasses the corresponding salts, such
as, for example, the potassium or sodium salts of palmitic acid or
the salts of palmitic acid with amines such as diethylamine.
[3593] [0051.0.5.8] and [0052.0.0.8] for the disclosure of the
paragraphs [0051.0.5.8] and [0052.0.0.8] see paragraphs
[0051.0.0.0] and [0052.0.0.0] above.
[3594] [0053.0.8.8] In one embodiment, the process of the present
invention comprises one or more of the following steps [3595] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention or the nucleic acid molecule or the
polypeptide used in the method of the invention, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 82 to 88 and 463 to 467 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 82 to 88
and 463 to 467, having herein-mentioned the fine chemical
increasing activity; [3596] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention, e.g. of a polypeptide having an activity
of a protein as indicated in Table II, column 3, lines 82 to 88 and
463 to 467 or its homologs activity, e.g. as indicated in Table II,
column 7, lines 82 to 88 and 463 to 467 or of a mRNA encoding the
polypeptide of the present invention having herein-mentioned
palmitic acid increasing activity; [3597] c) increasing the
specific activity of a protein conferring the increased expression
of a protein encoded by the nucleic acid molecule of the invention
or of the polypeptide of the present invention or the nucleic acid
molecule or the polypeptide used in the method of the invention,
having herein-mentioned palmitic acid increasing activity, e.g. of
a polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 82 to 88 and 463 to 467 or its homologs
activity, e.g. as indicated in Table II, column 7, lines 82 to 88
and 463 to 467, or decreasing the inhibiitory regulation of the
polypeptide of the invention or of the polypeptide used in the
method of the invention; [3598] d) generating or increasing the
expression of an endogenous or artificial transcription factor
mediating the expression of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention or of the polypeptide of the invention or the polypeptide
used in the method of the invention having herein-mentioned
palmitic acid increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 82
to 88 and 463 to 467 or its homolog's activity, e.g. as indicated
in Table II, column 7, lines 82 to 88 and 463 to 467; [3599] e)
stimulating activity of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
present invention or a polypeptide of the present invention having
herein-mentioned palmitic acid increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 82 to 88 and 463 to 467 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 82 to 88
and 463 to 467, by adding one or more exogenous inducing factors to
the organisms or parts thereof; [3600] f) expressing a transgenic
gene encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned palmitic acid increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 82 to 88 and 463 to 467 or its homolog's
activity, e.g. as indicated in Table II, column 7, lines 82 to 88
and 463 to 467; [3601] g) increasing the copy number of a gene
conferring the increased expression of a nucleic acid molecule
encoding a polypeptide encoded by the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention or the polypeptide of the invention or the polypeptide
used in the method of the invention having herein-mentioned
palmitic acid increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 82
to 88 and 463 to 467 or its homolog's activity, e.g. as indicated
in Table II, column 7, lines 82 to 88 and 463 to 467; [3602] h)
increasing the expression of the endogenous gene encoding the
polypeptide of the invention or the polypeptide used in the method
of the invention, e.g. a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 82 to 88 and 463
to 467 or its homolog's activity, e.g. as indicated in Table II,
column 7, lines 82 to 88 and 463 to 467, by adding positive
expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; [3603] i)
Modulating growth conditions of an organism in such a manner, that
the expression or activity of the gene encoding the protein of the
invention or the protein itself is enhanced for example
microorganisms or plants can be grown for example under a higher
temperature regime leading to an enhanced expression of heat shock
proteins, which can lead an enhanced fine chemical production.
[3604] j) selecting of organisms with especially high activity of
the proteins of the invention from natural or from mutagenized
resources and breeding them into the target organisms, eg the elite
crops.
[3605] [0054.0.8.8] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of palmitic acid after
increasing the expression or activity of the encoded polypeptide or
having the activity of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 82 to 88 and 463
to 467 or its homolog's activity, e.g. as indicated in Table II,
columns 5 or 7, lines 82 to 88 and 463 to 467.
[3606] [0055.0.0.8] to [0067.0.0.8] for the disclosure of the
paragraphs [0055.0.0.8] to [0067.0.0.8] see paragraphs [0055.0.0.0]
to [0067.0.0.0] above.
[3607] [0068.0.5.8] and [0069.0.5.8] for the disclosure of the
paragraphs [0068.0.5.8] and [0069.0.5.8] see paragraphs
[0068.0.0.0] and [0069.0.0.0] above.
[3608] [0070.0.6.8] and [0071.0.5.8] for the disclosure of the
paragraphs [0070.0.6.8] and [0071.0.5.8] see paragraphs
[0070.0.5.5] and [0071.0.0.0] above.
[3609] [0072.0.8.8] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to palmitic acid, triglycerides, lipids, oils and/or fats
containing palmitic acid compounds such as palmitate, palmitoleate,
stearate, oleate, .alpha.-linolenic acid and/or linoleic acid.
[3610] [0073.0.8.8] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[3611] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [3612] b) increasing the activity of a
protein having the activity of a polypeptide of the invention or
the polypeptide used in the method of the invention or a homolog
thereof, e.g. as shown in Table II, columns 5 or 7, lines 82 to 88
and 463 to 467 or of a polypeptide being encoded by the nucleic
acid molecule of the present invention and described below, i.e.
conferring an increase of the respective fine chemical in the
organism, preferably a microorganism, the a non-human animal, a
plant or animal cell, a plant or animal tissue or the plant, [3613]
c) growing the organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or the
plant under conditions which permit the production of the fine
chemical in the organism, preferably a microorganism, a plant cell,
a plant tissue or the plant; and [3614] d) if desired, recovering,
optionally isolating, the free and/or bound the respective fine
chemical and, optionally further free and/or bound fatty acids
synthetized by the organism, the microorganism, the non-human
animal, the plant or animal cell, the plant or animal tissue or the
plant.
[3615] [0074.0.5.8] for the disclosure of this paragraph see
[0074.0.5.5] above.
[3616] [0075.0.0.8] to [0084.0.0.8] for the disclosure of the
paragraphs [0075.0.0.8] to [0084.0.0.8] see paragraphs [0075.0.0.0]
to [0084.0.0.0] above.
[3617] [0085.0.8.8] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [3618] a) the nucleic acid sequence as
shown in table I, lines 82 to 88 and 463 to 467, columns 5 and 7 or
a derivative thereof, or [3619] b) a genetic regulatory element,
for example a promoter, which is functionally linked to the nucleic
acid sequence as shown table I, lines 82 to 88 and 463 to 467,
columns 5 and 7 or a derivative thereof, or [3620] c) (a) and (b)
is/are not present in its/their natural genetic environment or
has/have been modified by means of genetic manipulation methods, it
being possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[3621] [0086.0.0.8] and [0087.0.0.8] for the disclosure of the
paragraphs [0086.0.0.8] and [0087.0.0.8] see paragraphs
[0086.0.0.0] and [0087.0.0.0] above.
[3622] [0088.0.8.8] In an advantageous embodiment of the invention,
the organism takes the form of a plant whose fatty acid content is
modified advantageously owing to the nucleic acid molecule of the
present invention expressed. This is important for plant breeders
since, for example, the nutritional value of plants for poultry is
dependent on the abovementioned essential fatty acids and the
general amount of fatty acids as energy source in feed. After the
activity of a protein as shown in Table II, columns 5 or 7, lines
82 to 88 and 463 to 467 has been increased or generated, or after
the expression of nucleic acid molecule or polypeptide according to
the invention has been generated or increased, the transgenic plant
generated thus is grown on or in a nutrient medium or else in the
soil and subsequently harvested.
[3623] [0088.1.0.8], [0089.0.0.8], [0090.0.0.8] and [0091.0.5.8]
for the disclosure of the paragraphs [0088.1.0.8], [0089.0.0.8],
[0090.0.0.8] and [0091.0.5.8] see paragraphs [0088.1.0.0],
[0089.0.0.0], [0090.0.0.0] and [0091.0.0.0] above.
[3624] [0092.0.0.8] to [0094.0.0.8] for the disclosure of the
paragraphs [0092.0.0.8] to [0094.0.0.8] see paragraphs [0092.0.0.0]
to [0094.0.0.0] above.
[3625] [0095.0.5.8], [0096.0.5.8] and [0097.0.5.8] for the
disclosure of the paragraphs [0095.0.5.8], [0096.0.5.8] and
[0097.0.5.8] see paragraphs [0095.0.5.5], [0096.0.5.5] and
[0097.0.0.0] above.
[3626] [0098.0.8.8] In a preferred embodiment, the fine chemical
(palmitic acid) is produced in accordance with the invention and,
if desired, is isolated. The production of further fatty acids such
as stearic acid, palmitoleic acid, oleic acid, .alpha.-linolenic
acid and/or linoleic acid mixtures thereof or mixtures of other
fatty acids by the process according to the invention is
advantageous.
[3627] [0099.0.5.8] and [0100.0.5.8] for the disclosure of the
paragraphs [0099.0.5.8] and [0100.0.5.8] see paragraphs
[0099.0.5.5] and [0100.0.5.5] above.
[3628] [0101.0.5.8] and [0102.0.5.8] for the disclosure of the
paragraphs [0101.0.5.8] and [0102.0.5.8] see [0101.0.0.0] and
[0102.0.5.5] above.
[3629] [0103.0.8.8] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [3630]
a) nucleic acid molecule encoding, preferably at least the mature
form, of a polypeptide having a sequence as shown in Table II,
columns 5 or 7, lines 82 to 88 and 463 to 467 in or a fragment
thereof, which confers an increase in the amount of the respective
fine chemical in an organism or a part thereof; [3631] b) nucleic
acid molecule comprising, preferably at least the mature form, of
the nucleic acid molecule having a sequence as shown in Table I,
columns 5 or 7, lines 82 to 88 and 463 to 467, [3632] c) nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as result
of the degeneracy of the genetic code and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[3633] d) nucleic acid molecule encoding a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [3634] e) nucleic acid molecule which hybridizes with
a nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [3635] f)
nucleic acid molecule encoding a polypeptide, the polypeptide being
derived by substituting, deleting and/or adding one or more amino
acids of the amino acid sequence of the polypeptide encoded by the
nucleic acid molecules (a) to (d), preferably to (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [3636] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to [3637] (c) and and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [3638] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primer pairs
having a sequence as shown in Table III, column 7, lines 82 to 88
and 463 to 467 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [3639]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [3640] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequence as shown in Table IV, column 7, lines 82
to 88 and 463 to 467 and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[3641] k) nucleic acid molecule comprising one or more of the
nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of a polypeptide as shown in Table
II, columns 5 or 7, lines 82 to 88 and 463 to 467 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; and [3642] l) nucleic acid molecule
which is obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably to (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; or which comprises a sequence which is complementary
thereto.
[3643] [0103.1.0.8] and [0103.2.0.8] for the disclosure of the
paragraphs [0103.1.0.8] and [0103.2.0.8] see paragraphs
[0103.1.0.0] and [0103.2.0.0] above.
[3644] [0104.0.8.8] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence shown in Table
I, columns 5 or 7, lines 82 to 88 and 463 to 467, preferably over
the sequences as shown in Table IA, columns 5 or 7, lines 82 to 88
and 463 to 467 by one or more nucleotides or does not consist of
the sequence shown in Table I, columns 5 or 7, lines 82 to 88 and
463 to 467, preferably not of the sequences as shown in Table IA,
columns 5 or 7, lines 82 to 88 and 463 to 467. In one embodiment,
the nucleic acid molecule of the present invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to the sequence shown
in Table I, columns 5 or 7, lines 82 to 88 and 463 to 467,
preferably to the sequences as shown in Table IA, columns 5 or 7,
lines 82 to 88 and 463 to 467. In another embodiment, the nucleic
acid molecule does not encode a polypeptide of the sequence shown
in Table II, columns 5 or 7, lines 82 to 88 and 463 to 467,
preferably of the sequences as shown in Table IA, columns 5 or 7,
lines 82 to 88 and 463 to 467.
[3645] [0105.0.0.8] to [0107.0.0.8] for the disclosure of the
paragraphs [0105.0.0.8] to [0107.0.0.8] see paragraphs [0105.0.0.0]
and [0107.0.0.0] above.
[3646] [0108.0.8.8] Nucleic acid molecules with the sequence shown
in Table I, columns 5 or 7, lines 82 to 88 and 463 to 467, nucleic
acid molecules which are derived from the amino acid sequences
shown in Table II, columns 5 or 7, lines 82 to 88 and 463 to 467 or
from polypeptides comprising the consensus sequence shown in Table
IV, column 7, lines 82 to 88 and 463 to 467, or their derivatives
or homologues encoding polypeptides with the enzymatic or
biological activity of a protein as shown in Table II, columns 5 or
7, lines 82 to 88 and 463 to 467 or e.g. conferring a linoleic acid
increase after increasing its expression or activity are
advantageously increased in the process according to the
invention.
[3647] [0109.0.5.8] for the disclosure of this paragraph see
[0109.0.0.0] above.
[3648] [0110.0.8.8] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide used in the method
of the invention or used in the process of the invention, e.g. of a
protein as shown in Table II, columns 5 or 7, lines 82 to 88 and
463 to 467 or being encoded by a nucleic acid molecule indicated in
Table I, columns 5 or 7, lines 82 to 88 and 463 to 467 or of its
homologs, e.g. as indicated in Table II, columns 5 or 7, lines 82
to 88 and 463 to 467 can be determined from generally accessible
databases.
[3649] [0111.0.0.8] for the disclosure of this paragraph see
[0111.0.0.0] above.
[3650] [0112.0.8.8] The nucleic acid molecules used in the process
according to the invention take the form of isolated nucleic acid
sequences, which encode polypeptides with an activity of a
polypeptide as indicated in Table II, column 3, lines lines 82 to
88 and 463 to 467 or having the sequence of a polypeptide as
indicated in Table II, columns 5 and 7, lines 82 to 88 and 463 to
467 and conferring an increase of the respective fine chemical.
[3651] [0113.0.0.8] to [0120.0.0.8] for the disclosure of the
paragraphs [0113.0.0.8] to [0120.0.0.8] see paragraphs [0113.0.0.0]
and [0120.0.0.0] above.
[3652] [0121.0.8.8] However, it is also possible to use artificial
sequences, which differ in one or more bases from the nucleic acid
sequences found in organisms, or in one or more amino acid
molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences shown in Table II,
columns 5 or 7, lines 82 to 88 and 463 to 467 or the functional
homologues thereof as described herein, preferably conferring
above-mentioned activity, i.e. conferring an increase of the
respective fine chemical after increasing its activity.
[3653] [0122.0.0.8] to [0127.0.0.8] for the disclosure of the
paragraphs [0122.0.0.8] to [0127.0.0.8] see paragraphs [0122.0.0.0]
and [0127.0.0.0] above.
[3654] [0128.0.8.8] Synthetic oligonucleotide primers for the
amplification, e.g. as shown in Table III, column 7, lines 82 to 88
and 463 to 467, by means of polymerase chain reaction can be
generated on the basis of a sequence shown herein, for example the
sequence shown in Table I, columns 5 or 7, lines 82 to 88 and 463
to 467 or the sequences as shown in Table II, columns 5 or 7, lines
82 to 88 and 463 to 467.
[3655] [0129.0.8.8] Moreover, it is possible to identify conserved
regions from various organisms by carrying out protein sequence
alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequences shown in Table IV, column
7, lines 82 to 88 and 463 to 467 are derived from said
alignments.
[3656] [0130.0.8.8] for the disclosure of this paragraph see
[0130.0.0.0].
[3657] [0131.0.0.8] to [0138.0.0.8] for the disclosure of the
paragraphs [0131.0.0.8] to [0138.0.0.8] see paragraphs [0131.0.0.0]
to [0138.0.0.0] above.
[3658] [0139.0.8.8] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical increase, derived from
other organisms, can be encoded by other DNA sequences which
hybridize to the sequences shown in Table I, columns 5 or 7, lines
82 to 88 and 463 to 467, preferably of Table I B, columns 5 or 7,
lines 82 to 88 and 463 to 467 under relaxed hybridization
conditions and which code on expression for peptides having the
palmitic acid increasing activity.
[3659] [0140.0.0.8] to [0146.0.0.8] for the disclosure of the
paragraphs [0140.0.0.8] to [0146.0.0.8] see paragraphs [0140.0.0.0]
and [0146.0.0.0] above.
[3660] [0147.0.8.8] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 82 to 88 and 463 to 467, preferably in
Table I B, columns 5 or 7, lines 82 to 88 and 463 to 467 is one
which is sufficiently complementary to one of said nucleotide
sequences such that it can hybridize to one of said nucleotide
sequences thereby forming a stable duplex. Preferably, the
hybridisation is performed under stringent hybridization
conditions. However, a complement of one of the herein disclosed
sequences is preferably a sequence complement thereto according to
the base pairing of nucleic acid molecules well known to the
skilled person. For example, the bases A and G undergo base pairing
with the bases T and U or C, resp. and visa versa. Modifications of
the bases can influence the base-pairing partner.
[3661] [0148.0.8.8] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines
82 to 88 and 463 to 467, preferably of Table I B, columns 5 or 7,
lines 82 to 88 and 463 to 467, or a functional portion thereof and
preferably has above mentioned activity, in particular has
the--fine-chemical-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homologs.
[3662] [0149.0.8.8] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 82 to 88 and 463 to 467, preferably of Table I B, columns 5
or 7, lines 82 to 88 and 463 to 467 or a portion thereof and
encodes a protein having above-mentioned activity as indicated in
Table II, columns 5 or 7, lines 82 to 88 and 463 to 467, preferably
of Table II B, columns 5 or 7, lines 82 to 88 and 463 to 467, e.g.
conferring an increase of the respective fine chemical.
[3663] [00149.1.0.8] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 82 to 88 and 463 to 467, preferably of Table
I B, columns 5 or 7, lines 82 to 88 and 463 to 467 has further one
or more of the activities annotated or known for the a protein as
indicated in Table II, column 3, lines 82 to 88 and 463 to 467.
[3664] [0150.0.8.8] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table I, columns 5 or 7, lines 82 to 88 and 463 to 467, preferably
of Table I B, columns 5 or 7, lines 82 to 88 and 463 to 467, for
example a fragment which can be used as a probe or primer or a
fragment encoding a biologically active portion of the polypeptide
of the present invention or of a polypeptide used in the process of
the present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of methionine if its activity is increased.
The nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences indicated in Table I, columns 5 or 7, lines 82 to
88 and 463 to 467, an anti-sense sequence of one of the sequences
indicated in Table I, columns 5 or 7, lines 82 to 88 and 463 to
467, or naturally occurring mutants thereof. Primers based on a
nucleotide sequence of the invention can be used in PCR reactions
to clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table III, column 7, lines 82 to 88 and 463 to 467 will result in a
fragment of a polynucleotide sequence as indicated in Table I,
columns 5 or 7, lines 82 to 88 and 463 to 467. Preferred is Table I
B, column 7, lines 82 to 88 and 463 to 467.
[3665] [0151.0.0.8] for the disclosure of this paragraph see
[0151.0.0.0] above.
[3666] [0152.0.8.8] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to the amino acid
sequence shown in Table II, columns 5 or 7, lines 82 to 88 and 463
to 467 such that the protein or portion thereof maintains the
ability to participate in the fine chemical production, in
particular a palmitic acid increasing the activity as mentioned
above or as described in the examples in plants or microorganisms
is comprised.
[3667] [0153.0.8.8] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence shown in Table II, columns 5 or 7, lines 82 to 88 and
463 to 467 such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
shown in Table II, columns 5 or 7, lines 82 to 88 and 463 to 467
has for example an activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 82 to 88 and 463 to 467.
[3668] [0154.0.8.8] In one embodiment, the nucleic acid molecule of
the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as shown in Table II, columns 5 or 7, lines 82 to 88 and
463 to 467 and having above-mentioned activity, e.g. conferring
preferably the increase of the respective fine chemical.
[3669] [0155.0.0.8] and [0156.0.0.8] for the disclosure of the
paragraphs [0155.0.0.8] and [0156.0.0.8] see paragraphs
[0155.0.0.0] and [0156.0.0.0] above.
[3670] [0157.0.8.8] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 82 to 88 and 463 to 467
(and portions thereof) due to degeneracy of the genetic code and
thus encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides comprising the consensus sequences as indicated
in Table IV, column 7, lines 82 to 88 and 463 to 467 or of the
polypeptide as indicated in Table II, columns 5 or 7, lines 82 to
88 and 463 to 467 or their functional homologues. Advantageously,
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention comprises, or in an
other embodiment has, a nucleotide sequence encoding a protein
comprising, or in an other embodiment having, a consensus sequences
as indicated in Table IV, column 7, lines 82 to 88 and 463 to 467
or of the polypeptide as indicated in Table II, columns 5 or 7,
lines 82 to 88 and 463 to 467 or the functional homologues. In a
still further embodiment, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention encodes a full length protein which is substantially
homologous to an amino acid sequence comprising a consensus
sequence as indicated in Table IV, column 7, lines 82 to 88 and 463
to 467 or of a polypeptide as indicated in Table II, columns 5 or
7, lines 82 to 88 and 463 to 467 or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table I, columns 5 or 7, lines 82 to 88 and 463 to
467, preferably as indicated in Table I A, columns 5 or 7, lines 82
to 88 and 463 to 467. Preferably the nucleic acid molecule of the
invention is a functional homologue or identical to a nucleic acid
molecule indicated in Table I B, columns 5 or 7, lines 82 to 88 and
463 to 467.
[3671] [0158.0.0.8] to [0160.0.0.8] for the disclosure of the
paragraphs [0158.0.0.8] to [0160.0.0.8] see paragraphs [0158.0.0.0]
to [0160.0.0.0] above.
[3672] [0161.0.8.8] Accordingly, in another embodiment, a nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising the
sequence shown in Table I, columns 5 or 7, lines 82 to 88 and 463
to 467. The nucleic acid molecule is preferably at least 20, 30,
50, 100, 250 or more nucleotides in length.
[3673] [0162.0.0.8] for the disclosure of this paragraph see
paragraph [0162.0.0.0] above.
[3674] [0163.0.8.8] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 82 to 88 and 463 to
467 corresponds to a naturally-occurring nucleic acid molecule of
the invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[3675] [0164.0.0.8] for the disclosure of this paragraph see
paragraph [0164.0.0.0] above.
[3676] [0165.0.8.8] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as shown in
Table I, columns 5 or 7, lines 82 to 88 and 463 to 467.
[3677] [0166.0.0.8] and [0167.0.0.8] for the disclosure of the
paragraphs [0166.0.0.8] and [0167.0.0.8] see paragraphs
[0166.0.0.0] and [0167.0.0.0] above.
[3678] [0168.0.8.8] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organism or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 82 to 88 and 463 to 467 yet retain said activity described
herein. The nucleic acid molecule can comprise a nucleotide
sequence encoding a polypeptide, wherein the polypeptide comprises
an amino acid sequence at least about 50% identical to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 82 to
88 and 463 to 467 and is capable of participation in the increase
of production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to the
sequence as indicated in Table II, columns 5 or 7, lines 82 to 88
and 463 to 467, more preferably at least about 70% identical to one
of the sequences as indicated in Table II, columns 5 or 7, lines 82
to 88 and 463 to 467, even more preferably at least about 80%, 90%,
95% homologous to the sequence as indicated in Table II, columns 5
or 7, lines 82 to 88 and 463 to 467, and most preferably at least
about 96%, 97%, 98%, or 99% identical to the sequence as indicated
in Table II, columns 5 or 7, lines 82 to 88 and 463 to 467.
[3679] Accordingly, the invention relates to nucleic acid molecules
encoding a polypeptide having above-mentioned activity, e.g.
conferring an increase in the the respective fine chemical in an
organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 82 to 88 and 463 to 467, preferably of Table II B, column 7,
lines 82 to 88 and 463 to 467 yet retain said activity described
herein. The nucleic acid molecule can comprise a nucleotide
sequence encoding a polypeptide, wherein the polypeptide comprises
an amino acid sequence at least about 50% identical to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 82 to
88 and 463 to 467, preferably of Table II B, column 7, lines 82 to
88 and 463 to 467 and is capable of participation in the increase
of production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table II, columns 5 or 7, lines 82 to 88
and 463 to 467, preferably of Table II B, column 7, lines 82 to 88
and 463 to 467, more preferably at least about 70% identical to one
of the sequences as indicated in Table II, columns 5 or 7, lines 82
to 88 and 463 to 467, preferably of Table II B, column 7, lines 82
to 88 and 463 to 467, even more preferably at least about 80%, 90%,
or 95% homologous to a sequence as indicated in Table II, columns 5
or 7, lines 82 to 88 and 463 to 467, preferably of Table II B,
column 7, lines 82 to 88 and 463 to 467, and most preferably at
least about 96%, 97%, 98%, or 99% identical to the sequence as
indicated in Table II, columns 5 or 7, lines 82 to 88 and 463 to
467, preferably of Table II B, column 7, lines 82 to 88 and 463 to
467.
[3680] [0169.0.0.8] to [0175.0.5.8] for the disclosure of the
paragraphs [0169.0.0.8] to [0175.0.5.8] see paragraphs [0169.0.0.0]
to [0175.0.0.0] above.
[3681] [0176.0.8.8] Functional equivalents derived from one of the
polypeptides as shown in Table II, columns 5 or 7, lines 82 to 88
and 463 to 467 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as shown in Table II, columns
5 or 7, lines 82 to 88 and 463 to 467 according to the invention
and are distinguished by essentially the same properties as the
polypeptide as shown in Table II, columns 5 or 7, lines 82 to 88
and 463 to 467.
[3682] [0177.0.8.8] Functional equivalents derived from the nucleic
acid sequence as shown in Table I, columns 5 or 7, lines 82 to 88
and 463 to 467 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as shown in Table II, columns
5 or 7, lines 82 to 88 and 463 to 467 according to the invention
and encode polypeptides having essentially the same properties as
the polypeptide as shown in Table II, columns 5 or 7, lines 82 to
88 and 463 to 467.
[3683] [0178.0.0.8] for the disclosure of this paragraph see
[0178.0.0.0] above.
[3684] [0179.0.8.8] A nucleic acid molecule encoding a homologous
protein to a protein sequence of as indicated in Table II, columns
5 or 7, lines 82 to 88 and 463 to 467, preferably of Table II B,
column 7, lines 82 to 88 and 463 to 467 can be created by
introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of the nucleic acid molecule
of the present invention, in particular as indicated in Table I,
columns 5 or 7, lines 82 to 88 and 463 to 467 such that one or more
amino acid substitutions, additions or deletions are introduced
into the encoded protein. Mutations can be introduced into the
encoding sequences for example into a sequences as indicated in
Table I, columns 5 or 7, lines 82 to 88 and 463 to 467 by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[3685] [0180.0.0.8] to [0183.0.0.8] for the disclosure of the
paragraphs [0180.0.0.8] to [0183.0.0.8] see paragraphs [0180.0.0.0]
to [0183.0.0.0] above.
[3686] [0184.0.8.8] Homologues of the nucleic acid sequences used,
with a sequence as indicated in Table I, columns 5 or 7, lines 82
to 88 and 463 to 467, preferably of Table I B, column 7, lines 82
to 88 and 463 to 467, or of the nucleic acid sequences derived from
a sequences as indicated in Table II, columns 5 or 7, lines 82 to
88 and 463 to 467, preferably of Table II B, column 7, lines 82 to
88 and 463 to 467, comprise also allelic variants with at least
approximately 30%, 35%, 40% or 45% homology, by preference at least
approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
I, columns 5 or 7, lines 82 to 88 and 463 to 467, or from the
derived nucleic acid sequences, the intention being, however, that
the enzyme activity or the biological activity of the resulting
proteins synthesized is advantageously retained or increased.
[3687] [0185.0.8.8] In one embodiment of the present invention, the
nucleic acid molecule of the invention or used in the process of
the invention comprises one or more sequences as indicated in Table
I, columns 5 or 7, lines 82 to 88 and 463 to 467, preferably of
Table I B, column 7, lines 82 to 88 and 463 to 467. In one
embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotide sequences not
shown in any one of sequences as indicated in Table I, columns 5 or
7, lines 82 to 88 and 463 to 467, preferably of Table I B, column
7, lines 82 to 88 and 463 to 467. In one embodiment, the nucleic
acid molecule comprises less than 500, 400, 300, 200, 100, 90, 80,
70, 60, 50 or 40 further nucleotides. In a further embodiment, the
nucleic acid molecule comprises less than 30, 20 or 10 further
nucleotides. In one embodiment, a nucleic acid molecule used in the
process of the invention is identical to a sequences as indicated
in Table I, columns 5 or 7, lines 82 to 88 and 463 to 467,
preferably of Table I B, column 7, lines 82 to 88 and 463 to
467.
[3688] [0186.0.8.8] Also preferred is that one or more nucleic acid
molecule(s) used in the process of the invention encode a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 82 to 88 and 463 to 467, preferably of Table II B,
column 7, lines 82 to 88 and 463 to 467. In one embodiment, the
nucleic acid molecule encodes less than 150, 130, 100, 80, 60, 50,
40 or 30 further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table II, columns 5 or 7, lines 82 to 88 and 463 to 467,
preferably of Table II B, column 7, lines 82 to 88 and 463 to
467.
[3689] [0187.0.8.8] In one embodiment, the nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table II, columns 5 or 7,
lines 82 to 88 and 463 to 467, preferably of Table II B, column 7,
lines 82 to 88 and 463 to 467 and comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a sequences as indicated in Table II, columns 5
or 7, lines 82 to 88 and 463 to 467, preferably of Table II B,
column 7, lines 82 to 88 and 463 to 467.
[3690] [0188.0.8.8] Polypeptides (=proteins), which still have the
essential biological activity of the polypeptide of the present
invention conferring an increase of the fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide shown in Table II, columns 5 or 7, lines 82 to 88
and 463 to 467 expressed under identical conditions.
[3691] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, columns 7, lines 82 to 88 and 463 to 467.
[3692] [0189.0.8.8] Homologues of sequences as indicated in Table
I, columns 5 or 7, lines 82 to 88 and 463 to 467 or of the derived
sequences shown in Table II, columns 5 or 7, lines 82 to 88 and 463
to 467 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[3693] [0190.0.0.8], [0191.0.5.8], [00191.1.0.8] and [0192.0.0.8]
to [0203.0.0.8] for the disclosure of the paragraphs [0190.0.0.8],
[0191.0.5.8], [0191.1.0.8] and [0192.0.0.8] to [0203.0.0.8] see
paragraphs [0190.0.0.0], [0191.0.5.5], [0191.1.0.0] and
[0192.0.0.0] to [0203.0.0.0] above.
[3694] [0204.0.8.8] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [3695] a) nucleic
acid molecule encoding, preferably at least the mature form, of the
polypeptide shown in Table II, columns 5 or 7, lines 82 to 88 and
463 to 467; preferably of Table II B, column 7, lines 82 to 88 and
463 to 467; or a fragment thereof conferring an increase in the
amount of the fine chemical in an organism or a part thereof [3696]
b) nucleic acid molecule comprising, preferably at least the mature
form, of the nucleic acid molecule shown in Table I, columns 5 or
7, lines 82 to 88 and 463 to 467 preferably of Table I B, column 7,
lines 82 to 88 and 463 to 467; or a fragment thereof conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [3697] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [3698] d) nucleic
acid molecule encoding a polypeptide whose sequence has at least
50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [3699] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [3700] f) nucleic acid molecule encoding a polypeptide,
the polypeptide being derived by substituting, deleting and/or
adding one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[3701] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [3702] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using the primers or primer
pairs as indicated in Table III, column 7, lines 82 to 88 and 463
to 467 and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [3703] i) nucleic
acid molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [3704] j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence shown in Table IV,
column 7, lines 82 to 88 and 463 to 467 and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [3705] k) nucleic acid molecule encoding the amino
acid sequence of a polypeptide encoding a domaine of the
polypeptide shown in Table II, columns 5 or 7, lines 82 to 88 and
463 to 467, preferably of Table II B, column 7, lines 82 to 88 and
463 to 467; and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[3706] l) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment of at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (h) or of the
nucleic acid molecule shown in Table I, columns 5 or 7, lines 82 to
88 and 463 to 467 or a nucleic acid molecule encoding, preferably
at least the mature form of, the polypeptide shown in Table II,
columns 5 or 7, lines 82 to 88 and 463 to 467, and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; or which encompasses a sequence which
is complementary thereto; whereby, preferably, the nucleic acid
molecule according to (a) to (l) distinguishes over the sequence as
depicted in Table IA or IB, columns 5 or 7, lines 82 to 88 and 463
to 467 by one or more nucleotides. In one embodiment, the nucleic
acid molecule of the invention does not consist of the sequence
shown in Table IA or IB, columns 5 or 7, lines 82 to 88 and 463 to
467. In an other embodiment, the nucleic acid molecule of the
present invention is at least 30% identical and less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to the sequence shown in
Table IA or IB, columns 5 or 7, lines 82 to 88 and 463 to 467. In a
further embodiment the nucleic acid molecule does not encode the
polypeptide sequence shown in Table IIA or IIB, columns 5 or 7,
lines 82 to 88 and 463 to 467. Accordingly, in one embodiment, the
nucleic acid molecule of the present invention encodes in one
embodiment a polypeptide which differs at least in one or more
amino acids from the polypeptide as depicted in Table IIA or IIB,
columns 5 or 7, lines 82 to 88 and 463 to 467 and therefore does
not encode a protein of the sequence shown in Table IIA or IIB,
columns 5 or 7, lines 82 to 88 and 463 to 467. Accordingly, in one
embodiment, the protein encoded by a sequence of a nucleic acid
according to (a) to (l) does not consist of the sequence shown in
Table IIA or IIB, columns 5 or 7, lines 82 to 88 and 463 to 467. In
a further embodiment, the protein of the present invention is at
least 30% identical to protein sequence depicted in Table IIA or
IIB, columns 5 or 7, lines 82 to 88 and 463 to 467 and less than
100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 985, 97%, 96% or 95% identical to the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 82 to 88
and 463 to 467.
[3707] [0205.0.0.8] to [0226.0.0.8] for the disclosure of the
paragraphs [0205.0.0.8] to [0226.0.0.8] see paragraphs [0205.0.0.0]
to [0226.0.0.0] above.
[3708] [0227.0.8.8] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[3709] In addition to the sequence mentioned in Table I, columns 5
or 7, lines 82 to 88 and 463 to 467 or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the fatty acid biosynthetic pathway such as for
palmitate, palmitoleate, stearate and/or oleate is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine the sequences shown in Table I, columns 5
or 7, lines 82 to 88 and 463 to 467 with genes which generally
support or enhances to growth or yield of the target organisms, for
example genes which lead to faster growth rate of microorganisms or
genes which produces stress-, pathogen, or herbicide resistant
plants.
[3710] [0228.0.5.8] to [0230.0.5.8] for the disclosure of the
paragraphs [0228.0.5.8] to [0230.0.5.8] see paragraphs [0228.0.0.0]
to [0230.0.0.0] above.
[3711] [0231.0.8.8] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a palmitic acid degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[3712] [0232.0.0.8] to [0276.0.0.8] for the disclosure of the
paragraphs [0232.0.0.8] to [0276.0.0.8] see paragraphs [0232.0.0.0]
to [0276.0.0.0] above.
[3713] [0277.0.5.8] for the disclosure of this paragraph see
paragraph [0277.0.5.5] above.
[3714] [0278.0.0.8] to [0282.0.0.8] for the disclosure of the
paragraphs [0278.0.0.8] to [0282.0.0.8] see paragraphs [0278.0.0.0]
to [0282.0.0.0] above.
[3715] [0283.0.8.8] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an antibody against polypeptides as shown in
Table II, columns 5 or 7, lines 82 to 88 and 463 to 467 or an
antigenic part thereof, which can be produced by standard
techniques utilizing polypeptides comprising or consisting of
abovementioned sequences, e.g. the polypeptid of the present
invention or fragment thereof. Preferred are monoclonal antibodies
specifically binding to polypeptides as indicated in Table II,
columns 5 or 7, lines 82 to 88 and 463 to 467.
[3716] [0284.0.0.8] for the disclosure of this paragraph see
[0284.0.0.0] above.
[3717] [0285.0.8.8] In one embodiment, the present invention
relates to a polypeptide having the sequence shown in Table II,
columns 5 or 7, lines 82 to 88 and 463 to 467 or as coded by the
nucleic acid molecule shown in Table I, columns 5 or 7, lines 82 to
88 and 463 to 467 or functional homologues thereof.
[3718] [0286.0.8.8] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 82 to 88 and 463 to 467 and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table IV, column 7, lines 82 to 88 and 463 to 467, whereby 20 or
less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred
5 or 4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a poylpeptide or to a polypeptide comprising more than
one consensus sequences as indicated in Table IV, column 7, lines
82 to 88 and 463 to 467.
[3719] [0287.0.0.8] to [0290.0.0.8] for the disclosure of the
paragraphs [0287.0.0.8] to [0290.0.0.8] see paragraphs [0287.0.0.0]
to [0290.0.0.0] above.
[3720] [0291.0.8.8] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of a
plant or microorganism specific consensus sequences.
[3721] In one embodiment, said polypeptide of the invention
distinguishes over the sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 82 to 88 and 463 to 467 by one or more amino
acids. In one embodiment, polypeptide distinguishes form the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 82 to 88
and 463 to 467 by more than 5, 6, 7, 8 or 9 amino acids, preferably
by more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred
are more than 40, 50, or 60 amino acids and, preferably, the
sequence of the polypeptide of the invention distinguishes from the
sequence shown in Table IIA or IIB, columns 5 or 7, lines 82 to 88
and 463 to 467 by not more than 80% or 70% of the amino acids,
preferably not more than 60% or 50%, more preferred not more than
40% or 30%, even more preferred not more than 20% or 10%. In
another embodiment, said polypeptide of the invention does not
consist of the sequence shown in Table IIA or IIB, columns 5 or 7,
lines 82 to 88 and 463 to 467.
[3722] [0292.0.0.8] for the disclosure of this paragraph see
[0292.0.0.0] above.
[3723] [0293.0.8.8] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the
process.
[3724] In one embodiment, the polypeptide of the invention has a
sequence, which distinguishes from the sequence as shown in Table
IIA or IIB, columns 5 or 7, lines 82 to 88 and 463 to 467 by one or
more amino acids. In another embodiment, said polypeptide of the
invention does not consist of the sequence shown in Table IIA or
IIB, columns 5 or 7, lines 82 to 88 and 463 to 467. In a further
embodiment, said polypeptide of the present invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical. In one embodiment,
said polypeptide does not consist of the sequence encoded by the
nucleic acid molecules shown in Table IA or IB, columns 5 or 7,
lines 82 to 88 and 463 to 467.
[3725] [0294.0.8.8] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 82 to 88 and 463 to 467,
which distinguishes over the sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 82 to 88 and 463 to 467 by one or more
amino acids, preferably by more than 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, evenmore
preferred are more than 40, 50, or 60 amino acids but even more
preferred by less than 70% of the amino acids, more preferred by
less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[3726] [0295.0.0.8], [0296.0.0.8] and [0297.0.5.8] for the
disclosure of the paragraphs [0295.0.0.8], [0296.0.0.8] and
[0297.0.5.8] see paragraphs [0295.0.0.0] to [0297.0.0.0] above.
[3727] [00297.1.0.8] Non-polypeptide of the invention-chemicals are
e.g. polypeptides having not the activity and/or the amino acid
sequence of a polypeptide indicated in Table II, columns 3, 5 or 7,
lines 82 to 88 and 463 to 467.
[3728] [0298.0.8.8] A polypeptide of the invention can participate
in the process of the present invention. The polypeptide or a
portion thereof comprises preferably an amino acid sequence which
is sufficiently homologous to an amino acid sequence as indicated
in Table II, columns 5 or 7, lines 82 to 88 and 463 to 467 such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table II, columns 5 or 7, lines 82 to 88 and 463 to 467.
[3729] [0299.0.8.8] Further, the polypeptide can have an amino acid
sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences of Table II, columns 5 or 7, lines 82 to 88
and 463 to 467. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence of Table I, columns
5 or 7, lines 82 to 88 and 463 to 467 or which is homologous
thereto, as defined above.
[3730] [0300.0.8.8] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 82 to 88 and 463 to 467 in amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 82 to 88 and 463 to 467.
[3731] [0301.0.0.8] for the disclosure of this paragraph see
[0301.0.0.0] above.
[3732] [0302.0.8.8] Biologically active portions of an polypeptide
of the present invention include peptides comprising amino acid
sequences derived from the amino acid sequence of the polypeptide
of the present invention or used in the process of the present
invention, e.g., an amino acid sequence shown in Table II, columns
5 or 7, lines 82 to 88 and 463 to 467 or the amino acid sequence of
a protein homologous thereto, which include fewer amino acids than
a full length polypeptide of the present invention or used in the
process of the present invention or the full length protein which
is homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[3733] [0303.0.0.8] for the disclosure of this paragraph see
[0303.0.0.0] above.
[3734] [0304.0.8.8] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, columns 5 or 7, lines 82 to 88 and 463 to 467 but having
differences in the sequence from said wild-type protein. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity in relation to the wild type protein.
[3735] [0305.0.5.8], [0306.0.5.8] and [0306.1.0.8] for the
disclosure of the paragraphs [0305.0.5.8], [0306.0.5.8] and
[0306.1.0.8] see paragraphs [0305.0.0.0], [0306.0.0.0] and
[0306.1.0.0] above.
[3736] [0307.0.0.8] and [0308.0.0.8] for the disclosure of the
paragraphs [0307.0.0.8] and [0308.0.0.8] see paragraphs [0307.0.0.0
and [0308.0.0.0] above.
[3737] [0309.0.8.8] In one embodiment, a reference to protein
(=polypeptide)" of the invention e.g. as indicated in Table II,
columns 5 or 7, lines 82 to 88 and 463 to 467 refers to a
polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas a "non-polypeptide" or "other polypeptide" e.g.
not indicated in Table II, columns 5 or 7, lines 82 to 88 and 463
to 467 refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to
a polypeptide of the invention, preferably which is not
substantially homologous to a polypeptide having a protein
activity, e.g., a protein which does not confer the activity
described herein and which is derived from the same or a different
organism.
[3738] [0310.0.0.8] to [0334.0.0.8] for the disclosure of the
paragraphs [0310.0.0.8] to [0334.0.0.8] see paragraphs [0310.0.0.0]
to [0334.0.0.0] above.
[3739] [0335.0.8.8] The dsRNAi method has proved to be particularly
effective and advantageous for reducing the expression of a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 82 to
88 and 463 to 467 and/or homologs thereof. As described inter alia
in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table I,
columns 5 or 7, lines 82 to 88 and 463 to 467 and/or homologs
thereof. In a double-stranded RNA molecule for reducing the
expression of an protein encoded by a nucleic acid sequence of one
of the sequences as indicated in Table I, columns 5 or 7, lines 82
to 88 and 463 to 467 and/or homologs thereof, one of the two RNA
strands is essentially identical to at least part of a nucleic acid
sequence, and the respective other RNA strand is essentially
identical to at least part of the complementary strand of a nucleic
acid sequence.
[3740] [0336.0.0.8] to [0342.0.0.8] for the disclosure of the
paragraphs [0336.0.0.8] to [0342.0.0.8] see paragraphs [0336.0.0.0]
to [0342.0.0.0] above.
[3741] [0343.0.8.8] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 82 to 88
and 463 to 467 or its homolog is not necessarily required in order
to bring about effective reduction in the expression. The advantage
is, accordingly, that the method is tolerant with regard to
sequence deviations as may be present as a consequence of genetic
mutations, polymorphisms or evolutionary divergences. Thus, for
example, using the dsRNA, which has been generated starting from a
sequence of one of the sequences as indicated in Table I, columns 5
or 7, lines 82 to 88 and 463 to 467 or homologs thereof of the one
organism, may be used to suppress the corresponding expression in
another organism.
[3742] [0344.0.0.8] to [0350.0.0.8], [0351.0.5.8] and [0352.0.0.8]
to [0361.0.0.8] for the disclosure of the paragraphs [0344.0.0.8]
to [0350.0.0.8], [0351.0.5.8] and [0352.0.0.8] to [0361.0.0.8] see
paragraphs [0344.0.0.0] to [0361.0.0.0] above.
[3743] [0362.0.8.8] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, the nucleic acid construct of
the invention, the antisense molecule of the invention, the vector
of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. encoding a polypeptide having an
activity of a protein such as the polypeptides as indicated in
Table II, column 3, lines 82 to 88 and 463 to 467. Due to the above
mentioned activity the fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a protein such
as the polypeptides as indicated in Table II, column 3, lines 82 to
88 and 463 to 467. Activity means herein that due to modulation or
manipulation of the genome, the activity of polypeptide having an
activity of a protein such as the polypeptides as indicated in
Table II, column 3, lines 82 to 88 and 463 to 467 or a similar
activity, which is increased in the cell or organism or part
thereof. Examples are described above in context with the process
of the invention.
[3744] [0363.0.0.8], [0364.0.5.8] and [0365.0.0.8] to [0379.0.5.8]
for the disclosure of the paragraphs [0363.0.0.8], [0364.0.5.8] and
[0365.0.0.8] to [0379.0.5.8] see paragraphs [0363.0.0.0] to
[0379.0.0.0] above.
[3745] [0380.0.5.8], [0381.0.0.8] and [0382.0.0.8] for the
disclosure of the paragraphs [0380.0.5.8], [0381.0.0.8] and
[0382.0.0.8] see paragraphs [0380.0.5.5], [0381.0.0.0] and
[0382.0.0.0] above.
[3746] [0383.0.5.8], [0384.0.0.8], [0385.0.5.8] and [0386.0.5.8]
for the disclosure of the paragraphs [0383.0.5.8], [0384.0.0.8],
[0385.0.5.8] and [0386.0.5.8] see paragraphs [0383.0.5.5],
[0384.0.0.0], [0385.0.5.5] and [0386.0.5.5] above.
[3747] [0387.0.0.8] to [0392.0.0.8] for the disclosure of the
paragraphs [0387.0.0.8] to [0392.0.0.8] see paragraphs [0387.0.0.0]
to [0392.0.0.0] above.
[3748] [0393.0.8.8] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [3749] (a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the respective fine chemical after expression, with the
nucleic acid molecule of the present invention; [3750] (b)
identifying the nucleic acid molecules, which hybridize under
relaxed stringent conditions with the nucleic acid molecule of the
present invention in particular to the nucleic acid molecule
sequence shown in Table I, columns 5 or 7, lines 82 to 88 and 463
to 467, preferably in Table I B, columns 5 or 7, lines 82 to 88 and
463 to 467 and, optionally, isolating the full length cDNA clone or
complete genomic clone; [3751] (c) introducing the candidate
nucleic acid molecules in host cells, preferably in a plant cell or
a microorganism, appropriate for producing the respective fine
chemical; [3752] (d) expressing the identified nucleic acid
molecules in the host cells; [3753] (e) assaying the respective
fine chemical level in the host cells; and [3754] (f) identifying
the nucleic acid molecule and its gene product which expression
confers an increase in the respective fine chemical level in the
host cell after expression compared to the wild type.
[3755] [0394.0.0.8] to [0415.0.0.8] and [0416.0.5.8] for the
disclosure of the paragraphs [0394.0.0.8] to [0415.0.0.8] and
[0416.0.5.8] see paragraphs [0394.0.0.0] to [0416.0.0.0] above.
[3756] [0417.0.5.8] and [0418.0.0.8] to [0430.0.0.8] for the
disclosure of the paragraphs [0417.0.5.8] and [0418.0.0.8] to
[0430.0.0.8] see paragraphs [0417.0.5.5] and [0418.0.0.0] to
[0430.0.0.0] above.
[3757] [0431.0.5.8], [0432.0.5.8], [0433.0.0.8] and [0434.0.0.8]
for the disclosure of the paragraphs [0431.0.5.8], [0432.0.5.8],
[0433.0.0.8] and [0434.0.0.8] see paragraphs [0431.0.0.0] to
[0434.0.0.0] above.
[3758] [0435.0.5.8] to [0440.0.5.8] for the disclosure of the
paragraphs [0435.0.5.8] to [0440.0.5.8] see paragraphs [0435.0.5.5]
to [0440.0.5.5] above.
[3759] [0441.0.0.8] and [0442.0.5.8] for the disclosure of the
paragraphs [0441.0.0.8] and [0442.0.5.8] see [0441.0.0.0] and
[0442.0.5.5] above.
[3760] [0443.0.0.8] for the disclosure of this paragraph see
[0443.0.0.0] above.
[3761] [0444.0.5.8] and [0445.0.5.8] for the disclosure of the
paragraphs [0444.0.5.8] and [0445.0.5.8] see [0444.0.5.5] and
[0445.0.5.5] above.
[3762] [0446.0.0.8] to [0453.0.0.8] for the disclosure of the
paragraphs [0446.0.0.8] to [0453.0.0.8] see paragraphs [0446.0.0.0]
to [0453.0.0.0] above.
[3763] [0454.0.5.8] and [0455.0.5.8] for the disclosure of the
paragraphs [0454.0.5.8] and [0455.0.5.8] see [0454.0.5.5] and
[0455.0.5.5] above.
[3764] [0456.0.0.8] for the disclosure of this paragraph see
[0456.0.0.0] above.
[3765] [0457.0.5.8] to [0460.0.5.8] for the disclosure of the
paragraphs [0457.0.5.8] to [0460.0.6.8] see paragraphs [0457.0.5.5]
to [0460.0.5.5] above.
[0461.0.8.8] Example 10
Cloning SEQ ID NO: 7435 for the Expression in Plants
[3766] [0462.0.0.8] for the disclosure of this paragraph see
[0462.0.0.0] above.
[3767] [0463.0.8.8] SEQ ID NO: 7435 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[3768] [0464.0.5.8], [0465.0.5.8] and [0466.0.0.8] for the
disclosure of the paragraphs [0464.0.5.8], [0465.0.5.8] and
[0466.0.0.8] see paragraphs [0464.0.5.5], [0465.0.5.5] and
[0466.0.0.0] above.
[3769] [0467.0.8.8] The following primer sequences were selected
for the gene SEQ ID NO: 7435:
TABLE-US-00029 i) forward primer (SEQ ID NO: 7569) atggagacca
atttttcctt cgact ii) reverse primer (SEQ ID NO: 7570) ctattgaaat
accggcttca atattt
[3770] [0468.0.0.8] to [0479.0.0.8] for the disclosure of the
paragraphs [0468.0.0.8] to [0479.0.0.8] see paragraphs [0468.0.0.0]
to [0479.0.0.0] above.
[0480.0.8.8] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 7435
[3771] [0481.0.0.8] for the disclosure of this paragraph see
[0481.0.0.0] above.
[3772] [0482.0.0.8] to [0513.0.0.8] for the disclosure of the
paragraphs [0482.0.0.8] to [0513.0.0.8] see paragraphs [0482.0.0.0]
to [0513.0.0.0] above.
[3773] [0514.0.8.8] As an alternative, the amino acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55).
[3774] The results of the different plant analyses can be seen from
the table, which follows:
TABLE-US-00030 TABLE 1 ORF Metabolite Method Min Max YDR513W
Palmitic Acid (C16:0) GC 1.17 1.74 YDR447C Palmitic Acid (C16:0) GC
1.19 2.81 YBR089C-A Palmitic Acid (C16:0) GC 1.55 1.95 b3256
Palmitic Acid (C16:0) GC 1.15 1.17 YGR126W Palmitic Acid (C16:0) GC
1.34 2.67 YPL099C Palmitic Acid (C16:0) GC 1.21 1.55 b0399 Palmitic
Acid (C16:0) GC 1.20 1.27 b0849 Palmitic Acid (C16:0) GC 1.17 1.303
b3457 Palmitic Acid (C16:0) GC 1.16 1.36 b3578 Palmitic Acid
(C16:0) GC 1.16 1.271 b3644 Palmitic Acid (C16:0) GC 1.16 1.42
b4129 Palmitic Acid (C16:0) GC 1.33 1.36
[3775] [0515.0.5.8] Column 2 shows the fatty acid analyzed. Columns
4 and 5 shows the ratio of the analyzed fatty acid between the
transgenic plants and the wild type; Increase of the metabolites:
Max: maximal x-fold (normalised to wild type)-Min: minimal x-fold
(normalised to wild type). Decrease of the metabolites: Max:
maximal x-fold (normalised to wild type) (minimal decrease), Min:
minimal x-fold (normalised to wild type) (maximal decrease). Column
3 indicates the analytical method.
[3776] [0516.0.0.8] and [0517.0.5.8] for the disclosure of the
paragraphs [0516.0.0.8] and [0517.0.5.8] see paragraphs
[0516.0.0.0] and [0517.0.0.0] above.
[3777] [0518.0.0.8] to [0529.0.0.8] and [0530.0.5.8] for the
disclosure of the paragraphs [0518.0.0.8] to [0529.0.0.8] and
[0530.0.5.8] see paragraphs [0518.0.0.0] to [0530.0.0.0] above.
[3778] [0530.1.0.8] to [0530.6.0.8] for the disclosure of the
paragraphs [0530.1.0.8] to [0530.6.0.8] see paragraphs [0530.1.0.0]
to [0530.6.0.0] above.
[3779] [0531.0.0.8] to [0533.0.0.8] and [0534.0.5.8] for the
disclosure of the paragraphs [0531.0.0.8] to [0533.0.0.8] and
[0534.0.5.8] see paragraphs [0531.0.0.0] to [0534.0.0.0] above.
[3780] [0535.0.0.8] to [0537.0.0.8] and [0538.0.5.8] for the
disclosure of the paragraphs [0535.0.0.8] to [0537.0.0.8] and
[0538.0.5.8] see paragraphs [0535.0.0.0] to [0538.0.0.0] above.
[3781] [0539.0.0.8] to [0542.0.0.8] and [0543.0.5.8] for the
disclosure of the paragraphs [0539.0.0.8] to [0542.0.0.8] and
[0543.0.5.8] see paragraphs [0539.0.0.0] to [0543.0.0.0] above.
[3782] [0544.0.0.8] to [0547.0.0.8] and [0548.0.5.8] to
[0552.0.0.8] for the disclosure of the paragraphs [0544.0.0.8] to
[0547.0.0.8] and [0548.0.5.8] to [0552.0.0.8] see paragraphs
[0544.0.0.0] to [0552.0.0.0] above.
[0552.1.8.8] Example 15
Metabolite Profiling Info from Zea mays
[3783] Zea mays plants were engineered, grown and analyzed as
described in Example 14c. The results of the different Zea mays
plants analysed can be seen from Table 2 which follows:
TABLE-US-00031 TABLE 2 ORF_NAME Metabolite Min Max b3644 Palmitic
aicid (C16:0) 1.23 1.34
[3784] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in palmitic acid in
genetically modified corn plants expressing the E. coli nucleic
acid sequence b3644 resp.
[3785] In one embodiment, in case the activity of the E. coli
protein b3644 or its homologs, e.g. "an uncharacterized
stress-induced protein", is increased in corn plants, preferably,
an increase of the fine chemical palmitic acid between 23% and 34%
is conferred.
[3786] [0552.2.0.8] for the disclosure of this paragraph see
[0552.2.0.0] above.
[3787] [0553.0.8.8] [3788] 1. A process for the production of
palmitic acid, which comprises [3789] (a) increasing or generating
the activity of a protein as indicated in Table II, columns 5 or 7,
lines 82 to 88 and 463 to 467 or a functional equivalent thereof in
a non-human organism, or in one or more parts thereof; and [3790]
(b) growing the organism under conditions which permit the
production of palmitic acid in said organism. [3791] 2. A process
for the production of palmitic acid, comprising the increasing or
generating in an organism or a part thereof the expression of at
least one nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [3792] a) nucleic acid
molecule encoding of a polypeptide as indicated in Table II,
columns 5 or 7, lines 82 to 88 and 463 to 467 or a fragment
thereof, which confers an increase in the amount of palmitic acid
in an organism or a part thereof; [3793] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 82 to 88 and 463 to 467; [3794] c) nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as a
result of the degeneracy of the genetic code and conferring an
increase in the amount of palmitic acid in an organism or a part
thereof; [3795] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of palmitic
acid in an organism or a part thereof; [3796] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under stringent hybridisation conditions and conferring an
increase in the amount of palmitic acid in an organism or a part
thereof; [3797] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, column 7, lines
82 to 88 and 463 to 467 and conferring an increase in the amount of
palmitic acid in an organism or a part thereof; [3798] g) nucleic
acid molecule encoding a polypeptide which is isolated with the aid
of monoclonal antibodies against a polypeptide encoded by one of
the nucleic acid molecules of (a) to (f) and conferring an increase
in the amount of palmitic acid in an organism or a part thereof;
[3799] h) nucleic acid molecule encoding a polypeptide comprising a
consensus sequence as indicated in Table IV, column 7, lines 82 to
88 and 463 to 467 and conferring an increase in the amount of
palmitic acid in an organism or a part thereof; and [3800] i)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment thereof having at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (k) and
conferring an increase in the amount of palmitic acid in an
organism or a part thereof. [3801] or comprising a sequence which
is complementary thereto. [3802] 3. The process of claim 1 or 2,
comprising recovering of the free or bound palmitic acid. [3803] 4.
The process of any one of claims 1 to 3, comprising the following
steps: [3804] (a) selecting an organism or a part thereof
expressing a polypeptide encoded by the nucleic acid molecule
characterized in claim 2; [3805] (b) mutagenizing the selected
organism or the part thereof; [3806] (c) comparing the activity or
the expression level of said polypeptide in the mutagenized
organism or the part thereof with the activity or the expression of
said polypeptide of the selected organisms or the part thereof;
[3807] (d) selecting the mutated organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
[3808] (e) optionally, growing and cultivating the organisms or the
parts thereof; and [3809] (f) recovering, and optionally isolating,
the free or bound palmitic acid produced by the selected mutated
organisms or parts thereof. [3810] 5. The process of any one of
claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. [3811] 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [3812] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 82 to
88 and 463 to 467 or a fragment thereof, which confers an increase
in the amount of palmitic acid in an organism or a part thereof;
[3813] b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 82 to 88
and 463 to 467; [3814] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of palmitic acid in
an organism or a part thereof; [3815] d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of palmitic acid in an organism or a part thereof; [3816] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of palmitic acid in an
organism or a part thereof; [3817] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table III, column
7, lines 82 to 88 and 463 to 467 and conferring an increase in the
amount of palmitic acid in an organism or a part thereof; [3818] g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of palmitic acid in an organism or a part
thereof; [3819] h) nucleic acid molecule encoding a polypeptide
comprising a consensus sequence as indicated in Table IV, column 7,
lines 82 to 88 and 463 to 467 and conferring an increase in the
amount of palmitic acid in an organism or a part thereof; and
[3820] i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of palmitic acid in an
organism or a part thereof. [3821] whereby the nucleic acid
molecule distinguishes over the sequence as indicated in Table I A,
columns 5 or 7, lines 82 to 88 and 463 to 467 by one or more
nucleotides. [3822] 7. A nucleic acid construct which confers the
expression of the nucleic acid molecule of claim 6, comprising one
or more regulatory elements. [3823] 8. A vector comprising the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7. [3824] 9. The vector as claimed in claim 8,
wherein the nucleic acid molecule is in operable linkage with
regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic, host. [3825] 10. A
host cell, which has been transformed stably or transiently with
the vector as claimed in claim 8 or 9 or the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 2 to 5. [3826] 11.
The host cell of claim 10, which is a transgenic host cell. [3827]
12. The host cell of claim 10 or 11, which is a plant cell, an
animal cell, a microorganism, or a yeast cell, a fungus cell, a
prokaryotic cell, an eukaryotic cell or an archaebacterium. [3828]
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. [3829] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table II A, columns 5 or 7, lines 82 to 88 and 463 to
467 by one or more amino acids [3830] 15. An antibody, which binds
specifically to the polypeptide as claimed in claim 14. [3831] 16.
A plant tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [3832] 17. A method for screening for agonists
and antagonists of the activity of a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of palmitic acid in an organism or a part thereof
comprising: [3833] (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of palmitic acid in an organism or a part thereof with a
candidate compound or a sample comprising a plurality of compounds
under conditions which permit the expression the polypeptide;
[3834] (b) assaying the palmitic acid level or the polypeptide
expression level in the cell, tissue, plant or microorganism or the
media the cell, tissue, plant or microorganisms is cultured or
maintained in; and [3835] (c) identifying a agonist or antagonist
by comparing the measured palmitic acid level or polypeptide
expression level with a standard of palmitic acid or polypeptide
expression level measured in the absence of said candidate compound
or a sample comprising said plurality of compounds, whereby an
increased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an agonist and
a decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[3836] 18. A process for the identification of a compound
conferring increased palmitic acid production in a plant or
microorganism, comprising the steps: [3837] (a) culturing a plant
cell or tissue or microorganism or maintaining a plant expressing
the polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of palmitic acid in an
organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with dais readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of palmitic acid in an
organism or a part thereof; [3838] (b) identifying if the compound
is an effective agonist by detecting the presence or absence or
increase of a signal produced by said readout system. [3839] 19. A
method for the identification of a gene product conferring an
increase in palmitic acid production in a cell, comprising the
following steps: [3840] (a) contacting the nucleic acid molecules
of a sample, which can contain a candidate gene encoding a gene
product conferring an increase in palmitic acid after expression
with the nucleic acid molecule of claim 6; [3841] (b) identifying
the nucleic acid molecules, which hybridise under relaxed stringent
conditions with the nucleic acid molecule of claim 6; [3842] (c)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing palmitic acid; [3843] (d) expressing the
identified nucleic acid molecules in the host cells; [3844] (e)
assaying the palmitic acid level in the host cells; and [3845] (f)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the palmitic acid level in the
host cell in the host cell after expression compared to the wild
type. [3846] 20. A method for the identification of a gene product
conferring an increase in palmitic acid production in a cell,
comprising the following steps: [3847] (a) identifying in a data
bank nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the palmitic acid amount or level in an organism or a part thereof
after expression, and which are at least 20% homolog to the nucleic
acid molecule of claim 6; [3848] (b) introducing the candidate
nucleic acid molecules in host cells appropriate for producing
palmitic acid; [3849] (c) expressing the identified nucleic acid
molecules in the host cells; [3850] (d) assaying the palmitic acid
level in the host cells; and [3851] (e) identifying nucleic acid
molecule and its gene product which expression confers an increase
in the palmitic acid level in the host cell after expression
compared to the wild type. [3852] 21. A method for the production
of an agricultural composition comprising the steps of the method
of any one of claims 17 to 20 and formulating the compound
identified in any one of claims 17 to 20 in a form acceptable for
an application in agriculture. [3853] 22. A composition comprising
the nucleic acid molecule of claim 6, the polypeptide of claim 14,
the nucleic acid construct of claim 7, the vector of any one of
claim 8 or 9, an antagonist or agonist identified according to
claim 17, the compound of claim 18, the gene product of claim 19 or
20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. [3854] 23. Use of the nucleic acid molecule as
claimed in claim 6 for the identification of a nucleic acid
molecule conferring an increase of palmitic acid after expression.
[3855] 24. Use of the polypeptide of claim 14 or the nucleic acid
construct claim 7 or the gene product identified according to the
method of claim 19 or 20 for identifying compounds capable of
conferring a modulation of palmitic acid levels in an organism.
[3856] 25. Food or feed composition comprising the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the harvested material of
claim 16, the host cell of claim 10 to 12 or the gene product
identified according to the method of claim 19 or 20.
[3857] 26. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the host cell of claim 10 to 12 or the gene
product identified according to the method of claim 19 or 20 for
the protection of a plant against a palmitic acid synthesis
inhibiting herbicide.
[3858] [0554.0.0.8] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[3859] [0000.0.0.9] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
DESCRIPTION
[3860] [0001.0.0.9] see [0001.0.0.0]
[3861] [0002.0.9.9] Due to their plastids, plants possess some
biosynthetic pathways, which are, besides in cyanobacteria, unique
in living organisms. Some plastidic compounds are indispensable for
human and animal nutrition and are therefore called vitamins. Two
essential lipophilic components for nutrition are provitamin A
(beta-carotene) and vitamin E.
[3862] Vitamin E is classified by its pharmacological effect and
chromanol ring structure and not by biosynthesis. It comprises a
class of 8 lipid-soluble components, being subdivided into
tocopherols and tocotrienols. While tocopherols share an isoprenoid
side chain derived from phytyl-PP, tocotrienol side chains are
derivates of geranylgeranyl-PP. The .alpha., .beta., .gamma. and
.delta.-members of these subclasses differ in their degree of
methylation in the 6-chromanol-ring structure.
[3863] The tocopherol group (1a-d) has a saturated side chain, and
the tocotrienol group (2a-d) has an unsaturated side chain:
##STR00002##
[3864] In the present invention, vitamin E means all of the
aforementioned tocopherols and tocotrienols with vitamin E
activity.
[3865] [0003.0.9.9] The four major forms of tocopherols, .alpha.,
.beta., .gamma., and .delta., differ in the position and number of
methyl groups. The predominant form in the leaves of higher plants
is .alpha.-tocopherol, whereas in seeds .gamma.-tocopherol is often
the major isoform. Tocopherols predominantly function as
antioxidants in vivo in photosynthetic organisms and in animals, as
well as in isolated compounds such as oils. The antioxidant
properties of tocopherols derive from their ability to quench free
radicals and different tocopherols may be optimal as antioxidants
for different biological systems. For human and animal utility,
.alpha.-tocopherol has the highest vitamin E activity and has been
implicated in a variety of health areas, including possible
benefits in preventing cardiovascular disease, certain cancers, and
cataract formation. The amounts of vitamin E needed to achieve
these effects are often quite high, 100 to 400 International Units
(I.U.) and even up to 800 I.U. compared with the recommended daily
allowance of 40 I.U. In fats and oils, tocopherols protect
unsaturated fatty acids from oxidation. In these systems,
.gamma.-tocopherol appears to have the greater utility. In fact,
tocopherols are often included in processed oils to help stabilize
the fatty acids. For human health as well as food and feed utility,
it is desirable to have plants with increased tocopherol content
along with those where the tocopherol composition is
customized.
[3866] [0004.0.9.9] Tocopherols contain an aromatic head group,
which is derived from homogentisic acid (HGA) and a hydrocarbon
portion, which arises from phytyldiphosphate (phytyl-DP). HGA is
derived from the shikimic acid pathway and phytyl-DP is generated
from the condensation of four isoprenoid units. The isoprenoid
contribution to tocopherol biosynthesis is thought to come
primarily from the plastidal methyl-erythritol phosphate pathway,
and not the cytosolic mevalonic acid pathway. The condensation of
HGA and phytyl-DP to form 2-methyl-6-phytylplastoquinol, the first
committed step in tocopherol biosynthesis, is a prenyltransferase
reaction that is performed by a homogentisate phytyltransferase
(HPT). Subsequent cyclization and methylation reactions result in
the formation of the four major tocopherols. The enzymatic
reactions in tocopherol biosynthesis were identified 15 to 20 years
ago, but cloning of the genes encoding these enzymes has only
occurred in the last few years.
[3867] [0005.0.9.9] Tocopherol biosynthesis takes place in the
plastid and the enzymes are associated with the chloroplast
envelope. The membrane association of the enzymes has made
purification difficult. With the advent of genomics and the
availability of complete genome sequences of a number of organisms,
including Synechocystis sp. PCC 6803 and Arabidopsis, it has become
possible to use bioinformatics techniques to identify and clone
additional genes in the tocopherol pathway.
[3868] The first enzyme cloned in the tocopherol pathway,
.gamma.-tocopherol methyl transferase (.gamma.-TMT), was identified
in Synechocystis sp. PCC 6803 and Arabidopsis using bioinformatics.
In that study, the Arabidopsis .gamma.-TMT was shown to alter seed
tocopherol composition when overexpressed in Arabidopsis.
.gamma.-Tocopherol, normally the predominant tocopherol isomer in
Arabidopsis seeds, was almost completely converted to
.alpha.-tocopherol.
[3869] HPT catalyzes the first committed reaction in the tocopherol
pathway, and was unidentified previously. Concomitant with this
study, slr1736 was found to encode a HPT in Synechocystis sp. PCC
6803 and the Arabidopsis HTP was identified.
[3870] There are prenyltransferases that condense prenyl groups
with allylic chains and those that condense prenyl chains with
aromatic groups. The prenyltransferases that catalyze sequential
condensations of isopentenylpyrophosphate with allylic chains share
common features, including Asp-rich motifs, and lead to the
formation of compounds with two isoprenoid units, such as
geranylpyrophosphate, or to much longer molecules, such as rubber,
which contains greater than 1,000 isoprenoid units.
Prenyltransferases that catalyze condensations with nonisoprenoid
groups have an Asp-rich motif distinct from that of the allylic
class, and include UbiA, which attaches a prenyl group to
4-hydroxybenzoic acid, and chlorophyll synthase, which attaches a
prenyl group to chlorophyllide.
[3871] [0006.0.9.9] The first committed step in tocopherol
biosynthesis is catalyzed by an aromatic prenyltransferase that
transfers a phytyl chain to HGA
[3872] Classification by head groups would arrange tocopherols,
tocotrienols and plastoquinones in one group, being quinones with
antioxidant properties and having homogentisic acid as a precursor.
Plastoquinones are important components of the quinone-pool in the
photosynthetic electron transport chains of plastids, also
interfering in the biosynthesis of provitamin A (beta-carotene;
Norris S R, (1995). Plant Cell 7, 2139-2149).
[3873] [0007.0.9.9] Vitamin E is predominantly delivered by the
ingestion of vegetable oils. It plays an important role as a
membrane-associated antioxidant scavenger. During past years
several additional functions of vitamin E as
anti-hypercholesterolemic and immunostimulatory agent in humans
have been proposed (Beharka (1997). Methods Enzymol. 282,
247-263).
[3874] These compounds with vitamin E activity are important
natural fat-soluble antioxidants. A vitamin E deficiency leads to
pathophysiological situations in humans and animals. Vitamin E
compounds therefore are of high economical value as additives in
the food and feed sectors, in pharmaceutical formulations and in
cosmetic applications.
[3875] [0008.0.9.9] In plastids of plants many isoprenoid pathways
are localized, which are interconnected by their substrates, end
products and by regulation. These are, e.g. monoterpene-,
diterpene-, giberillic acid-, abscisic acid-, chlorophyll-,
phylloquinone-, carotenoid-, tocopherol-, tocotrienol- and
plastoquinone-biosynthesis. In all these pathways
prenyltransferases are involved in the biosynthesis of these
compounds. With respect to the length of their side chains
diterpenes, chlorophylls, phylloquinones, tocopherols and
tocotrienols can be arranged into a C.sub.20-group of isoprenoids.
Another classification by degree of desaturation of the side chain,
would arrange e.g. chlorophylls, phylloquinones and tocopherols
into a phytyl-group and e.g. diterpenes, tocotrienols,
plastoquinones and carotenoids into a group of desaturated
isoprenoid compounds.
[3876] [0009.0.9.9] An economical method for producing vitamin E or
its precursor and food- and feedstuffs with increased vitamin E
content are therefore very important. Particularly economical
methods are biotechnological methods utilizing vitamin E-producing
organisms which are either natural or optimized by genetic
modification.
[3877] There is a constant need for providing novel enzyme
activities or direct or indirect regulators and thus alternative
methods with advantageous properties for producing vitamin E or its
precursor in organisms, e.g. in transgenic organisms.
[3878] Attempts are known to achieve an increase in the flow of
metabolites so as to increase the tocopherol and/or tocotrienol
content by overexpressing Phytyl/prenyltransferasegenes in
transgenic organisms; WO 00/63391, WO 00/68393, WO 01/62781 and WO
02/33060.
[3879] [0010.0.9.9] Therefore improving the quality of foodstuffs
and animal feeds is an important task of the food-and-feed
industry. This is necessary since, for example, Vitamin E, which
occur in plants and some microorganisms are limited with regard to
the supply of mammals. Especially advantageous for the quality of
foodstuffs and animal feeds is as balanced as possible a vitamin
profile in the diet since a great excess of some vitamins above a
specific concentration in the food has only some or little or no
positive effect. A further increase in quality is only possible via
addition of further vitamins, which are limiting.
[3880] [0011.0.9.9] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add one or a plurality of
vitamins in a balanced manner to suit the organism.
[3881] [0012.0.9.9] Accordingly, there is still a great demand for
new and more suitable genes which encode enzymes which participate
in the biosynthesis of vitamins, in particular vitamin E and make
it possible to produce them specifically on an industrial scale
without unwanted byproducts forming. In the selection of genes for
or regulators of biosynthesis two characteristics above all are
particularly important. On the one hand, there is as ever a need
for improved processes for obtaining the highest possible contents
of vitamins like vitamin E; on the other hand as less as possible
byproducts should be produced in the production process.
[3882] [0013.0.0.9] see [0013.0.0.0]
[3883] [0014.0.9.9] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is Vitamin E. Accordingly, in
the present invention, the term "the fine chemical" as used herein
relates to a "Vitamin E". Further, in an other embodiment the term
"the fine chemicals" as used herein also relates to compositions of
the fine chemicals comprising Vitamin E.
[3884] [0015.0.9.9] In one embodiment, the term "Vitamin E" or "the
fine chemical" or "the respective fine chemical" means at least one
chemical compound with vitamin E activity selected from the group
"alpha-tocopherol, beta-tocopherol, gamma-tocopherol,
delta-tocopherol, alpha-tocotrienol, beta-tocotrienol,
gamma-tocotrienol and delta-tocotrienol". In an preferred
embodiment, the term "the fine chemical" or the term "Vitamin E" or
the term "the respective fine chemical" means at least one chemical
compound with vitamin E activity selected from the group
"alpha-tocopherol, beta-tocopherol, and gamma-tocopherol".
[3885] An increased vitamin E content normally means an increased
total vitamin E content. However, an increased vitamin E content
also means, in particular, a modified content of the
above-described 8 compounds with vitamin E activity, without the
need for an inevitable increase in the total vitamin E content. In
a preferred embodiment, the term "the fine chemical" means vitamin
E in free form or its salts or its ester or bound. In one
embodiment, the term "the fine chemical" and the term "the
respective fine chemical" mean at least one chemical compound with
an activity of the above mentioned fine chemical.
[3886] [0016.0.9.9] Accordingly, the present invention relates to a
process for the production of Vitamin E comprising [3887] (a)
increasing or generating the activity of one or more [3888]
YFL019C, YPL268W, YFL013C, b1829, and/or b1827-protein(s); [3889]
b0161, b0970, b3160, b4063, b2699, b0112, and/or b1829-protein(s);
[3890] b0785, b3938, YFL053W, b1827, b0986, b1829 and/or
b0175-protein(s); and/or [3891] b0175, b0785, b0986, b3838, YFL053W
and/or b1829-protein(s); [3892] in a non-human organism in one or
more parts thereof; and [3893] (b) growing the organism under
conditions which permit the production of the fine chemical, thus,
vitamin E, in particular alpha-tocopherol, beta-tocopherol, and/or
gamma-tocopherol and/or the vitamin E precursor
2,3-Dimethyl-5-pythylquinol, respectively, in said organism.
[3894] In particular, the present invention relates to a process
for the production of 2,3-Dimethyl-5-phytylquinol,
alpha-Tocopherol, beta-Tocopherol, and/or gamma-Tocopherol
comprising [3895] (a) increasing or generating the activity of one
or more [3896] YFL019C, YPL268W, YFL013C, b1829, and/or
b1827-protein(s) for 2,3-Dimethyl-5-phytylquinol; [3897] b0161,
b0970, b3160, b4063, b2699, b0112, and/or b1829-protein(s) for
alpha-Tocopherol, [3898] b0785, b3938, YFL053W, b1827, b0986, b1829
and/or b0175-protein(s) for beta-Tocopherol; and/or [3899] b0175,
b0785, b0986, b3838, YFL053W and/or b1829-protein(s) for
gamma-Tocopherol; [3900] in a non-human organism in one or more
parts thereof; and [3901] (b) growing the organism under conditions
which permit the production of alpha-tocopherol, beta-tocopherol,
and/or gamma-tocopherol or the vitamin E precursor
2,3-Dimethyl-5-pythylquinol, respectively, in said organism.
[3902] Accordingly, the present invention relates to a process for
the production of vitamin E comprising [3903] (a) increasing or
generating the activity of one or more proteins having the activity
of a protein indicated in Table II, column 3, lines 89 to 102 or
lines 472 to 482 or having the sequence of a polypeptide encoded by
a nucleic acid molecule indicated in Table I, column 5 or 7, lines
89 to 102 or lines 472 to 482, in a non-human organism in one or
more parts thereof and [3904] (b) growing the organism under
conditions which permit the production of the fine chemical, thus,
vitamin E, in particular alpha-tocopherol, beta-tocopherol, and/or
gamma-tocopherol and/or the vitamin E precursor
2,3-Dimethyl-5-pythylquinol, resp.
[3905] Particularly, the present invention relates to a process for
the production of vitamin E comprising [3906] (a) increasing or
generating the activity of one or more proteins having the activity
of a protein indicated in [3907] Table II, column 3, lines 89 to 92
or 482 for 2,3-Dimethyl-5-phytylquinol; [3908] Table II, column 3,
lines 93 to 95 or 472 to 475 for alpha-Tocopherol; [3909] Table II,
column 3, lines 96 to 100 or 476 to 477 for beta-Tocopherol; and/or
[3910] Table II, column 3, lines 101 to 102 or 478 to 481 for
gamma-Tocopherol; e.g. having the sequence of a polypeptide encoded
by a nucleic acid molecule indicated in [3911] Table I, column 5 or
7, lines 89 to 92 or 482 for 2,3-Dimethyl-5-phytylquinol; [3912]
Table I, column 5 or 7, lines 93 to 95 or 472 to 475 for
alpha-Tocopherol; [3913] Table I, column 5 or 7, lines 96 to 100 or
476 to 477 for beta-Tocopherol; and/or [3914] Table I, column 5 or
7, lines 101 to 102 or 478 to 481 for gamma-Tocopherol; in a
non-human organism in one or more parts thereof and [3915] (b)
growing the organism under conditions which permit the production
of the fine chemical, in particular alpha-tocopherol,
beta-tocopherol, and/or gamma-tocopherol and/or the vitamin E
precursor 2,3-Dimethyl-5-pythylquinol.
[3916] [0016.1.9.9] Accordingly, the term "the fine chemical" means
in one embodiment "2,3-dimethyl-5-pythylquinol" in relation to all
sequences listed in Table I to IV, lines 89 to 92 or 482 or
homologs thereof and means in one embodiment "alpha-tocopherol" in
relation to all sequences listed in Tables I to IV, lines 93 to 95
or 472 to 475 or homologs thereof and means in one embodiment
"beta-tocopherol" in relation to all sequences listed in Table I,
lines 96 to 100 or 476 to 477, and means in one embodiment
"gamma-tocopherol" in relation to all sequences listed in Table I
to IV, lines 101 to 102 or 478 to 481.
[3917] In one embodiment, the method of the present invention
confers the increase of the content of more than one of the
respective fine chemicals, i.e. of alpha-, beta-, gamma-tocopherol
or 2,3-dimethyl-5-pythylquinol. Accordingly, in one embodiment the
term "the fine chemical" means "2,3-dimethyl-5-phytylquinol",
"beta-tocopherol" and "gamma-tocopherol" in relation to all
sequences listed in Table I to IV, lines 91, 94, 100 or 102, i.e.
the method of the present invention confers the increase of the
content of more than one of the respective fine chemicals, i.e. of
2,3-dimethyl-5-phytylquinol, alpha-, beta- and gamma-tocopherol.
Preferably the method of the invention confers the increase the
content of 2,3-dimethyl-5-phytylquinol, alpha-, beta- and
gamma-tocopherol. In one embodiment the term "the fine chemical"
means "2,3-dimethyl-5-phytylquinol" and "beta-tocopherol" in
relation to all sequences listed in Table I to IV, lines 92 or 97,
i.e. the method of the present invention confers the increase of
the content of 2,3-dimethyl-5-phytylquinol and beta-tocopherol. In
one embodiment the term "the fine chemical" means "beta-tocopherol"
and "gamma-tococpherol in relation to all sequences listed in Table
I to IV, lines 96, 98, 99, 101, 476, 477, 478, 479, 480, or
481.
[3918] Accordingly, the term "the fine chemical" can mean
"2,3-dimethyl-5-pythylquinol", "alpha-tocopherol",
"beta-tocopherol", and/or "gamma-tocopherol", owing to
circumstances and the context. In order to illustrate that the
meaning of the term "the fine chemical" means
"2,3-dimethyl-5-pythylquinol", "alpha-tocopherol",
"beta-tocopherol", and/or "gamma-tocopherol" the term "the
respective fine chemical" is also used.
[3919] [0017.0.0.9] to [0018.0.0.9]: see [0017.0.0.0] to
[0018.0.0.0]
[3920] [0019.0.9.9] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the respective fine chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table II, column 3, lines 89 to 102 and 472 to 482 or
encoded by nucleic acid molecule indicated in Table I, columns 5 or
7, lines 89 to 102 and 472 to 482.
[3921] [0020.0.9.9] Surprisingly it was found, that the transgenic
expression of at least one of the proteins of the Saccharomyces
cerevisiae protein(s) YFL019C, YPL268W and/or YFL013C, and/or the
Escherichia coli K12 protein(s) b1829, and/or b1827 in Arabidopsis
thaliana conferred an increase in the 2,3-dimethyl-5-phytylquinol,
which is a precursor in the biosynthesis of vitamin E, in
particular of gamma-tocopherol and thus alpha-tocopherol. Thus, an
increase in the level of this precursor of the tocopherol
biosynthesis can be advantageous for the production of vitamin E.
For example, in one embodiment the level of
2,3-dimethyl-5-phytylquinol is increased in combination with the
modulation of the expression of other genes of the biosynthesis of
vitamin E, in particular of genes encoding enzymes metabolising
2,3-dimethyl-5-phytylquinol to produce vitamin E or a precursor
thereof, such as the 2,3-dimethyl-5-phytylquinol-Cyclase and/or
gamma-tocopherol-methyltransferasellor with genes encoding the
polypeptides indicated in Table II, columns 5 or 7, lines 89 to 102
or 472 to 482.
[3922] Surprisingly, it was found that the transgenic expression of
the Escherichia coli K12 protein(s) b0161, b0970, b3160, b4063,
b2699, b1829, and/or b0112 in Arabidopsis thaliana conferred an
increase in the alpha-tocopherol content of the transformed
plants.
[3923] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein YFL053W and/or of the
Escherichia coli K12 protein(s) b0785, b3938, b1827, b1829, b0986,
and/or b0175 in Arabidopsis thaliana conferred an increase in the
beta-tocopherol content of the transformed plants.
[3924] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein YFL053W, and/or the
Escherichia coli K12 protein(s) b0175, b0785, b03938, b0986, b1829
in Arabidopsis thaliana conferred an increase in gamma-tocopherol
content of the transformed plants.
[3925] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein b1829 in Arabidopsis
thaliana conferred an increase in 2,3-dimethyl-5-phytylquinol,
alpha-tocopherol, beta-tocopherol, and/or gamma-tocopherol (or the
respective fine chemical) content of the transformed plants. Thus,
in one embodiment, said protein or its homologs are used for the
production of 2,3-Dimethyl-5-phytylquinol; in one embodiment, said
protein or its homologs are used for the production of
alpha-tocopherol; in one embodiment, said protein or its homologs
are used for the production of beta-tocopherol; in one embodiment,
said protein or its homologs are used for the production of
gamma-tocopherol; in one embodiment, said protein or its homologs
are used for the production of one or more fine chemical selected
from the group consisting of: 2,3-Dimethyl-5-phytylquinol,
alpha-tocopherol, beta-tocopherol, and gamma-tocopherol.
[3926] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein b1827 in Arabidopsis
thaliana conferred an increase in 2,3-Dimethyl-5-phytylquinol,
and/or beta-tocopherol (or the respective fine chemical) content of
the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of
2,3-Dimethyl-5-phytylquinol; in one embodiment, said protein or its
homologs are used for the production of beta-tocopherol; in one
embodiment, said protein or its homologs are used for the
production of 2,3-Dimethyl-5-phytylquinol and beta-tocopherol.
[3927] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein YFL053W and the E. coli
protein b0785, b3938, b0986 or b0175 in Arabidopsis thaliana
conferred an increase in beta-tocopherol and/or gamma-tocopherol
(or the respective fine chemical) content of the transformed
plants. Thus, in one embodiment, said protein or its homologs are
used for the production of beta-tocopherol; in one embodiment, said
protein or its homologs are used for the production of
gamma-tocopherol, in one embodiment, said protein or its homologs
are used for the production of beta-tocopherol and
gamma-tocopherol.
[3928] [0021.0.0.9] see [0021.0.0.0]
[3929] [0022.0.9.9] The sequence of b1829 from Escherichia coli K12
has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as a heat shock
protein with protease activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein of
the stress response, the pheromone response, the mating type
determination, protein modification or having a proteolytic
degradation activity or being a sex specific protein, in particular
the use of a protease, in particular of a heat shock protein with
protease activity, preferably of the superfamily of the heat shock
protein htpX, e.g. as shown herein, for the production of the
respective fine chemical, meaning of vitamin E, e.g.
alpha-tocopherol, beta-tocopherol and/or gamma-tocopherol, e.g. via
increasing of the vitamin E biosynthesis precursor
2,3-dimethyl-5-phytylquinol, preferably in free or bound form, in
an organism or a part thereof, as mentioned. In one further
embodiment the b1829 protein expression is increased together with
the increase of another gene of the vitamin E biosynthesis pathway,
preferably with a gene encoding a protein being involved in the
production of vitamin E via transformation of the intermediate
2,3-dimethyl-5-phytylquiol. In one embodiment, in the process of
the present invention the activity of a protease, in particular of
a heat shock protein with protease activity, preferably of the
superfamily of the heat shock protein htpX, is increased or
generated, e.g. from E. coli or a homolog thereof.
[3930] The sequence of b1827 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative transcriptional
repressor with DNA-binding Winged helix domain (IcIR family),
preferably of the acetate operon repressor superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein of the acetate operon
repressor superfamily, preferably such protein is having a
transcriptional control activity, in particular an activity of a
putative transcriptional repressor with DNA-binding Winged helix
domain (IcIR family), e.g. as shown herein, for the production of
the respective fine chemical, meaning of vitamin E, e.g.
beta-tocopherol, e.g. via increasing the biosynthesis of the
vitamin E precursor 2,3-dimethyl-5-phytylquinol, preferably in free
or bound form, in an organism or a part thereof, as mentioned. In
one further embodiment the b1827 protein expression is increased
together with the increase of another gene of the vitamin E
biosynthesis pathway, preferably with a gene encoding a protein
being involved in the production of vitamin E from the intermediate
2,3-dimethyl-5-phytylquiol. In one embodiment, in the process of
the present invention said activity, e.g. the activity of a
putative transcriptional repressor with DNA-binding Winged helix
domain (IcIR family), preferably of the acetate operon repressor
superfamily is increased or generated from E. coli or a homolog
thereof.
[3931] The sequence of b0785 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a Molybdenum cofactor
biosynthesis protein E. Accordingly, in one embodiment, the process
of the present invention comprises the use of a protein involved in
the metabolism of vitamins, cofactors, and prosthetic groups; other
metal binding; protein binding; molybdopterin binding, preferably
of the molybdopterin biosynthesis protein E chain superfamily,
preferably of a Molybdenum cofactor biosynthesis protein E, e.g.
from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning vitamin E, in
particular beta-tocopherol and/or gamma-tocopherol, preferably in
free or bound form, in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention said
activity, e.g. the activity of a Molybdenum cofactor biosynthesis
protein E is increased or generated, e.g. from E. coli or a homolog
thereof.
[3932] The sequence of b3938 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a transcriptional repressor
for methionine biosynthesis. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein of
the metJ protein superfamily, preferably of a transcriptional
repressor for methionine biosynthesis, e.g from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning vitamin E, in particular beta-tocopherol
and/or gamma-tocopherol, preferably in free or bound form, in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention said activity, e.g. the activity
of a transcriptional repressor for methionine biosynthesis is
increased or generated, e.g. from E. coli or a homolog thereof.
[3933] The sequence of b0175 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a CDP-diglyceride synthase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein involved in the
nucleotide-metabolism, phospholipid biosynthesis, lipid, fatty-acid
or isoprenoids metabolism, cellular communication, signal
transduction, or cellular sensing and response, preferably of the
phophatidate cytidylyltransferase superfamily, preferably of a
CDP-diglyceride synthase, e.g from E. coli or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning vitamin E, in particular beta-tocopherol and/or
gamma-tocopherol, preferably in free or bound, form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention said activity, e.g. the activity of a
phosphatidate cytidylytransf erase, in particular of a
CDP-diglyceride synthase, is increased or generated, e.g. from E.
coli or a homolog thereof.
[3934] The sequence of b2699 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a DNA strand exchange and
recombination protein with protease and nuclease activity of the
superfamily of the recombination protein recA. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein with a DNA recombination and DNA repair activity, a
pheromone response activity, a mating-type determination activity,
a sex-specific protein activity, a nucleotide binding activity
and/or a protease and nuclease activity, in particular a DNA strand
exchange and recombination protein with protease and nuclease
activity, in particular of the superfamily of the recombination
protein recA from E. coli or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, meaning of vitamin
E, in particular for increasing the amount of alpha-tocopherol,
preferably in free or bound form, in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention said activity, e.g. the activity of a protease and
nuclease activity, in particular a DNA strand exchange and
recombination protein with protease and nuclease activity, in
particular of the superfamily of the recombination protein recA is
increased or generated, e.g. from E. coli or a homolog thereof.
[3935] The sequence of b3160 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative monooxygenase with
luciferase-like ATPase activity. Accordingly, in one embodiment,
the process of the present invention comprises the use of a
putative monooxygenase with luciferase-like ATPase activity from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of alpha-tocopherol, in particular for
increasing the amount of alpha-tocopherol, preferably
alpha-tocopherol in free or bound form, in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a putative monooxygenase with
luciferase-like ATPase activity is increased or generated, e.g.
from E. coli or a homolog thereof.
[3936] The sequence of b0161 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a periplasmic serine protease
and/or heat shock protein. Accordingly, in one embodiment, the
process of the present invention comprises the use of
extracellular/secretion proteins, or proteins involved in cell
growth/morphogenesis, proteolytic degradation, protein binding,
intracellular signalling, preferably being a protein of the
superfamily of Helicobacter serine proteinase, preferably a
periplasmic serine protease (heat shock protein) from E. coli or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of alpha-tocopherol, in particular for increasing
the amount of alpha-tocopherol, preferably alpha-tocopherol in free
or bound form, in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a periplasmic serine protease (heat shock protein) is
increased or generated, e.g. from E. coli or a homolog thereof.
[3937] The sequence of b0970 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as probable glutamate receptor.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein being involved in
apoptosis (type I programmed cell death), transmembrane signal
transduction, channel/pore class transporters, unspecified signal
transduction and/or protein binding, preferably from the
Escherichia coli ybhL protein superfamily, in particular of a
probable glutamate receptor from E. coli or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of vitamin E, in particular for increasing the amount of
alpha-tocopherol, preferably in free or bound form, in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention said activity, e.g. an probable glutamate
receptor, in particular of the superfamily of Escherichia coli ybhL
protein, is increased or generated, e.g. from E. coli or a homolog
thereof.
[3938] The sequence of b4063 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as transcriptional activator for
superoxide response. Accordingly, in one embodiment, the process of
the present invention comprises the use of a protein being involved
in transcriptional control, DNA binding, inorganic chemical agent
resistance (e.g. heavy metals), regulation of amino acid metabolism
and/or regulation of nitrogen and sulphur utilization, in
particular of a transcriptional activator for superoxide response
from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of vitamin E,
in particular for increasing the amount of alpha-tocopherol,
preferably in free or bound form, in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention said activity, e.g. a transcriptional activator for
superoxide response, in particular of the superfamily of
Escherichia coli ybhL protein, is increased or generated, e.g. from
E. coli or a homolog thereof.
[3939] The sequence of b0112 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as aromatic amino acid transport
protein (APC family). Accordingly, in one embodiment, the process
of the present invention comprises the use of a protein with a
amino acid transporter activity, a cellular transport activity,
preferably from the arginine permease superfamily, in particular of
a aromatic amino acid transport protein (APC family) from E. coli
or its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of vitamin E, in particular for
increasing the amount of alpha-tocopherol, preferably in free or
bound form, in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention said activity,
e.g. the activity of a protein of the APC family, in particular an
aromatic amino acid transport protein, in particular of the
superfamily of arginine permease, is increased or generated, e.g.
from E. coli or a homolog thereof.
[3940] The sequence of b0986 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a lipoprotein. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a lipoprotein, preferably from the superfamily of the
hypothetical protein b1706, from E. coli or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of vitamin E, in particular for increasing the amount of
beta-tocopherol and/or gamma-tocopherol, preferably in free or
bound form, in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention said activity,
e.g. the activity of a lipoprotein, in particular of the
superfamily the hypothetical protein b1706, is increased or
generated, e.g. from E. coli or a homolog thereof.
[3941] The sequence of YFL019C from Saccharomyces cerevisiae has
been published in Murakami Y. et al., Nat. Genet. 10:261-268(1995)
and its activity has not been characterized yet. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a protein with a YFL019C activity or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of vitamin E, e.g. via increasing the biosynthesis of the
vitamin E precursor 2,3-dimethyl-5-phytylquinol, preferably in free
or bound form, in an organism or a part thereof, as mentioned. In
one further embodiment the YFL019C protein expression is increased
together with the increase of another gene of the vitamin E
biosynthesis pathway, preferably with a gene encoding a protein
being involved in the production of vitamin E from the intermediate
2,3-Dimethyl-5-phytylquiol. In one embodiment, in the process of
the present invention said YFL019C activity is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[3942] The sequence of YFL013C from Saccharomyces cerevisiae has
been published in Murakami, Y et al., Nat. Genet. 10 (3), 261-268
(1995), and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is being defined as subunit of the INO80 chromatin
remodelling complex. Accordingly, in one embodiment, the process of
the present invention comprises the use of a protein having the
activity of a subunit of the INO80 chromation remodelling complex,
in particular of a subunit of the INO80 chromation remodelling
complex of the superfamily of the probable membrane protein
YFL013c, e.g. from E. coli or its homolog, for the production of
the respective fine chemical, meaning vitamin E, e.g. via
increasing the biosynthesis of the vitamin E precursor
2,3-dimethyl-5-phytylquinol, preferably in free or bound form, in
an organism or a part thereof, as mentioned. In one further
embodiment the YFL013C protein expression is increased together
with the increase of another gene of the vitamin E biosynthesis
pathway, preferably with a gene encoding a protein being involved
in the production of vitamin E from the intermediate
2,3-Dimethyl-5-phytylquiol. In one embodiment, in the process of
the present invention said activity, e.g. the activity of a subunit
of the INO80 chromation remodelling complex is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[3943] The sequence of YPL268W from Saccharomyces cerevisiae has
been published in Bussey, H. et al., Nature 387 (6632 Suppl),
103-105 (1997), and Goffeau et al., Science 274 (5287), 546-547,
1996, and its activity is being defined as phophoinositide
phospholipase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein involved in the
breakdown of lipids, fatty acids and isoprenoids, cell growth and
morphogenesis, mitotic cell cycle, cell cycle control,
intracellular signal transduction activities, or DNA processing, in
particular of the superfamiliy of the yeast
1-phosphatidylinostiol-4,5-bisphophate phosphodiesterase,
preferably having a phophoinositide phospholipase activity or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of vitamin E, e.g. via increasing the
biosynthesis of the vitamin E precursor
2,3-dimethyl-5-phytylquinol, preferably in free or bound form, in
an organism or a part thereof, as mentioned. In one further
embodiment the YPL268W protein expression is increased together
with the increase of another gene of the vitamin E biosynthesis
pathway, preferably with a gene encoding a protein being involved
in the production of vitamin E from the intermediate
2,3-Dimethyl-5-phytylquiol. In one embodiment, in the process of
the present invention said activity, e.g. of a protein of the yeast
1-phosphatidylinostiol-4,5-bisphophate phosphodiesterase
superfamily or preferably having a phophoinositide phospholipase
activity is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof.
[3944] The sequence of YFL053W from Saccharomyces cerevisiae has
been published in Murakami, Y et al., Nat. Genet. 10 (3), 261-268
(1995), and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is being defined as dihydroxyacetone kinase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein involved in the
utilization of c-compound and carbohydrate, stress response,
modification by phosphorylation or dephosphorylation, in particular
of the superfamiliy glyerone kinase DAK1, preferably having a
dihydroxyacetone kinase activity, or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of vitamin E, e.g. gamma- and/or beta-tocopherol, preferably in
free or bound form, in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention said
activity, e.g. of a protein of the superfamiliy glyerone kinase
DAK1, preferably having a dihydroxyacetone kinase activity, is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[3945] [0023.0.9.9] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
Further, in the present invention, the term "homologue" relates to
the sequence of an organism having the highest sequence homology to
the herein mentioned or listed sequences of all expressed sequences
of said organism. However, the person skilled in the art knows,
that, preferably, the homologue has said
the--fine-chemical-increasing activity and, if known, the same
biological function or activity in the organism as at least one of
the protein(s) indicated in Table II, Column 3, lines 89 to 102
and/or 472 to 482, e.g. having the sequence of a polypeptide
encoded by a nucleic acid molecule comprising the sequence
indicated in Table I, Column 5 or 7, lines 89 to 102 and/or 472 to
482.
[3946] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 89 or 90 or 482,
resp. is a homolog having the same or a similar activity, resp. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of vitamin E via its precursor
2,3-dimethyl-5-phytylquinol. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
line 89 or 90 or 482, resp. In one embodiment, the homolog of one
of the polypeptides indicated in Table II, column 3, line 89 or 90
or 482 resp., is derived from an eukaryotic. In one embodiment, the
homolog is derived from Fungi. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 89 or 90 or 482,
resp., is derived from Ascomyceta. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, line 89 or 90 or
482, resp., is derived from Saccharomycotina. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, line
89 or 90 or 482, resp., is derived from Saccharomycetes. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 89 or 90 or 482, resp., is a homolog being derived
from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 89 or 90 or 482,
resp., is a homolog having the same or a similar activity being
derived from Saccharomycetaceae. In one embodiment, the homolog of
a polypeptide indicated in Table II, column 3, line 89 or 90 or
482, resp., is a homolog having the same or a similar activity
being derived from Saccharomycetes.
[3947] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 96 is a homolog
having the same or a similar activity. In particular an increase of
activity confers an increase in the content of the respective fine
chemical in the organisms, preferably of vitamin E, preferably of
beta-tocopherol. In one embodiment, the homolog is a homolog with a
sequence as indicated in Table I or II, column 7, line 96. In one
embodiment, the homolog of one of the polypeptides indicated in
Table II, column 3, line 96., is derived from an eukaryotic. In one
embodiment, the homolog is derived from Fungi. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, line
96, is derived from Ascomyceta. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 96, is derived
from Saccharomycotina. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 96, is derived
from Saccharomycetes. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 96, is a homolog
being derived from Saccharomycetales. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 96,
is a homolog having the same or a similar activity being derived
from Saccharomycetaceae. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 96, is a homolog
having the same or a similar activity being derived from
Saccharomycetes.
[3948] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 101. is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms, preferably of vitamin E,
preferably of gamma-tocopherol. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
line 101. In one embodiment, the homolog of one of the polypeptides
indicated in Table II, column 3, line 101., is derived from an
eukaryotic. In one embodiment, the homolog is derived from Fungi.
In one embodiment, the homolog of a polypeptide indicated in Table
II, column 3, line 101, is derived from Ascomyceta. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 101, is derived from Saccharomycotina. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 101, is derived from Saccharomycetes. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 101, is a homolog being derived from
Saccharomycetales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 101, is a homolog having the
same or a similar activity being derived from Saccharomycetaceae.
In one embodiment, the homolog of a polypeptide indicated in Table
II, column 3, line 101, is a homolog having the same or a similar
activity being derived from Saccharomycetes.
[3949] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 91 or 92 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms preferably of vitamin E
via its precursor 2,3-dimethyl-5-phytylquinol. In one embodiment,
the homolog is a homolog with a sequence as indicated in Table I or
II, column 7, lines 91 or 92, resp. In one embodiment, the homolog
of one of the polypeptides indicated in Table II, column 3, 91 or
92 is derived from an bacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 91 or 92 is
derived from Proteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 91 or 92 is a
homolog having the same or a similar activity being derived from
Gammaproteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 91 and 92 is
derived from Enterobacteriales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 91 or 92 is a
homolog being derived from Enterobacteriaceae. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
91 or 92 is a homolog having the same or a similar activity and
being derived from Escherichia.
[3950] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 93, 94, 95,
472, 473, 474, or 475 is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of the respective fine chemical in the organisms,
preferably of alpha-tocopherol. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
lines 93, 94, 95, 472, 473, 474, or 475, resp. In one embodiment,
the homolog of one of the polypeptides indicated in Table II,
column 3, lines 93, 94, 95, 472, 473, 474, or 475 is derived from
an bacteria. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 93, 94, 95, 472, 473, 474,
or 475 is derived from Proteobacteria. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 93,
94, 95, 472, 473, 474, or 475 is a homolog having the same or a
similar activity being derived from Gammaproteobacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 93, 94, 95, 472, 473, 474, or 475 is derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 93, 94, 95, 472, 473, 474,
or 475 is a homolog being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 93, 94, 95, 472, 473, 474, or 475 is a homolog
having the same or a similar activity and being derived from
Escherichia.
[3951] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 97 to 100 or
476 or 477 is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms preferably
beta-tocopherol. In one embodiment, the homolog is a homolog with a
sequence as indicated in Table I or II, column 7, lines 97 to 100
or 476 or 477, resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table II, column 3, lines 97 to 100 or
476 or 477 is derived from an bacteria. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 97
to 100 or 476 or 477 is derived from Proteobacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 97 to 100 or 476 or 477 is a homolog having the
same or a similar activity being derived from Gammaproteobacteria.
In one embodiment, the homolog of a polypeptide indicated in Table
II, column 3, lines 97 to 100 or 476 or 477 is derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 97 to 100 or 476 or 477 is a
homolog being derived from Enterobacteriaceae. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
97 to 100 or 476 or 477 is a homolog having the same or a similar
activity, being derived from Escherichia.
[3952] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 102 or lines 478
to 481 is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms preferably
gamma-tocopherol. In one embodiment, the homolog is a homolog with
a sequence as indicated in Table I or II, column 7, line 102 or
lines 478 to 481, resp. In one embodiment, the homolog of one of
the polypeptides indicated in Table II, column 3, line 102 or lines
478 to 481 is derived from an bacteria. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 102
or lines 478 to 481 is derived from Proteobacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 102 or lines 478 to 481 is a homolog having the same
or a similar activity being derived from Gammaproteobacteria. In
one embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 102 or lines 478 to 481 is derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 102 or lines 478 to 481 is a
homolog being derived from Enterobacteriaceae. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, line
102 or lines 478 to 481 is a homolog having the same or a similar
activity, being derived from Escherichia.
[3953] [0023.1.9.9] Homologs of the polypeptide indicated in Table
II, column 3, lines 89 to 102 or 472 to 482 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 89 to 102 or 472 to 482, resp., or may be the polypeptides
indicated in Table II, column 7, lines 89 to 102 or 472 to 482,
resp. Homologs of the polypeptides indicated in Table II, column 3,
lines 89 to 102 or 472 to 482 may be the polypeptides encoded by
the nucleic acid molecules indicated in Table I, column 7, lines 89
to 102 or 472 to 482, resp., or may be the polypeptides indicated
in Table II, column 7, lines 89 to 102 or 472 to 482.
[3954] Homologs of the polypeptides indicated in Table II, column
3, lines 89 to 92 or 482 may be the polypeptides encoded by the
nucleic acid molecules indicated in Table I, column 7, lines 89 to
92 or 482, respectively or may be the polypeptides indicated in
Table II, column 7, lines 89 to 92 or 482, having a
2,3-Dimethyl-5-phytylquinol content- and/or amount-increasing
activity. Homologs of the polypeptides indicated in Table II,
column 3, lines 89 to 92 or 482 may be the polypeptides encoded by
the nucleic acid molecules indicated in Table I, column 7, lines 89
to 92 or 482 or may be the polypeptides indicated in Table II,
column 7, lines 89 to 92 or 482 having a
2,3-Dimethyl-5-phytylquinol content- and/or amount-increasing
activity.
[3955] Homologs of the polypeptides indicated in Table II, column
3, lines 93 to 95 or 472 to 475 may be the polypeptides encoded by
the nucleic acid molecules indicated in Table I, column 7, lines 93
to 95 or 472 to 475, respectively or may be the polypeptides
indicated in Table II, column 7, lines 93 to 95 or 472 to 475,
having a alpha-tocopherol content- and/or amount-increasing
activity. Homologs of the polypeptides indicated in Table II,
column 3, lines 93 to 95 or 472 to 475 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 93 to 95 or 472 to 475 or may be the polypeptides
indicated in Table II, column 7, lines 93 to 95 or 472 to 475
having a alpha-tocopherol content- and/or amount-increasing
activity.
[3956] Homologs of the polypeptides indicated in Table II, column
3, lines 96 to 100 or 476 to 477 may be the polypeptides encoded by
the nucleic acid molecules indicated in Table I, column 7, 96 to
100 or 476 to 477, respectively or may be the polypeptides
indicated in Table II, column 7, 96 to 100 or 476 to 477, having a
beta-tocopherol content- and/or amount-increasing activity.
Homologs of the polypeptides indicated in Table II, column 3, 96 to
100 or 476 to 477 may be the polypeptides encoded by the nucleic
acid molecules indicated in Table I, column 7, 96 to 100 or 476 to
477 or may be the polypeptides indicated in Table II, column 7,
lines 96 to 100 or 476 to 477 having a beta-tocopherol content-
and/or amount-increasing activity.
[3957] Homologs of the polypeptides indicated in Table II, column
3, lines 101 to 102 or 478 to 481 may be the polypeptides encoded
by the nucleic acid molecules indicated in Table I, column 7, 101
to 102 or 478 to 481, respectively or may be the polypeptides
indicated in Table II, column 7, 101 to 102 or 478 to 481, having a
gamma-tocopherol content- and/or amount-increasing activity.
Homologs of the polypeptides indicated in
[3958] Table II, column 3, 101 to 102 or 478 to 481 may be the
polypeptides encoded by the nucleic acid molecules indicated in
Table I, column 7, 101 to 102 or 478 to 481 or may be the
polypeptides indicated in Table II, column 7, lines 101 to 102 or
478 to 481 having a gamma-tocopherol content- and/or
amount-increasing activity.
[3959] [0024.0.0.9] see [0024.0.0.0]
[3960] [0025.0.9.9] In accordance with the invention, a protein or
polypeptide has the "activity of an protein of the invention", e.g.
the activity of a protein indicated in Table II, column 3, lines 89
to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp., if
its de novo activity, or its increased expression directly or
indirectly leads to an increased vitamin E or its precursor
2,3-dimethyl-5-phytylquinol level, in particular to a increased
alpha-, beta-, and/or gamma-tocopherol, resp., in the organism or a
part thereof, preferably in a cell of said organism. In a preferred
embodiment, the protein or polypeptide has the above-mentioned
additional activities of a protein indicated in Table II, column 3,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481.
Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of any one of the proteins indicated in Table II, column
3, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp. or which has at least 10% of the original enzymatic activity,
preferably 20%, particularly preferably 30%, most particularly
preferably 40% in comparison to any one of the proteins indicated
in Table II, column 3, lines 89, 90, 96, 101, or 482 of
Saccharomyces cerevisiae and/or any one of the proteins indicated
in Table II, column 3, lines 91 to 95, 97 to 100, or 102 or 472 to
481 of E. coli K12.
[3961] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[3962] [0025.1.0.9] see [0025.1.0.0]
[3963] [0025.2.0.9] see [0025.2.0.0]
[3964] [0025.3.9.9] In one embodiment, the polypeptide of the
invention or used in the method of the invention confers said
activity, e.g. the increase of the respective fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[3965] [0026.0.0.9] to [0033.0.0.9]: see [0026.0.0.0] to
[0033.0.0.0]
[3966] [0034.0.9.9] Preferably, the reference, control or wild type
differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. as result of
an increase in the level of the nucleic acid molecule of the
present invention or an increase of the specific activity of the
polypeptide of the invention or the polypeptide used in the method
of the invention. E.g., it differs by or in the expression level or
activity of an protein having the activity of a protein as
indicated in Table II, column 3, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 and/or 102 and/or lines 478 to 481, resp., or being
encoded by a nucleic acid molecule indicated in Table I, column 5,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 and/or 102 and/or
lines 478 to 481, resp., or its homologs, e.g. as indicated in
Table I, column 7, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp., its biochemical or genetical causes and
therefore shows the increased amount of the respective fine
chemical.
[3967] [0035.0.0.9] to [0044.0.0.9]: see [0035.0.0.0] to
[0044.0.0.0]
[3968] [0045.0.9.9] In case the activity of the Escherichia coli
K12 protein b0161 or its homologs, as indicated in Table I, columns
5 or 7, line 472, e.g. protein with an activity as defined in
[0022.0.9.9], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of alpha-tocopherol
between 50% and 138% or more is conferred.
[3969] In case the activity of the Escherichia coli K12 protein
b0970 or its homologs, as indicated in Table I, columns 5 or 7,
line 473, e.g. protein with an activity as defined in [0022.0.9.9],
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of alpha-tocopherol between 47% and 54%
or more is conferred.
[3970] In case the activity of the Escherichia coli K12 protein
b4063 or its homologs, as indicated in Table I, columns 5 or 7,
line 475, e.g. protein with an activity as defined in [0022.0.9.9],
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of alpha-tocopherol between 16% and 47%
or more is conferred.
[3971] In case the activity of the Escherichia coli K12 protein
b0785 or its homologs, as indicated in Table I, columns 5 or 7,
line 476 or 479, e.g. protein with an activity as defined in
[0022.0.9.9], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of beta- and/or
gamma-tocopherol between 34% and 40% or more is conferred.
[3972] In case the activity of the Escherichia coli K12 protein
b3938 or its homologs, as indicated in Table I, columns 5 or 7,
line 477 or 481, e.g. protein with an activity as defined in
[0022.0.9.9], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of beta- and/or
gamma-tocopherol between 50% and 120% or more is conferred.
[3973] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3160, as indicated in Table I, columns 5 or 7,
line 474, or a protein with the activity defined as putative
monooxygenase with luciferase-like ATPase activity or its homologs,
e.g. transcriptional regulator, is increased, preferably, in one
embodiment an increase of the fine chemical alpha-tocopherol
between 47% and 91% is conferred.
[3974] In one embodiment the activity of the Escherichia coli K12
protein b1829 or its homologs, e.g. having a heat shock protein or
a protease activity, e.g. as indicated in Table I, columns 5 or 7,
line 91, is increased conferring an increase of the respective fine
chemical, preferably of the vitamin E precursor
2,3-dimethyl-5-phytylquinol, between 48% and 283% or more.
[3975] In one embodiment, the activity of the Escherichia coli K12
protein b1829 or its homologs, e.g. with a heat shock protein with
protease activity, e.g. as indicated in Table I, columns 5 or 7,
line 94, is increased conferring an increase of the respective fine
chemical, preferably of vitamin E, in particular of
alpha-tocopherol, between 39% and 90% or more.
[3976] In one embodiment, the activity of the Escherichia coli K12
protein b1829 or its homologs, e.g. with a heat shock protein with
protease activity, e.g. as indicated in Table I, columns 5 or 7,
line 100 or 102, is increased conferring an increase of the
respective fine chemical, preferably of the vitamin E in particular
of beta- and/or gamma-tocopherol between 40% and 325% or more.
[3977] In one embodiment, the activity of the Escherichia coli K12
protein b1827 or its homologs, e.g. with a putative transcriptional
repressor with DNA-binding Winged helix domain (IcIR family)
activity, e.g. as indicated in Table I, columns 5 or 7, line 92, is
increased conferring an increase of the respective fine chemical,
preferably of the vitamin E precursor 2,3-dimethyl-5-phytylquinol,
between 76% and 91% or more.
[3978] In one embodiment, the activity of the Escherichia coli K12
protein b1827 or its homologs, e.g. with a putative transcriptional
repressor with DNA-binding Winged helix domain (IcIR family)
activity, e.g. as indicated in Table I, columns 5 or 7, line 97, is
increased conferring an increase of the respective fine chemical,
preferably of the vitamin E, in particular beta-tocopherol, between
34% and 116% or more.
[3979] In one embodiment, the activity of the Escherichia coli K12
protein b2699 or its homologs, e.g. a protein with a DNA strand
exchange and recombination protein with protease and
nuclease-activity, e.g. as indicated in Table I, columns 5 or 7,
line 93, is increased conferring an increase of the respective fine
chemical, preferably of the vitamin E, in particular of
alpha-tocopherol, between 51% and 196% or more.
[3980] In one embodiment, the activity of the Escherichia coli K12
protein b0112 or its homologs, e.g. a protein with an aromatic
amino acid transport protein (APC family)-activity, e.g. as
indicated in Table I, columns 5 or 7, line 95, is increased
conferring an increase of the respective fine chemical, preferably
of the vitamin E, in particular of alpha-tocopherol, between 43%
and 63% or more.
[3981] In one embodiment, the activity of the Escherichia coli K12
protein b0175 or its homologs, e.g. a CDP-diglyceride
synthase-activity, e.g. as indicated in Table I, columns 5 or 7,
line 99 or 478, is increased conferring an increase of the
respective fine chemical, preferably of the vitamin E, in
particular of beta- and/or gamma-tocopherol, between 34% and 40% or
more.
[3982] In one embodiment, the activity of the Escherichia coli K12
protein b0986 or its homologs, e.g. a lipoprotein, e.g. as
indicated in Table I, columns 5 or 7, line 98 and 480, is increased
conferring an increase of the respective fine chemical, preferably
of the vitamin E, in particular of beta and/or gamma-tocopherol,
between 35% and 61% or more.
[3983] In case the activity of the Saccharomyces cerevisiae protein
YFL019C or its homologs, as indicated in Table I, columns 5 or 7,
line 482, e.g. protein with an activity as defined in [0022.0.9.9],
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of 2,3-Dimethyl-5-pythylquinol between
97% and 440% or more is conferred.
[3984] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YFL013C or its homologs, e.g. an activity of a
subunit of the INO80 chroamtion remodeling complex, in particular,
of the superfamiliy of the membrane protein YFL013c, e.g. as
indicated in Table I, columns 5 or 7, 90, is increased conferring
an increase of the respective fine chemical, preferably of the
vitamin E precursor 2,3-dimethyl-5-phytyhlquinol between 97% and
440% or more.
[3985] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YPL268W or its homologs, e.g. an activity of a
phophoinositide phopholipase, in particular, of the superfamiliy of
the yeast 1-phosphatidylinositol-4,5-bisphosphate phophodiesterase,
e.g. as indicated in Table I, columns 5 or 7, 89, is increased
conferring an increase of the respective fine chemical, preferably
of the vitamin E precursor 2,3-dimethyl-5-phytyhlquinol between 44%
and 51% or more.
[3986] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YFL053W or its homologs, e.g. an activity of a
dihydroxaacetone kinase, in particular, of the superfamiliy of
theglyerone kinase DAK1, e.g. as indicated in Table I, columns 5 or
7, 96 and 101, is increased conferring an increase of the
respective fine chemical, preferably of vitamin E, particularly of
gamma and/or beta-tocopherol, between 45% and 123% or more.
[3987] [0046.0.9.9] In one embodiment the activity of a Escherichia
coli K12 protein b0161 as disclosed in [0016.0.9.9] or its
homologs, as indicated in Table I, columns 5 or 7, lines 472, e.g.
a protein with an activity as defined in [0022.0.9.9], is increased
conferring, preferably, an increase of the respective fine
chemical, preferably of alpha-tocopherol and of further vitamin E
activity-having compounds or their precursors.
[3988] In one embodiment the activity of a Escherichia coli K12
protein b0970 as disclosed in [0016.0.9.9] or its homologs, as
indicated in Table I, columns 5 or 7, lines 473, e.g. a protein
with an activity as defined in [0022.0.9.9], is increased
conferring preferably, an increase of the respective fine chemical,
preferably of alpha-tocopherol and of further vitamin E
activity-having compounds or their precursors.
[3989] In one embodiment the activity of a Escherichia coli K12
protein b3160 as disclosed in [0016.0.9.9] or its homologs, as
indicated in Table I, columns 5 or 7, lines 474, e.g. a protein
with an activity as defined in [0022.0.9.9], is increased
conferring preferably, an increase of the respective fine chemical,
preferably of alpha-tocopherol and of further vitamin E
activity-having compounds or their precursors.
[3990] In one embodiment the activity of a Escherichia coli K12
protein b4063 as disclosed in [0016.0.9.9] or its homologs, as
indicated in Table I, columns 5 or 7, lines 475, e.g. a protein
with an activity as defined in [0022.0.9.9], is increased
conferring, preferably, an increase of the respective fine
chemical, preferably of alpha-tocopherol and of further vitamin E
activity-having compounds or their precursors.
[3991] In one embodiment the activity of a Escherichia coli K12
protein b0785 as disclosed in [0016.0.9.9] or its homologs, as
indicated in Table I, columns 5 or 7, lines 476 and 479, e.g. a
protein with an activity as defined in [0022.0.9.9] is increased
conferring, preferably, an increase of the respective fine
chemical, preferably of beta-tocopherol and/or gamma-tocopherol and
of further vitamin E activity-having compounds or their
precursors.
[3992] In one embodiment, the activity of a Escherichia coli K12
protein b3938 as disclosed in [0016.0.9.9] or its homologs, as
indicated in Table I, columns 5 or 7, lines 477 and 481, e.g. a
protein with an activity as defined in [0022.0.9.9] is increased
conferring, preferably, an increase of the respective fine
chemical, preferably of beta-tocopherol and/or gamma-tocopherol and
of further vitamin E activity-having compounds or their
precursors.
[3993] In one embodiment, the activity of the Escherichia coli K12
protein b1829 or its homologs, e.g. having a heat shock protein
with protease activity, e.g. as indicated in Table I, columns 5 or
7, line 91, 94, or 100, is increased conferring an increase of the
respective fine chemical, preferably of alpha-, beta- and/or
gamma-tocopherol and/or 2,3-Dimethyl-5-phyhtylquinol, and of
further vitamin E activity-having compounds or their
precursors.
[3994] In one embodiment, the activity of the Escherichia coli K12
protein b1827 or its homologs, e.g. with a putative transcriptional
repressor with DNA-binding Winged helix domain (IcIR family)
activity, e.g. as indicated in Table I, columns 5 or 7, line 92 or
97 is increased, conferring preferably, an increase of the
respective fine chemical, preferably of beta-tocopherol and/or
2,3-Dimethyl-5-phyhtylquinol, and of further vitamin E
activity-having compounds or their precursors.
[3995] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2699 or its homologs, e.g. a protein with a DNA
strand exchange and recombination protein with protease and
nuclease-activity, e.g. as indicated in Table I, columns 5 or 7,
line 93, is increased conferring, preferably, an increase of the
respective fine chemical, preferably of alpha-tocopherol and of
further vitamin E activity-having compounds or their
precursors.
[3996] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0112 or its homologs, e.g. a protein with an
aromatic amino acid transport protein (APC family)-activity, e.g.
as indicated in Table I, columns 5 or 7, line 95, is increased
conferring, preferably, an increase of the respective fine
chemical, preferably of alpha-tocopherol, and of further vitamin E
activity-having compounds or their precursors.
[3997] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0175 or its homologs, e.g. a CDP-diglyceride
synthase-activity, e.g. as indicated in Table I, columns 5 or 7,
line 99 or 478, is increased conferring, preferably, an increase of
the respective fine chemical, preferably of beta-tocopherol and/or
gamma-tocopherol, and of further vitamin E activity-having
compounds or their precursor.
[3998] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0986 or its homologs, e.g. a lipoprotein, e.g. as
indicated in Table I, columns 5 or 7, line 98 and 480, is increased
conferring, preferably, an increase of the respective fine
chemical, preferably of beta-tocopherol and/or gamma-tocopherol,
and of further vitamin E-activity having compounds or their
precursors.
[3999] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YFL013C or its homologs, e.g. an activity of a
subunit of the INO80 chroamtion remodeling complexes, in
particular, of the superfamiliy of the membrane protein YFL013c,
e.g. as indicated in Table I, columns 5 or 7, 90, is increased
conferring, preferably, an increase of the respective fine
chemical, preferably of 2,3-Dimethyl-5-phyhtylquinol, and of
further vitamin E activity having-compounds or their
precursors.
[4000] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YFL053W or its homologs, e.g. an activity of a
dihydroxaacetone kinase, in particular, of the superfamiliy of
theglyerone kinase DAK1, e.g. as indicated in Table I, columns 5 or
7, 96 or 101, is increased conferring, preferably, an increase of
the respective fine chemical, preferably of beta- and/or
gamma-tocopherol, and of further vitamin E activity-having
compounds or their precursors.
[4001] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YPL268W or its homologs, e.g. an activity of a
phophoinositide phopholipase, in particular, of the superfamiliy of
the yeast 1-phosphatidylinositol-4,5-bisphosphate phophodiesterase,
e.g. as indicated in Table I, columns 5 or 7, 89, is increased
conferring, preferably, an increase of the respective fine
chemical, preferably of 2,3-Dimethyl-5-phyhtylquinol, and of
further vitamin E activity-having compounds or their
precursors.
[4002] [0047.0.0.9] to [0048.0.0.9]: see [0047.0.0.0] to
[0048.0.0.0]
[4003] [0049.0.9.9] A protein having an activity conferring an
increase in the amount or level of the 2,3-dimethyl-5-phytylquinol
preferably has the structure of the polypeptide described herein.
In a particular embodiment, the polypeptides used in the process of
the present invention or the polypeptide of the present invention
comprises the sequence of a consensus sequence as indicated in
Table IV, columns 7, lines 89 to 92 or 482 and/or the sequence of a
polypeptide as indicated in Table II, columns 5 or 7, lines 89 to
92 or 482, or of a functional homologue thereof as described
herein, or of a polypeptide which is encoded by the nucleic acid
molecule characterized herein or the nucleic acid molecule
according to the invention, for example by a nucleic acid molecule
as indicated in Table I, columns 5 or 7, lines 89 to 92 or 482 or
its herein described functional homologues and has the herein
mentioned activity conferring an increase in the
beta-2,3-dimethyl-5-phytylquinol level.
[4004] A protein having an activity conferring an increase in the
amount or level of the alpha-tocopherol preferably has the
structure of the polypeptide described herein. In a particular
embodiment, the polypeptides used in the process of the present
invention or the polypeptide of the present invention comprises the
sequence of a consensus sequence as indicated in Table IV, columns
7, lines 93 to 95 or 472 to 475 and/or the sequence of a
polypeptide as indicated in Table II, columns 5 or 7, lines 93 to
95 or 472 to 475 or of a functional homologue thereof as described
herein, or of a polypeptide which is encoded by the nucleic acid
molecule characterized herein or the nucleic acid molecule
according to the invention, for example by a nucleic acid molecule
as indicated in Table I, columns 5 or 7, lines 93 to 95 or 472 to
475 or its herein described functional homologues and has the
herein mentioned activity conferring an increase in the
alpha-tocopherol level.
[4005] A protein having an activity conferring an increase in the
amount or level of the beta-tocopherol preferably has the structure
of the polypeptide described herein. In particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence as indicated in Table IV, columns 7, lines 96 to
100 or 476 to 477 and/or the sequence of a polypeptide as indicated
in Table II, columns 5 or 7, lines 96 to 100 or 476 to 477 or of a
functional homologue thereof as described herein, or of a
polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, lines 96 to 100 or 476 to 477 or its herein described
functional homologues and has the herein mentioned activity
conferring an increase in the beta-tocopherol level.
[4006] A protein having an activity conferring an increase in the
amount or level of the gamma-tocopherol preferably has the
structure of the polypeptide described herein. In a particular
embodiment, the polypeptides used in the process of the present
invention or the polypeptide of the present invention comprises the
sequence of a consensus sequence as indicated in Table IV, columns
7, lines 101 to 102 or 478 to 481 and/or the sequence of a
polypeptide as indicated in Table II, columns 5 or 7, lines 101 to
102 or 478 to 481 or of a functional homologue thereof as described
herein, or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 101 to 102 or 478 to 481 or its
herein described functional homologues and has the herein mentioned
activity conferring an increase in the gamma-tocopherol level.
[4007] [0050.0.9.9] For the purposes of the present invention, the
term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of vitamin E or its precursor 2,3-dimethyl-5-phytylquinol,
resp., or their ester.
[4008] [0051.0.9.9] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g compositions comprising vitamin E or
its precursor 2,3-dimethyl-5-phytylquinol, resp., Depending on the
choice of the organism used for the process according to the
present invention, for example a microorganism or a plant,
compositions or mixtures of various vitamin E compounds or their
precursor 2,3-dimethyl-5-phytylquinol, resp., can be produced.
[4009] [0052.0.0.9] see [0052.0.0.0]
[4010] [0053.0.9.9] In one embodiment, the process of the present
invention comprises one or more of the following steps [4011] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention or the polypeptide used in the
method of the invention, e.g. of a polypeptide having an activity
of a protein as indicated in Table II, column 3, lines 89 to 92 or
482 or lines 93 to 95 or 472 to 475 or lines 96 to 100 or 476 to
477 or lines 101 to 102 or 478 to 481 or its homologs, e.g. as
indicated in Table II, columns 5 or 7, lines 89 to 92 or 482 or
lines 93 to 95 or 472 to 475 or lines 96 to 100 or 476 to 477 or
line 101 to102 or lines 478 to 481, activity having
herein-mentioned the respective fine chemical-increasing activity;
[4012] b) stabilizing a mRNA conferring the increased expression of
a protein encoded by the nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention, e.g.
of a polypeptide having an activity of a protein as indicated in
Table II, column 3, lines 89 to 92 or 482 or lines 93 to 95 or 472
to 475 or lines 96 to 100 or 476 to 477 or lines 101 to 102 or 478
to 481 or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 or lines 93 to 95 or 472 to
475 or lines 96 to 100 or 476 to 477 or lines 101 to 102 or 478 to
481, or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned the respective fine chemical-increasing
activity; [4013] c) increasing the specific activity of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
present invention or the nucleic acid molecule or polypeptide used
in the method of the invention having herein-mentioned the
respective fine chemical-increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 89 to 92 or 482 or lines 93 to 95 or 472 to 475 or lines 96
to 100 or 476 to 477 or lines 101 to 102 or 478 to 481 or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 89 to 92 or 482 or lines 93 to 95 or 472 to 475 or lines 96
to 100 or 476 to 477 or lines 101 to 102 or 478 to 481, or
decreasing the inhibitory regulation of the polypeptide of the
invention or the polypeptide used in the method of the invention;
[4014] d) generating or increasing the expression of an endogenous
or artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention or of the polypeptide
of the invention or the polypeptide used in the method of the
invention having herein-mentioned the respective fine
chemical-increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 89
to 92 or 482 or lines 93 to 95 or 472 to 475 or lines 96 to 100 or
476 to 477 or lines 101 to 102 or 478 to 481 or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 89
to 92 or 482 or lines 93 to 95 or 472 to 475 or lines 96 to 100 or
476 to 477 or lines 101 to 102 or 478 to 481, [4015] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned the respective fine chemical-increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 89 to 92 or 482 or lines 93 to 95 or
472 to 475 or lines 96 to 100 or 476 to 477 or lines 101 to 102 or
478 to 481 or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 or lines 93 to 95 or 472 to
475 or lines 96 to 100 or 476 to 477 or lines 101 to 102 or 478 to
481, by adding one or more exogenous inducing factors to the
organism or parts thereof; [4016] f) expressing a transgenic gene
encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned the respective fine chemical-increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 89 to 92 or 482 or lines 93 to 95 or
472 to 475 or lines 96 to 100 or 476 to 477 or lines 101 to 102 or
478 to 481 or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 or lines 93 to 95 or 472 to
475 or lines 96 to 100 or 476 to 477 or lines 101 to 102 or 478 to
481, and/or [4017] g) increasing the copy number of a gene
conferring the increased expression of a nucleic acid molecule
encoding a polypeptide encoded by the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention or the polypeptide of the invention or the polypeptide
used in the method of the invention having herein-mentioned the
respective fine chemical-increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 89 to 92 or 482 or lines 93 to 95 or 472 to 475 or lines 96
to 100 or 476 to 477 or lines 101 to 102 or 478 to 481 or its
homologs, e.g. as indicated in Table II, columns 5 or 7, lines 89
to 92 or 482 or lines 93 to 95 or 472 to 475 or lines 96 to 100 or
476 to 477 or lines 101 to 102 or 478 to 481, activity. [4018] h)
Increasing the expression of the endogenous gene encoding the
polypeptide of the invention or the polypeptide used in the method
of the invention, e.g. a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 89 to 92 or 482
or lines 93 to 95 or 472 to 475 or lines 96 to 100 or 476 to 477 or
lines 101 to 102 or 478 to 481 or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 89 to 92 or 482 or
lines 93 to 95 or 472 to 475 or lines 96 to 100 or 476 to 477 or
lines 101 to 102 or 478 to 481, by adding positive expression or
removing negative expression elements, e.g. homologous
recombination can be used to either introduce positive regulatory
elements like for plants the 35S enhancer into the promoter or to
remove repressor elements form regulatory regions. Further gene
conversion methods can be used to disrupt repressor elements or to
enhance to activity of positive elements. Positive elements can be
randomly introduced in plants by T-DNA or transposon mutagenesis
and lines can be identified in which the positive elements have be
integrated near to a gene of the invention, the expression of which
is thereby enhanced; [4019] i) Modulating growth conditions of an
organism in such a manner, that the expression or activity of the
gene encoding the protein of the invention or the protein itself is
enhanced for example microorganisms or plants can be grown for
example under a higher temperature regime leading to an enhanced
expression of heat shock proteins, which can lead an enhanced fine
chemical production and/or [4020] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, e.g. the elite crops.
[4021] [0054.0.9.9] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table II, column 5, lines 89
to 92 or 482 or lines 93 to 95 or 472 to 475 or lines 96 to 100 or
476 to 477 or lines 101 to 102 or 478 to 481, resp., or its
homologs activity, e.g. as indicated in Table II, column 7, lines
89 to 92 or 482 or lines 93 to 95 or 472 to 475 or lines 96 to 100
or 476 to 477 or lines 101 to 102 or 478 to 481, resp.
[4022] [0055.0.0.9] to [0067.0.0.9]: see [0055.0.0.0] to
[0067.0.0.0]
[4023] [0068.0.9.9] The mutation is introduced in such a way that
the production of the vitamin E or its precursor
2,3-dimethyl-5-phytylquinol is not adversely affected.
[4024] [0069.0.0.9] see [0069.0.0.0]
[4025] [0070.0.9.9] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention or
the nucleic acid molecule used in the method of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention or the polypeptide used in the method of
the invention into an organism alone or in combination with other
genes, it is possible not only to increase the biosynthetic flux
towards the end product, but also to increase, modify or create de
novo an advantageous, preferably novel metabolites composition in
the organism, e.g. an advantageous composition of vitamins
comprising a higher content of (from a viewpoint of nutritional
physiology limited) of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol.
[4026] [0071.0.0.9] see [0071.0.0.0]
[4027] [0072.0.9.9] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol, further vitamins or provitamins or
carotenoids.
[4028] [0073.0.9.9] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[4029] e) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [4030] f) increasing an activity of a
polypeptide of the invention or the polypeptide used in the method
of the invention or a homolog thereof, e.g. as indicated in Table
II, columns 5 or 7, lines 89 to 92 or 482 or lines 93 to 95 or 472
to 475 or lines 96 to 100 or 476 to 477 or lines 101 to 102 or 478
to 481, or of a polypeptide being encoded by the nucleic acid
molecule of the present invention and described below, e.g.
conferring an increase of the respective fine chemical in an
organism, preferably in a microorganism, a non-human animal, a
plant or animal cell, a plant or animal tissue or a plant, [4031]
g) growing an organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a plant
under conditions which permit the production of the respective fine
chemical in the organism, preferably the microorganism, the plant
cell, the plant tissue or the plant; and [4032] h) if desired,
recovering, optionally isolating, the free and/or bound the
respective fine chemical and, optionally further free and/or bound
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol synthesized
by the organism, the microorganism, the non-human animal, the plant
or animal cell, the plant or animal tissue or the plant.
[4033] [0074.0.9.9] The organism, in particular the microorganism,
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant is advantageously grown in such a way that it
is not only possible to recover, if desired isolate the free or
bound the respective fine chemical or the free and bound the
respective fine chemical but as option it is also possible to
produce, recover and, if desired isolate, other free or/and bound
of vitamin E or its precursor 2,3-dimethyl-5-phytylquinol.
[4034] [0075.0.0.9] to [0077.0.0.9]: see [0075.0.0.0] to
[0077.0.0.0]
[4035] [0078.0.9.9] The organism such as microorganisms or plants
or the recovered, and if desired isolated, respective fine chemical
can then be processed further directly into foodstuffs or animal
feeds or for other applications, for example according to the
disclosures made in U.S. Pat. No. 6,399,059: Thermally stable
enzyme composition and method of preparing the same, U.S. Pat. No.
6,361,800: Multi-vitamin and mineral supplement, U.S. Pat. No.
6,348,200: Cosmetic composition, U.S. Pat. No. 6,338,854:
Photoaging skin-care preparation and method of treating wrinkled
skin, U.S. Pat. No. 6,323,188: Treatment and prevention of
cardiovascular diseases, heart attack, and stroke, primary and
subsequent, with help of aspirin and certain vitamins, U.S. Pat.
No. 6,299,896: Multi-vitamin and mineral supplement, U.S. Pat. No.
6,262,279: Preparation of tocopherols, U.S. Pat. No. 6,362,221:
Compositions containing natural lycopene and natural tocopherol,
U.S. Pat. No. 6,358,997: Tocopherol and tocotrienol compositions,
U.S. Pat. No. 6,344,573: Process for extraction and concentration
of liposoluble vitamins and provitamins, growth factors and animal
and vegetable hormones from residues and by-products of
industrialized animal and vegetable products, U.S. Pat. No.
6,242,227: Method of vitamin production, U.S. Pat. No. 6,207,187:
Compositions based on tocopherols, U.S. Pat. No. 6,177,114:
Refining of edible oil rich in natural carotenes and Vitamin E
which are expressly incorporated herein by reference. The
fermentation broth, fermentation products, plants or plant products
can be purified as described in above mentioned applications or by
other methods known to the person skilled in the art and described
herein below.
[4036] In the method for producing vitamin E or its precursor
2,3-dimethyl-5-phytylquinol according to the invention, the
cultivation step of the genetically modified organisms, also
referred to as transgenic organisms herein below, is preferably
followed by harvesting said organisms and isolating vitamin E from
said organisms.
[4037] The organisms are harvested in a manner known per se and
appropriate for the particular organism. Microorganisms such as
bacteria, mosses, yeasts and fungi or plant cells which are
cultured in liquid media by fermentation may be removed, for
example, by centrifugation, decanting or filtration. Plants are
grown on solid media in a manner known per se and harvested
accordingly.
[4038] Vitamin E or its precursor 2,3-dimethyl-5-phytylquinol are
isolated from the harvested biomass in a manner known per se, for
example by extraction and, where appropriate, further chemical or
physical purification processes such as, for example, precipitation
methods, crystallography, thermal separation methods such as
rectification methods or physical separation methods such as, for
example, chromatography.
[4039] Vitamin E is isolated from oil-containing plants, for
example, preferably by chemical conversion and distillation from
vegetable oils or from the steam distillates obtained in the
deodorization of vegetable oils (deodorizer condensates).
[4040] Further methods of isolating vitamin E from deodorizer
condensates are described, for example, in DE 31 26 110 A1, EP 171
009 A2, GB 2 145 079, EP 333 472 A2 and WO 94/05650.
[4041] Products of these different work-up procedures are vitamin E
or its precursor 2,3-dimethyl-5-phytylquinol, e.g. alpha, beta, or
gamma-tocopherol or vitamin E or its precursor
2,3-dimethyl-5-phytylquinol comprising compositions, e.g.
compositions comprising alpha, beta, or gamma-tocopherol which
still comprise fermentation broth, plant particles and cell
components in different amounts, advantageously in the range of
from 0 to 99% by weight, preferably below 80% by weight, especially
preferably between below 50% by weight.
[4042] [0079.0.0.9] to [0084.0.0.9]: see [0079.0.0.0] to
[0084.0.0.0]
[4043] [0084.1.9.9] The invention also contemplates embodiments in
which the vitamin E or its precursor 2,3-dimethyl-5-phytylquinol,
or other vitamin E precursor compounds in the production of the
respective fine chemical, is present in the phytosynthetically
active organisms chosen as the host; for example, cyanobacteria,
mosses, algae or plants which, even as a wild type, are capable of
producing vitamin E. The invention also contemplates embodiments in
which a host lacks vitamin E or its precursor
2,3-dimethyl-5-phytylquinol or other vitamin E or its precursor
2,3-dimethyl-5-phytylquinol precursors, such as the vinca. In a
plant of the latter type, the inserted DNA includes genes that code
for proteins producing vitamin E precursors (compounds that can be
converted biologically into a compound with vitamin E activity) and
one or more modifying enzymes which were originally absent in such
a plant.
[4044] Preferred monocotyledonous plants are selected in particular
from the monocotyledonous crop plants such as, for example, of the
family of Gramineae such as rice, corn, wheat or other cereals
species such as barley, millet, rye, triticale or oats, and
sugarcane, and all grass species. The invention is particularly
applied to dicotyledonous plant organisms. Preferred dicotyledonous
plants are in particular selected from the dicotyledonous crop
plants such as, for example, [4045] Asteraceae such as sunflower,
Tagetes or Calendula and many others, [4046] Compositae, especially
the genus Lactuca, very especially the species sativa (lettuce) and
many others, [4047] Cruciferae, especially the genus Brassica, very
especially the species napus (oilseed rape), campestris (beet),
oleracea (e.g. cabbage, cauliflower or broccoli and other cabbage
types); and of the genus Arabidopsis, very especially the species
thaliana, and cress or canola and many others, [4048] Cucurbitaceae
such as melon, pumpkin or zucchini and many others, [4049]
Leguminosae especially the genus Glycine, very especially the
species max (soybean) soja and alfalfa, pea, beans or peanut and
many others, [4050] Rubiaceae, preferably of the subclass Lamiidae
such as, for example, Coffea arabica or Coffea liberica (coffee
plant) and many others, [4051] Solanaceae especially the genus
Lycopersicon, very especially the species esculentum (tomato) and
the genus Solanum, very especially the species tuberosum (potato)
and melongena (aubergine) and the genus Capsicum, very especially
the species annum (paprika), and tobacco and many others, [4052]
Sterculiaceae, preferably of the subclass Dilleniidae such as, for
example, Theobroma cacao (cocoa plant) and many others, [4053]
Theaceae, preferably of the subclass Dilleniidae such as, for
example, Camellia sinensis or Thea sinensis (tea plant) and many
others, [4054] Umbelliferae, especially the genus Daucus (very
especially the species carota (carrot)) and Apium (very especially
the species graveolens dulce (celeriac)) and many others; and
linseed, soybean, cotton, hemp, flax, cucumber, spinach, carrot,
sugarbeet and the various tree, nut and grape species, especially
banana and kiwi.
[4055] Also comprised are ornamental plants, productive or
ornamental trees, flowers, cut flowers, shrubs or turf.
Non-restrictive examples which may be mentioned are angiosperms,
bryophytes such as, for example, Hepaticae (liverworts) and Musci
(mosses); pteridophytes such as ferns, horsetail and club mosses;
gymnosperms such as conifers, cycads, ginkgo and gnetales, the
families of Rosaceae such as rose, Ericaceae such as rhododendrons
and azaleas, Euphorbiaceae such as poinsettias and croton,
Caryophyllaceae such as carnations, Solanaceae such as petunias,
Gesneriaceae such as African violet, Balsaminaceae such as
touch-me-not, Orchidaceae such as orchids, Iridaceae such as
gladioli, iris, freesia and crocus, Compositae such as marigold,
Geraniaceae such as geraniums, Liliaceae such as the dragon tree,
Moraceae such as ficus, Araceae such as philodendron and many
others.
[4056] Particularly preferred plant organisms are those selected
from the group of oil plants consisting of Borago officinalis,
Brassica campestris, Brassica napus, Brassica rapa, Cannabis
sativa, Carthamus tinctorius, Cocos nucifera, Crambe abyssinica,
Cuphea species, Elaeis guineensis, Ekeis oleiferu, Glycine max,
Gossypium hirsitum, Gossypium barbadense, Gossypium herbaceum,
Helianthus annus, Linum usitatissimum, Oenothera biennis, Ozea
europea, Oryza sativa, Ricinus communis, Sesamum indicum, Triticum
species, Zea maize, walnut and almond.
[4057] In one embodiment, preferred plants include, but are not
limited to: Tagetes erecta, Brassica napus, Nicotinana tabacum,
sunflower, canola, Solanum sp. (potato), and soybean.
[4058] Preferred cyanobacteria are cyanobacteria of the genus
Synechocystis, e.g. Synechocystis sp. PCC 6803, or Physcometrella
patens or Nostoc punctiforme ATCC 29133, Anabaena sp. PCC7120.
[4059] Preferred algae are green algae such as, for example, algae
of the genus Chlorella, Haematococcus, Phaedactylum tricornatum,
Volvox or Dunaliella, in particular e.g. Spongiococcum sp, e.g.
Spongiococcum exentricum, Chlorella sp., Haematococcus,
Phaedactylum tricornatum.
[4060] [0085.0.9.9] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [4061] a) a nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., or a derivative
thereof, or [4062] b) a genetic regulatory element, for example a
promoter, which is functionally linked to the nucleic acid sequence
as indicated in Table I, columns 5 or 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp. or a derivative
thereof, or [4063] c) (a) and (b) is/are not present in its/their
natural genetic environment or has/have been modified by means of
genetic manipulation methods, it being possible for the
modification to be, by way of example, a substitution, addition,
deletion, inversion or insertion of one or more nucleotide.
"Natural genetic environment" means the natural chromosomal locus
in the organism of origin or the presence in a genomic library. In
the case of a genomic library, the natural, genetic environment of
the nucleic acid sequence is preferably at least partially still
preserved. The environment flanks the nucleic acid sequence at
least on one side and has a sequence length of at least 50 bp,
preferably at least 500 bp, particularly preferably at least 1000
bp, very particularly preferably at least 5000 bp.
[4064] [0086.0.0.9] to [0087.0.0.9]: see [0086.0.0.0] to
[0087.0.0.0]
[4065] [0088.0.9.9] In an advantageous embodiment of the invention,
the organism takes the form of a plant whose content the respective
fine chemical is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for poultry is dependent on the abovementioned vitamin E and the
general amount ofvitamin E as source in feed. Further, this is also
important for the production of cosmetic compostions since, for
example, the antioxidant level of plant extracts is depending on
the abovementioned vitamin E and the general amount of vitamins
e.g. as antioxidants.
[4066] [0088.1.0.9] see [0088.1.0.0]
[4067] [0089.0.0.9] to [0090.0.0.9]: see [0089.0.0.0] to
[0090.0.0.0]
[4068] [0091.0.9.9] Thus, the content of plant components and
preferably also further impurities is as low as possible, and the
abovementioned vitamin E or its precursor
2,3-dimethyl-5-phytylquinol are obtained in as pure form as
possible. In these applications, the content of plant components
advantageously amounts to less than 10%, preferably 1%, more
preferably 0.1%, very especially preferably 0.01% or less.
[4069] [0092.0.0.9] to [0094.0.0.9]: see [0092.0.0.0] to
[0094.0.0.0]
[4070] [0095.0.9.9] It may be advantageous to increase the pool of
said vitamin E or its precursor 2,3-dimethyl-5-phytylquinol in the
transgenic organisms by the process according to the invention in
order to isolate high amounts of the essentially pure fine
chemical.
[4071] [0096.0.9.9] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol in the
organism, is useful to increase the production of the respective
fine chemical.
[4072] [0097.0.0.9] see [0097.0.0.0]
[4073] [0098.0.9.9] In a preferred embodiment, the respective fine
chemical (vitamin E or its precursor 2,3-dimethyl-5-phytylquinol)
is produced in accordance with the invention and, if desired, is
isolated. The production of further vitamins, provitamins or
carotenoids, e.g. carotenes or xanthophylls, or mixtures thereof or
mixtures with other compounds by the process according to the
invention is advantageous.
[4074] [0099.0.9.9] In the case of the fermentation of
microorganisms, the abovementioned vitamin E or its precursor
2,3-dimethyl-5-phytylquinolmay accumulate in the medium and/or the
cells. If microorganisms are used in the process according to the
invention, the fermentation broth can be processed after the
cultivation. Depending on the requirement, all or some of the
biomass can be removed from the fermentation broth by separation
methods such as, for example, centrifugation, filtration, decanting
or a combination of these methods, or else the biomass can be left
in the fermentation broth. The fermentation broth can subsequently
be reduced, or concentrated, with the aid of known methods such as,
for example, rotary evaporator, thin-layer evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. Afterwards
advantageously further compounds for formulation can be added such
as corn starch or silicates. This concentrated fermentation broth
advantageously together with compounds for the formulation can
subsequently be processed by lyophilization, spray drying, and
spray granulation or by other methods. Preferably the respective
fine chemical or the vitamin E or its precursor
2,3-dimethyl-5-phytylquinol comprising compositions are isolated
from the organisms, such as the microorganisms or plants or the
culture medium in or on which the organisms have been grown, or
from the organism and the culture medium, in the known manner, for
example via extraction, distillation, crystallization,
chromatography or a combination of these methods. These
purification methods can be used alone or in combination with the
aforementioned methods such as the separation and/or concentration
methods.
[4075] [0100.0.9.9] Transgenic plants which comprise the vitamin E
or its precursor 2,3-dimethyl-5-phytylquinolsuch as alpha, beta, or
gamma-tocopherol, synthesized in the process according to the
invention can advantageously be marketed directly without there
being any need for the oils, lipids or fatty acids synthesized to
be isolated. Plants for the process according to the invention are
listed as meaning intact plants and all plant parts, plant organs
or plant parts such as leaf, stem, seeds, root, tubers, anthers,
fibers, root hairs, stalks, embryos, calli, cotelydons, petioles,
harvested material, plant tissue, reproductive tissue and cell
cultures which are derived from the actual transgenic plant and/or
can be used for bringing about the transgenic plant. In this
context, the seed comprises all parts of the seed such as the seed
coats, epidermal cells, seed cells, endosperm or embryonic
tissue.
[4076] The site of vitamin E biosynthesis in plants is, inter alia,
the leaf tissue so that the isolation of leafs makes sense.
However, this is not limiting, since the expression may also take
place in a tissue-specific manner in all of the remaining parts of
the plant, in particular in fat-containing seeds. A further
preferred embodiment therefore relates to a seed-specific isolation
of vitamin E or its precursor 2,3-dimethyl-5-phytylquinol.
[4077] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, in the form of their oils, fats,
lipids, as extracts, e.g. ether, alcohol, or other organic solvents
or water containing extract and/or free vitamin E or its precursor
2,3-dimethyl-5-phytylquinol. The respective fine chemical produced
by this process can be obtained by harvesting the organisms, either
from the crop in which they grow, or from the field. This can be
done via pressing or extraction of the plant parts, e.g. in the
plant seeds. To increase the efficiency of extraction it is
beneficial to clean, to temper and if necessary to hull and to
flake the plant material especially the seeds. E.g., oils, fats,
and/or lipids comprising vitamin E or its precursor
2,3-dimethyl-5-phytylquinolcan be obtained by what is known as cold
beating or cold pressing without applying heat. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pre-treated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[4078] Because vitamin E or its precursor
2,3-dimethyl-5-phytylquinol in microorganisms may be localized
intracellular, their recovery essentials comes down to the
isolation of the biomass. Well-established approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Determination of
tocopherols in cells has been described by Tan and Tsumura 1989,
see also Biotechnology of Vitamins, Pigments and Growth Factors,
Edited by Erik J. Vandamme, London, 1989, p. 96 to 103. Many
further methods to determine the tocopherol content are known to
the person skilled in the art.
[4079] [0101.0.0.9] see [0101.0.0.0]
[4080] [0102.0.9.9] Vitamin E or its precursor
2,3-dimethyl-5-phytylquinol can for example be analyzed
advantageously via HPLC or GC separation methods and detected by MS
or MSMS methods. The unambiguous detection for the presence of
Vitamin E or its precursor 2,3-dimethyl-5-phytylquinol containing
products can be obtained by analyzing recombinant organisms using
analytical standard methods: GC, GC-MS or TLC, as described on
several occasions by Christie and the references therein (1997, in:
Advances on Lipid Methodology, Fourth Edition: Christie, Oily
Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). The
material to be analyzed can be disrupted by sonication, grinding in
a glass mill, liquid nitrogen and grinding, cooking, or via other
applicable methods; see also Biotechnology of Vitamins, Pigments
and Growth Factors, Edited by Erik J. Vandamme, London, 1989, p. 96
to 103.
[4081] [0103.0.9.9] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [4082] a) nucleic acid molecule encoding, preferably
at least the mature form, of the polypeptide having a sequence as
indicated in Table II, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp. or a fragment thereof,
which confers an increase in the amount of the respective fine
chemical in an organism or a part thereof; [4083] b) nucleic acid
molecule comprising, preferably at least the mature form, of a
nucleic acid molecule having a sequence as indicated in Table I,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp. [4084] c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as result of the degeneracy of the
genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [4085]
d) nucleic acid molecule encoding a polypeptide which has at least
50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [4086] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [4087] f) nucleic acid molecule encoding a polypeptide,
the polypeptide being derived by substituting, deleting and/or
adding one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[4088] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [4089] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers pairs having a sequence as indicated in Table
III, column 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp. and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[4090] i) nucleic acid molecule encoding a polypeptide which is
isolated, e.g. from an expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (h), preferably to (a) to (c), and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [4091] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequences as indicated in Table IV, column 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [4092] k) nucleic
acid molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of the polypeptide indicated in Table II, columns 5 or 7, lines 89
to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp. and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; and [4093] l) nucleic
acid molecule which is obtainable by screening a suitable library
under stringent conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k), preferably to
(a) to (c), or with a fragment of at least 15 nt, preferably 20 nt,
30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k), preferably to (a) to (c), and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which comprises a
sequence which is complementary thereto.
[4094] [0103.1.9.9] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I A, columns 5 or 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp., by one or
more nucleotides. In one embodiment, the nucleic acid molecule used
in the process of the invention does not consist of the sequence
indicated in Table I A, columns 5 or 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp.: In one
embodiment, the nucleic acid molecule used in the process of the
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I A, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp. In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table II A, columns
5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp.
[4095] [0103.2.9.9.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over a or more
sequence(s) indicated in Table I B, column 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp., by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of a or more
sequence(s) indicated in Table I B, column 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp. In one
embodiment, the nucleic acid molecule used in the process of the
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I B, column 7, lines 89
to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp. In
another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II B, column 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp.
[4096] [0104.0.9.9] In one embodiment, the nucleic acid molecule of
the invention or used in the process distinguishes over the
sequence indicated in Table I, columns 5 or 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp., by one or
more nucleotides. In one embodiment, the nucleic acid molecule used
in the process of the invention or the nucleic acid used in the
process of the invention does not consist of the sequence indicated
in Table I, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93
to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp., In one embodiment, the
nucleic acid molecule of the present invention is less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to the sequence indicated
in Table I, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93
to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp.
[4097] [0105.0.0.9] to [0107.0.0.9] see [0105.0.0.0] to
[0107.0.0.0]
[4098] [0108.0.9.9] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., nucleic acid molecules
which are derived from an amino acid sequences as indicated in
Table II, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp., or from polypeptides comprising
the consensus sequence as indicated in Table IV, column 7, lines 89
to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp. or
their derivatives or homologues encoding polypeptides with the
enzymatic or biological activity of an activity of a polypeptide as
indicated in Table II, column 3, 5 or 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp., or conferring
an increase of the respective fine chemical, meaning Vitamin E or
its precursor 2,3-dimethyl-5-phytylquinol., in particular, alpha-,
beta-, and/or gamma-tocopherol after increasing its expression or
activity are advantageously increased in the process according to
the invention.
[4099] [0109.0.0.9] see [0109.0.0.0]
[4100] [0110.0.9.9] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide of the invention or
the polypeptide used in the method of the invention or used in the
process of the invention, e.g. of a protein as indicated in Table
II, column 5, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to
475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or
478 to 481, resp or being encoded by a nucleic acid molecule
indicated in Table I, column 5, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp, or of its homologs, e.g. as
indicated in Table I or Table II, column 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp., can be
determined from generally accessible databases.
[4101] [0111.0.0.9] see [0111.0.0.0]
[4102] [0112.0.9.9] The nucleic acid molecules used in the process
according to the invention take the form of isolated nucleic acid
sequences, which encode polypeptides with an activity of a
polypeptide as indicated in Table II, column 3, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp., or having
the sequence of a polypeptide as indicated in Table II, columns 5
and 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., and conferring an increase in the level of vitamin E
or its precursor 2,3-dimethyl-5-phytylquinol resp., in particular,
of alpha-, beta-, and/or gamma-tocopherol.
[4103] [0113.0.0.9] to [0120.0.0.9]: see [0113.0.0.0] to
[0120.0.0.0]
[4104] [0120.1.9.9]: Production strains which are also
advantageously selected in the process according to the invention
are microorganisms selected from the group of green algae, like
Spongioccoccum exentricum, Chlorella sorokiniana (pyrenoidosa,
7-11-05), or algae of the genus Haematococcus, Phaedactylum
tricornatum, Volvox or Dunaliella or form the group of fungi like
fungi belonging to the Daccrymycetaceae family, or
non-photosynthetic bacteria, like methylotrophs, flavobacteria,
actinomycetes, like streptomyces chrestomyceticus, Mycobacteria
like Mycobacterim phlei, or Rhodobacter capsulatus. Thus, the
invention also contemplates embodiments in which a host lacks
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol or other
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol precursors,
such as the vinca. In a plant of the latter type, the inserted DNA
includes genes that code for proteins producing vitamin E
precursors (compounds that can be converted biologically into a
cmpound with vitamin E activity) and one or more modifiying enzymes
which were originally absent in such a plant.
[4105] The invention also contemplates embodiments in which the
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol, or other
vitamin E precursor compounds in the production of the respective
fine chemical, is present in the phytosynthetically active
organisms chosen as the host; for example, cyanobacteria, mosses,
algae or plants which, even as a wild type, are capable of
producing vitamin E.
[4106] [0121.0.9.9] However, it is also possible to use artificial
sequences, which differ in one or more bases from the nucleic acid
sequences found in organisms, or in one or more amino acid
molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., or the functional homologues thereof as
described herein, preferably conferring above-mentioned activity,
i.e. conferring an increase in the level of
2,3-dimethyl-5-phytyhlquinol after increasing the activity of the
polypeptide sequences indicated in Table II, columns 5 or 7, lines
89 to 92 or 482; or conferring increase in the level of
alpha-tocopherol after increasing the activity of the polypeptide
sequences indicated in Table II, columns 5 or 7, lines 93 to 95 or
472 to 475; or conferring increase in the level of beta-tocopherol
after increasing the activity of the polypeptide sequences
indicated in Table II, columns 5 or 7, lines 96 to 100 or 476 to
477; or conferring increase in the level of gamma-tocopherol after
increasing the activity of the polypeptide sequences indicated in
Table II, columns 5 or 7, lines 101 to 102 or 478 to 481.
[4107] [0122.0.0.9] to [0127.0.0.9]: see [0122.0.0.0] to
[0127.0.0.0]
[4108] [0128.0.9.9] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, column 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp. by means of polymerase chain reaction can be generated on the
basis of a sequence shown herein, for example the sequence as
indicated in Table I, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp. or the sequences
derived from a sequence as indicated in Table II, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp.
[4109] [0129.0.9.9] Moreover, it is possible to identify conserved
regions from various organisms by carrying out protein sequence
alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention or the polypeptide used in the method of the invention,
from which conserved regions, and in turn, degenerate primers can
be derived. Conserved region for the polypeptide of the invention
or the polypeptide used in the method of the invention are
indicated in the alignments shown in the figures. Conserved regions
are those, which show a very little variation in the amino acid
sequence in one particular position of several homologs from
different origin. The consensus sequence indicated in Table IV,
column 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp. is derived from said alignments.
[4110] [0130.0.9.9] Degenerated primers can then be utilized by PCR
for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of vitamin E
or its precursor 2,3-dimethyl-5-phytylquinol resp., in particular,
of alpha-, beta-, and/or gamma-tocopherol after increasing the
expression or activity of the protein comprising said fragment.
[4111] [0131.0.0.9] to [0138.0.0.9]: see [0131.0.0.0] to
[0138.0.0.0]
[4112] [0139.0.9.9] Polypeptides having above-mentioned activity,
i.e. conferring the increase of the respective fine chemical level,
derived from other organisms, can be encoded by other DNA sequences
which hybridize to a sequences indicated in Table I, columns 5 or
7, lines 89 to 92 or 482, preferably Table I B, column 7, lines 89
to 92 or 482 for 2,3-dimethyl-5-phytylquinol or indicated in Table
I, columns 5 or 7, lines 93 to 95 or 472 to 475, preferably Table I
B, column 7, lines 93 to 95 or 472 to 475 for alpha-tocopherol or
indicated in Table I, columns 5 or 7, lines 96 to 100 or 476 to
477, preferably Table I B, column 7, lines 96 to 100 or 476 to 477
for beta-tocopherol or indicated in Table I, columns 5 or 7, lines
101 to 102 or 478 to 481, preferably Table I B, column 7, lines 96
to 100 or 476 to 477 for gamma-tocopherol under relaxed
hybridization conditions and which code on expression for peptides
having the respective fine chemical, i.e. vitamin E or its
precursor 2,3-dimethyl-5-phytylquinol resp., in particular, of
alpha-, beta-, and/or gamma-tocopherol, resp., increasing
activity.
[4113] [0140.0.0.9] to [0146.0.0.9]: see [0140.0.0.0] to
[0146.0.0.0]
[4114] [0147.0.9.9] Further, the nucleic acid molecule of the
invention or used in the method of the invention comprises a
nucleic acid molecule, which is a complement of one of the
nucleotide sequences of above mentioned nucleic acid molecules or a
portion thereof. A nucleic acid molecule which is complementary to
one of the nucleotide sequences indicated in Table I, columns 5 or
7, lines 89 to 92 or 482, and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., preferably Table I B, column 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp., is one
which is sufficiently complementary to one of said nucleotide
sequences such that it can hybridize to one of said nucleotide
sequences, thereby forming a stable duplex. Preferably, the
hybridisation is performed under stringent hybridization
conditions. However, a complement of one of the herein disclosed
sequences is preferably a sequence complement thereto according to
the base pairing of nucleic acid molecules well known to the
skilled person. For example, the bases A and G undergo base pairing
with the bases T and U or C, resp. and visa versa. Modifications of
the bases can influence the base-pairing partner.
[4115] [0148.0.9.9] The nucleic acid molecule of the invention or
used in the method of the invention comprises a nucleotide sequence
which is at least about 30%, 35%, 40% or 45%, preferably at least
about 50%, 55%, 60% or 65%, more preferably at least about 70%,
80%, or 90%, and even more preferably at least about 95%, 97%, 98%,
99% or more homologous to a nucleotide sequence indicated in Table
I, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp., preferably Table I B, column 7, lines 89
to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp. or a
portion thereof and preferably has above mentioned activity, in
particular having a vitamin E or its precursor
2,3-dimethyl-5-phytylquinol resp., in particular, of alpha-, beta-,
and/or gamma-tocopherol increasing activity after increasing the
activity or an activity of a product of a gene encoding said
sequences or their homologs.
[4116] [0149.0.9.9] The nucleic acid molecule of the invention or
used in the method of the invention comprises a nucleotide sequence
which hybridizes, preferably hybridizes under stringent conditions
as defined herein, to one of the nucleotide sequences indicated in
Table I, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp. or a portion thereof and encodes a
protein having above-mentioned activity, e.g. conferring increase
of vitamin E or its precursor 2,3-dimethyl-5-phytylquinol resp., in
particular, of alpha-, beta-, and/or gamma-tocopherol, resp., and
optionally, the activity of a protein indicated in Table II.
[4117] [00149.1.9.9] Optionally, in one embodiment, the nucleotide
sequence, which hybridises to one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., preferably Table I B,
columns 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to
475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or
478 to 481, resp., has further one or more of the activities
annotated or known for the a protein as indicated in Table II,
column 3, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481.
[4118] [0150.0.9.9] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table I, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp., preferably Table I B, column 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp. for example a fragment which can be used as a probe or primer
or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol resp., in particular, of alpha-, beta-,
and/or gamma-tocopherol resp., if its activity is increased. The
nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table I, columns
5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., preferably Table I B, column 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp. an
anti-sense sequence of one of the sequences, e.g., as indicated in
Table I, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp., preferably Table I B, column 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp. or naturally occurring mutants thereof. Primers based on a
nucleotide of invention can be used in PCR reactions to clone
homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table III, column 7, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp., can result in a fragment of a
polynucleotide sequence as indicated in Table I, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., preferably Table I B, column 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., or its gene
product.
[4119] [0151.0.0.9] see [0151.0.0.0]
[4120] [0152.0.9.9] The nucleic acid molecule of the invention or
the nucleic acid used in the method of the invention encodes a
polypeptide or portion thereof which includes an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table II, columns 5 or 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp., preferably
Table II B, column 7, lines 89 to 92 or 482 and/or lines 93 to 95
or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101
to 102 or 478 to 481, resp., such that the protein or portion
thereof maintains the ability to participate in the respective fine
chemical production, in particular an activity increasing the level
of vitamin E or its precursor 2,3-dimethyl-5-phytylquinol resp., in
particular, of alpha-, beta-, and/or gamma-tocopherol, resp., as
mentioned above or as described in the examples in plants or
microorganisms is comprised.
[4121] [0153.0.9.9] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp., such
that the protein or portion thereof is able to participate in the
increase of the respective fine chemical production. In one
embodiment, a protein or portion thereof as indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., has for example an activity of a polypeptide
as indicated in Table II, column 3, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp.
[4122] [0154.0.9.9] In one embodiment, the nucleic acid molecule of
the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 89 to 92
or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100
or 476 to 477 and/or line 101 to 102 or 478 to 481, resp., and has
above-mentioned activity, e.g. conferring preferably the increase
of the respective fine chemical.
[4123] [0155.0.0.9] to [0156.0.0.9]: see [0155.0.0.0] to
[0156.0.0.0]
[4124] [0157.0.9.9] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences as
indicated in Table I, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., (and portions thereof)
due to degeneracy of the genetic code and thus encode a polypeptide
of the present invention, in particular a polypeptide having above
mentioned activity, e.g. conferring an increase in the respective
fine chemical in a organism, e.g. as that polypeptides comprising a
consensus sequence as indicated in Table IV, column 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp., or
of the polypeptide as indicated in Table II, columns 5 or 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
or the functional homologues.
[4125] Advantageously, the nucleic acid molecule of the invention
or used in the method of the invention comprises, or in an other
embodiment has, a nucleotide sequence encoding a protein
comprising, or in an other embodiment having, an amino acid
sequence of a consensus sequences as indicated in Table IV, column
7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., or of the polypeptide as indicated in Table II, columns 5 or
7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., or the functional homologues. In a still further embodiment,
the nucleic acid molecule of the invention encodes a full length
protein which is substantially homologous to an amino acid sequence
comprising a consensus sequence as indicated in Table IV, column 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., or of a polypeptide as indicated in Table II, columns 5 or
7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., or the functional homologues. However, in a preferred
embodiment, the nucleic acid molecule of the present invention does
not consist of a sequence as indicated in Table I, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., preferably of Table I A, columns 5 or 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481. Preferably, the
nucleic acid molecule of the invention is a functional homologue or
identical to a nucleic acid molecule indicated in Table I B, column
7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to
481.
[4126] [0158.0.0.9] to [0160.0.0.9]: see [0158.0.0.0] to
[0160.0.0.0]
[4127] [0161.0.9.9] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp. The nucleic
acid molecule is preferably at least 20, 30, 50, 100, 250 or more
nucleotides in length.
[4128] [0162.0.0.9] see [0162.0.0.0]
[4129] [0163.0.9.9] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp., corresponds to
a naturally-occurring nucleic acid molecule of the invention. As
used herein, a "naturally-occurring" nucleic acid molecule refers
to an RNA or DNA molecule having a nucleotide sequence that occurs
in nature (e.g., encodes a natural protein). Preferably, the
nucleic acid molecule encodes a natural protein having
above-mentioned activity, e.g. conferring the respective fine
chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[4130] [0164.0.0.9] see [0164.0.0.0]
[4131] [0165.0.9.9] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, preferably Table I B, columns 5 or 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp.
[4132] [0166.0.0.9] to [0167.0.0.9]: see [0166.0.0.0] to
[0167.0.0.0]
[4133] [0168.0.9.9] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., preferably Table II B, column 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp., yet retain said
activity described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table II, columns 5 or 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
preferably Table II B, column 7, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp., and is capable of
participation in the increase of production of the respective fine
chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., preferably Table II B, column 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp. more
preferably at least about 70% identical to one of the sequences as
indicated in Table II, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp. even more preferably at
least about 80%, 90%, 95% homologous to a sequence as indicated in
Table II, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp., preferably Table II B, column 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp. and most preferably at least about 96%, 97%, 98%, or 99%
identical to the sequence as indicated in Table II, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., preferably Table II B, column 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481.
[4134] [0169.0.0.9] to [0172.0.0.9]: see [0169.0.0.0] to
[0172.0.0.0]
[4135] [0173.0.9.9] For example a sequence, which has 80% homology
with sequence SEQ ID NO: 8673 at the nucleic acid level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 8673 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[4136] [0174.0.0.9] see [0174.0.0.0]
[4137] [0175.0.9.9] For example a sequence which has a 80% homology
with sequence SEQ ID NO: 8674 at the protein level is understood as
meaning a sequence which, upon comparison with the sequence SEQ ID
NO: 8674 by the above program algorithm with the above parameter
set, has a 80% homology.
[4138] [0176.0.9.9] Functional equivalents derived from one of the
polypeptides as indicated in Table II, columns 5 or 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
according to the invention by substitution, insertion or deletion
have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%,
60%, 65% or 70% by preference at least 80%, especially preferably
at least 85% or 90%, 91%, 92%, 93% or 94%, very especially
preferably at least 95%, 97%, 98% or 99% homology with one of the
polypeptides as indicated in Table II, columns 5 or 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
according to the invention and are distinguished by essentially the
same properties as a polypeptide as indicated in Table II, columns
5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp.
[4139] [0177.0.9.9] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
according to the invention by substitution, insertion or deletion
have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%,
60%, 65% or 70% by preference at least 80%, especially preferably
at least 85% or 90%, 91%, 92%, 93% or 94%, very especially
preferably at least 95%, 97%, 98% or 99% homology with one of the
polypeptides as indicated in Table II, columns 5 or 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
according to the invention and encode polypeptides having
essentially the same properties as a polypeptide as indicated in
Table II, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp.
[4140] [0178.0.0.9] see [0178.0.0.0]
[4141] [0179.0.9.9] A nucleic acid molecule encoding an homolog to
a protein sequence as indicated in Table II, columns 5 or 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
preferably Table II B, column 7, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp., can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table I, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., preferably Table I B, column 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., such that one or more
amino acid substitutions, additions or deletions are introduced
into the encoded protein. Mutations can be introduced into the
encoding sequences of a sequences as indicated in Table I, columns
5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., preferably Table I B, column 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp., by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[4142] [0180.0.0.9] to [0183.0.0.9]: see [0180.0.0.0] to
[0183.0.0.0]
[4143] [0184.0.9.9] Homologues of the nucleic acid sequences used,
with a sequence as indicated in Table I, columns 5 or 7, lines 89
to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp., or
of the nucleic acid sequences derived from a sequences as indicated
in Table II, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93
to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp. comprise also allelic variants
with at least approximately 30%, 35%, 40% or 45% homology, by
preference at least approximately 50%, 60% or 70%, more preferably
at least approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
I, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp., preferably Table I B, column 7, lines 89
to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp., or
from the derived nucleic acid sequences, the intention being,
however, that the enzyme activity or the biological activity of the
resulting proteins synthesized is advantageously retained or
increased.
[4144] [0185.0.9.9] In one embodiment of the present invention, the
nucleic acid molecule of the invention or used in the process of
the invention comprises one or more sequences as indicated in Table
I, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp., preferably Table I B, column 7, lines 89
to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp., In
one embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotides not shown in any
one of sequences as indicated in Table I, columns 5 or 7, lines 89
to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
preferably Table I B, column 7, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp., In one embodiment, the
nucleic acid molecule comprises less than 500, 400, 300, 200, 100,
90, 80, 70, 60, 50 or 40 further nucleotides. In a further
embodiment, the nucleic acid molecule comprises less than 30, 20 or
10 further nucleotides. In one embodiment, a nucleic acid molecule
used in the process of the invention is identical to a sequences as
indicated in Table I, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., preferably Table I B,
column 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp.,
[4145] [0186.0.9.9] Also preferred is that one or more nucleic acid
molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., preferably Table II B, column 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp. In one
embodiment, the nucleic acid molecule encodes less than 150, 130,
100, 80, 60, 50, 40 or 30 further amino acids. In a further
embodiment, the encoded polypeptide comprises less than 20, 15, 10,
9, 8, 7, 6 or 5 further amino acids. In one embodiment, the encoded
polypeptide used in the process of the invention is identical to
the sequences as indicated in Table II, columns 5 or 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
preferably Table II B, column 7, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp
[4146] [0187.0.9.9] In one embodiment, a nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., preferably Table IIB, column 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp, comprises less than 100
further nucleotides. In a further embodiment, said nucleic acid
molecule comprises less than 30 further nucleotides. In one
embodiment, the nucleic acid molecule used in the process is
identical to a coding sequence as indicated in Table II, columns 5
or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., preferably Table II B, column 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp.
[4147] [0188.0.9.9] Polypeptides (=proteins), which still have the
essential biological or enzymatic activity of the polypeptide of
the present invention conferring an increase of the respective fine
chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp., and is expressed under identical
conditions.
[4148] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, column 7, lines 89 to 92 or 482 and/or lines 93 to 95
or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101
to 102 or 478 to 481.
[4149] [0189.0.9.9] Homologues of a sequences as indicated in Table
I, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp., or of a derived sequences as indicated in
Table II, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp., also mean truncated sequences,
cDNA, single-stranded DNA or RNA of the coding and noncoding DNA
sequence. Homologues of said sequences are also understood as
meaning derivatives, which comprise noncoding regions such as, for
example, UTRs, terminators, enhancers or promoter variants. The
promoters upstream of the nucleotide sequences stated can be
modified by one or more nucleotide substitution(s), insertion(s)
and/or deletion(s) without, however, interfering with the
functionality or activity either of the promoters, the open reading
frame (=ORF) or with the 3'-regulatory region such as terminators
or other 3' regulatory regions, which are far away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[4150] [0190.0.0.9] to [0191.0.0.9] see [0190.0.0.0] to
[0191.0.0.0]
[4151] [0191.1.9.9] -/-
[4152] [0192.0.0.9] to [0203.0.0.9] see [0192.0.0.0] to
[0203.0.0.0]
[4153] [0204.0.9.9] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [4154] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, columns 5 or 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
preferably Table II B, column 7, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp.; or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, i.e. vitamin E or its precursor
2,3-dimethyl-5-phytylquinol (lines 89 to 92 or 482), resp., in
particular, of alpha- (lines 93 to 95 or 472 to 475), beta- (lines
96 to 100 or 476 to 477), and/or gamma-tocopherol (lines 101 to 102
or 478 to 481), resp., in an organism or a part thereof [4155] b)
nucleic acid molecule comprising, preferably at least the mature
form, of a nucleic acid molecule as indicated in Table I, columns 5
or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., preferably Table I B, column 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp., or a
fragment thereof conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [4156]
c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [4157] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [4158] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [4159]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c),
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [4160] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[4161] h) nucleic acid molecule comprising a nucleic acid molecule
which is obtained by amplifying a cDNA library or a genomic library
using primers or primer pairs as indicated in Table III, column 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., and conferring an increase in the amount of the respective
fine chemical, i.e. vitamin E or its precursor
2,3-dimethyl-5-phytylquinol (lines 89 to 92 or 482), resp., in
particular, of alpha- (lines 93 to 95 or 472 to 475), beta- (lines
96 to 100 or 476 to 477), and/or gamma-tocopherol (lines 101 to 102
or 478 to 481), resp. in an organism or a part thereof; [4162] i)
nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from a expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (g), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [4163] j) nucleic acid molecule which
encodes a polypeptide comprising a consensus sequence as indicated
in Table IV, columns 7, lines 89 to 92 or 482 and/or lines 93 to 95
or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101
to 102 or 478 to 481, resp., and conferring an increase in the
amount of the respective fine chemical, i.e. vitamin E or its
precursor 2,3-dimethyl-5-phytylquinol (lines 89 to 92 or 482),
resp., in particular, of alpha- (lines 93 to 95 or 472 to 475),
beta- (lines 96 to 100 or 476 to 477), and/or gamma-tocopherol
(lines 101 to 102 or 478 to 481), resp., in an organism or a part
thereof; [4164] k) nucleic acid molecule encoding the amino acid
sequence of a polypeptide encoding a domain of a polypeptide as
indicated in Table II, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., preferably Table II B,
column 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., and conferring an increase in the amount of the
respective fine chemical, i.e. vitamin E or its precursor
2,3-dimethyl-5-phytylquinol (lines 89 to 92 or 482), resp., in
particular, of alpha- (lines 93 to 95 or 472 to 475), beta- (lines
96 to 100 or 476 to 477), and/or gamma-tocopherol (lines 101 to 102
or 478 to 481), resp., in an organism or a part thereof; and [4165]
l) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment of at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (h) or of a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., preferably Table I B, column 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., or a nucleic acid
molecule encoding, preferably at least the mature form of, a
polypeptide as indicated in Table II, columns 5 or 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
preferably Table II B, column 7, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp. and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over a sequence as indicated in Table I
A, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp., by one or more nucleotides. In one
embodiment, the nucleic acid molecule of the invention does not
consist of the sequence as indicated in Table I, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., In an other embodiment, the nucleic acid molecule of the
present invention is at least 30 identical and less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence as indicated
in Table I A, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93
to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp. In a further embodiment the
nucleic acid molecule does not encode a polypeptide sequence as
indicated in Table II A, columns 5 or 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp. Accordingly, in
one embodiment, the nucleic acid molecule of the present invention
encodes in one embodiment a polypeptide which differs at least in
one or more amino acids from a polypeptide indicated in Table II A,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., does not encode a protein of a sequence as
indicated in Table II A, columns 5 or 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp. Accordingly, in
one embodiment, the protein encoded by a sequences of a nucleic
acid according to (a) to (l) does not consist of a sequence as
indicated in Table II A, columns 5 or 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp. In a further
embodiment, the protein of the present invention is at least 30
identical to a protein sequence indicated in Table II A, columns 5
or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., and less than 100%, preferably less than 99.999%,
99.99% or 99.9%, more preferably less than 99%, 985, 97%, 96% or
95% identical to a sequence as indicated in Table II A, columns 5
or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp. whereby, in a further embodiment, the nucleic acid
molecule according to (a) to (l) distinguishes over a sequence as
indicated in Table I B, column 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., by one or more
nucleotides. In one embodiment, the nucleic acid molecule of the
invention does not consist of the sequence as indicated in Table I
B, column 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to
475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or
478 to 481, resp., In an further embodiment, the nucleic acid
molecule of the present invention is at least 30 identical and less
than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence as
indicated in Table I B, column 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp. In a further embodiment
the nucleic acid molecule does not encode a polypeptide sequence as
indicated in Table II B, column 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp. Accordingly, in one
embodiment, the nucleic acid molecule of the present invention
encodes in one embodiment a polypeptide which differs at least in
one or more amino acids from a polypeptide indicated in Table II B,
column 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., does not encode a protein of a sequence as indicated
in Table II B, columns 5 or 7, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp. Accordingly, in one further
embodiment, the protein encoded by a sequences of a nucleic acid
according to [4166] (a) to (l) does not consist of a sequence as
indicated in Table II B, column 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp. In a further
embodiment, the protein of the present invention is at least 30%
identical to a protein sequence indicated in Table II B, column 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., and less than 100%, preferably less than 99.999%, 99.99% or
99.9%, more preferably less than 99%, 985, 97%, 96% or 95%
identical to a sequence as indicated in Table II B, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp.
[4167] [0205.0.0.9] to [0206.0.0.9]: see [0205.0.0.0] to
[0206.0.0.0]
[4168] [0207.0.9.9] As described herein, the nucleic acid construct
can also comprise further genes, which are to be introduced into
the organisms or cells. It is possible and advantageous to
introduce into, and express in, the host organisms regulatory genes
such as genes for inductors, repressors or enzymes, which, owing to
their enzymatic activity, engage in the regulation of one or more
genes of a biosynthetic pathway. These genes can be of heterologous
or homologous origin. Moreover, further biosynthesis genes may
advantageously be present, or else these genes may be located on
one or more further nucleic acid constructs. Genes, which are
advantageously employed as biosynthesis genes, are genes of the
tocopherol metabolism, the tocotriene metabolism, the carotenoid
metabolism, the amino acid metabolism, of glycolysis, of the
tricarboxylic acid metabolism or their combinations. As described
herein, regulator sequences or factors can have a positive effect
on preferably the gene expression of the genes introduced, thus
increasing it. Thus, an enhancement of the regulator elements may
advantageously take place at the transcriptional level by using
strong transcription signals such as promoters and/or enhancers. In
addition, however, an enhancement of translation is also possible,
for example by increasing mRNA stability or by inserting a
translation enhancer sequence.
[4169] [0208.0.0.9] to [0226.0.0.9]: see [0208.0.0.0] to
[0226.0.0.0]
[4170] [0227.0.9.9] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[4171] In addition to a sequence indicated in Table I, columns 5 or
7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., preferably Table I B, column 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., or its derivatives, it
is advantageous additionally to express and/or mutate further genes
in the organisms. Especially advantageously, additionally at least
one further gene of the tocopherol biosynthetic pathway such as for
a vitamin E precursor, is expressed in the organisms such as plants
or microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the amino acids desired since, for example, feedback
regulations no longer exist to the same extent or not at all. In
addition it might be advantageously to combine one or more of the
sequences indicated in Table I, columns 5 or 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp., with genes
which generally support or enhances to growth or yield of the
target organism, for example genes which lead to faster growth rate
of microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[4172] [0228.0.9.9] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the tocopherol
metabolism, in particular in synthesis of alpha-, beta-, and/or
gamma-tocopherol.
[4173] [0229.0.9.9] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the tocopherol biosynthetic
pathway, such as the homogentisate phytyltransferase (HPT) or the
enzymes catalysing the subsequent cyclization and methylation
reactions, .gamma.-tocopherol methyl transf erase (.gamma.-TMT),
prenyltransferases that condense prenyl groups with allylic chains
and those that condense prenyl chains with aromatic groups and
others. These genes can lead to an increased synthesis of the
essential vitamin E or its precursor 2,3-dimethyl-5-phytylquinol
resp., in particular, of alpha-, beta-, and/or gamma-tocopherol,
resp.
[4174] [0230.0.0.9] see [0230.0.0.0]
[4175] [0231.0.9.9] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a protein degrading vitamin E or its
precursor 2,3-dimethyl-5-phytylquinol resp., in particular, alpha-,
beta-, and/or gamma-tocopherol, resp., is attenuated, in particular
by reducing the rate of expression of the corresponding gene.
[4176] [0232.0.0.9] to [0276.0.0.9]: see [0232.0.0.0] to
[0276.0.0.0]
[4177] [0277.0.9.9] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
is familiar. For example, via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously. The respective fine chemical produced by this process
can be obtained by harvesting the organisms, either from the crop
in which they grow, or from the field. This can be done via
pressing or extraction of the plant parts.
[4178] [0278.0.0.9] to [0282.0.0.9]: see [0278.0.0.0] to
[0282.0.0.0]
[4179] [0283.0.9.9] Moreover, a native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table II,
column 3, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., or an antibody against a polypeptide as indicated in
Table II, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp. or an antigenic part thereof, which
can be produced by standard techniques utilizing the polypeptid of
the present invention or fragment thereof, i.e., the polypeptide of
this invention. Preferred are monoclonal antibodies specifically
binding to polypeptide as indicated in Table II, columns 5 or 7,
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp.
[4180] [0284.0.0.9]: see [0284.0.0.0]
[4181] [0285.0.9.9] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp., or as encoded by a nucleic acid molecule
as indicated in Table I, columns 5 or 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp., or functional
homologues thereof.
[4182] [0286.0.9.9] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp. In another embodiment, the present
invention relates to a polypeptide comprising or consisting of a
consensus sequence as indicated in Table IV, column 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
whereby 20 or less, preferably 15 or 10, preferably 9, 8, 7, or 6,
more preferred 5 or 4, even more preferred 3, even more preferred
2, even more preferred 1, most preferred 0 of the amino acids
positions indicated can be replaced by any amino acid or, in an
further embodiment, can be replaced and/or absent. In one
embodiment, the present invention relates to the method of the
present invention comprising a polypeptide or to a polypeptide
comprising more than one consensus sequences (of an individual
line) as indicated in Table IV, column 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp
[4183] [0287.0.0.9] to [0290.0.0.9]: see [0287.0.0.0] to
[0290.0.0.0]
[4184] [0291.0.9.9] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[4185] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table II A or II B,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., by one or more amino acids. In one
embodiment, polypeptide distinguishes from a sequence as indicated
in Table II A or II B, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., by more than 5, 6, 7,
8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or 30
amino acids, even more preferred are more than 40, 50, or 60 amino
acids and, preferably, the sequence of the polypeptide of the
invention distinguishes from a sequence as indicated in Table II A
or II B, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp., by not more than 80% or 70% of the
amino acids, preferably not more than 60% or 50%, more preferred
not more than 40% or 30%, even more preferred not more than 20% or
10%. In an other embodiment, said polypeptide of the invention does
not consist of a sequence as indicated in Table II A orll B,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp.
[4186] [0292.0.0.9] see [0292.0.0.0]
[4187] [0293.0.9.9] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, said polypeptide
is having a sequence which distinguishes from a sequence as
indicated in Table II A or II B, columns 5 or 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp., by one or
more amino acids. In an other embodiment, the polypeptide of the
invention does not consist of the sequence as indicated in Table II
A or II B, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp. In a further embodiment, the
polypeptide of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical.
[4188] [0294.0.9.9] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp., which distinguishes over a
sequence as indicated in Table II A or II B, columns 5 or 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
by one or more amino acids, preferably by more than 5, 6, 7, 8 or 9
amino acids, preferably by more than 10, 15, 20, 25 or 30 amino
acids, even more preferred are more than 40, 50, or 60 amino acids
but even more preferred by less than 70% of the amino acids, more
preferred by less than 50%, even more preferred my less than 30% or
25%, more preferred are 20% or 15%, even more preferred are less
than 10%.
[4189] [0295.0.0.9] to [0297.0.0.9]: see [0295.0.0.0] to
[0297.0.0.0]
[4190] [0297.1.9.9] Non polypeptide of the invention-chemicals are
e.g. polypeptides having not the activity of a polypeptide
indicated in Table II, columns 3, 5 or 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp.
[4191] [0298.0.9.9] A polypeptide of the invention can participate
in the process of the present invention. The polypeptide or a
portion thereof comprises preferably an amino acid sequence which
is sufficiently homologous to an amino acid sequence as indicated
in Table II, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93
to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp. The portion of the protein is
preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table II, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp.
[4192] [0299.0.9.9] Further, the polypeptide can have an amino acid
sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.
The preferred polypeptide of the present invention preferably
possesses at least one of the activities according to the invention
and described herein. A preferred polypeptide of the present
invention includes an amino acid sequence encoded by a nucleotide
sequence which hybridizes, preferably hybridizes under stringent
conditions, to a nucleotide sequence as indicated in Table I,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., or which is homologous thereto, as defined
above.
[4193] [0300.0.9.9] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., in amino acid sequence due to natural
variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprise an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence of a sequence as indicated in Table II A or II
B, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp.
[4194] [0301.0.0.9] see [0301.0.0.0]
[4195] [0302.0.9.9] Biologically active portions of an polypeptide
of the present invention include peptides comprising amino acid
sequences derived from the amino acid sequence of the polypeptide
of the present invention or used in the process of the present
invention, e.g., an amino acid sequence as indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., or the amino acid sequence of a protein
homologous thereto, which include fewer amino acids than a full
length polypeptide of the present invention or used in the process
of the present invention or the full length protein which is
homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[4196] [0303.0.0.9]: see [0303.0.0.0]
[4197] [0304.0.9.9] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to
475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or
478 to 481, resp., but having differences in the sequence from said
wild-type protein. These proteins may be improved in efficiency or
activity, may be present in greater numbers in the cell than is
usual, or may be decreased in efficiency or activity in relation to
the wild type protein.
[4198] [0305.0.0.9] to [0306.0.0.9]: see [0305.0.0.0] to
[0306.0.0.0]
[4199] [0306.1.9.9] Preferably, the compound is a composition
comprising the essentially pure fine chemical, i.e. Vitamin E, i.e.
alpha-tocopherol, beta-tocopherol, and/or gamma-tocopherol or the
vitamin E precursor 2,3-Dimethyl-5-pythylquinol, respectively or a
recovered or isolated Vitamin E, i.e. alpha-tocopherol,
beta-tocopherol, and/or gamma-tocopherol or the vitamin E precursor
2,3-Dimethyl-5-pythylquinol, respectively, e.g. in free or in
protein- or membrane-bound form.
[4200] [0307.0.0.9] to [0308.0.0.9]: see [0307.0.0.0] to
[0308.0.0.0]
[4201] [0309.0.9.9] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., refers to a polypeptide having an amino acid
sequence corresponding to the polypeptide of the invention or used
in the process of the invention, whereas a "non-polypeptide of the
invention" or "other polypeptide" not being indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., refers to a polypeptide having an amino acid
sequence corresponding to a protein which is not substantially
homologous a polypeptide of the invention, preferably which is not
substantially homologous to a polypeptide as indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., e.g., a protein which does not confer the
activity described herein or annotated or known for as indicated in
Table II, column 3, lines 89 to 92 or 482 and/or lines 93 to 95 or
472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to
102 or 478 to 481, resp., and which is derived from the same or a
different organism. In one embodiment, a "non-polypeptide of the
invention" or "other polypeptide" not being indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., does not confer an increase of the respective
fine chemical in an organism or part thereof.
[4202] [0310.0.0.9] to [0334.0.0.9]: see [0310.0.0.0] to
[0334.0.0.0]
[4203] [0335.0.9.9] The dsRNAi method has proved to be particularly
effective and advantageous for reducing the expression of a nucleic
acid sequences as indicated in Table I, columns 5 or 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
and/or homologs thereof. As described inter alia in WO 99/32619,
dsRNAi approaches are clearly superior to traditional antisense
approaches. The invention therefore furthermore relates to
double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived therefrom), bring about altered
metabolic activity by the reduction in the expression of a nucleic
acid sequences as indicated in Table I, columns 5 or 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
and/or homologs thereof. In a double-stranded RNA molecule for
reducing the expression of an protein encoded by a nucleic acid
sequence sequences as indicated in Table I, columns 5 or 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
and/or homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[4204] [0336.0.0.9] to [0342.0.0.9]: see [0336.0.0.0] to
[0342.0.0.0]
[4205] [0343.0.9.9] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 89 to 92 or
482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or
476 to 477 and/or line 101 to 102 or 478 to 481, resp., or its
homolog is not necessarily required in order to bring about
effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table I, columns 5 or 7, lines 89 to 92 or 482 and/or
lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477
and/or line 101 to 102 or 478 to 481, resp., or homologs thereof of
the one organism, may be used to suppress the corresponding
expression in another organism.
[4206] [0344.0.0.9] to [0361.0.0.9]: see [0344.0.0.0] to
[0361.0.0.0]
[4207] [0362.0.9.9] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, the nucleic acid construct of
the invention, the antisense molecule of the invention, the vector
of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp. e.g. encoding a polypeptide having
protein activity, as indicated in Table II, column 3, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp. Due
to the above mentioned activity the respective fine chemical
content in a cell or an organism is increased. For example, due to
modulation or manipulation, the cellular activity of the
polypeptide of the invention or nucleic acid molecule of the
invention is increased, e.g. due to an increased expression or
specific activity of the subject matters of the invention in a cell
or an organism or a part thereof. In one embodiment, transgenic for
a polypeptide having an activity of a polypeptide as indicated in
Table II, columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to
95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or line
101 to 102 or 478 to 481, resp., means herein that due to
modulation or manipulation of the genome, an activity as annotated
for a polypeptide as indicated in Table II, column 3, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp. e.g.
having a sequence as indicated in Table II, columns 5 or 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
is increased in a cell or an organism or a part thereof. Examples
are described above in context with the process of the
invention
[4208] [0363.0.0.9]: see [0363.0.0.0]
[4209] [0364.0.9.9] A naturally occurring expression cassette--for
example the naturally occurring combination of a promoter of a
polypeptide of the invention as indicated in Table II, column 3,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp., with the corresponding protein-encoding sequence as
indicated in Table I, column 3, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp., --becomes a transgenic
expression cassette when it is modified by non-natural, synthetic
"artificial" methods such as, for example, mutagenization. Such
methods have been described (U.S. Pat. No. 5,565,350; WO 00/15815;
also see above).
[4210] [0365.0.0.9] to [0373.0.0.9]: see [0365.0.0.0] to
[0373.0.0.0]
[4211] [0374.0.9.9] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. Vitamin E or its precursor
2,3-dimethyl-5-phytylquinol resp., in particular, alpha-, beta-,
and/or gamma-tocopherol resp., produced in the process according to
the invention may, however, also be isolated from the plant in the
form of their free vitamin E or bound in or to compounds or
moieties. Vitamin E or its precursor 2,3-dimethyl-5-phytylquinol
resp., in particular, alpha-, beta-, and/or gamma-tocopherol resp.,
produced by this process can be harvested by harvesting the
organisms either from the culture in which they grow or from the
field. This can be done via expressing, grinding and/or extraction,
salt precipitation and/or ion-exchange chromatography or other
chromatographic methods of the plant parts, preferably the plant
seeds, plant fruits, plant tubers and the like.
[4212] [0375.0.0.9] to [0376.0.0.9]: see [0375.0.0.0] to
[0376.0.0.0]
[4213] [0377.0.9.9] Accordingly, the present invention relates also
to a process according to the present invention whereby the
produced vitamin E comprising composition or the produced the
respective fine chemical is isolated.
[4214] [0378.0.9.9]: In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the vitamin E or
its precursor 2,3-dimethyl-5-phytylquinol resp., in particular,
alpha-, beta-, and/or gamma-tocopherol, resp., produced in the
process can be isolated. The resulting recovered, isolated or
purified vitamin E or its precursor 2,3-dimethyl-5-phytylquinol
resp., in particular, alpha-, beta-, and/or gamma-tocopherol resp.,
e.g. compositions comprising the former, can, if appropriate,
subsequently be further purified, if desired mixed with other
active ingredients such as vitamins, amino acids, carbohydrates,
antibiotics and the like, and, if appropriate, formulated.
[4215] [0379.0.9.9] In one embodiment, the vitamin E or its
precursor 2,3-dimethyl-5-phytylquinol resp., in particular, alpha-,
beta-, and/or gamma-tocopherol resp., is a mixture comprising of
one or more the respective fine chemicals. In one embodiment, the
respective fine chemical means here vitamin E, in particular
alpha-, beta-, or gamma-tocopherol. In one embodiment, vitamin E
means here a mixture of the respective fine chemicals.
[4216] [0380.0.9.9] The vitamin E or its precursor
2,3-dimethyl-5-phytylquinol, resp., in particular, alpha-, beta-,
and/or gamma-tocopherol resp., obtained in the process are suitable
as starting material for the synthesis of further products of
value. For example, they can be used in combination with each other
or alone for the production of pharmaceuticals, foodstuffs, animal
feeds or cosmetics. Accordingly, the present invention relates a
method for the production of pharmaceuticals, food stuff, animal
feeds, nutrients or cosmetics comprising the steps of the process
according to the invention, including the isolation of the Vitamin
E- or its precursor 2,3-dimethyl-5-phytylquinol-comprising
composition produced or the respective fine chemical produced if
desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the Vitamin E or its precursor
2,3-dimethyl-5-phytylquinol resp., produced in the process or of
the transgenic organisms in animal feeds, foodstuffs, medicines,
food supplements, cosmetics or pharmaceuticals.
[4217] [0381.0.0.9] to [0382.0.0.9]: see [0381.0.0.0] to
[0382.0.0.0]
[4218] [0383.0.9.9]: ./.
[4219] [0384.0.0.9]: see [0384.0.0.0]
[4220] [0385.0.9.9] The fermentation broths obtained in this way,
containing in particular vitamin E or its precursor
2,3-dimethyl-5-phytylquinol, resp., in particular, alpha-, beta-,
and/or gamma-tocopherol, resp., in mixtures with other compounds,
in particular with other vitamins or e.g. with carotenoids, e.g.
with astaxanthin, or fatty acids or containing microorganisms or
parts of microorganisms, like plastids, containing vitamin E or its
precursor 2,3-dimethyl-5-phytylquinol resp., in particular, alpha-,
beta-, and/or gamma-tocopherol resp., in mixtures with other
compounds, e.g. with carotenoids, normally have a dry matter
content of from 7.5 to 25% by weight. Sugar-limited fermentation is
additionally advantageous, e.g. at the end, for example over at
least 30% of the fermentation time. This means that the
concentration of utilizable sugar in the fermentation medium is
kept at, or reduced to, 0 to 3 g/l during this time. The
fermentation broth is then processed further. Depending on
requirements, the biomass can be removed or isolated entirely or
partly by separation methods, such as, for example, centrifugation,
filtration, decantation, coagulation/flocculation or a combination
of these methods, from the fermentation broth or left completely in
it.
[4221] The fermentation broth can then be thickened or concentrated
by known methods, such as, for example, with the aid of a rotary
evaporator, thin-film evaporator, falling film evaporator, by
reverse osmosis or by nanofiltration. This concentrated
fermentation broth can then be worked up by freeze-drying, spray
drying, spray granulation or by other processes.
[4222] As vitamin E is often localized in membranes or plastids, in
one embodiment it is advantageous to avoid a leaching of the cells
when the biomass is isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth. The dry biomass can directly
be added to animal feed, provided the vitamin E concentration is
sufficiently high and no toxic compounds are present. In view of
the instability of vitamin E, conditions for drying, e.g. spray or
flash-drying, can be mild and can be avoiding oxidation and
cis/trans isomerization. For example antioxidants, e.g. BHT,
ethoxyquin or other, can be added. In case the vitamin E
concentration in the biomass is to dilute, solvent extraction can
be used for their isolation, e.g. with alcohols, ether or other
organic solvents, e.g. with methanol, ethanol, acetone, alcoholic
potassium hydroxide, glycerol-fenol, liquefied fenol or for example
with acids or bases, like trichloroacetatic acid or potassium
hydroxide. A wide range of advantageous methods and techniques for
the isolation of vitamin E can be found in the state of the
art.
[4223] [0386.0.9.9] Accordingly, it is possible to further purify
the produced vitamin E or its precursor 2,3-dimethyl-5-phytylquinol
resp., in particular, alpha-, beta-, and/or gamma-tocopherol resp.
For this purpose, the product-containing composition, e.g. a total
or partial lipid extraction fraction using organic solvents, e.g.
as described above, is subjected for example to a saponification to
remove triglycerides, partition between e.g. hexane/methanol
(separation of non-polar epiphase from more polar hypophasic
derivates) and separation via e.g. an open column chromatography or
HPLC in which case the desired product or the impurities are
retained wholly or partly on the chromatography resin. These
chromatography steps can be repeated if necessary, using the same
or different chromatography resins. The skilled worker is familiar
with the choice of suitable chromatography resins and their most
effective use.
[4224] [0387.0.0.9] to [0392.0.0.9]: see [0387.0.0.0] to
[0392.0.0.0]
[4225] [0393.0.9.9] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [4226] (b) contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [4227] (c) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table I, preferably I B, columns
5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478
to 481, resp., and, optionally, isolating the full length cDNA
clone or complete genomic clone; [4228] (d) introducing the
candidate nucleic acid molecules in host cells, preferably in a
plant cell or a microorganism, appropriate for producing the
respective fine chemical; [4229] (e) expressing the identified
nucleic acid molecules in the host cells; [4230] (f) assaying the
respective fine chemical level in the host cells; and [4231] (g)
identifying the nucleic acid molecule and its gene product which
expression confers an increase in the respective the respective
fine chemical level in the host cell after expression compared to
the wild type.
[4232] [0394.0.0.9] to [0398.0.0.9]: see [0394.0.0.0] to
[0398.0.0.0]
[4233] [0399.0.9.9] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increased respective fine chemical production in a plant
or microorganism, comprising the steps:
(g) culturing a cell or tissue or microorganism or maintaining a
plant expressing the polypeptide according to the invention or a
nucleic acid molecule encoding said polypeptide and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and the polypeptide of the present invention or used
in the process of the invention; and (h) identifying if the
compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout
system.
[4234] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in fine chemical production.
[4235] [00399.1.9.9] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or line 101 to 102
or 478 to 481, resp., or a homolog thereof, e.g. comparing the
phenotype of nearly identical organisms with low and high activity
of a protein as indicated in Table II, columns 5 or 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.,
after incubation with the drug.
[4236] [0400.0.0.9] to [0416.0.0.9]: see [0400.0.0.0] to
[0416.0.0.0]
[4237] [0417.0.9.9] The nucleic acid molecule of the invention or
used in the method of the invention, the vector of the invention or
the nucleic acid construct of the invention may also be useful for
the production of organisms resistant to inhibitors of the vitamin
E production biosynthesis pathways. In particular, the
overexpression of the polypeptide of the present invention may
protect an organism such as a microorganism or a plant against
inhibitors, which block the vitamin E, in particular the respective
fine chemical synthesis in said organism.
[4238] As vitamin E can protect organisms against damages of
oxidative stress, especially singlet oxygen, a increased level of
the respective fine chemical can protect plants against herbicides
which cause the toxic build-up of oxidative compounds, e.g. singlet
oxygen. For example, inhibition of the protoporphorineogen oxidase
(Protox), an enzyme important in the synthesis of chlorophyll and
heme biosynthesis results in the loss of chlorophyll and
carotenoids and in leaky membranes; the membrane destruction is due
to creation of free oxygen radicals (which is also reported for
other classic photosynthetic inhibitor herbicides).
[4239] Accordingly, in one embodiment, the increase of the level of
the respective fine chemical is used to protect plants against
herbicides destroying membranes due to the creation of free oxygen
radicals.
[4240] Examples of inhibitors or herbicides building up oxidative
stress are aryl triazion, e.g. sulfentrazone, carfentrazone; or
diphenylethers, e.g. acifluorfen, lactofen, or oxyfluorfen; or
N-Phenylphthalimide, e.g. flumiclorac or flumioxazin; substituted
ureas, e.g. fluometuron, tebuthiuron, diuron, or linuron;
triazines, e.g. atrazine, prometryn, ametryn, metributzin,
prometon, simazine, or hexazinone: or uracils, e.g. bromacil or
terbacil.
[4241] [0418.0.0.9] to [0423.0.0.9]: see [0418.0.0.0] to
[0423.0.0.0]
[4242] [0424.0.9.9] Accordingly, the nucleic acid of the invention
or the nucleic acid molecule used in the method of the invention,
the polypeptide of the invention or the polypeptide used in the
method of the invention, the nucleic acid construct of the
invention, the organisms, the host cell, the microorganisms, the
plant, plant tissue, plant cell, or the part thereof of the
invention, the vector of the invention, the agonist identified with
the method of the invention, the nucleic acid molecule identified
with the method of the present invention, can be used for the
production of the respective fine chemical or of the respective
fine chemical and one or more other carotenoids, vitamins or fatty
acids. In one embodiment, in the process of the present invention,
the produced vitamin E is used to protect fatty acids against
oxidization, e.g. it is in a further step added in a pure form or
only partly isolated to a composition comprising fatty acids.
[4243] Accordingly, the nucleic acid of the invention or the
nucleic acid molecule used in the method of the invention, or the
nucleic acid molecule identified with the method of the present
invention or the complement sequences thereof, the polypeptide of
the invention or the polypeptide used in the method of the
invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the respective fine chemical in a organism or part
thereof, e.g. in a cell.
[4244] [0424.1.9.9] In a further embodiment the present invention
relates to the use of the antagonist of the present invention, the
plant of the present invention or a part thereof, the microorganism
or the host cell of the present invention or a part thereof for the
production a cosmetic composition or a pharmaceutical composition.
Such a composition has an antioxidative activity, photoprotective
activity, can be used to protect, treat or heal the above mentioned
diseases, e.g. rhypercholesterolemic or cardiovascular diseases,
certain cancers, and cataract formation or as immunostimulatory
agent.
[4245] The vitamin E can be also used as stabilizer of other
colours or oxygen sensitive compounds, like fatty acids, in
particular unsaturated fatty acids.
[4246] [0425.0.0.9] to [0434.0.0.9]: see [0425.0.0.0] to
[0434.0.0.0]
[0435.0.9.9] Example 3
In-Vivo and In-Vitro Mutagenesis
[4247] [0436.0.9.9] An in vivo mutagenesis of organisms such as
algae (e.g. Spongiococcum sp, e.g. Spongiococcum exentricum,
Chlorella sp., Haematococcus, Phaedactylum tricornatum, Volvox or
Dunaliella), Synechocystis sp. PCC 6803, Physcometrella patens,
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of vitamin E can be carried
out by passing a plasmid DNA (or another vector DNA) containing the
desired nucleic acid sequence or nucleic acid sequences, e.g. the
nucleic acid molecule of the invention or the vector of the
invention, through E. coli and other microorganisms (for example
Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are
not capable of maintaining the integrity of its genetic
information. Usual mutator strains have mutations in the genes for
the DNA repair system [for example mutHLS, mutD, mutT and the like;
for comparison, see Rupp, W. D. (1996) DNA repair mechanisms in
Escherichia coli and Salmonella, pp. 2277-2294, ASM: Washington].
The skilled worker knows these strains. The use of these strains is
illustrated for example in Greener, A. and Callahan, M. (1994)
Strategies 7; 32-34.
[4248] In-vitro mutation methods such as increasing the spontaneous
mutation rates by chemical or physical treatment are well known to
the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widely used as
chemical agents for random in-vitro mutagenesis. The most common
physical method for mutagenesis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[4249] Site-directed mutagenesis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagenesis. The cloud of this method involves cloning of the
nucleic acid sequence of the invention into an M13 or phagemid
vector, which permits recovery of single-stranded recombinant
nucleic acid sequence. A mutagenic oligonucleotide primer is then
designed whose sequence is perfectly complementary to nucleic acid
sequence in the region to be mutated, but with a single difference:
at the intended mutation site it bears a base that is complementary
to the desired mutant nucleotide rather than the original. The
mutagenic oligonucleotide is then allowed to prime new DNA
synthesis to create a complementary full-length sequence containing
the desired mutation. Another site-directed mutagenesis method is
the PCR mismatch primer mutagenesis method also known to the
skilled person. Dpnl site-directed mutagenesis is a further known
method as described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[4250] Positive mutation events can be selected by screening the
organisms for the production of the desired fine chemical.
[4251] [0436.1.0.9] see [0436.1.0.0]
[4252] [0437.0.9.9] to [0445.0.9.9]: see [0437.0.5.5] to
[0445.0.5.5]
[4253] [0445.1.9.9] Synechocystis sp. PCC 6803 is a unicellular,
non-nitrogen-fixing cyanobacterium which has undergone thorough
genetic investigation (Churin et al. (1995) J Bacteriol 177:
3337-3343), can easily be transformed (Williams (1988) Methods
Enzymol 167:766-778) and has a very active homologous recombination
potential. The strain PCC 6803 was isolated as Aphanocapsa N-1 from
fresh water in California, USA, by R. Kunisawa in 1968 and is now
obtainable through the "Pasteur Culture Collection of Axenic
Cyanobacterial Strains" (PCC), Unite de Physiologie Microbienne,
Paris, France. The complete genomic sequence of Synechocystis sp.
PCC 6803 has been published from 1995 (Kaneko et al. (1995) DNA
Research 2:153-166; Kaneko et al. (1995) DNA Research 2:191-198;
Kaneko et al. (1996) DNA Research 3:109-136; Kaneko et al. (1996)
DNA Research 3:185-209; Kaneko and Tabata (1997) Plant Cell Physiol
38:1171-1176; Kotani and Tabata (1998) Annu Rev Plant Physiol
49:151-171) and is published on the Internet
(http://www.kazusa.or.jp/cyano/cyano.html) under the name
"CyanoBase". Efficient expression systems for Synechocystis 6803
are described in the literature (Mermet-Bouvier et al. (1993) Curr
Microbiol 27:323-327; Mermet-Bouvier and Chauvat (1993) Curr
Microbiol 28:145-148; Murphy and Stevens (1992) Appl Environ
Microbiol 58:1650-1655; Takeshima et al. (1994) Proc Natl Acad Sci
USA 91:9685-9689; Xiaoqiang et al. (1997) Appl Environ Microbiol
63:4971-4975; Ren et al. (1998) FEMS Microbiol Lett
158:127-132).
[4254] [0445.2.9.9] Growing synechocystis
[4255] The cells of Synechocystis sp. PCC 6803 can be normally
cultured autotrophically in BG11 medium. They have a diameter of
2.3 to 2.5 .mu.m. For example, a cyanobacterium Synechocystis sp.
PCC 6803 strain which is glucose-tolerant can be used, i.e. it is
also able to grow heterotrophically in the dark with only a few
minutes of weak blue light illumination per day. The culture
conditions were developed by Anderson and McIntosh (Anderson and
McIntosh (1991) J Bacteriol 173:2761-2767) and called
light-activated heterotrophic growth (LANG). This makes it possible
to cultivate these cyanobacteria without continuous photosynthesis
and thus without production of oxygen.
[4256] BG 11 culture medium for Synechocystis
[4257] Stock solution 100.times.BG11:
TABLE-US-00032 NaNO.sub.3 1.76M = 149.58 g MgSO.sub.4 .times.
7H.sub.2O 30.4 mM = 7.49 g CaCl.sub.2 .times. 2H.sub.2O 24.5 mM =
3.6 g Citric acid 3.12 mM = 0.6 g Na EDTA pH 8 0.279 mM = 0.104
g
[4258] The weighed substances can be dissolved in 900 ml of
H.sub.2O and made up to 1000 ml with 100 ml of the trace metal mix
stock 1000.times.. The solution thus obtained is used as stock
solution.
[4259] Trace Metal Mix Stock 1000.times.:
TABLE-US-00033 H.sub.3BO.sub.3 46.3 mM = 2.86 g/l MnCl.sub.2
.times. 4 H.sub.2O 4.15 mM = 1.81 g/l ZnSO.sub.4 .times. 7 H.sub.2O
0.77 mM = 0.222 g/l Na.sub.2MoO.sub.4 .times. 2 H.sub.2O 1.61 mM =
0.39 g/l CuSO.sub.4 .times. 5 H.sub.2O 0.32 mM = 0.079 g/l
Co(NO.sub.3).sub.2 .times. 6 H.sub.2O 0.17 mM = 0.0494 g/l
[4260] The following solutions are required for 1 liter of BG11
culture solution:
1. 10 ml of stock solution 100.times.BG 11
2. 1 ml Na.sub.2CO.sub.3 (189 mM)
3. 5 ml TES (1 M, pH 8)
4. 1 ml K.sub.2PO.sub.4 (175 mM)
[4261] Whereas solution 2. and 3. ought to be sterilized by
filtration, solution 4 must be autoclaved. The complete BG11
culture solution must be autoclaved before use and then be mixed
with 1 ml of iron ammonium citrate (6 mg/ml) which has previously
been sterilized by filtration. The iron ammonium citrate should
never be autoclaved. For agar plates, 1.5% (w/v) bacto agar are
added per liter of BG11 medium.
[4262] [0445.3.9.9] Amplification and cloning of DNA from
Synechocystis spec. PCC 6803
[4263] The DNA can be amplified by the polymerase chain reaction
(PCR) from Synechocystis spec. PCC 6803 by the method of Crispin A.
Howitt (Howitt C A (1996) BioTechniques 21:32-34).
[4264] [0445.4.9.9] Tocopherol production in Synechocystis spec.
PCC 6803
[4265] The cells of each of independent Synechocystis spec. PCC
6803 strains cultured on the BG-11 km agar medium, and
untransformed wild-type cells (on BG11 agar medium without
kanamycin) can be used to inoculate liquid cultures. For this,
cells of a mutant or of the wild-type Synechocystis spec. PCC 6803
are transferred from plate into 10 ml of liquid culture in each
case. These cultures are cultivated at 28.degree. C. and 30 .mu.mol
photons*(m.sup.2*s).sup.-1 (30 .mu.E) for about 3 days. After
determination of the OD.sub.730 of the individual cultures, the
OD.sub.730 of all cultures is synchronized by appropriate dilutions
with BG-11 (wild types) or e.g. BG-11 km (mutants). These cell
density-synchronized cultures are used to inoculate three cultures
of the mutant and of the wild-type control. It is thus possible to
carry out biochemical analyses using in each case three
independently grown cultures of a mutant and of the corresponding
wild types. The cultures are grown until the optical density was
OD.sub.730=0.3.
[4266] The cell culture medium is removed by centrifugation in an
Eppendorf bench centrifuge at 14000 rpm twice. The subsequent
disruption of the cells and extraction of the tocopherols or
vitamin E take place by incubation in an Eppendorf shaker at
30.degree. C., 1000 rpm in 100% methanol for 15 minutes twice,
combining the supernatants obtained in each case.
[4267] In order to avoid oxidation, the resulting extracts can be
analyzed immediate after the extraction with the aid of a Waters
Allience 2690 HPLC system. Tocopherols and vitamin E is separated
on a reverse phase column (ProntoSil 200-3-C30, Bischoff) with a
mobile phase of 100% methanol, and identified by means of a
standard (Merck). The fluorescence of the substances (excitation
295 nm, emission 320 nm), which is detected with the aid of a Jasco
FP 920 fluorescence detector, can serve as detection system.
[4268] [0446.0.0.9] to [0450.0.0.9]: see [0446.0.0.0] to
[0450.0.0.0]
[4269] [0451.0.0.9]: see [0451.0.5.5]
[4270] [0452.0.0.9] to [0454.0.0.9]: see [0452.0.0.0] to
[0454.0.0.0]
[4271] [0455.0.9.9] Characterization of the Transgenic Plants.
[4272] In order to confirm that vitamin E biosynthesis in the
transgenic plants is influenced by the expression of the
polypeptides described herein, the tocopherol and vitamin E
contents in leaves and seeds of the plants transformed with the
described constructs
[4273] (Arabidopsis thaliana, Brassica napus and Nicotiana tabacum)
are analyzed. For this purpose, the transgenic plants are grown in
a greenhouse, and plants which express the gene coding for
polypeptide of the invention or used in the method of the invention
are identified at the Northern level. The tocopherol content or the
vitamin E content in leaves and seeds of these plants is measured.
In all, the tocopherol concentration is raised by comparison with
untransformed plants.
[4274] [0456.0.0.9]: see [0456.0.0.0]
[0457.0.9.9] Example 9
Purification of the Vitamin E or its Precursor
2,3-Dimethyl-5-phytylquinol
[4275] [0458.0.9.9] Abbreviations: GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[4276] The unambiguous detection for the presence of vitamin E,
like alpha-, beta-, or gamma-tocopherol, or its precursor
2,3-dimethyl-5-phytylquinol can be obtained by analyzing
recombinant organisms using analytical standard methods: GC, GC-MS
or TLC, as described (1997, in: Advances on Lipid Methodology,
Fourth Edition: Christie, Oily Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353).
[4277] The total vitamin E produced in the organism for example in
yeasts used in the inventive process can be analysed for example
according to the following procedure: The material such as yeasts,
E. coli or plants to be analyzed can be disrupted by sonication,
grinding in a glass mill, liquid nitrogen and grinding or via other
applicable methods.
[4278] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[4279] A typical sample pretreatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography. E.g.:
[4280] [0459.0.9.9]: If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the vitamin E, like alpha-, beta-, or
gamma-tocopherol, or its precursor 2,3-dimethyl-5-phytylquinol, can
be further purified with a so-called RTHPLC. As eluent
acetonitrile/water or chloroform/acetonitrile mixtures can be used.
If necessary, these chromatography steps may be repeated, using
identical or other chromatography resins. The skilled worker is
familiar with the selection of suitable chromatography resin and
the most effective use for a particular molecule to be
purified.
[4281] [0460.0.9.9] see [0460.0.0.0]
[0461.0.9.9] Example 10
Cloning SEQ ID NO: 8673 for the Expression in Plants
[4282] [0462.0.0.9]: see [0462.0.0.0]
[4283] [0463.0.9.9] SEQ ID NO: 8673 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[4284] [0464.0.0.9] to [0466.0.0.9]: see [0464.0.0.0] to
[0466.0.0.0]
[4285] [0467.0.9.9] The following primer sequences were selected
for the gene SEQ ID NO: 8673:
TABLE-US-00034 i) forward primer (SEQ ID NO: 8677) atggggaaga
gagtatacga tcca ii) reverse primer (SEQ ID NO: 8678) tcactccagc
ttaaacatgg cgg
[4286] [0468.0.0.9] to [0479.0.0.9]: see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.9.9]: Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 8673
[4287] [0481.0.0.9] to [0513.0.0.9]: see [0481.0.0.0] to
[0513.0.0.0]
[4288] [0514.0.9.9]: As an alternative, the vitamin E, like alpha-,
beta-, or gamma-tocopherol, or its precursor
2,3-dimethyl-5-phytylquinol, can be detected advantageously as
described in Deli, J. & Molnar, P., Paprika carotenoids:
Analysis, isolation, structure elucidation. Curr. Org. Chem. 6,
1197-1219 (2004) or Fraser, P. D., Pinto, M. E., Holloway, D. E.
& Bramley, P. M. Technical advance: application of
high-performance liquid chromatography with photodiode array
detection to the metabolic profiling of plant isoprenoids. Plant J.
24, 551-558 (2000).
[4289] The results of the different plant analyses can be seen from
the table 1, which follows:
TABLE-US-00035 TABLE 1 ORF Metabolite Method Min Max YPL268W
2,3-Dimethy1-5- LC 1,44 1,51 phytylquinol YFL013C 2,3-Dimethy1-5-
LC 1,97 5,40 phytylquinol b1829 2,3-Dimethy1-5- LC 1,48 3,83
phytylquinol b1827 2,3-Dimethy1-5- LC 1,76 1,91 phytylquinol
YFL019C 2,3-Dimethy1-5- LC 1,97 5,40 phytylquinol b2699
alpha-Tocopherol GC 1,51 2,96 b1829 alpha-Tocopherol LC 1,39 1,90
b0112 alpha-Tocopherol LC 1,43 1,63 b0161 alpha-Tocopherol GC 1,50
2,38 b0970 alpha-Tocopherol LC 1,47 1,54 b3160 alpha-Tocopherol GC
1,47 1,91 b4063 alpha-Tocopherol LC 1,16 1,47 b1827 beta-Tocopherol
LC 1,34 2,16 b0986 gamma + beta- LC 1,35 1,61 Tocopherol b0175
gamma + beta- LC 1,34 1,40 Tocopherol b0785 gamma + beta- LC 1,34
1,85 Tocopherol b3938 gamma + beta- LC 1,50 2,20 Tocopherol YFL053W
gamma + beta- LC 1,45 2,23 Tocopherol b1829 gamma + beta- LC 1,40
4,25 Tocopherol
[4290] [0515.0.0.9] to [0552.0.0.9]: see [0515.0.0.0] to
[0552.0.0.0] including [0530.1.0.0] to [0530.6.0.0]
[0552.1.9.9] Example 15
Metabolite Profiling Info from Zea mays
[4291] Zea mays plants were engineered, grown and analyzed as
described in Example 14c.
[4292] The results of the different Zea mays plants analysed can be
seen from Table 2 which follows:
TABLE-US-00036 TABLE 2 ORF_NAME Metabolite Min Max b0112
alpha-Tocopherol 1.61 2.33 b0970 alpha-Tocopherol 1.13 2.45 b0986
beta/gamma-Tocopherol 1.84 4.62 b1829 alpha-Tocopherol 1.22 1.86
b1829 beta/gamma-Tocopherol 1.62 2.74 b1829 2,3-Dimethyl-5- 1.64
2.39 phytylquinol b2699 alpha-Tocopherol 1.71 1.93
[4293] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in alpha-Tocopherol and/or
beta/gamma-Tocopherol and/or 2,3-Dimethyl-5-phytylquinol in
genetically modified corn plants expressing the E. coli nucleic
acid sequence b0112, b0970, b0986, b1829 or b2699 resp.
[4294] In one embodiment, in case the activity of the E. coli
protein b0112 or its homologs, e.g. "a protein with an aromatic
amino acid transport protein (APC family)-activity", is increased
in corn plants, preferably, an increase of the fine chemical
alpha-Tocopherol between 61% and 133% is conferred.
[4295] In one embodiment, in case the activity of the E. coli
protein b0970 or its homologs, e.g. "its activity is being defined
as probable glutamate receptor", is increased in corn plants,
preferably, an increase of the fine chemical alpha-Tocopherol
between 13% and 145% is conferred.
[4296] In one embodiment, in case the activity of the E. coli
protein b0986 or its homologs, e.g. "a lipoprotein", is increased
in corn plants, preferably, an increase of the fine chemical beta
and/or gamma-Tocopherol between 84% and 362% is conferred.
[4297] In one embodiment, in case the activity of the E. coli
protein b1829 or its homologs, e.g. "having a heat shock protein
with protease activity", is increased in corn plants, preferably,
an increase of the fine chemical alpha-Tocopherol between 22% and
86% is conferred and/or an increase of the fine chemical beta
and/or gamma-Tocopherol between 62% and 174% is conferred and/or an
increase of the fine chemical 2,3-Dimethyl-5-phytylquinol between
64% and 139% is conferred.
[4298] In one embodiment, in case the activity of the E. coli
protein b2699 or its homologs, e.g. "a protein with a DNA strand
exchange and recombination protein with protease and
nuclease-activity", is increased in corn plants, preferably, an
increase of the fine chemical alpha-Tocopherol between 71% and 93%
is conferred.
[4299] [0552.2.0.9]: see [0552.2.0.0]
[4300] [0553.0.9.9] [4301] 1. A process for the production of
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol, which
comprises [4302] (a) increasing or generating the activity of one
or more proteins as indicated in Table II, columns 5 or 7, lines 89
to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or lines 101 to 102 or 478 to 481 resp.,
preferably Table II B, column 7, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
line 101 to 102 or 478 to 481, resp., or a functional equivalent
thereof in a non-human organism, or in one or more parts thereof;
and [4303] (b) growing the organism under conditions which permit
the production of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol in said organism. [4304] 2. A process
for the production of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol, comprising the increasing or
generating in an organism or a part thereof the expression of at
least one nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [4305] a) nucleic acid
molecule encoding of a polypeptide as indicated in Table II,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or lines 101 to 102
or 478 to 481, resp., preferably Table II B, column 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp., or a
fragment thereof, which confers an increase in the amount of
vitamin E or its precursor 2,3-dimethyl-5-phytylquinolin an
organism or a part thereof; [4306] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472
to 475 and/or lines 96 to 100 or 476 to 477 and/or lines 101 to 102
or 478 to 481, resp., preferably Table I B, column 7, lines 89 to
92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96 to
100 or 476 to 477 and/or line 101 to 102 or 478 to 481, resp.;
[4307] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol in an organism or a part thereof;
[4308] d) nucleic acid molecule which encodes a polypeptide which
has at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol in an organism or a part thereof;
[4309] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of vitamin E or
its precursor 2,3-dimethyl-5-phytylquinol in an organism or a part
thereof; [4310] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, column 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or lines 101 to 102 or 478 to 481 and
conferring an increase in the amount of vitamin E or its precursor
2,3-dimethyl-5-phytylquinolin an organism or a part thereof; [4311]
g) nucleic acid molecule encoding a polypeptide which is isolated
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (f) and conferring
an increase in the amount of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol in an organism or a part thereof;
[4312] h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table IV, column 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or lines 101 to 102 or 478 to 481 and conferring an
increase in the amount of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol in an organism or a part thereof; and
[4313] i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of vitamin E or its
precursor 2,3-dimethyl-5-phytylquinol in an organism or a part
thereof. [4314] or comprising a sequence which is complementary
thereto. [4315] 3. The process of claim 1 or 2, wherein
2,3-dimethyl-5-phytylquinol is isolated. [4316] 4. The process of
any one of claims 1 to 3, comprising the following steps: [4317]
(a) selecting an organism or a part thereof expressing a
polypeptide encoded by the nucleic acid molecule characterized in
claim 2; [4318] (b) mutagenizing the selected organism or the part
thereof; [4319] (c) comparing the activity or the expression level
of said polypeptide in the mutagenized organism or the part thereof
with the activity or the expression of said polypeptide of the
selected organisms or the part thereof; [4320] (d) selecting the
mutated organisms or parts thereof, which comprise an increased
activity or expression level of said polypeptide compared to the
selected organism or the part thereof; [4321] (e) optionally,
growing and cultivating the organisms or the parts thereof; and
[4322] (f) recovering, and optionally isolating, the free or bound
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol produced by
the selected mutated organisms or parts thereof. [4323] 5. The
process of any one of claims 1 to 4, wherein the activity of said
protein or the expression of said nucleic acid molecule is
increased or generated transiently or stably. [4324] 6. An isolated
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: [4325] a) nucleic acid molecule
encoding of a polypeptide as indicated in Table II, columns 5 or 7,
lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or lines 101 to 102 or 478 to 481
resp., preferably Table II B, column 7, lines 89 to 92 or 482
and/or lines 93 to 95 or 472 to 475 and/or lines 96 to 100 or 476
to 477 and/or line 101 to 102 or 478 to 481, resp., or a fragment
thereof, which confers an increase in the amount of vitamin E or
its precursor 2,3-dimethyl-5-phytylquinol in an organism or a part
thereof; [4326] b) nucleic acid molecule comprising of a nucleic
acid molecule as indicated in
[4327] Table I, columns 5 or 7, lines 89 to 92 or 482 and/or lines
93 to 95 or 472 to 475 and/or lines 96 to 100 or 476 to 477 and/or
lines 101 to 102 or 478 to 481, resp., preferably Table I B, column
7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or
lines 96 to 100 or 476 to 477 and/or line 101 to 102 or 478 to 481,
resp.,; [4328] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of vitamin E or its
precursor 2,3-dimethyl-5-phytylquinol in an organism or a part
thereof; [4329] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of vitamin E or
its precursor 2,3-dimethyl-5-phytylquinol in an organism or a part
thereof; [4330] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of vitamin E or
its precursor 2,3-dimethyl-5-phytylquinol in an organism or a part
thereof; [4331] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, column 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or lines 101 to 102 or 478 to 481 and
conferring an increase in the amount of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol in an organism or a part thereof;
[4332] g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of vitamin E or its
precursor 2,3-dimethyl-5-phytylquinol in an organism or a part
thereof; [4333] h) nucleic acid molecule encoding a polypeptide
comprising a consensus as indicated in Table IV, columns 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or lines 101 to 102 or 478 to 481 and
conferring an increase in the amount of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol in an organism or a part thereof; and
[4334] i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of vitamin E or its
precursor 2,3-dimethyl-5-phytylquinol in an organism or a part
thereof. [4335] whereby the nucleic acid molecule distinguishes
over the sequence as indicated in Table I A, columns 5 or 7, lines
89 to 92 or 482 and/or lines 93 to 95 or 472 to 475 and/or lines 96
to 100 or 476 to 477 and/or lines 101 to 102 or 478 to 481 by one
or more nucleotides. [4336] 7. A nucleic acid construct which
confers the expression of the nucleic acid molecule of claim 6,
comprising one or more regulatory elements. [4337] 8. A vector
comprising the nucleic acid molecule as claimed in claim 6 or the
nucleic acid construct of claim 7. [4338] 9. The vector as claimed
in claim 8, wherein the nucleic acid molecule is in operable
linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [4339] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [4340] 11. The host cell of claim 10, which is a
transgenic host cell. [4341] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [4342] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [4343] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table II A, columns 5
or 7, lines 89 to 92 or 482 and/or lines 93 to 95 or 472 to 475
and/or lines 96 to 100 or 476 to 477 and/or lines 101 to 102 or 478
to 481 by one or more amino acids [4344] 15. An antibody, which
binds specifically to the polypeptide as claimed in claim 14.
[4345] 16. A plant tissue, propagation material, harvested material
or a plant comprising the host cell as claimed in claim 12 which is
plant cell or an Agrobacterium. [4346] 17. A method for screening
for agonists and antagonists of the activity of a polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol in an organism or a part thereof
comprising: [4347] (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of vitamin E or its precursor 2,3-dimethyl-5-phytylquinol in
an organism or a part thereof with a candidate compound or a sample
comprising a plurality of compounds under conditions which permit
the expression the polypeptide; [4348] (b) assaying the vitamin E
level or its precursor 2,3-dimethyl-5-phytylquinol level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [4349] (c)
identifying a agonist or antagonist by comparing the measured
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol level or
polypeptide expression level with a standard vitamin E or its
precursor 2,3-dimethyl-5-phytylquinol acid or polypeptide
expression level measured in the absence of said candidate compound
or a sample comprising said plurality of compounds, whereby an
increased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an agonist and
a decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[4350] 18. A process for the identification of a compound
conferring increased vitamin E or its precursor
2,3-dimethyl-5-phytylquinol production in a plant or microorganism,
comprising the steps: [4351] (a) culturing a plant cell or tissue
or microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of vitamin
E or its precursor 2,3-dimethyl-5-phytylquinol in an organism or a
part thereof; [4352] (b) identifying if the compound is an
effective agonist by detecting the presence or absence or increase
of a signal produced by said readout system. [4353] 19. A method
for the identification of a gene product conferring an increase in
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol production
in a cell, comprising the following steps: [4354] (a) contacting
the nucleic acid molecules of a sample, which can contain a
candidate gene encoding a gene product conferring an increase in
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol after
expression with the nucleic acid molecule of claim 6; [4355] (b)
identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; [4356] (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing vitamin E or its
precursor 2,3-dimethyl-5-phytylquinol; [4357] (d) expressing the
identified nucleic acid molecules in the host cells; [4358] (e)
assaying the vitamin E or its precursor 2,3-dimethyl-5-phytylquinol
level in the host cells; and [4359] (f) identifying nucleic acid
molecule and its gene product which expression confers an increase
in the vitamin E or its precursor 2,3-dimethyl-5-phytylquinol level
in the host cell in the host cell after expression compared to the
wild type. [4360] 20. A method for the identification of a gene
product conferring an increase in vitamin E or its precursor
2,3-dimethyl-5-phytylquinol production in a cell, comprising the
following steps: [4361] (a) identifying in a data bank nucleic acid
molecules of an organism; which can contain a candidate gene
encoding a gene product conferring an increase in the vitamin E or
its precursor 2,3-dimethyl-5-phytylquinol amount or level in an
organism or a part thereof after expression, and which are at least
20% homolog to the nucleic acid molecule of claim 6; [4362] (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing vitamin E or its precursor
2,3-dimethyl-5-phytylquinol; [4363] (c) expressing the identified
nucleic acid molecules in the host cells; [4364] (d) assaying the
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol level in the
host cells; and [4365] (e) identifying nucleic acid molecule and
its gene product which expression confers an increase in the
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol level in the
host cell after expression compared to the wild type. [4366] 21. A
method for the production of an agricultural composition comprising
the steps of the method of any one of claims 17 to 20 and
formulating the compound identified in any one of claims 17 to 20
in a form acceptable for an application in agriculture. [4367] 22.
A composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [4368] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
vitamin E or its precursor 2,3-dimethyl-5-phytylquinol after
expression. [4369] 24. Use of the polypeptide of claim 14 or the
nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of vitamin E or its precursor
2,3-dimethyl-5-phytylquinol levels in an organism. [4370] 25.
Cosmetic, pharmaceutical, food or feed composition comprising the
nucleic acid molecule of claim 6, the polypeptide of claim 14, the
nucleic acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20. [4371] 26. The method of any one of claims 1 to 5, the nucleic
acid molecule of claim 6, the polypeptide of claim 14, the nucleic
acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20, wherein the vitamin E is alpha-, beta-, or gamma-tocopherol,
resp. [4372] 27. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claims 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the protection of a plant against
a oxidative stress. [4373] 28. Use of the nucleic acid molecule of
claim 6, the polypeptide of claim 14, the nucleic acid construct of
claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the harvested material of claim
16, the host cell of claim 10 to 12 or the gene product identified
according to the method of claim 19 or 20 for the protection of a
plant against a oxidative stress causing herbicide. [4374] 29. Use
of the agonist identified according to claim 17, the plant or plant
tissue of claim 16, the harvested material of claim 16, or the host
cell of claim 10 to 12 for the production of a cosmetic or
pharmaceutical composition.
[4375] [0554.0.0.9] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[4376] [0000.0.0.10] see [0000.0.0.1]
[4377] [0001.0.0.10] see [0001.0.0.0]
[4378] [0002.0.10.10] Carotenoids are red, yellow and orange
pigments that are widely distributed in nature. Although specific
carotenoids have been identified in photosynthetic centers in
plants, in bird feathers, in crustaceans and in marigold petals,
they are especially abundant in yellow-orange fruits and vegetables
and dark green, leafy vegetables. Of the more than 700 naturally
occurring carotenoids identified thus far, as many as 50 may be
absorbed and metabolized by the human body. To date, only 14
carotenoids have been identified in human serum.
[4379] In animals some carotenoids (particularly beta-carotene)
serve as dietary precursors to Vitamin A, and many of them may
function as fat-soluble antioxidants. In plants carotenes serve for
example as antioxidants to protect the highly reactive photosystems
and act as accessory photopigments. In vitro experiments have shown
that lycopene, alpha-carotene, zeaxanthin, lutein and cryptoxanthin
quench singlet oxygen and inhibit lipid peroxidation. The isolation
and identification of oxidized metabolites of lutein, zeaxanthin
and lycopene provide direct evidence of the antioxidant action of
these carotenoids.
[4380] Carotenoids are 40-carbon (C.sub.40) terpenoids generally
comprising eight isoprene (C.sub.5) units joined together. Linking
of the units is reversed at the center of the molecule.
"Ketocarotenoid" is a general term for carotenoid pigments that
contain a keto group in the ionene ring portion of the molecule,
whereas "hydroxycarotenoid" refers to carotenoid pigments that
contain a hydroxyl group in the ionene ring. Trivial names and
abbreviations will be used throughout this disclosure, with
IUPAC-recommended semi-systematic names usually being given in
parentheses after first mention of a trivial name.
[4381] Carotenoids are synthesized from a five carbon atom
metabolic precursor, isopentenyl pyrophosphate (IPP). There are at
least two known biosynthetic pathways in the formation of IPP, the
universal isoprene unit. One pathway begins with mevalonic acid,
the first specific precursor of terpenoids, formed from acetyl-CoA
via HMG-CoA (3-hydroxy-3-methylglutaryl-CoA), that is itself
converted to isopentenyl pyrophosphate (IPP). Later, condensation
of two geranylgeranyl pyrophosphate (GGPP) molecules with each
other produces colorless phytoene, which is the initial carotenoid.
Studies have also shown the existence of an alternative,
mevalonate-independent pathway for IPP formation that was
characterized initially in several species of eubacteria, a green
alga, and in the plastids of higher plants. The first reaction in
this alternative pathway is the transketolase-type condensation
reaction of pyruvate and D-glyceraldehylde-3-phosphate to yield
1-deoxy-D-xylulose-5-phosphate (DXP) as an intermediate.
[4382] Through a series of desaturation reactions, phytoene is
converted to phytofluene, -carotene, neurosporene and finally to
lycopene. Subsequently, lycopene is converted by a cyclization
reaction to .beta.-carotene that contains two .beta.-ionene rings.
A keto-group and/or a hydroxyl group are introduced into each ring
of .beta.-carotene to thereby synthesize canthaxanthin, zeaxanthin,
astaxanthin. A hydroxylase enzyme has been shown to convert
canthaxanthin to astaxanthin. Similarly, a ketolase enzyme has been
shown to convert zeaxanthin to astaxanthin. The ketolase also
converts .beta.-carotene to canthaxanthin and the hydroxylase
converts .beta.-carotene to zeaxanthin.
[4383] Carotenoids absorb light in the 400-500 nm region of the
visible spectrum. This physical property imparts the characteristic
red/yellow color of the pigments. A conjugated backbone composed of
isoprene units is usually inverted at the center of the molecule,
imparting symmetry. Changes in geometrical configuration about the
double bonds result in the existence of many cis- and
trans-isomers. Hydroxylated, oxidized, hydrogenated or
ring-containing derivatives also exist. Hydrocarbon carotenoids are
classified as carotenes while those containing oxygen are known as
xanthophylls.
[4384] In animals, carotenoids are absorbed from the intestine with
the aid of dietary fat and incorporated into chylomicrons for
transport in the serum. The different structural features possessed
by carotenoids account for selective distribution in organ tissue,
biological activity and pro-vitamin A potency, or in vivo
conversion to vitamin A. Due to the hydrophobic character,
carotenoids are associated with lipid portions of human tissues,
cells, and membranes. In general, 80-85% of carotenoids are
distributed in adipose tissue, with smaller amounts found in the
liver, muscle, adrenal glands, and reproductive organs.
Approximately 1% circulate in the serum on high and low density
lipoproteins. Serum concentrations are fairly constant and slow to
change during periods of low intake. The estimated half-life was
estimated to be 11-14 days for lycopene, -carotene, -carotene,
lutein and zeaxanthin. Evidence for the existence of more than one
body pool has been published. The major serum carotenoids are
-carotene, -carotene, lutein, zeaxanthin, lycopene and
cryptoxanthin. Smaller amounts of polyenes such as phytoene and
phytofluene are also present.
[4385] Human serum levels reflect lifestyle choices and dietary
habits within and between cultures. Approximately only 15 circulate
in the blood, on HDL and LDL. Variations can be attributed to
different intakes, unequal abilities to absorb certain carotenoids,
and different rates of metabolism and tissue uptake. Decreased
serum levels occur with alcohol consumption, the use of oral
contraceptives, smoking and prolonged exposure to UV light.
[4386] -Carotene, -carotene and -cryptoxanthin can be converted to
retinol or vitamin A in the intestine and liver by the enzyme
15-15'-b-carotenoid dioxygenase. Such in vivo formation of retinol
appears to be homeostatically controlled, such that conversion to
retinol is limited in persons having adequate vitamin A status.
[4387] [0003.0.10.10] The established efficacy of beta-carotene in
quenching singlet oxygen and intercepting deleterious free radicals
and reactive oxygen species makes it part of the diverse
antioxidant defense system in humans. Reactive oxygen species have
been implicated in the development of many diseases, including
ischemic heart disease, various cancers, cataracts and macular
degeneration. Because the conjugated polyene portion of
beta-carotene confers its antioxidant capability and all
carotenoids possess this structural feature, research efforts have
been directed at evaluating the efficacy of other carotenoids in
the prevention of free radical-mediated diseases. Indeed, in vitro
experiments have demonstrated that lycopene, alpha-carotene,
zeaxanthin, lutein and cryptoxanthin quench singlet oxygen and
inhibit lipid peroxidation. The isolation and identification of
oxidized metabolites of lutein, zeaxanthin and lycopene may provide
direct evidence of the antioxidant action of these carotenoids.
[4388] In addition to antioxidant capability, other biological
actions of carotenoids include the ability to enhance
immunocompetence and in vitro gap junction communication, reduce or
inhibited mutagenesis and inhibit cell transformations in
vitro.
[4389] Many epidemiological studies have established an inverse
correlation between dietary intake of yellow-orange fruit and dark
green, leafy vegetables and the incidence of various cancers,
especially those of the mouth, pharynx, larynx, esophagus, lung,
stomach, cervix and bladder. While a number of protective compounds
may be responsible for this observation, the co-incidence of
carotenoids in these foods has been noted. Because nutritionists
and medical professionals currently recognize the occurrence of a
large number of distinct carotenoids in food, interest in their
functions and biological impact on health is burgeoning.
[4390] Lutein exists in the retina. It functions to protect
photoreceptor cells from light-generated oxygen radicals, and thus
plays a key role in preventing advanced macular degeneration.
Lutein possesses chemopreventive activity, induces gap junction
communication between cells and inhibits lipid peroxidation in
vitro more effectively than beta-carotene, alpha-carotene and
lycopene. High levels of lutein in serum have been inversely
correlated with lung cancer.
[4391] In addition to lutein, zeaxanthin exists in the retina and
confers protection against macular degeneration. Zeaxanthin is also
prevalent in ovaries and adipocyte tissue. This xanthophyll does
not possess provitamin A activity.
[4392] Alcohol consumption has been shown to influence lipid
peroxidation. Anhydrolutein, an oxidative by-product of lutein and
zeaxanthin, was higher in plasma after alcohol ingestion, while
concentrations of these xanthophylls were reduced. Lutein and
zeaxanthin may therefore have protective effects against LDL
oxidation.
[4393] The all-trans isomer of Lycopene is typically quantified in
serum, although signals for 9-, 13- and 15-cis isomers are
detectable and account for as much as 50% of the total lycopene. In
experiments performed in vitro, lycopene quenched singlet oxygen
more efficiently than alpha-carotene, beta-carotene, zeaxanthin,
lutein and cryptoxanthin. Lycopene induces gap junction
communication, inhibits lipid peroxidation and has displays
chemopreventive activity. Serum levels of lycopene have been
inversely related to the risk of cancer in the pancreas and cervix.
This carotenoid has been identified in tissues of the thyroid,
kidneys, adrenals, spleen, liver, heart, testes and pancreas.
Lycopene is not converted to retinol in vivo.
[4394] beta-Cryptoxanthin is capable of quenching singlet oxygen.
beta-Cryptoxanthin is used to color butter. beta-Cryptoxanthin
exhibits provitamin A activity.
[4395] The all-trans isomer of this carotenoid is the major source
of dietary retinoids, due to its high provitamin A activity. One
molecule of trans-beta-carotene can theoretically provide two
molecules of trans retinaldehyde in vivo. Signals for 13- and
15-cis isomers of beta-carotene are also observed in the carotenoid
profile and account for 10% or less of the total beta-carotene in
serum. beta-Carotene quenches singlet oxygen, induces gap junction
communication and inhibits lipid peroxidation. High serum levels of
beta-carotene are correlated with low incidences of cancer in the
mouth, lung, breast, cervix, skin and stomach. beta-Carotene has
been identified in tissues of the thyroid, kidney, spleen, liver,
heart, pancreas, fat, ovaries and adrenal glands.
[4396] alpha-Carotene is similar to beta-carotene in its biological
activity, but quenches singlet oxygen more effectively.
alpha-Carotene improves gap junction communication, prevents lipid
peroxidation and inhibits the formation and uptake of carcinogens
in the body. High serum levels have been associated with lower
risks of lung cancer. With one half the provitamin A potency of
beta-carotene, alpha-carotene also restores normal cell growth and
differentiation. Serum levels are usually between 10 and 20% of the
values for total beta-carotene.
[4397] Alpha-Carotene, beta-carotene and beta-cryptoxanthin can be
converted to Vitamin A in the intestine and liver. Vitamin A is
essential for the immune response and is also involved in other
defenses against infectious agents. Nevertheless, in many
individuals, this conversion is slow and ineffectual, particularly
for older. Some individuals are known as non or low-responders
because they do not convert beta-carotene to Vitamin A at the rate
as expected. A number of factors can inhibit this conversion of
beta-carotene to Vitamin A. The major reason why so many Americans
have a poor vitamin A status is the regular use of excessive
alcohol. Intestinal parasites can be a factor. And, any
prescription drug that requires liver metabolism will decrease the
liver conversion of beta-carotene to retinol in the liver.
Diabetics and individuals with hypothyroidism or even borderline
hypothyroidism are likely to be low-responders.
[4398] [0004.0.10.10] In plants, approximately 80-90% of the
carotenoids present in green, leafy vegetables such as broccoli,
kale, spinach and brussel sprouts are xanthophylls, whereas 10-20%
are carotenes. Conversely, yellow and orange vegetables including
carrots, sweet potatoes and squash contain predominantly carotenes.
Up to 60% of the xanthophylls and 15% of the carotenes in these
foods are destroyed during microwave cooking. Of the xanthophylls,
lutein appears to be the most stable.
[4399] Lutein occurs in mango, papaya, oranges, kiwi, peaches,
squash, peas, lima beans, green beans, broccoli, brussel sprouts,
cabbage, kale, lettuce, prunes, pumpkin, sweet potatoes and
honeydew melon. Commercial sources are obtained from the extraction
of marigold petals. Lutein does not possess provitamin A
activity.
[4400] Dietary sources of Zeaxanthin include peaches, squash,
apricots, oranges, papaya, prunes, pumpkin, mango, kale, kiwi,
lettuce, honeydew melon and yellow corn.
[4401] The red color of fruits and vegetables such as tomatoes,
pink grapefruit, the skin of red grapes, watermelon and red guavas
is due to lycopene. Other dietary sources include papaya and
apricots.
[4402] beta-Cryptoxanthin occurs in oranges, mango, papaya,
cantaloupe, peaches, prunes, squash.
[4403] Dietary sources of beta-Carotene include mango, cantaloupe,
carrots, pumpkin, papaya, peaches, prunes, squash, sweet potato,
apricots, cabbage, lima beans, green beans, broccoli, brussel
sprouts, kale, kiwi, lettuce, peas, spinach, tomatoes, pink
grapefruit, honeydew melon and oranges.
[4404] Dietary sources of alpha-Carotene include sweet potatoes,
apricots, pumpkin, cantaloupe, green beans, lima beans, broccoli,
brussel sprouts, cabbage, kale, kiwi, lettuce, peas, spinach,
prunes, peaches, mango, papaya, squash and carrots.
[4405] [0005.0.10.10] Some carotenoids occur particularly in a wide
variety of marine animals including fish such as salmonids and sea
bream, and crustaceans such as crab, lobster, and shrimp. Because
animals generally cannot biosynthesize carotenoids, they obtain
those carotenoids present in microorganisms or plants upon which
they feed.
[4406] Carotenoids e.g. xanthophylls, e.g. as astaxanthin, supplied
from biological sources, such as crustaceans, yeast, and green alga
is limited by low yield and costly extraction methods when compared
with that obtained by organic synthetic methods. Usual synthetic
methods, however, produce by-products that can be considered
unacceptable. It is therefore desirable to find a relatively
inexpensive source of carotenoids, in particular xantophylls, to be
used as a feed supplement in aquaculture and as a valuable chemical
for other industrial uses and for diets. Sources of Xanthophylls
include crustaceans such as a krill in the Antarctic Ocean,
cultured products of the yeast Phaffia, cultured products of a
green alga Haematococcus pluvialis, and products obtained by
organic synthetic methods. However, when crustaceans such as a
krill or the like are used, a great deal of work and expense are
required for the isolation of xanthophylls from contaminants such
as lipids and the like during the harvesting and extraction.
Moreover, in the case of the cultured product of the yeast Phaffia,
a great deal of expense is required for the gathering and
extraction of astaxanthin because the yeast has rigid cell walls
and produces xantophylls only in a low yield. One approach to
increase the productivity of some xantophylls' production in a
biological system is to use genetic engineering technology.
[4407] [0006.0.10.10] In many plants, lycopene is a branch point in
carotenoid biosynthesis. Thus, some of the plant's lycopene is made
into beta-carotene and zeaxanthin, and sometimes zeaxanthin
diglucoside, whereas remaining portions of lycopene are formed into
alpha-carotene and lutein (3,3'-dihydroxy-.alpha.-carotene),
another hydroxylated compound. Carotenoids in higher plants; i.e.,
angiosperms, are found in plastids; i.e., chloroplasts and
chromoplasts. Plastids are intracellular storage bodies that differ
from vacuoles in being surrounded by a double membrane rather than
a single membrane. Plastids such as chloroplasts can also contain
their own DNA and ribosomes, can reproduce independently and
synthesize some of their own proteins. Plastids thus share several
characteristics of mitochondria. In leaves, carotenoids are usually
present in the grana of chloroplasts where they provide a
photoprotective function. Beta-carotene and lutein are the
predominant carotenoids, with the epoxidized carotenoids
violaxanthin and neoxanthin being present in smaller amounts.
[4408] Carotenoids accumulate in developing chromoplasts of flower
petals, usually with the disappearance of chlorophyll. As in flower
petals, carotenoids appear in fruit chromoplasts as they develop
from chloroplasts. Most enzymes that take part in conversion of
phytoene to carotenes and xanthophylls are labile,
membrane-associated proteins that lose activity upon
solubilization. In maize, cartonoids were present in horny
endosperm (74% to 86%), floury endosperm (9%-23%) and in the germ
and bran of the kernel.
[4409] [0007.0.10.10] At the present time only a few plants are
widely used for commercial colored carotenoid production. However,
the productivity of colored carotenoid synthesis in most of these
plants is relatively low and the resulting carotenoids are
expensively produced.
[4410] Dried marigold petals and marigold petal concentrates
obtained from so-called xanthophyll marigolds are used as feed
additives in the poultry industry to intensify the yellow color of
egg yolks and broiler skin. The pigmenting ability of marigold
petal meal resides largely in the carotenoid fraction known as the
xanthophylls, primarily lutein esters. The xanthophyll zeaxanthin,
also found in marigold petals, has been shown to be effective as a
broiler pigmenter, producing a highly acceptable yellow to
yellow-orange color. Of the xanthophylls, the pigments lutein and
zeaxanthin are the most abundant in commercially available hybrids.
Structural formulas for lutein and zeaxanthin are shown below.
[4411] Carotenoids have been found in various higher plants in
storage organs and in flower petals. For example, marigold flower
petals accumulate large quantities of esterified lutein as their
predominant xanthophyll carotenoid (about 75 to more than 90
percent), with smaller amounts of esterified zeaxanthin. Besides
lutein and zeaxanthin, marigold flower petals also typically
exhibit a small accumulation of .beta.-carotene and epoxidized
xanthophylls, but do not produce or accumulate canthaxanthin or
astaxanthin because a 4-keto-.beta.-ionene ring-forming enzyme is
absent in naturally-occurring marigolds or their hybrids.
[4412] [0008.0.10.10] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce colored carotenoids generally and,
with the use of recent advances in determining carotenoid
biosynthesis from .beta.-carotene to xanthophylls to control the
production of carotenoids. That type of production permits control
over quality, quantity and selection of the most suitable and
efficient producer organisms. The latter is especially important
for commercial production economics and therefore availability to
consumers.
[4413] Methods of recombinant DNA technology have been used for
some years to improve the production of Xanthophylls in
microorganisms, in particular algae or in plants by amplifying
individual xanthophyll biosynthesis genes and investigating the
effect on xanthophyll production. It is for example reported, that
the five ketocarotenoids, e.g. the xanthophyll astaxanthin could be
produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. The Pds gene was said by
those workers to direct transcription and expression in
chloroplasts and/or chromoplast-containing tissues of plants. Those
results indicated that about 75 percent of the carotenoids found in
the flower of the transformed plant contained a keto group.
Further, in maize the phytonene synthase (Psy), Phytone desaturase
(Pds), and the -carotene desaturase were identified and it was
shown, that PSY activity is an important control point for the
regulation of the flux.
[4414] Genes suitable for conversion of microorganisms have also
been reported (U.S. Pat. No. 6,150,130 WO 99/61652). Two different
genes that can convert a carotenoid .beta.-ionene ring compound
into astaxanthin have been isolated from the green alga
Haematococcus pluvialis. Zeaxanthin or -carotene were also found in
the marine bacteria Agrobacterium aurantiacum, Alcaligenes PC-1,
Erwinia uredovora. An A. aurantiacum crtZ gene was introduced to an
E. coli transformant that accumulated all-trans-.beta.-carotene.
The transformant so formed produced zeaxanthin. A gene cluster
encoding the enzymes for a carotenoid biosynthesis pathway has been
also cloned from the purple photosynthetic bacterium Rhodobacter
capsulatus. A similar cluster for carotenoid biosynthesis from
ubiquitous precursors such as farnesyl pyrophosphate and geranyl
pyrophosphate has been cloned from the non-photosynthetic bacteria
Erwinia herbicola. Yet another carotenoid biosynthesis gene cluster
has been cloned from Erwinia uredovora. It is yet unknown and
unpredictable as to whether enzymes encoded by other organisms
behave similarly to that of A. aurantiacum in vitro or in vivo
after transformation into the cells of a higher plant.
[4415] [0009.0.10.10] In addition to the above said about the
biological importance of carotenoids, e.g. in vision, bone growth,
reproduction, immune function, gene expression, emboryonic
expression, cell division and cell differation, and respiration, it
should be mentioned that in the world, the prevalence of vitamin A
deficiency ranges from 100 to 250 million children and an estimated
250.000 to 500.000 children go blind each year from vitamin A
deficiency.
[4416] Thus, it would be advantageous if an algae or other
microorganism were available who produce large amounts of
.beta.-carotene, beta-cryptoxanthin, lutein, zeaxanthin, or other
carotenoids. It might be advantageous that only small amounts or no
lutein is produced so that such organisms could be transformed with
e.g. one or more of an appropriate hydroxylase gene and/or an
appropriate ketolase gene to produce cryptoxanthin, zeaxanthin or
astaxanthin. The invention discussed hereinafter relates in some
embodiments to such transformed prokaryotic or eukaryotic
microorganisms. It would also be advantageous if a marigold or
other plants were available whose flowers produced large amounts of
.beta.-carotene, beta-cryptoxanthin, lutein, zeaxanthin, or other
carotenoids. It might be advantageous that only small amounts or no
lutein is produced so that such plants could be transformed with
one or more of an appropriate hydroxylase gene and an appropriate
ketolase gene to produce cryptoxanthin, zeaxanthin or astaxanthin
from e.g the flowers of the resulting transformants. The invention
discussed hereinafter relates in some embodiments to such
transformed plants.
[4417] [0010.0.10.10] Therefore improving the quality of foodstuffs
and animal feeds is an important task of the food-and-feed
industry. This is necessary since, for example, as mentioned above
xanthophylls, which occur in plants and some microorganisms are
limited with regard to the supply of mammals. Especially
advantageous for the quality of foodstuffs and animal feeds is as
balanced as possible a carotenoids profile in the diet since a
great excess of some carotenoids above a specific concentration in
the food has only some positive effect. A further increase in
quality is only possible via addition of further carotenoids, which
are limiting.
[4418] [0011.0.10.10] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add one or a plurality of
carotenoids in a balanced manner to suit the organism.
[4419] [0012.0.10.10] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes which
participate in the biosynthesis of carotenoids, e.g. xanthophylls,
e.g. like beta-crypotxanthin, or zeaxanthin, or astaxanthin, and
make it possible to produce them specifically on an industrial
scale without unwanted byproducts forming. In the selection of
genes for biosynthesis two characteristics above all are
particularly important. On the one hand, there is as ever a need
for improved processes for obtaining the highest possible contents
of carotenoids like xanthophylls; on the other hand as less as
possible byproducts should be produced in the production
process.
[4420] [0013.0.0.10] see [0013.0.0.0]
[4421] [0014.0.10.10] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a xanthophyll. Accordingly,
in the present invention, the term "the fine chemical" as used
herein relates to a "Xanthophyll". Further, in an other embodiment
the term "the fine chemicals" as used herein also relates to
compositions of fine chemicals comprising xanthophylls.
[4422] [0015.0.10.10] In one embodiment, the term xanthophylls" or
"the fine chemical" or "the respective fine chemical" means at
least one chemical compound with xanthophylls activity selected
from the group comprising zeaxanthin or cryptoxanthin.
[4423] In one embodiment, the term "the fine chemical" means a
xanthophyll. In one embodiment, the term "the fine chemical" means
beta-cryptoxanthin or zeaxanthin depending on the context in which
the term is used. Throughout the specification the term "the fine
chemical" means a xanthophyll, in particular beta cryptoxanthin or
zeaxanthin, its salts, ester, thioester or in free form or bound to
other compounds such sugars or sugarpolymers, like glucoside, e.g.
diglucoside. In one embodiment, the term "the fine chemicals" means
beta-cryptoxanthin and zeaxanthin, in free form or their salts or
their ester or bound to a glucoside, e.g a diglucoside. In one
embodiment, the term "the fine chemical" and the term "the
respective fine chemical" mean at least one chemical compound with
an activity of the above mentioned fine chemical.
[4424] [0016.0.10.10] Accordingly, the present invention relates to
a process comprising [4425] (a) increasing or generating the
activity of one or more b0050, b0851, b2211, b3926, b0986, b3684,
b4401, and/or YCL040W protein(s) or a b2699 and/or YHR055C
protein(s) or of a protein having the sequence of a polypeptide
encoded by a nucleic acid molecule indicated in Table II, column 3,
lines 103 to 108 or 468 to 471 in a non-human organism in one or
more parts thereof; and [4426] (b) growing the organism under
conditions which permit the production of the fine chemical, thus,
beta-cryptoxanthin or zeaxanthin, resp., or fine chemicals
comprising beta-cryptoxanthin or zeaxanthin, resp., in said
organism.
[4427] Accordingly, the present invention relates to a process
comprising [4428] (a) increasing or generating the activity of one
or more proteins having the activity of a protein indicated in
Table II, column 3, lines 103 to 108 or 468 to 471, resp., or
having the sequence of a polypeptide encoded by a nucleic acid
molecule indicated in Table I, column 5 or 7, lines 103 to 108 or
468 to 471, resp., in a non-human organism in one or more parts
thereof; and [4429] (b) growing the organism under conditions which
permit the production of the fine chemical, thus,
beta-cryptoxanthin or zeaxanthin, resp., or fine chemicals
comprising beta-cryptoxanthin or zeaxanthin, resp., in said
organism.
[4430] [0016.1.0.10] Accordingly, the term "the fine chemical"
means "beta-cryptoxanthin" in relation to all sequences listed in
Table I to Table IV, lines 103 to 106 and/or 468 to 471 or homologs
thereof and means "zeaxanthin" in relation to all sequences listed
in Table I to Table IV, lines 107 and 108 or homologs thereof.
Accordingly, the term "the fine chemical" can mean "zeaxanthin" or
"cryptoxanthin", owing to circumstances and the context. In order
to illustrate that the meaning of the term "the respective fine
chemical" means "cryptoxanthin", and/or "zeaxanthin" owing to the
sequences listed in the context the term "the respective fine
chemical" is also used.
[4431] The terms "beta-cryptoxanthin" and "cryptoxanthin" are used
as equivalent terms.
[4432] [0017.0.0.10] to [0018.0.0.10]: see [0017.0.0.0] to
[0018.0.0.0]
[4433] [0019.0.10.10] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the respective fine chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table II, column 3, lines 103 to 108 or 468 to 471 or
encoded by nucleic acid molecule indicated in Table I, columns 5 or
7, lines 103 to 108 or 468 to 471.
[4434] [0020.0.10.10] Surprisingly it was found, that the
transgenic expression of at least one of the proteins of the
Saccharomyces cerevisiae protein YCL040W, and/or the Escherichia
coli K12 protein b0050, b0851, b2211, b3926, b0986, b3684 and/or
b4401 in Arabidopsis thaliana conferred an increase in the
beta-cryptoxanthin ("the fine chemical" or "the fine respective
chemical" in respect to said proteins and their homologs as wells
as the encoding nucleic acid molecules, in particular as indicated
in Table I or II, column 3, lines 103 to 106 and/or 468 to 471)
content of the transformed plants.
[4435] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein YHR055C, and/or the
Escherichia coli K12 protein b2699 in Arabidopsis thaliana
conferred an increase in the zeaxanthin ("the fine chemical" or
"the fine respective chemical" in respect to said proteins and
their homologs as wells as the encoding nucleic acid molecules, in
particular as indicated in Table I or II, column 3, lines 107 and
108 of) content of the transformed plants.
[4436] [0021.0.0.10] see [0021.0.0.0]
[4437] [0022.0.10.10] The sequence of b0050 from Escherichia coli
K12 has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as conserved
protein potentially involved in protein-protein interaction of the
superfamily of apaG protein. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
with an activity for proteolytic degradation, protein modification,
modification by ubiquitination, deubiquitination, and/or protein
binding, in particular being a conserved protein potentially
involved in protein-protein interaction, in particular being a
conserved protein potentially involved in protein-protein
interaction of the superfamily of apaG protein from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of xanthophylls, in particular for
increasing the amount of beta-cryptoxanthin, preferably in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a conserved protein potentially involved in protein-protein
interaction of the superfamily of apaG protein is increased or
generated, e.g. from E. coli or a homolog thereof.
[4438] The sequence of b0851 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as oxygen insensitive NADPH
nitroreductase of the superfamily of NADPH-flavin oxidoreductase
homologs. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein with an activity
for nitrogen metabolism, sulfur metabolism, electron transport and
membrane-associated energy conservation, stress response,
electron/hydrogen carrier, C-compound and carbohydrate utilization,
and/or utilization of vitamins, cofactors, and prosthetic groups
and/or as a resistance protein, in particular being a oxygen
insensitive NADPH nitroreductase, in particular being a oxygen
insensitive NADPH nitroreductase of the superfamily of NADPH-flavin
oxidoreductase homologs from E. coli or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of xanthophylls, in particular for increasing the amount of
beta-cryptoxanthin, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a oxygen insensitive NADPH
nitroreductase of the superfamily of NADPH-flavin oxidoreductase
homologs is increased or generated, e.g. from E. coli or a homolog
thereof.
[4439] The sequence of b2211 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as ATP-binding transport protein
of the ABC superfamily of the superfamily of unassigned ATP-binding
cassette proteins. Accordingly, in one embodiment, the process of
the present invention comprises the use of a protein with an
activity for nucleotide binding, ABC transporters, and/or CELLULAR
TRANSPORT AND TRANSPORT MECHANISMS, in particular being a
ATP-binding transport protein of the ABC superfamily, in particular
being a ATP-binding transport protein of the ABC superfamily of the
superfamily of unassigned ATP-binding cassette proteins from E.
coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of xanthophylls, in
particular for increasing the amount of beta-cryptoxanthin,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of an ATP-binding transport protein of the
ABC superfamily of the superfamily of unassigned ATP-binding
cassette proteins is increased or generated, e.g. from E. coli or a
homolog thereof.
[4440] The sequence of b3926 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as glycerol kinase of the
superfamily of xylulokinase. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
with an activity in the C-compound and carbohydrate catabolism,
phospholipid biosynthesis, glycolipid biosynthesis, unspecified
signal transduction, enzyme mediated signal transduction, and/or
phosphotransferase system, in particular being a glycerol kinase,
in particular being a glycerol kinase of the superfamily of
xylulokinase from E. coli or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, meaning of
xanthophylls, in particular for increasing the amount of
beta-cryptoxanthin, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a glycerol kinase of the
superfamily of xylulokinase is increased or generated, e.g. from E.
coli or a homolog thereof.
[4441] The sequence of b0986 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a lipoprotein of the
superfamily of the hypothetical protein b1706. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a lipoprotein, in particular of a lipoprotein of the superfamily
of the hypothetical protein b1706 from E. coli or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, meaning of xanthophylls, in particular for increasing the
amount of beta-cryptoxanthin, preferably in free or bound form in
an organism or a part thereof, as mentioned. In one embodiment, in
the process of the present invention the activity of a lipoprotein,
in particular of a lipoprotein of the superfamily of the
hypothetical protein b1706 is increased or generated, e.g. from E.
coli or a homolog thereof.
[4442] The sequence of b3684 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a transcriptional regulator of
the GntR family. Accordingly, in one embodiment, the process of the
present invention comprises the use of protein having a
transcriptional control activity, a regulation of C-compound and
carbohydrate utilization activity, a DNA binding activity, a
transcriptional repressor activity, a regulation of lipid,
fatty-acid and/or isoprenoid metabolism activity, and/or a
regulation of C-compound and carbohydrate utilization activity,
e.g. of a transcriptional regulator, in particular of a
transcriptional regulator of the GntR family from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of xanthophylls, in particular for
increasing the amount of beta-cryptoxanthin, preferably in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention said activity,
e.g. the activity of a transcriptional regulator, in particular of
a transcriptional regulator of the GntR family is increased or
generated, e.g. from E. coli or a homolog thereof.
[4443] The sequence of b4401 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a response regulator of the
OmpR family. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein of a
transcriptional control activity, a unspecified signal transduction
activity, a two-component signal transduction system (e.g. response
regulator component activity, a transcriptional activator activity,
a regulation of respiration activity, an aerobic respiration
activity, and/or an anaerobic respiration activity, e.g. of a
response regulator, in particular of a response regulator of the
OmpR family from E. coli or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, meaning of
xanthophylls, in particular for increasing the amount of
beta-cryptoxanthin, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention said activity, e.g. the activity of a
response regulator, in particular of a response regulator of the
OmpR family is increased or generated, e.g. from E. coli or a
homolog thereof.
[4444] The sequence of b2699 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a DNA strand exchange and
recombination protein with protease and nuclease activity of the
superfamily of the recombination protein recA. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein with a DNA recombination and DNA repair activity, a
pheromone response activity, a mating-type determination activity,
a sex-specific protein activity, a nucleotide binding activity
and/or a protease and nuclease activity, in particular a DNA strand
exchange and recombination protein with protease and nuclease
activity, in particular of the superfamily of the recombination
protein recA from E. coli or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, meaning of
xanthophylls, in particular for increasing the amount of
zeaxanthin, preferably in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention said activity, e.g. the activity of a
protease and nuclease activity, in particular a DNA strand exchange
and recombination protein with protease and nuclease activity, in
particular of the superfamily of the recombination protein recA is
increased or generated, e.g. from E. coli or a homolog thereof.
[4445] The sequence of YCL040W from Saccharomyces cerevisiae has
been published in Oliver, S. G., et al., Nature 357 (6373), 38-46
(1992), and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is being defined as glucokinase of the superfamily
hexokinase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein having a
C-compound and carbohydrate utilization activity, a C-compound or
carbohydrate transport activity, a activity in the glycolysis
and/or a activity in the gluconeogenesis, and/or a cellular import
activity, in particular of a hexokinase, preferably of a glucinase
or its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of xanthophylls, in particular
for increasing the amount of beta-cryptoxanthin in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention said activity,
e.g. the activity of a glucokinase is increased or generated, e.g.
from Saccharomyces cerevisiae or a homolog thereof. The sequence of
YHR055C from Saccharomyces cerevisiae has been published in
Johnston, Metal., Science 265 (5181), 2077-2082 (1994), and Goffeau
et al., Science 274 (5287), 546-547, 1996, and its activity is
being defined as copper binding metallothionein of the superfamily
metallothionein. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein having a
detoxification activity, particular of a metallothionein,
preferably of copper binding metallothionein or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, meaning of xanthophylls, in particular for increasing the
amount of zeaxanthin in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention said activity, e.g. the activity of a
metellothionein, preferably of a copper binding metellothionein is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[4446] [0023.0.10.10] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
Further, in the present invention, the term "homologue" relates to
the sequence of an organism having the highest sequence homology to
the herein mentioned or listed sequences of all expressed sequences
of said organism. However, the person skilled in the art knows,
that, preferably, the homologue has said
the--fine-chemical-increasing activity and, if known, the same
biological function or activity in the organism as at least one of
the protein(s) indicated in Table II, Column 3, lines 103 to 106
and/or 468 to 471, e.g. having the sequence of a polypeptide
encoded by a nucleic acid molecule comprising the sequence
indicated in Table I, Column 5 or 7, lines 103 to 106 and/or 468 to
471.
[4447] In one embodiment, the homolog of any one of the
polypeptides indicated in Table II, column 3, line 103 is a homolog
having the same or a similar activity. In particular an increase of
activity confers an increase in the content of the respective fine
chemical in the organisms preferably beta-cryptoxanthin. In one
embodiment, the homologue is a homolog with a sequence as indicated
in Table I or II, column 7, lines 103, resp. In one embodiment, the
homologue of one of the polypeptides indicated in Table II, column
3, line 103 is derived from an eukaryotic. In one embodiment, the
homologue is derived from Fungi. In one embodiment, the homologue
of a polypeptide indicated in Table II, column 3, line 103 is
derived from Ascomyceta. In one embodiment, the homologue of a
polypeptide indicated in Table II, column 3, line 103 is derived
from Saccharomycotina. In one embodiment, the homologue of a
polypeptide indicated in Table II, column 3, line 103 is derived
from Saccharomycetes. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 103 is a
homologue being derived from Saccharomycetales. In one embodiment,
the homologue of a polypeptide indicated in Table II, column 3,
line 103 is a homologue having the same or a similar activity being
derived from Saccharomycetaceae. In one embodiment, the homologue
of a polypeptide indicated in Table II, column 3, line 103 is a
homologue having the same or a similar activity being derived from
Saccharomycetes.
[4448] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 104 to 106 or
468 to 471 is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms preferably
cryptoxanthin. In one embodiment, the homolog is a homolog with a
sequence as indicated in Table I or II, column 7, lines 104 to 106
or 468 to 471, resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table II, column 3, lines 104 to 106 or
468 to 471 is derived from an bacteria. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 104
to 106 or 468 to 471 is derived from Proteobacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 104 to 106 or 468 to 471 is a homolog having the
same or a similar activity being derived from Gammaproteobacteria.
In one embodiment, the homolog of a polypeptide indicated in Table
II, column 3, lines 104 to 106 or 468 to 471 is derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 104 to 106 or 468 to 471 is
a homolog being derived from Enterobacteriaceae. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
104 to 106 or 468 to 471 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the beta-cryptoxanthin in the organisms or part
thereof being derived from Escherichia.
[4449] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 107 is a homolog
having the same or a similar activity. In particular an increase of
activity confers an increase in the content of the respective fine
chemical in the organisms preferably zeaxanthin. In one embodiment,
the homolog is a homolog with a sequence as indicated in Table I or
II, column 7, line 107, resp. In one embodiment, the homolog of one
of the polypeptides indicated in Table II, column 3, line 107 is
derived from an eukaryotic. In one embodiment, the homolog is
derived from Fungi. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 107 is derived from
Ascomyceta. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 1071 ines 65 to 67 is derived
from Saccharomycotina. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines line 107 is
derived from Saccharomycetes. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 107 is a homolog
being derived from Saccharomycetales. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 107
is a homolog having the same or a similar activity being derived
from Saccharomycetaceae. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 107 is a homolog
having the same or a similar activity being derived from
Saccharomycetes.
[4450] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 108 is a homolog
having the same or a similar activity. In particular an increase of
activity confers an increase in the content of the respective fine
chemical in the organisms preferably zeaxanthin. In one embodiment,
the homolog is a homolog with a sequence as indicated in Table I or
II, column 7, line 108, resp. In one embodiment, the homolog of one
of the polypeptides indicated in Table II, column 3, line 108 is
derived from an bacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 108 is derived
from Proteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 108 is a homolog
having the same or a similar activity being derived from
Gammaproteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 108 is derived
from Enterobacteriales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 108 is a homolog
being derived from Enterobacteriaceae. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 108
is a homolog having the same or a similar activity, in particular
an increase of activity confers an increase in the content of the
zeaxanthin in the organisms or part thereof being derived from
Escherichia.
[4451] [0023.1.0.10] Homologs of the polypeptides indicated in
Table II, column 3, lines 103 to 108 or 468 to 471 may be the
polypeptides encoded by the nucleic acid molecules indicated in
Table I, column 7, lines 103 to 108 or 468 to 471 or may be the
polypeptides indicated in Table II, column 7, lines 103 to 108 or
468 to 471. Homologs of the polypeptides indicated in Table II,
column 3, lines 103 to 108 or 468 to 471 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 103 to 108 or 468 to 471 or may be the polypeptides
indicated in Table II, column 7, lines 103 to 108 or 468 to
471.
[4452] Homologs of the polypeptides indicated in Table II, column
3, lines 103 to 106 and/or 468 to 471 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 103 to 106 and/or 468 to 471, respectively or may be the
polypeptides indicated in Table II, column 7, lines 103 to 106
and/or 468 to 471, having a cryptoxanthin content and/or amount
increasing activity. Homologs of the polypeptides indicated in
Table II, column 3, lines 103 to 106 and/or 468 to 471 may be the
polypeptides encoded by the nucleic acid molecules indicated in
Table I, column 7, lines 103 to 106 and/or 468 to 471 or may be the
polypeptides indicated in Table II, column 7, lines 103 to 106
and/or 468 to 471 having a cryptoxanthin content and/or amount
increasing activity.
[4453] Homologs of the polypeptides indicated in Table II, column
3, lines 107 and/or 108 may be the polypeptides encoded by the
nucleic acid molecules indicated in Table I, column 7, lines 107
and/or 108, respectively or may be the polypeptides indicated in
Table II, column 7, lines 107 and/or 108, having a zeaxanthin
content and/or amount increasing activity. Homologs of the
polypeptides indicated in Table II, column 3, lines 107 and/or 108
may be the polypeptides encoded by the nucleic acid molecules
indicated in Table I, column 7, lines 107 and/or 108 or may be the
polypeptides indicated in Table II, column 7, lines 107 and/or 108
having a zeaxanthin content and/or amount increasing activity.
[4454] [0024.0.0.10] see [0024.0.0.0]
[4455] [0025.0.10.10] In accordance with the invention, a protein
or polypeptide has the "activity of an protein of the invention",
e.g. the activity of a protein indicated in Table II, column 3,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108 if its
de novo activity, or its increased expression directly or
indirectly leads to an increased xanthophylls level, in particular
to a increased beta-cryptoxanthin level or zeaxanthin level, resp.,
in the organism or a part thereof, preferably in a cell of said
organism. In a preferred embodiment, the protein or polypeptide has
the above-mentioned additional activities of a protein indicated in
Table II, column 3, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108. Throughout the specification the activity or preferably
the biological activity of such a protein or polypeptide or an
nucleic acid molecule or sequence encoding such protein or
polypeptide is identical or similar if it still has the biological
or enzymatic activity of any one of the proteins indicated in Table
II, column 3, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, or which has at least 10% of the original enzymatic
activity, preferably 20%, particularly preferably 30%, most
particularly preferably 40% in comparison to any one of the
proteins indicated in
[4456] Table II, column 3, line 103 or line 107 of Saccharomyces
cerevisiae and/or any one of the proteins indicated in Table II,
column 3, lines 104 to 106 or line 108 and/or lines 468 to 471 of
E. coli K12.
[4457] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[4458] [0025.1.0.10] see [0025.1.0.0]
[4459] [0025.2.0.10] see [0025.2.0.0]
[4460] [0026.0.0.10] to [0033.0.0.10]: see [0026.0.0.0] to
[0033.0.0.0]
[4461] [0034.0.10.10] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. as result of
an increase in the level of the nucleic acid molecule of the
present invention or an increase of the specific activity of the
polypeptide of the invention or the polypeptide used in the method
of the invention. E.g., it differs by or in the expression level or
activity of an protein having the activity of a protein as
indicated in Table II, column 3, lines 103 to 106 and/or 468 to 471
or lines 107 and/or 108 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, lines 103 to 106 and/or 468 to 471
or lines 107 and/or 108 or its homologs, e.g. as indicated in Table
I, column 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or
108, its biochemical or genetic causes. It therefore shows the
increased amount of the respective fine chemical.
[4462] [0035.0.0.10] to [0044.0.0.10]: see [0035.0.0.0] to
[0044.0.0.0]
[4463] [0045.0.10.10] In one embodiment, the activity of the
Escherichia coli K12 protein b0050 or its homologs e.g. a conserved
protein potentially involved in protein-protein interaction, in
particular of a conserved protein potentially involved in
protein-protein interaction of the superfamily of apaG proteins,
e.g. as indicated in Table I, columns 5 or 7, line 468, is
increased conferring preferably an increase of the respective fine
chemical, preferably of beta-cryptoxanthin of between 42% and 84%
or more. In one embodiment, the activity of the Escherichia coli
K12 protein b0851 or its homologs, e.g. oxygen insensitive NADPH
nitroreductase, in particular of a oxygen insensitive NADPH
nitroreductase of the superfamily of NADPH-flavin oxidoreductase
homologs, e.g. as indicated in Table I, columns 5 or 7, line 469,
is increased conferring preferably an increase of the respective
fine chemical, preferably of beta-cryptoxanthin of between 57% and
76% or more.
[4464] In one embodiment, the activity of the Escherichia coli K12
protein b2211 or its homologs, e.g. an ATP-binding transport
protein of the ABC superfamily, in particular of an ATP-binding
transport proteins of the ABC superfamily of the superfamily of
unassigned ATP-binding cassette proteins, e.g. as indicated in
Table I, columns 5 or 7, line 470, is increased conferring
preferably an increase of the respective fine chemical, preferably
of beta-cryptoxanthin of between 5% and 26% or more.
[4465] In one embodiment, the activity of the Escherichia coli K12
protein b3926 or its homologs, e.g. a glycerol kinase, in
particular of a glycerol kinase of the superfamily of
xylulokinases, e.g. as indicated in Table I, columns 5 or 7, line
471, is increased conferring preferably an increase of the
respective fine chemical, preferably of beta-cryptoxanthin of
around 33% or more.
[4466] In one embodiment, the activity of the Escherichia coli K12
protein b0986 or its homologs e.g. a lipoprotein, in particular of
a lipoprotein of the superfamily of the hypothetical protein b1706,
e.g. as indicated in Table I, columns 5 or 7, line 104, is
increased, conferring preferably, the increase of the respective
fine chemical, preferably of beta-cryptoxanthin between 33% and 40%
or more.
[4467] Thus, in one embodiment, the activity of the Escherichia
coli K12 protein b3684 or its homologs is increased. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a protein having a transcriptional control activity, a
regulation of C-compound and carbohydrate utilization activity, a
DNA binding activity, a transcriptional repressor activity, a
regulation of lipid, fatty-acid and/or isoprenoid metabolism
activity, and/or a regulation of C-compound and carbohydrate
utilization activity, e.g. of a transcriptional regulator, in
particular of a transcriptional regulator of the GntR family, e.g.
as indicated in Table I, columns 5 or 7, line 105, is increased,
preferably, conferring the increase of the respective fine
chemical, preferably of beta-cryptoxanthin between 35% and 67% or
more.
[4468] In one embodiment, the activity of the Escherichia coli K12
protein b4401 or its homologs e.g. a protein of a transcriptional
control activity, a unspecified signal transduction activity, a
two-component signal transduction system (e.g. response regulator
component activity, a transcriptional activator activity, a
regulation of respiration activity, an aerobic respiration
activity, and/or an anaerobic respiration activity, e.g. of a
response regulator, in particular of a response regulator of the
OmpR family, e.g. as indicated in Table I, columns 5 or 7, line
106, is increased, preferably, conferring the increase of the
respective fine chemical, preferably of beta-cryptoxanthin between
48% and 60% or more.
[4469] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YCL040W or its homologs, e.g. a protein having a
C-compound and carbohydrate utilization activity, a C-compound or
carbohydrate transport activity, a activity in the glycolysis
and/or a activity in the gluconeogenesis, and/or a cellular import
activity, in particular of a hexokinase, preferably of a glucinase,
e.g. as indicated in Table I, columns 5 or 7, line 103, is
increased, preferably, conferring an increase of the respective
fine chemical, preferably of beta-cryptoxanthin between 36% and 51%
or more.
[4470] In one embodiment, the activity of the Escherichia coli K12
protein b2699 or its homologs e.g. a DNA recombination and DNA
repair activity, a pheromone response activity, a mating-type
determination activity, a sex-specific protein activity, a
nucleotide binding activity and/or a protease and nuclease
activity, in particular a DNA strand exchange and recombination
protein with protease and nuclease activity, in particular of the
superfamily of the recombination protein recA, e.g. as indicated in
Table I, columns 5 or 7, line 108, is increased, preferably,
conferring the increase of the respective fine chemical, preferably
of zeaxanthin between 25% and 48% or more. In one embodiment, the
activity of the Saccharomyces cerevisiae protein YHR055C or its
homologs, e.g. a protein having a detoxification activity,
particular of a metallothionein, preferably of copper binding
metallothionein or its homolog, e.g. as indicated in Table I,
columns 5 or 7, line 107, is increased, preferably, conferring an
increase of the respective fine chemical, preferably of zeaxanthin
between 05% and 38% or more.
[4471] [0046.0.10.10] In one embodiment, the activity of the
Escherichia coli K12 protein b0050 or its homologs e.g. a conserved
protein potentially involved in protein-protein interaction, in
particular of a conserved protein potentially involved in
protein-protein interaction of the superfamily of apaG proteins,
e.g. as indicated in Table I, columns 5 or 7, line 468, is
increased conferring preferably an increase of the respective fine
chemical and of further carotenoids, preferably xanthophylls.
[4472] In one embodiment, the activity of the Escherichia coli K12
protein b0851 or its homologs, e.g. oxygen insensitive NADPH
nitroreductase, in particular of a oxygen insensitive NADPH
nitroreductase of the superfamily of NADPH-flavin oxidoreductase
homologs, e.g. as indicated in Table I, columns 5 or 7, line 469,
is increased conferring preferably an increase of the respective
fine chemical and of further carotenoids, preferably
xanthophylls.
[4473] In one embodiment, the activity of the Escherichia coli K12
protein b2211 or its homologs, e.g. an ATP-binding transport
protein of the ABC superfamily, in particular of an ATP-binding
transport proteins of the ABC superfamily of the superfamily of
unassigned ATP-binding cassette proteins, e.g. as indicated in
Table I, columns 5 or 7, line 470, is increased conferring
preferably an increase of the respective fine chemical and of
further carotenoids, preferably xanthophylls.
[4474] In one embodiment, the activity of the Escherichia coli K12
protein b3926 or its homologs, e.g. a glycerol kinase, in
particular of a glycerol kinase of the superfamily of
xylulokinases, e.g. as indicated in Table I, columns 5 or 7, line
471, is increased conferring preferably an increase of the
respective fine chemical and of further carotenoids, preferably
xanthophylls.
[4475] In one embodiment, the activity of the Escherichia coli K12
protein b0986 or its homologs e.g. lipoprotein, in particular of a
lipoprotein of the superfamily of the hypothetical protein b1706,
e.g. as indicated in Table I, columns 5 or 7, line 104, is
increased; conferring preferably an increase of the respective fine
chemical and of further carotenoids, preferably xanthophylls.
[4476] In one embodiment, the activity of the Escherichia coli K12
protein b3684 or its homologs, meaning e.g. a transcriptional
regulator of the GntR family, is increased; preferably an increase
of the respective fine chemical and of further carotenoids,
preferably xanthophylls is conferred. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein having a transcriptional control activity, a
regulation of C-compound and carbohydrate utilization activity, a
DNA binding activity, a transcriptional repressor activity, a
regulation of lipid, fatty-acid and/or isoprenoid metabolism
activity, and/or a regulation of C-compound and carbohydrate
utilization activity, e.g. the activity of a transcriptional
regulator, in particular of a transcriptional regulator of the GntR
family, e.g. as indicated in Table I, columns 5 or 7, line 105,
preferably conferring an increase of the respective fine chemical
and of further carotenoids, preferably xanthophylls.
[4477] In one embodiment, the activity of the Escherichia coli K12
protein b4401 or its homologs e.g. a protein of a transcriptional
control activity, a unspecified signal transduction activity, a
two-component signal transduction system (e.g. response regulator
component activity, a transcriptional activator activity, a
regulation of respiration activity, an aerobic respiration
activity, and/or an anaerobic respiration activity, e.g. of a
response regulator, in particular of a response regulator of the
OmpR family, e.g. as indicated in Table I, columns 5 or 7, line
106, is increased, preferably conferring an increase of the
respective fine chemical and of further carotenoids, preferably
xanthophylls.
[4478] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YCL040W or its homologs, e.g. a protein having a
C-compound and carbohydrate utilization activity, a C-compound or
carbohydrate transport activity, a activity in the glycolysis
and/or a activity in the gluconeogenesis, and/or a cellular import
activity, in particular of a hexokinase, preferably of a glucinase,
e.g. as indicated in Table I, columns 5 or 7, line 103, is
increased, preferably conferring an increase of the respective fine
chemical and of further carotenoids, preferably xanthophylls.
[4479] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2699 or its homologs e.g. a DNA recombination and
DNA repair activity, a pheromone response activity, a mating-type
determination activity, a sex-specific protein activity, a
nucleotide binding activity and/or a protease and nuclease
activity, in particular a DNA strand exchange and recombination
protein with protease and nuclease activity, in particular of the
superfamily of the recombination protein recA, e.g. as indicated in
Table I, columns 5 or 7, line 108, is increased, preferably
conferring an increase of the respective fine chemical and of
further carotenoids, preferably xanthophylls.
[4480] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YHR055C or its homologs, e.g. a protein having a
detoxification activity, particular of a metallothionein,
preferably of copper binding metallothionein or its homolog, e.g.
as indicated in Table I, columns 5 or 7, line 107, is increased,
preferably conferring an increase of the respective fine chemical
and of further carotenoids, preferably xanthophylls is
conferred.
[4481] [0047.0.0.10] to [0048.0.0.10]: see [0047.0.0.0] to
[0048.0.0.0]
[4482] [0049.0.10.10] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
cryptoxanthin preferably has the structure of the polypeptide
described herein. In a particular embodiment, the polypeptides used
in the process of the present invention or the polypeptide of the
present invention comprises the sequence of a consensus sequence as
indicated in Table IV, column 7, lines 103 to 107 or and/or 468 to
471 or of a polypeptide as indicated in Table II, columns 5 or 7,
lines 103 to 106 and/or 468 to 471, or of a functional homologue
thereof as described herein, or of a polypeptide encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or its herein described functional homologues and
has the herein mentioned activity conferring an increase in the
beta-cryptoxanthin level.
[4483] A protein having an activity conferring an increase in the
amount or level of the zeaxanthin preferably has the structure of
the polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence as indicated in Table IV, column 7, lines 107
and 108 or of a polypeptide as indicated in Table II, columns 5 or
7, line 107 or 108 or of a functional homologue thereof as
described herein, or of a polypeptide encoded by the nucleic acid
molecule characterized herein or the nucleic acid molecule
according to the invention, for example by a nucleic acid molecule
as indicated in Table I, columns 5 or 7, lines 107 or 108 or its
herein described functional homologues and has the herein mentioned
activity conferring an increase in the zeaxanthin level.
[4484] [0050.0.10.10] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of cryptoxanthin or zeaxanthin, resp., or their ester, or
glucoside thereof, e.g. the diglucoside thereof.
[4485] [0051.0.10.10] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g. compositions comprising xanthophylls,
in particular cryptoxanthin or zeaxanthin. Depending on the choice
of the organism used for the process according to the present
invention, for example a microorganism or a plant, compositions or
mixtures of various xanthophylls, in particular beta-cryptoxanthin
or zeaxanthin. can be produced.
[4486] [0052.0.0.10] see [0052.0.0.0]
[4487] [0053.0.10.10] In one embodiment, the process of the present
invention comprises one or more of the following steps [4488] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention or the polypeptide used in the
method of the invention, e.g. of a polypeptide having an activity
of a protein as indicated in Table II, column 3, lines 103 to 106
and/or 468 to 471 or line 107 and/or 108 or its homologs, e.g. as
indicated in Table II, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or line 107 and/or 108, activity having herein-mentioned the
respective fine chemical increasing activity; [4489] b) stabilizing
a mRNA conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 103 to 106 and/or 468 to 471 or line 107 and/or 108 or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or line 107 and/or 108, or of a
mRNA encoding the polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity;
[4490] c) increasing the specific activity of a protein conferring
the increased expression of a protein encoded by the nucleic acid
molecule of the invention or of the polypeptide of the present
invention or the nucleic acid molecule or polypeptide used in the
method of the invention having herein-mentioned the respective fine
chemical increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 103
to 106 and/or 468 to 471 or line 107 and/or 108 or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 103
to 106 and/or 468 to 471 or line 107 and/or 108, or decreasing the
inhibitory regulation of the polypeptide of the invention or the
polypeptide used in the method of the invention; [4491] d)
generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention or of the polypeptide
of the invention or the polypeptide used in the method of the
invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 103 to 106 and/or
468 to 471 or line 107 and/or 108 or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or line 107 and/or 108; [4492] e) stimulating activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the present invention or a polypeptide
of the present invention having herein-mentioned the respective
fine chemical increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 103
to 106 and/or 468 to 471 or line 107 and/or 108 or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 103
to 106 and/or 468 to 471 or line 107 and/or 108, by adding one or
more exogenous inducing factors to the organism or parts thereof;
[4493] f) expressing a transgenic gene encoding a protein
conferring the increased expression of a polypeptide encoded by the
nucleic acid molecule of the present invention or a polypeptide of
the present invention, having herein-mentioned the respective fine
chemical increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 103
to 106 and/or 468 to 471 or line 107 and/or 108 or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 103
to 106 and/or 468 to 471 or line 107 and/or 108, and/or [4494] g)
increasing the copy number of a gene conferring the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or the
nucleic acid molecule used in the method of the invention or the
polypeptide of the invention or the polypeptide used in the method
of the invention having herein-mentioned the respective fine
chemical increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 103
to 106 and/or 468 to 471 or line 107 and/or 108 or its homologs,
e.g. as indicated in Table II, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or line 107 and/or 108, activity. [4495] h)
Increasing the expression of the endogenous gene encoding the
polypeptide of the invention or the polypeptide used in the method
of the invention, e.g. a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 103 to 106 and/or
468 to 471 or line 107 and/or 108 or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or line 107 and/or 108, by adding positive expression or
removing negative expression elements, e.g. homologous
recombination can be used to either introduce positive regulatory
elements like for plants the 35S enhancer into the promoter or to
remove repressor elements form regulatory regions. Further gene
conversion methods can be used to disrupt repressor elements or to
enhance to activity of positive elements. Positive elements can be
randomly introduced in plants by T-DNA or transposon mutagenesis
and lines can be identified in which the positive elements have be
integrated near to a gene of the invention, the expression of which
is thereby enhanced; [4496] i) Modulating growth conditions of an
organism in such a manner, that the expression or activity of the
gene encoding the protein of the invention or the protein itself is
enhanced for example microorganisms or plants can be grown for
example under a higher temperature regime leading to an enhanced
expression of heat shock proteins, which can lead an enhanced
respective fine chemical production and/or [4497] j) selecting of
organisms with especially high activity of the proteins of the
invention from natural or from mutagenized resources and breeding
them into the target organisms, e.g. the elite crops.
[4498] [0054.0.10.10] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table II, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or line 107 and/or 108, resp.,
or its homologs activity, e.g. as indicated in Table II, columns 5
or 7, lines 103 to 106 and/or 468 to 471 or line 107 and/or 108,
resp.,
[4499] [0055.0.0.10] to [0067.0.0.10]: see [0055.0.0.0] to
[0067.0.0.0]
[4500] [0068.0.10.10] The mutation is introduced in such a way that
the production of the xanthophylls, in particular of
beta-cryptoxanthin or zeaxanthin is not adversely affected.
[4501] [0069.0.10.10] see [0069.0.0.0]
[4502] [0070.0.10.10] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention or the polypeptide of the invention or the
polypeptide used in the method of the invention, for example the
nucleic acid construct mentioned below, or encoding a protein of
the invention into an organism alone or in combination with other
genes, it is possible not only to increase the biosynthetic flux
towards the end product, but also to increase, modify or create de
novo an advantageous, preferably novel metabolites composition in
the organism, e.g. an advantageous composition of carotenoids, in
particular xanthophylls, e.g. comprising a higher content of (from
a viewpoint of nutritional physiology limited) carotenoids, in
particular xanthophylls, like beta-cryptoxanthin or zeaxanthin.
[4503] [0071.0.0.10] see [0071.0.0.0]
[4504] [0072.0.10.10] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to beta-cryptoxanthin or zeaxanthin further carotenoids,
e.g. carotenes or xanthophylls, in particular ketocarentoids, or
hydrocarotenoids, e.g. Lutein, lycopene, alpha-carotene, or
beta-carentene, or compounds for which beta-cryptoxanthin or
zeaxanthin are precursor compounds, in particular astaxanthin.
[4505] [0073.0.10.10] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
(f) providing a non-human organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant; (g) increasing an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
a homolog thereof, e.g. as indicated in Table II, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or line 107 and/or 108, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the respective fine chemical in an organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, (h) growing an organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant under conditions which permit the
production of the respective fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and (i) if desired, recovering, optionally isolating,
the free and/or bound the respective fine chemical and, optionally
further free and/or bound carotenoids, in particular xanthophylls,
e.g. astaxanthin, synthesized by the organism, the microorganism,
the non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[4506] [0074.0.10.10] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the respective fine chemical or the free and
bound the respective fine chemical but as option it is also
possible to produce, recover and, if desired isolate, other free
or/and bound carotenoids, in particular xanthophylls, e.g.
astaxanthin.
[4507] [0075.0.0.10] to [0077.0.0.10]: see [0075.0.0.0] to
[0077.0.0.0]
[4508] [0078.0.10.10] The organism such as microorganisms or plants
or the recovered, and if desired isolated, the respective fine
chemical can then be processed further directly into foodstuffs or
animal feeds or for other applications, for example according to
the disclosures made in U.S. Pat. No. 6,380,442; U.S. Pat. No.
6,329,557, U.S. Pat. No. 6,329,432, U.S. Pat. No. 6,316,012, U.S.
Pat. No. 6,309,883; U.S. Pat. No. 6,291,533, U.S. Pat. No.
6,291,294, U.S. Pat. No. 6,287,615, U.S. Pat. No. 6,262,284, U.S.
Pat. No. 6,262,284, U.S. Pat. No. 6,261,622, U.S. Pat. No.
6,261,598, U.S. Pat. No. 6,235,315, U.S. Pat. No. 6,224,876, U.S.
Pat. No. 6,221,417, U.S. Pat. No. 6,221,412, U.S. Pat. No.
6,218,436, U.S. Pat. No. 6,207,409, U.S. Pat. No. 6,319,872, U.S.
Pat. No. 6,132,790, U.S. Pat. No. 6,124,113 U.S. Pat. No.
6,110,478, U.S. Pat. No. 6,093,348, U.S. Pat. No. 6,087,152, or
U.S. Pat. No. 6,056,962. (Pharmaceutical and other compositions
comprising zeaxanthin are e.g. described in: U.S. Pat. No.
6,383,523, U.S. Pat. No. 6,368,621, U.S. Pat. No. 6,362,221, U.S.
Pat. No. 6,348,200, U.S. Pat. No. 6,316,012: Cosmetic or
pharmaceutical composition comprising, in combination, a peroxidase
and an anti-singlet oxygen agent, U.S. Pat. No. 6,296,880:
Pharmaceutical compositions and methods for managing skin
conditions U.S. Pat. No. 6,296,877: Stable, aqueous dispersions and
stable, water-dispersible dry xanthophyll powder, their production
and use, U.S. Pat. No. 6,261,598: Carotenoid formulations,
comprising a mixture of B-carotens, lycopene and lutein, U.S. Pat.
No. 6,248,378: Enhanced food products, U.S. Pat. No. 6,248,374:
Stabilized food additive. Processes for the isolation are described
e.g. in U.S. Pat. No. 6,380,442, U.S. Pat. No. 6,362,221:
Compositions containing natural lycopene and natural tocopherol,
U.S. Pat. No. 6,291,204: Fermentative carotenoid production, U.S.
Pat. No. 6,262,284: Process for extraction and purification of
lutein, zeaxanthin and rare carotenoids from marigold flowers and
plants, or U.S. Pat. No. 6,224,876: Isolation and formulations of
nutrient-rich carotenoids. The cited literatures describe some
preferred embodiments. Said applications describe some advantageous
embodiments without meant to be limiting. The fermentation broth,
fermentation products, plants or plant products can be purified as
described in above mentioned applications and other methods known
to the person skilled in the art, e.g. as described in Methods in
Enzymology: Carotenoids, Part A: Chemistry, Separation,
Quantitation and Antioxidation, by John N Abelson or Part B,
Metabolism, Genetics, and Biochemistry, or described herein below.
Products of these different work-up procedures are xanthophylls, in
particular zeaxanthin or beta-cryptoxanthin or xanthophylls, in
particular zeaxanthin or cryptoxanthin comprising compositions
which still comprise fermentation broth, plant particles and cell
components in different amounts, advantageously in the range of
from 0 to 99% by weight, preferably below 80% by weight, especially
preferably between below 50% by weight.
[4509] [0079.0.0.10] to [0084.0.0.10]: see [0079.0.0.0] to
[0084.0.0.0]
[4510] [0084.1.0.10] The invention also contemplates embodiments in
which the .beta.-carotene, or other carotenoid precursor compounds
in the production of the respective fine chemical, is present in
the flowers of the flowering plant chosen as the host (for example,
marigolds). The invention also contemplates embodiments in which a
host plant's flowers lack .beta.-carotene or other carotenoid
precursors, such as the vinca. In a plant of the latter type, the
inserted DNA includes genes that code for carotenoid precursors
(compounds that can be converted biologically into .beta.-carotene)
and a ketolase, as well as a hydroxylase, if otherwise absent. In
one embodiment, preferred flowering plants include, but are not
limited to: Amaryllidaceae (Allium, Narcissus); Apocynaceae
(Catharanthus); Asteraceae, alternatively Compositae (Aster,
Calendula, Callistephus, Cichorium, Coreopsis, Dahlia,
Dendranthema, Gazania, Gerbera, Helianthus, Helichrysum, Lactuca,
Rudbeckia, Tagetes, Zinnia); Balsaminaceae (Impatiens); Begoniaceae
(Begonia); Caryophyllaceae (Dianthus); Chenopodiaceae (Beta,
Spinacia); Cucurbitaceae (Citrullus, Curcurbita, Cucumis);
Cruciferae (Alyssum, Brassica, Erysimum, Matthiola, Raphanus);
Gentinaceae (Eustoma); Geraniaceae (Pelargonium); Graminae,
alternatively Poaceae, (Avena, Horedum, Oryza, Panicum, Pennisetum,
Poa, Saccharum, Secale, Sorghum, Triticum, Zea); Euphorbiaceae
(Poinsettia); Labiatae (Salvia); Leguminosae (Glycine, Lathyrus,
Medicago, Phaseolus, Pisum); Liliaceae (Cilium); Lobeliaceae
(Lobelia); Malvaceae (Abelmoschus, Gossypium, Melva);
Plumbaginaceae (Limonium); Polemoniaceae (Phlox); Primulaceae
(Cyclamen); Ranunculaceae (Aconitum, Anemone, Aquilegia, Caltha,
Delphinium, Ranunculus); Rosaceae (Rosa); Rubiaceae (Pentas);
Scrophulariaceae (Angelonia, Antirrhinum, Torenia); Solanaceae
(Capsicum, Lycopersicon, Nicotiana, Petunia, Solanum); Umbelliferae
(Apium, Daucus, Pastinaca); Verbenaceae (Verbena, Lantana);
Violaceae (Viola).
[4511] [0085.0.10.10] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [4512] a) a nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108, or a derivative thereof, or [4513]
b) a genetic regulatory element, for example a promoter, which is
functionally linked to the nucleic acid sequence as indicated in
Table I, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, or a derivative thereof, or [4514] c) (a) and
(b) is/are not present in its/their natural genetic environment or
has/have been modified by means of genetic manipulation methods, it
being possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[4515] [0086.0.0.10] to [0087.0.0.10]: see [0086.0.0.0] to
[0087.0.0.0]
[4516] [0088.0.10.10] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned
xanthophylls and the general amount of xanthohylls as energy source
in feed. Further, this is also important for the production of
cosmetic compostions since, for example, the antioxidant level of
plant extraction is dependent on the amount of th abovementioned of
xanthophylls and/or the amount of carotenoids as antioxidants.
[4517] [0088.1.0.10] see [0088.1.0.0]
[4518] [0089.0.0.10] to [0090.0.0.10]: see [0089.0.0.0] to
[0090.0.0.0]
[4519] [0091.0.10.10] Thus, the content of plant components and
preferably also further impurities is as low as possible, and the
abovementioned xanthophylls, in particular the fine chemical,
is/are obtained in as pure form as possible. In these applications,
the content of plant components advantageously amounts to less than
10%, preferably 1%, more preferably 0.1%, very especially
preferably 0.01% or less.
[4520] [0092.0.0.10] to [0094.0.0.10]: see [0092.0.0.0] to
[0094.0.0.0]
[4521] [0095.0.10.10] It may be advantageous to increase the pool
of said carotenoids, in particular xanthophylls, preferably
cryptoxanthin or zeaxanthin in the transgenic organisms by the
process according to the invention in order to isolate high amounts
of the essentially pure respective fine chemical.
[4522] [0096.0.10.10] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example
zeaxanthin or cryptoxanthin in the organism, is useful to increase
the production of the respective fine chemical. It has been
reported, that the inhibition of Lyopene production increases the
amount of other xanthophylls in the cell. Further it may be, that
the inhibition of enzymes using zeaxanthin or cryptoxanthin as
substrate increases the amount of said chemicals in a cell. For
example, in one embodiment, it can be advantageous to inhibit the
production of astaxanthin, if a high amount of cryptoxanthin or
zeaxanthin is desired.
[4523] [0097.0.10.10] Glucosides, in particular, dicglucosides of
the zeaxanthin and beta-cryptoxanthin as well as other modification
of zeaxanthin and cryptoxanthin are known to a person skilled in
the art. In may also be advantageous to increase the content of the
bound respective fine chemical, e.g. of modification of zeaxanthin
and cryptoxanthin, in particular its glucosides, e.g.
diglucosides.
[4524] [0098.0.10.10] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated. The production of further carotenoids, e.g.
carotenes or xanthophylls, in particular ketocarentoids or
hydrocarotenoids, e.g. lutein, lycopene, alpha-carotene, or
beta-carentene, or compounds for which the respective fine chemical
is a biosynthesis precursor compounds, e.g. astaxanthin, or
mixtures thereof or mixtures of other carotenoids, in particular of
xanthophylls, by the process according to the invention is
advantageous.
[4525] [0099.0.10.10] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[4526] [0100.0.10.10] Transgenic plants which comprise the
carotenoids such as said xanthophylls, e.g. cryptoxanthin or
zeaxanthin (or astaxanthin as it is synthesized from cryptoxanthin
or zeaxanthin) synthesized in the process according to the
invention can advantageously be marketed directly without there
being any need for the carotenoids synthesized to be isolated.
Plants for the process according to the invention are listed as
meaning intact plants and all plant parts, plant organs or plant
parts such as leaf, stem, seeds, root, tubers, anthers, fibers,
root hairs, stalks, embryos, calli, cotelydons, petioles, harvested
material, plant tissue, reproductive tissue and cell cultures which
are derived from the actual transgenic plant and/or can be used for
bringing about the transgenic plant. In this context, the seed
comprises all parts of the seed such as the seed coats, epidermal
cells, seed cells, endosperm or embryonic tissue. However, the
respective fine chemical produced in the process according to the
invention can also be isolated from the organisms, advantageously
plants, (in the form of their oils, fats, lipids, as extracts, e.g.
ether, alcohol, or other organic solvents or water containing
extract and/or free xanthophylls. The respective fine chemical
produced by this process can be obtained by harvesting the
organisms, either from the crop in which they grow, or from the
field. This can be done via pressing or extraction of the plant
parts. To increase the efficiency of extraction it is beneficial to
clean, to temper and if necessary to hull and to flake the plant
material.E.g., oils, fats, and/or lipids comprising xanthophylls
can be obtained by what is known as cold beating or cold pressing
without applying heat. To allow for greater ease of disruption of
the plant parts, specifically the seeds, they can previously be
comminuted, steamed or roasted. Seeds, which have been pretreated
in this manner can subsequently be pressed or extracted with
solvents such as warm hexane. The solvent is subsequently removed.
In the case of microorganisms, the latter are, after harvesting,
for example extracted directly without further processing steps or
else, after disruption, extracted via various methods with which
the skilled worker is familiar. Thereafter, the resulting products
can be processed further, i.e. degummed and/or refined. In this
process, substances such as the plant mucilages and suspended
matter canb be first removed. What is known as desliming can be
affected enzymatically or, for example, chemico-physically by
addition of acid such as phosphoric acid.
[4527] Because carotenoids in microorganisms are localized
intracellular, their recovery essentials comes down to the
isolation of the biomass. Well-established approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Of the residual
hydrocarbon, adsorbed on the cells, has to be removed. Solvent
extraction or treatment with surfactants have been suggested for
this purpose. However, it can be advantageous to avoid this
treatment as it can result in cells devoid of most carotenoids.
[4528] [0101.0.10.10] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[4529] [0102.0.10.10] Xanthophylls, in particular
beta-cryptoxanthin or zeaxanthin can for example be detected
advantageously via HPLC, LC or GC separation methods. The
unambiguous detection for the presence of xanthophylls, in
particular beta-cryptoxanthin or zeaxanthin containing products can
be obtained by analyzing recombinant organisms using analytical
standard methods: LC, LC-MS, MS or TLC). The material to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding, cooking, or via other applicable
methods
[4530] [0103.0.10.10] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [4531] a) nucleic acid molecule encoding, preferably
at least the mature form, of the polypeptide having a sequence as
indicated in Table II, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108, or a fragment thereof, which
confers an increase in the amount of the respective fine chemical
in an organism or a part thereof; [4532] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule having a sequence as indicated in Table I, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108, [4533]
c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [4534] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[4535] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [4536]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [4537] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [4538] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers pairs
having a sequence as indicated in Table III, column 7, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [4539] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from an expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [4540] j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence having a sequences as
indicated in Table IV, column 7, lines 103 to 106 and/or 468 to 471
or lines 107 and/or 108 and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[4541] k) nucleic acid molecule comprising one or more of the
nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of the polypeptide indicated in Table
II, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[4542] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[4543] [0103.1.10.10] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108, by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of the sequence
indicated in Table I A, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108: In one embodiment, the nucleic acid
molecule used in the process of the invention is less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence indicated in
Table I A, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table II A, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108.
[4544] [0103.2.10.10.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108, by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of the sequence
indicated in Table I B, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108: In one embodiment, the nucleic acid
molecule used in the process of the invention is less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence indicated in
Table I B, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table II B, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108.
[4545] [0104.0.10.10] In one embodiment, the nucleic acid molecule
of the invention or used in the process distinguishes over the
sequence indicated in Table I, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108 by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process or the nucleic acid used in the process of the
invention of the invention does not consist of the sequence
indicated in Table I, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108 In one embodiment, the nucleic acid
molecule of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to the sequence indicated in Table
I, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108. In another embodiment, the nucleic acid molecule does
not encode a polypeptide of a sequence indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108.
[4546] [0105.0.0.10] to [0107.0.0.10]: see [0105.0.0.0] to
[0107.0.0.0]
[4547] [0108.0.10.10] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108, nucleic acid molecules which are
derived from an amino acid sequences as indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108 or from polypeptides comprising the consensus sequence
as indicated in Table IV, column 7, lines 103 to 106 and/or 468 to
471 or lines 107 and/or 108, or their derivatives or homologues
encoding polypeptides with the enzymatic or biological activity of
a polypeptide as indicated in Table II, column 3, 5 or 7, lines 103
to 106 and/or 468 to 471 or lines 107 and/or 108, e.g. conferring
the increase of the respective fine chemical, meaning
beta-cryptoxanthin or zeaxanthin, resp., after increasing its
expression or activity, are advantageously increased in the process
according to the invention.
[4548] [0109.0.10.10] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular
beta-cryptoxanthin or zeaxanthin, produced in the process according
to the invention.
[4549] [0110.0.10.10] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
used in the process of the invention, e.g. of a protein as
indicated in Table II, column 5, lines 103 to 106 and/or 468 to 471
or lines 107 and/or 108 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, lines 103 to 106 and/or 468 to 471
or lines 107 and/or 108 or of its homologs, e.g. as indicated in
Table I or Table II, column 7, lines 103 to 106 and/or 468 to 471
or lines 107 and/or 1088, can be determined from generally
accessible databases.
[4550] [0111.0.0.10] see [0111.0.0.0]
[4551] [0112.0.10.10] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table II, column 3, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108 or having the
sequence of a polypeptide as indicated in Table II, columns 5 and
7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108 and
conferring an increase in the cryptoxanthin or zeaxanthin
level.
[4552] [0113.0.0.10] to [01200.0.10]: see [0113.0.0.0] to
[0120.0.0.0]
[4553] [0120.1.10.10]: Production strains which are also
advantageously selected in the process according to the invention
are microorganisms selected from the group green algae, like
Spongioccoccum exentricum, Chlorella sorokiniana (pyrenoidosa,
7-11-05), or form the group of fungi like fungi belonging to the
Daccrymycetaceae family, or non-photosynthetic bacteria, like
methylotrophs, flavobacteria, actinomycetes, like streptomyces
chrestomyceticus, Mycobacteria like Mycobacterim phlei, or
Rhodobacter capsulatus.
[4554] [0121.0.10.10] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108 or the functional homologues thereof as described
herein, preferably conferring above-mentioned activity, i.e.
conferring a cryptoxanthin level increase after increasing the
activity of the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or conferring a
zeaxanthin level increase after increasing the activity of the
polypeptide sequences indicated in Table II, columns 5 or 7, lines
107 and/or 108.
[4555] [0122.0.0.10] to [0127.0.0.10]: see [0122.0.0.0] to
[0127.0.0.0]
[4556] [0128.0.10.10] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, column 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108, by
means of polymerase chain reaction can be generated on the basis of
a sequence shown herein, for example the sequence as indicated in
Table I, columns 5 or 7, lines 103 to 106 and/or 468 to 471, resp.,
or lines 107 and/or 108, resp. or the sequences derived from a
sequences as indicated in Table II, columns 5 or 7, lines 103 to
106 and/or 468 to 471, resp., or lines 107 and/or 108, resp.
[4557] [0129.0.10.10] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention or the polypeptide used in the method of the invention,
from which conserved regions, and in turn, degenerate primers can
be derived. Conserved region for the polypeptide of the invention
or the polypeptide used in the method of the invention are
indicated in the alignments shown in the figures. Conserved regions
are those, which show a very little variation in the amino acid in
one particular position of several homologs from different origin.
The consensus sequence indicated in Table IV, column 7, lines 103
to 106 and/or 468 to 471 or lines 107 and/or 108 is derived from
said alignments.
[4558] [0130.0.10.10] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of
zeaxanthin or cryptoxanthin after increasing the expression or
activity the protein comprising said fragment.
[4559] [0131.0.0.10] to [0138.0.0.10]: see [0131.0.0.0] to
[0138.0.0.0]
[4560] [0139.0.10.10] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical level increase,
derived from other organisms, can be encoded by other DNA sequences
which hybridise to a sequences indicated in Table I, columns 5 or
7, lines 103 to 106 and/or 468 to 471, preferably of Table I B,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 for
cryptoxanthin or indicated in Table I, columns 5 or 7, lines 107
and/or 108, preferably of Table I B, columns 5 or 7, lines 107
and/or 108 for zeaxanthin under relaxed hybridization conditions
and which code on expression for peptides having the respective
fine chemical, i.e. cryptoxanthin or zeaxanthin, resp.,
increasing-activity.
[4561] [0140.0.0.10] to [0146.0.0.10]: see [0140.0.0.0] to
[0146.0.0.0]
[4562] [0147.0.10.10] Further, the nucleic acid molecule of the
invention or used in the method of the invention comprises a
nucleic acid molecule, which is a complement of one of the
nucleotide sequences of above mentioned nucleic acid molecules or a
portion thereof. A nucleic acid molecule which is complementary to
one of the nucleotide sequences indicated in Table I, columns 5 or
7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
preferably of Table I B, columns 5 or 7, lines 103 to 106 and/or
468 to 471 or lines 107 and/or 108, is one which is sufficiently
complementary to one of said nucleotide sequences such that it can
hybridise to one of said nucleotide sequences, thereby forming a
stable duplex. Preferably, the hybridisation is performed under
stringent hybridisation conditions. However, a complement of one of
the herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[4563] [0148.0.10.10] The nucleic acid molecule of the invention or
used in the method of the invention comprises a nucleotide sequence
which is at least about 30%, 35%, 40% or 45%, preferably at least
about 50%, 55%, 60% or 65%, more preferably at least about 70%,
80%, or 90%, and even more preferably at least about 95%, 97%, 98%,
99% or more homologous to a nucleotide sequence indicated in Table
I, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, preferably of Table I B, columns 5 or 7, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108, or a portion thereof
and preferably has above mentioned activity, in particular having a
cryptoxanthin or zeaxanthin increasing activity after increasing
the activity or an activity of a product of a gene encoding said
sequences or their homologs.
[4564] [0149.0.10.10] The nucleic acid molecule of the invention or
used in the process of the invention comprises a nucleotide
sequence which hybridizes, preferably hybridizes under stringent
conditions as defined herein, to one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108, preferably of Table I B, columns 5
or 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
or a portion thereof and encodes a protein having above-mentioned
activity, e.g. conferring a of cryptoxanthin or zeaxanthin
increase, resp., and optionally, the activity of protein indicated
in Table II, column 5, lines 103 to 106 and/or 468 to 471, or lines
107 and/or 108, preferably of Table II B, columns 5 or 7, lines 103
to 106 and/or 468 to 471 or lines 107 and/or 108.
[4565] [00149.1.10.10] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table I, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108, preferably of Table I B,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, has further one or more of the activities annotated or
known for the a protein as indicated in Table II, column 3, lines
103 to 106 and/or 468 to 471 or lines 107 and/or 108.
[4566] [0150.0.10.10] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table I, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, preferably of Table I B, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108, for
example a fragment which can be used as a probe or primer or a
fragment encoding a biologically active portion of the polypeptide
of the present invention or of a polypeptide used in the process of
the present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of cryptoxanthin or zeaxanthin, resp., if
its activity is increased. The nucleotide sequences determined from
the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table I, columns
5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
an anti-sense sequence of one of the sequences, e.g., as indicated
in Table I, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, or naturally occurring mutants thereof.
Primers based on a nucleotide of invention can be used in PCR
reactions to clone homologues of the polypeptide of the invention
or of the polypeptide used in the process of the invention, e.g. as
the primers described in the examples of the present invention,
e.g. as shown in the examples. A PCR with the primer pairs
indicated in Table III, column 7, lines 103 to 106 and/or 468 to
471 or lines 107 and/or 108 can result in a fragment of a
polynucleotide sequence as indicated in Table I, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108 or its
gene product.
[4567] [0151.0.0.10]: see [0151.0.0.0]
[4568] [0152.0.10.10] The nucleic acid molecule of the invention or
the nucleic acid used in the method of the invention encodes a
polypeptide or portion thereof which includes an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table II, columns 5 or 7, lines 103 to 106 and/or
468 to 471 or lines 107 and/or 108 such that the protein or portion
thereof maintains the ability to participate in the respective fine
chemical production, in particular a cryptoxanthin (lines 103 to
106 and/or 468 to 471) or zeaxanthin (lines 107 and/or 108)
increasing activity as mentioned above or as described in the
examples in plants or microorganisms is comprised.
[4569] [0153.0.10.10] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 103
to 106 and/or 468 to 471 or lines 107 and/or 108 such that the
protein or portion thereof is able to participate in the increase
of the respective fine chemical production. In one embodiment, a
protein or portion thereof as indicated in Table II, columns 5 or
7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108 has
for example an activity of a polypeptide indicated in Table II,
column 3, lines 103 to 106 and/or 468 to 471 or lines 107 and/or
108.
[4570] [0154.0.10.10] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108 and has above-mentioned
activity, e.g. conferring preferably the increase of the respective
fine chemical.
[4571] [0155.0.0.10] to [0156.0.0.10]: see [0155.0.0.0] to
[0156.0.0.0]
[4572] [0157.0.10.10] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences as
indicated in Table I, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108 (and portions thereof) due to
degeneracy of the genetic code and thus encode a polypeptide of the
present invention, in particular a polypeptide having above
mentioned activity, e.g. conferring an increase in the respective
fine chemical in a organism, e.g. as polypeptides comprising a
consensus sequence as indicated in Table IV, column 7, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108 or as polypeptides
depicted in Table II, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108 or the functional homologues.
Advantageously, the nucleic acid molecule of the invention or used
in the method of the invention comprises, or in an other embodiment
has, a nucleotide sequence encoding a protein comprising, or in an
other embodiment having, an amino acid sequence of a consensus
sequences as indicated in Table IV, column 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108 or of the polypeptide as
indicated in Table II, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108, resp., or the functional
homologues. In a still further embodiment, the nucleic acid
molecule of the invention encodes a full length protein which is
substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table IV, column 7, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108 or of a polypeptide
as indicated in Table II, columns 5 or 7, lines 103 to 106 and/or
468 to 471 or lines 107 and/or 108 or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table I, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108, resp., preferably of Table I A,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108. Preferably the nucleic acid molecule of the invention
is a functional homologue or identical to a nucleic acid molecule
indicated in Table I B, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108.
[4573] [0158.0.0.10] to [0160.0.0.10]: see [0158.0.0.0] to
[0160.0.0.0]
[4574] [0161.0.10.10] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108. The nucleic acid
molecule is preferably at least 20, 30, 50, 100, 250 or more
nucleotides in length.
[4575] [0162.0.0.10] see [0162.0.0.0]
[4576] [0163.0.10.10] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 103 to 106 and/or
468 to 471 or lines 107 and/or 108 corresponds to a
naturally-occurring nucleic acid molecule of the invention. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein). Preferably, the nucleic
acid molecule encodes a natural protein having above-mentioned
activity, e.g. conferring the increase of the amount of the
respective fine chemical in a organism or a part thereof, e.g. a
tissue, a cell, or a compartment of a cell, after increasing the
expression or activity thereof or the activity of a protein of the
invention or used in the process of the invention.
[4577] [0164.0.0.10] see [0164.0.0.0]
[4578] [0165.0.10.10] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108, resp.,
[4579] [0166.0.0.10] to [0167.0.0.10]: see [0166.0.0.0] to
[0167.0.0.0]
[4580] [0168.0.10.10] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
preferably of Table II B, column 7, lines 103 to 106 and/or 468 to
471 or lines 107 and/or 108, resp., yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table II, columns 5 or 7, lines
103 to 106 and/or 468 to 471 or lines 107 and/or 108, preferably of
Table II B, column 7, lines 103 to 106 and/or 468 to 471 or lines
107 and/or 108, resp., and is capable of participation in the
increase of production of the respective fine chemical after
increasing its activity, e.g. its expression. Preferably, the
protein encoded by the nucleic acid molecule is at least about 60%
identical to a sequence as indicated in Table II, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
preferably of Table II B, column 7, lines 103 to 106 and/or 468 to
471 or lines 107 and/or 108, resp., more preferably at least about
70% identical to one of the sequences as indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, preferably of Table II B, column 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108, resp., even more
preferably at least about 80%, 90%, 95% homologous to a sequence as
indicated in Table II, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108, preferably of Table II B, column 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108, resp.,
and most preferably at least about 96%, 97%, 98%, or 99% identical
to the sequence as indicated in Table II, columns 5 or 7, lines 103
to 106 and/or 468 to 471 or lines 107 and/or 108, preferably of
Table II B, column 7, lines 103 to 106 and/or 468 to 471 or lines
107 and/or 108, resp.
[4581] [0169.0.0.10] to [0172.0.0.10]: see [0169.0.0.0] to
[0172.0.0.0]
[4582] [0173.0.10.10] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 10237 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 10237 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[4583] [0174.0.0.10]: see [0174.0.0.0]
[4584] [0175.0.10.10] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 10238 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 10238 by the above program algorithm with the
above parameter set, has a 80% homology.
[4585] [0176.0.10.10] Functional equivalents derived from one of
the polypeptides as indicated in Table II, columns 5 or 7, lines
103 to 106 and/or 468 to 471 or lines 107 and/or 108, resp.,
according to the invention by substitution, insertion or deletion
have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%,
60%, 65% or 70% by preference at least 80%, especially preferably
at least 85% or 90%, 91%, 92%, 93% or 94%, very especially
preferably at least 95%, 97%, 98% or 99% homology with one of the
polypeptides as indicated in Table II, columns 5 or 7, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108, resp., according to
the invention and are distinguished by essentially the same
properties as a polypeptide as indicated in Table II, columns 5 or
7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
resp.,
[4586] [0177.0.10.10] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108, resp., according to
the invention by substitution, insertion or deletion have at least
30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70%
by preference at least 80%, especially preferably at least 85% or
90%, 91%, 92%, 93% or 94%, very especially preferably at least 95%,
97%, 98% or 99% homology with one of the polypeptides as indicated
in Table II, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, resp., according to the invention and encode
polypeptides having essentially the same properties as a
polypeptide as indicated in Table II, columns 5 or 7, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108, resp.
[4587] [0178.0.0.10] see [0178.0.0.0]
[4588] [0179.0.10.10] A nucleic acid molecule encoding an
homologous to a protein sequence as indicated in Table II, columns
5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
preferably of Table II B, column 7, lines 103 to 106 and/or 468 to
471 or lines 107 and/or 108, resp., can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table I, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108, resp.,
such that one or more amino acid substitutions, additions or
deletions are introduced into the encoded protein. Mutations can be
introduced into the encoding sequences of a sequences for example
as indicated in Table I, columns 5 or 7, lines 103 to 106 and/or
468 to 471 or lines 107 and/or 108, resp., by standard techniques,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
[4589] [0180.0.0.10] to [0183.0.0.10]: see [0180.0.0.0] to
[0183.0.0.0]
[4590] [0184.0.10.10] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table I, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
preferably of Table I B, column 7, lines 103 to 106 and/or 468 to
471 or lines 107 and/or 108, resp., or of the nucleic acid
sequences derived from a sequences as indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, preferably of Table II B, column 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108, resp., comprise also
allelic variants with at least approximately 30%, 35%, 40% or 45%
homology, by preference at least approximately 50%, 60% or 70%,
more preferably at least approximately 90%, 91%, 92%, 93%, 94% or
95% and even more preferably at least approximately 96%, 97%, 98%,
99% or more homology with one of the nucleotide sequences shown or
the abovementioned derived nucleic acid sequences or their
homologues, derivatives or analogues or parts of these. Allelic
variants encompass in particular functional variants which can be
obtained by deletion, insertion or substitution of nucleotides from
the sequences shown, preferably from a sequence as indicated in
Table I, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, resp., or from the derived nucleic acid
sequences, the intention being, however, that the enzyme activity
or the biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[4591] [0185.0.10.10] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table I, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, preferably of Table I B, column 7, lines 103
to 106 and/or 468 to 471 or lines 107 and/or 108, resp., In one
embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotides or nucleotide
sequences not shown in any one of sequences as indicated in Table
I, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, preferably of Table I B, column 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108, resp., In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequence as indicated in Table I, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108, preferably of Table I B,
column 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or
108, resp.,
[4592] [0186.0.10.10] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
preferably of Table II B, column 7, lines 103 to 106 and/or 468 to
471 or lines 107 and/or 108, resp., In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table II, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, resp. preferably of Table II B, column 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108.
[4593] [0187.0.10.10] In one embodiment, a nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
preferably of Table II B, column 7, lines 103 to 106 and/or 468 to
471 or lines 107 and/or 108, resp., comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a sequences as indicated in Table II, columns 5
or 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
resp., preferably of Table II B, column 7, lines 103 to 106 and/or
468 to 471 or lines 107 and/or 108.
[4594] [0188.0.10.10] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, resp., preferably compared to a sequence as indicated
in Table II, column 3 and 5, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, and expressed under identical conditions.
[4595] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, column 7, lines 103 to 106 and/or 468 to 471 or lines
107 and/or 108.
[4596] [0189.0.10.10] Homologues of a sequences as indicated in
Table I, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, resp., or of a derived sequences as indicated
in Table II, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, resp., also mean truncated sequences, cDNA,
single-stranded DNA or RNA of the coding and noncoding DNA
sequence. Homologues of said sequences are also understood as
meaning derivatives, which comprise noncoding regions such as, for
example, UTRs, terminators, enhancers or promoter variants. The
promoters upstream of the nucleotide sequences stated can be
modified by one or more nucleotide substitution(s), insertion(s)
and/or deletion(s) without, however, interfering with the
functionality or activity either of the promoters, the open reading
frame (=ORF) or with the 3'-regulatory region such as terminators
or other 3' regulatory regions, which are far away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[4597] [0190.0.0.10]: see [0190.0.0.0]
[4598] [0191.0.0.10] see [0191.1.0.0]
[4599] [0192.0.0.10] to [0203.0.0.10]: see [0192.0.0.0] to
[0203.0.0.0]
[4600] [0204.0.10.10] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [4601] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, columns 5 or 7, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108, preferably of Table
II B, column 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108 resp.; or a fragment thereof conferring an increase in
the amount of the respective fine chemical, i.e. beta-cryptoxanthin
(lines 103 to 106 and/or 468 to 471) or zeaxanthin (lines 107
and/or 108), resp., in an organism or a part thereof [4602] b)
nucleic acid molecule comprising, preferably at least the mature
form, of a nucleic acid molecule as indicated in Table I, columns 5
or 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
preferably of Table I B, column 7, lines 103 to 106 and/or 468 to
471 or lines 107 and/or 108 resp., or a fragment thereof conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [4603] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [4604]
d) nucleic acid molecule encoding a polypeptide whose sequence has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [4605] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under stringent hybridisation conditions and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [4606] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [4607] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [4608]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
primers or primer pairs as indicated in Table III, column 7, lines
103 to 106 and/or 468 to 471 or lines 107 and/or 108 and conferring
an increase in the amount of the respective fine chemical, i.e.
beta-cryptoxanthin (lines 103 to 106 and/or 468 to 471) or
zeaxanthin (lines 107 and/or 108), resp., in an organism or a part
thereof; [4609] i) nucleic acid molecule encoding a polypeptide
which is isolated, e.g. from a expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (g), preferably to (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [4610] j) nucleic acid
molecule which encodes a polypeptide comprising a consensus
sequence as indicated in Table IV, column 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108 and conferring an
increase in the amount of the respective fine chemical, i.e.
beta-cryptoxanthin (lines 103 to 106 and/or 468 to 471) or
zeaxanthin (lines 107 and/or 108), resp., in an organism or a part
thereof; [4611] k) nucleic acid molecule encoding the amino acid
sequence of a polypeptide encoding a domain of a polypeptide as
indicated in Table II, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108, preferably of Table II B, column 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108, resp.,
and conferring an increase in the amount of the respective fine
chemical, i.e. beta-cryptoxanthin (lines 103 to 106 and/or 468 to
471) or zeaxanthin (lines 107 and/or 108), resp., in an organism or
a part thereof; and [4612] l) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, preferably of Table II B, column 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108, resp., or a nucleic acid
molecule encoding, preferably at least the mature form of, a
polypeptide as indicated in Table II, columns 5 or 7, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108, preferably of Table
II B, column 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108 resp., and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [4613]
or which encompasses a sequence which is complementary thereto;
whereby, preferably, the nucleic acid molecule according to (a) to
(l) distinguishes over the sequence indicated in Table IA, columns
5 or 7, lines 1 to 5 and/or lines 334 to 338, by one or more
nucleotides. In one embodiment, the nucleic acid molecule does not
consist of the sequence shown and indicated in Table I A or I B,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108: In one embodiment, the nucleic acid molecule is less
than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table I A or I B, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108. In another embodiment,
the nucleic acid molecule does not encode a polypeptide of a
sequence indicated in Table II A or II B, columns 5 or 7, lines 103
to 106 and/or 468 to 471 or lines 107 and/or 108. In an other
embodiment, the nucleic acid molecule of the present invention is
at least 30%, 40%, 50%, or 60% identical and less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence indicated in
Table I A or I B, columns 5 or 7, lines 103 to 106 and/or 468 to
471 or lines 107 and/or 108. In a further embodiment the nucleic
acid molecule does not encode a polypeptide sequence as indicated
in Table II A or II B, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108. Accordingly, in one embodiment, the
nucleic acid molecule of the differs at least in one or more
residues from a nucleic acid molecule indicated in Table I A or I
B, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108. Accordingly, in one embodiment, the nucleic acid
molecule of the present invention encodes a polypeptide, which
differs at least in one or more amino acids from a polypeptide
indicated in Table II A or II B, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108. In another embodiment, a
nucleic acid molecule indicated in Table I A or I B, columns 5 or
7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108 does
not encode a protein of a sequence indicated in Table II A or II B,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108. Accordingly, in one embodiment, the protein encoded by
a sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table II A or II B, columns 5
or 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108.
In a further embodiment, the protein of the present invention is at
least 30%, 40%, 50%, or 60% identical to a protein sequence
indicated in Table II A or II B, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108 and less than 100%,
preferably less than 99.999%, 99.99% or 99.9%, more preferably less
than 99%, 985, 97%, 96% or 95% identical to a sequence as indicated
in Table II A or II B, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108.
[4614] [0205.0.0.10] to [0206.0.0.10]: see [0205.0.0.0] to
[0206.0.0.0]
[4615] [0207.0.10.10] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the carotenoid metabolism, the
xanthophyll metabolism, the astaxanthin metabolism, the amino acid
metabolism, of glycolysis, of the tricarboxylic acid metabolism or
their combinations. As described herein, regulator sequences or
factors can have a positive effect on preferably the gene
expression of the genes introduced, thus increasing it. Thus, an
enhancement of the regulator elements may advantageously take place
at the transcriptional level by using strong transcription signals
such as promoters and/or enhancers. In addition, however, an
enhancement of translation is also possible, for example by
increasing mRNA stability or by inserting a translation enhancer
sequence.
[4616] [0208.0.0.10] to [0226.0.0.10]: see [0208.0.0.0] to
[0226.0.0.0]
[4617] [0227.0.10.10] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[4618] In addition to a sequence indicated in Table I, columns 5 or
7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108 or
its derivatives, it is advantageous to express and/or mutate
further genes in the organisms. Especially advantageously,
additionally at least one further gene of the xanthophyll
biosynthetic pathway such as for cryptoxanthin or zeaxanthin, e.g.
one of the above mentioned genes of this pathway, or e.g. for the
synthesis of astaxanthin or for another provitamin A or for another
carotenoids is expressed in the organisms such as plants or
microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the amino acids desired since, for example, feedback
regulations no longer exist to the same extent or not at all. In
addition it might be advantageously to combine one or more of the
sequences indicated in Table I, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108, resp., with genes which
generally support or enhances to growth or yield of the target
organism, for example genes which lead to faster growth rate of
microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[4619] [0228.0.10.10] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the xanthophylls
metabolism, in particular in synthesis of beta-cryptoxanthin,
zeaxanthin, astaxanthin or lutein.
[4620] [0229.0.10.10] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the carotenoids biosynthetic
pathway, such as phytoene synthase (Psy), which is an important
control point for the regulation of the flux (Fraser et al., 2002),
phytoene desaturase (Pds), z-carotene desaturase, above mentioned
enzymes (s. introduction of the application), e.g. hydroxylases
such as beta-carotene hydroxylase (U.S. Pat. No. 6,214,575),
ketolases, or cyclases such as the beta-cyclase (U.S. Pat. No.
6,232,530) or oxygenases such as the beta-C4-oxygenase described in
U.S. Pat. No. 6,218,599 or homologs thereof, astaxanthin synthase
(U.S. Pat. No. 6,365,386), or other genes as described in U.S. Pat.
No. 6,150,130. These genes can lead to an increased synthesis of
the essential carotenoids, in particular xanthophylls.
[4621] [0230.0.0.10] see [230.0.0.0].
[4622] [0231.0.10.10] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a zeaxanthin or cryptoxanthin
degrading protein is attenuated, in particular by reducing the rate
of expression of the corresponding gene. A person skilled in the
art knows for example, that the inhibition of the lutein synthesis
from carotene increases the amount of cryptoxanthin and zeaxanthin
in an organism, in particular in plants. In one embodiment, the
level of astaxanthin in the organism shall be increased. Thus,
astaxanthin degrading enzymes are attenuated but not enzymes
catalyzing the synthesis of astaxanthin from zeaxanthin or
cryptoxanthin.
[4623] [0232.0.0.10] to [0276.0.0.10]: see [0232.0.0.0] to
[0276.0.0.0]
[4624] [0277.0.10.10] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously. The fine chemical and other xanthophylls produced by
this process can be obtained by harvesting the organisms, either
from the crop in which they grow, or from the field. This can be
done via pressing or extraction of the plant parts
[4625] [0278.0.0.10] to [0282.0.0.10]: see [0278.0.0.0] to
[0282.0.0.0]
[4626] [0283.0.10.10] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table II,
column 3, lines 103 to 106 and/or 468 to 471 or lines 107 and/or
108, resp., or an antibody against a polypeptide as indicated in
Table II, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, resp., or an antigenic part thereof, which
can be produced by standard techniques utilizing polypeptides
comprising or consisting of above mentioned sequences, e.g. the
polypeptide of the present invention or a fragment thereof, i.e.,
the polypeptide of this invention. Preferred are monoclonal
antibodies specifically binding to polypeptide as indicated in
Table II, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108.
[4627] [0284.0.0.10] see [0284.0.0.0]
[4628] [0285.0.10.10] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, resp., or as coded by a nucleic acid molecule as
indicated in Table I, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108, resp., or functional homologues
thereof.
[4629] [0286.0.10.10] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108 and in one another embodiment, the present invention
relates to a polypeptide comprising or consisting of a consensus
sequence as indicated in Table IV, column 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108 whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid. or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table IV, column 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108.
[4630] [0287.0.0.10] to [0289.0.0.10]: see [0287.0.0.0] to
[0289.0.0.0]
[4631] [00290.0.10.10] The alignment was performed with the
Software AlignX (Sep. 25, 2002) a component of Vector NTI Suite
8.0, InforMax.TM., Invitrogen.TM. life science software, U.S. Main
Office, 7305 Executive Way, Frederick, Md. 21704, USA with the
following settings: For pairwise alignments: gap opening penalty:
10.0; gap extension penalty 0.1. For multiple alignments: Gap
opening penalty: 10.0; Gap extension penalty: 0.1; Gap separation
penalty range: 8; Residue substitution matrix: blosum62;
Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0.5;
Consensus calculation options: Residue fraction for consensus: 0.9.
Presettings were selected to allow also for the alignment of
conserved amino acids.
[4632] [0291.0.10.10] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of
plant or microorganism specific consensus sequences.
[4633] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table II A or IIB,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108 by one or more amino acids. In one embodiment,
polypeptide distinguishes form a sequence as indicated in Table II
A or IIB, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108 by more than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino
acids, preferably by more than 10, 15, 20, 25 or 30 amino acids,
even more preferred are more than 40, 50, or 60 amino acids and,
preferably, the sequence of the polypeptide of the invention
distinguishes from a sequence as indicated in Table II A or II B,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108 by not more than 80% or 70% of the amino acids,
preferably not more than 60% or 50%, more preferred not more than
40% or 30%, even more preferred not more than 20% or 10%. In an
other embodiment, said polypeptide of the invention does not
consist of a sequence as indicated in Table II A or II B, columns 5
or 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or
108.
[4634] [0292.0.0.10] see [0292.0.0.0]
[4635] [0293.0.10.10] In one embodiment, the invention relates to
polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or by a nucleic acid molecule used in the process of the
invention.
[4636] In one embodiment, the polypeptide of the invention has a
sequence which distinguishes from a sequence as indicated in Table
II A or II B, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108 by one or more amino acids. In an other
embodiment, said polypeptide of the invention does not consist of
the sequence as indicated in Table II A or II B, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108. In a
further embodiment, said polypeptide of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical. In one
embodiment, said polypeptide does not consist of the sequence
encoded by a nucleic acid molecules as indicated in Table I A or
IB, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108.
[4637] [0294.0.10.10] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 103 to 106 and/or 468 to 471
or lines 107 and/or 108, which distinguishes over a sequence as
indicated in Table II A or II B, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108 by one or more amino
acids, preferably by more than 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, even more
preferred are more than 40, 50, or 60 amino acids but even more
preferred by less than 70% of the amino acids, more preferred by
less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[4638] [0295.0.0.10] to [0297.0.0.10]: see [0295.0.0.0] to
[0297.0.0.0]
[4639] [00297.1.0.10] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity and/or amino acid
sequence of a polypeptide indicated in Table II, columns 3, 5 or 7,
lines 103 to 108 or 468 to 471, resp.,
[4640] [0298.0.10.10] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table II, columns 5 or 7, lines 103 to 106 and/or
468 to 471 or lines 107 and/or 108, resp. The portion of the
protein is preferably a biologically active portion as described
herein. Preferably, the polypeptide used in the process of the
invention has an amino acid sequence identical to a sequence as
indicated in Table II, columns 5 or 7, lines 103 to 106 and/or 468
to 471 or lines 107 and/or 108, resp.
[4641] [0299.0.10.10] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
103 to 106 and/or 468 to 471 or lines 107 and/or 108, resp., The
preferred polypeptide of the present invention preferably possesses
at least one of the activities according to the invention and
described herein. A preferred polypeptide of the present invention
includes an amino acid sequence encoded by a nucleotide sequence
which hybridizes, preferably hybridizes under stringent conditions,
to a nucleotide sequence as indicated in Table I, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108, resp.,
or which is homologous thereto, as defined above.
[4642] [0300.0.10.10] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, resp., in amino acid sequence due to natural variation
or mutagenesis, as described in detail herein. Accordingly, the
polypeptide comprise an amino acid sequence which is at least about
35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about
75%, 80%, 85% or 90, and more preferably at least about 91%, 92%,
93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%,
99% or more homologous to an entire amino acid sequence of as
indicated in Table II A or II B, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108, resp.,
[4643] [0301.0.0.10] see [0301.0.0.0]
[4644] [0302.0.10.10] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table II, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, resp., or the amino acid sequence of a
protein homologous thereto, which include fewer amino acids than a
full length polypeptide of the present invention or used in the
process of the present invention or the full length protein which
is homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[4645] [0303.0.0.10] see [0303.0.0.0]
[4646] [0304.0.10.10] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108 but having differences in the sequence from said
wild-type protein. These proteins may be improved in efficiency or
activity, may be present in greater numbers in the cell than is
usual, or may be decreased in efficiency or activity in relation to
the wild type protein.
[4647] [0305.0.0.10] to [0306.0.0.10]: see [0305.0.0.0] to
[0306.0.0.0]
[4648] [0306.1.0.10] Preferably, the compound is a composition
comprising the essentially pure cryptoxanthin or zeaxanthin or a
recovered or isolated cryptoxanthin or zeaxanthin, in particular,
the respective fine chemical, free or in protein-bound form.
[4649] [0307.0.0.10] to [0308.0.0.10]: see [0307.0.0.0] to
[0308.0.0.0]
[4650] [0309.0.10.10] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, resp., refers to a polypeptide having an amino acid
sequence corresponding to the polypeptide of the invention or used
in the process of the invention, whereas a "non-polypeptide of the
invention" or "other polypeptide" not being indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, resp., refers to a polypeptide having an amino acid
sequence corresponding to a protein which is not substantially
homologous a polypeptide of the invention, preferably which is not
substantially homologous to a polypeptide as indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, resp., e.g., a protein which does not confer the
activity described herein or annotated or known for as indicated in
Table II, column 3, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, resp., and which is derived from the same or a
different organism. In one embodiment, a "non-polypeptide of the
invention" or "other polypeptide" not being indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, resp., does not confer an increase of the respective
fine chemical in an organism or part thereof.
[4651] [0310.0.0.10] to [0334.0.0.10]: see [0310.0.0.0] to
[0334.0.0.0]
[4652] [0335.0.10.10] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table I, columns 5 or
7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108,
resp., and/or homologs thereof. As described inter alia in WO
99/32619, dsRNAi approaches are clearly superior to traditional
antisense approaches. The invention therefore furthermore relates
to double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived there from), bring about
altered metabolic activity by the reduction in the expression of a
nucleic acid sequences as indicated in Table I, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108, resp.,
and/or homologs thereof. In a double-stranded RNA molecule for
reducing the expression of an protein encoded by a nucleic acid
sequence sequences as indicated in Table I, columns 5 or 7, lines
103 to 106 and/or 468 to 471 or lines 107 and/or 108, resp., and/or
homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[4653] [0336.0.0.10] to [0342.0.0.10]: see [0336.0.0.0] to
[0342.0.0.0]
[4654] [0343.0.10.10] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 103 to 106
and/or 468 to 471 or lines 107 and/or 108, resp., or its homolog is
not necessarily required in order to bring about effective
reduction in the expression. The advantage is, accordingly, that
the method is tolerant with regard to sequence deviations as may be
present as a consequence of genetic mutations, polymorphisms or
evolutionary divergences. Thus, for example, using the dsRNA, which
has been generated starting from a sequence as indicated in Table
I, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, resp., or homologs thereof of the one organism, may be
used to suppress the corresponding expression in another
organism.
[4655] [0344.0.0.10] to [0361.0.0.10]: see [0344.0.0.0] to
[0361.0.0.0]
[4656] [0362.0.10.10] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, the nucleic acid construct of
the invention, the antisense molecule of the invention, the vector
of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, resp., e.g. encoding a polypeptide having
protein activity, as indicated in Table II, columns 3, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108, resp., Due to the
above mentioned activity the respective fine chemical content in a
cell or an organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. In one embodiment, transgenic for a polypeptide having an
activity of a polypeptide as indicated in Table II, columns 5 or 7,
lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108, resp.,
means herein that due to modulation or manipulation of the genome,
an activity as annotated for a polypeptide as indicated in Table
II, column 3, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, e.g. having a sequence as indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108, resp., is increased in a cell or an organism or a part
thereof. Examples are described above in context with the process
of the invention
[4657] [0363.0.0.10] see [0363.0.0.0]
[4658] [0364.0.10.10] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table II, column 3, lines 103 to 106 and/or 468 to 471
or lines 107 and/or 108, resp., with the corresponding
protein-encoding sequence as indicated in Table I, column 3, lines
103 to 106 and/or 468 to 471 or lines 107 and/or 108, resp.,
--becomes a transgenic expression cassette when it is modified by
non-natural, synthetic "artificial" methods such as, for example,
mutagenization. Such methods have been described (U.S. Pat. No.
5,565,350; WO 00/15815; also see above).
[4659] [0365.0.0.10] to [0373.0.0.10]: see [0365.0.0.0] to
[0373.0.0.0]
[4660] [0374.0.10.10] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. Xanthophylls, in particular
the respective fine chemical, produced in the process according to
the invention may, however, also be isolated from the plant in the
form of their free xanthophylls, in particular the free respective
fine chemical, or bound in or to compounds or moieties, like
glucosides, e.g. diglucosides. The respective fine chemical
produced by this process can be harvested by harvesting the
organisms either from the culture in which they grow or from the
field. This can be done via expressing, grinding and/or extraction,
salt precipitation and/or ion-exchange chromatography or other
chromatographic methods of the plant parts, preferably the plant
seeds, plant fruits, plant tubers and the like.
[4661] [0375.0.0.10] to [0376.0.0.10]: see [0375.0.0.0] to
[0376.0.0.0]
[4662] [0377.0.10.10] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced carotenoids comprising, in particular the xanthophylls
comprising composition is isolated. In one embodiment, the produced
respective fine chemical is isolated.
[4663] [0378.0.10.10] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the xanthophylls,
in particular the respective fine chemical produced in the process
can be isolated. The resulting recovered, isolated or purified
xanthophylls, e.g. the composition comprising xanthophylls can, if
appropriate, subsequently be further purified, if desired mixed
with other active ingredients such as vitamins, amino acids,
carbohydrates, antibiotics and the like, and, if appropriate,
formulated.
[4664] [0379.0.10.10] In one embodiment, the xanthophylls are a
mixture of the respective fine chemicals. In one embodiment, the
xanthophylls are the respective fine chemical. In one embodiment,
the xanthophylls are a mixture of the respective fine chemicals
with astaxanthin.
[4665] [0380.0.10.10] The xanthophylls, in particular the
respective fine chemicals obtained in the process are suitable as
starting material for the synthesis of further products of value.
For example, they can be used in combination with each other or
alone for the production of pharmaceuticals, foodstuffs, animal
feeds or cosmetics. Accordingly, the present invention relates a
method for the production of pharmaceuticals, food stuff, animal
feeds, nutrients or cosmetics comprising the steps of the process
according to the invention, including the isolation of the
carotenoids containing, in particular xanthophylls containing
composition produced or the respective fine chemical produced if
desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the carentoids or xanthophylls
produced in the process or of the transgenic organisms in animal
feeds, foodstuffs, medicines, food supplements, cosmetics or
pharmaceuticals or for the production of astaxanthin, e.g. in after
isolation of the respective fine chemical or without, e.g. in situ,
e.g. in the organism used for the process for the production of the
respective fine chemical.
[4666] [0381.0.0.10] to [0382.0.0.10]: see [0381.0.0.0] to
[0382.0.0.0]
[4667] [0383.0.10.10] ./.
[4668] [0384.0.0.10] see [0384.0.0.0]
[4669] [0385.0.10.10] The fermentation broths obtained in this way,
containing in particular zeaxanthin or beta-cryptoxanthin in
mixtures with other carotenoids, in particular with other
xanthophylls, e.g. with astaxanthin, or containing microorganisms
or parts of microorganisms, like plastids, containing zeaxanthin or
beta-cryptoxanthin in mixtures with other carotenoids, in
particular with other xanthophylls, e.g. with astaxanthin, normally
have a dry matter content of from 1 to 70% by weight, preferably
7.5 to 25% by weight. Sugar-limited fermentation is additionally
advantageous, e.g. at the end, for example over at least 30% of the
fermentation time. This means that the concentration of utilizable
sugar in the fermentation medium is kept at, or reduced to, 0 to 10
g/l, preferably to 0 to 3 g/l during this time. The fermentation
broth is then processed further. Depending on requirements, the
biomass can be removed or isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth or left completely in it.
[4670] The fermentation broth can then be thickened or concentrated
by known methods, such as, for example, with the aid of a rotary
evaporator, thin-film evaporator, falling film evaporator, by
reverse osmosis or by nanofiltration. This concentrated
fermentation broth can then be worked up by freeze-drying, spray
drying, spray granulation or by other processes.
[4671] As carotenoids are often localized in membranes or plastids,
in one embodiment it is advantageous to avoid a leaching of the
cells when the biomass is isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth. The dry biomass can directly
be added to animal feed, provided the carotenoids concentration is
sufficiently high and no toxic compounds are present. In view of
the instability of carentoids, conditions for drying, e.g. spray or
flash-drying, can be mild and can be avoiding oxidation and
cis/trans isomerization. For example antioxidants, e.g. BHT,
ethoxyquin or other, can be added. In case the carotenoids
concentration in the biomass is to dilute, solvent extraction can
be used for their isolation, e.g. with alcohols, ether or other
organic solvents, e.g. with methanol, ethanol, acetone, alcoholic
potassium hydroxide, glycerol-phenol, liquefied phenol or for
example with acids or bases, like trichloroacetatic acid or
potassium hydroxide. A wide range of advantageous Methods and
techniques for the isolation of carotenoids, in particular of
xanthophylls, in particular of zeaxanthin or cryptoxanthin can be
found in the state of the art. In case phenol is used it can for
example be removed with ether and water extraction and the dry
eluate comprises a mixture of the carotenoids of the biomass.
[4672] [0386.0.10.10] Accordingly, it is possible to purify the
carotenoids, in particular the xanthophylls produced according to
the invention further. For this purpose, the product-containing
composition, e.g. a total or partial lipid extraction fraction
using organic solvents, e.g. as described above, is subjected for
example to a saponification to remove triglycerides, partition
between e.g. hexane/methanol (separation of non-polar epiphase from
more polar hypophasic derivates) and separation via e.g. an open
column chromatography or HPLC in which case the desired product or
the impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use.
[4673] [0387.0.0.10] to [0392.0.0.10]: see [0387.0.0.0] to
[0392.0.0.0]
[4674] [0393.0.10.10] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [4675] (a) contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [4676] (b) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table I, columns 5 or 7, lines
103 to 106 and/or 468 to 471 or lines 107 and/or 108, preferably in
Table I B, columns 5 or 7, lines 103 to 106 and/or 468 to 471 or
lines 107 and/or 108, resp., and, optionally, isolating the full
length cDNA clone or complete genomic clone; [4677] (c) introducing
the candidate nucleic acid molecules in host cells, preferably in a
plant cell or a microorganism, appropriate for producing the
respective fine chemical; [4678] (d) expressing the identified
nucleic acid molecules in the host cells; [4679] (e) assaying the
respective fine chemical level in the host cells; and [4680] (f)
identifying the nucleic acid molecule and its gene product which
expression confers an increase in the respective fine chemical
level in the host cell after expression compared to the wild
type.
[4681] [0394.0.0.10] to [0398.0.0.10]: see [0394.0.0.0] to
[0398.0.0.0]
[4682] [0399.0.10.10] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increased the respective fine chemical production in a
plant or microorganism, comprising the steps: [4683] (h) culturing
a cell or tissue or microorganism or maintaining a plant expressing
the polypeptide according to the invention or a nucleic acid
molecule encoding said polypeptide and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and the
polypeptide of the present invention or used in the process of the
invention; and [4684] (i) identifying if the compound is an
effective agonist by detecting the presence or absence or increase
of a signal produced by said readout system.
[4685] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in respective fine chemical
production.
[4686] [00399.1.0.10] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108 or a homolog thereof, e.g. comparing the phenotype of
nearly identical organisms with low and high activity of a protein
as indicated in Table II, columns 5 or 7, lines 103 to 106 and/or
468 to 471 or lines 107 and/or 108 after incubation with the
drug.
[4687] [0400.0.0.10] to [0416.0.0.10]: see [0400.0.0.0] to
[0416.0.0.0]
[4688] [0417.0.10.10] The nucleic acid molecule of the invention or
used in the method of the invention, the vector of the invention or
the nucleic acid construct of the invention may also be useful for
the production of organisms resistant to inhibitors of the
carotenoids production biosynthesis pathways. In particular, the
overexpression of the polypeptide of the present invention may
protect an organism such as a microorganism or a plant against
inhibitors, which block the carotenoid synthesis, in particular the
respective fine chemical synthesis in said organism. Examples of
inhibitors or herbicides blocking the synthesis of carotenoids in
organism such as microorganism or plants are for example classified
in two groups. The first group consists of inhibitors that cause
the accumulation of early intermediates in the pathway,
particularly the colorless phytene, e.g. diphenylamine. Other
inhibitors preferentially block late reactions in the pathway,
notably the cyclization of lycopene. Inhibitors are e.g. nicotine,
2-(4-chlorophenylthio)-triethylamine and other substituted amines
as well as nitrogenous heterocyclic bases, e.g. imidazole.
[4689] As xanthophylls can protect organisms against damages of
oxidative stress, especially singlet oxygen, a increased level of
the respective fine chemical can protect plants against herbicides
which cause the toxic build-up of oxidative compounds, e.g. singlet
oxygen. For example, inhibition of the protoporphorineogen oxidase
(Protox), an enzyme important in the synthesis of chlorophyll and
heme biosynthesis results in the loss of chlorophyll and
carotenoids and in leaky membranes; the membrane destruction is due
to creation of free oxygen radicals (which is also reported for
other classic photosynthetic inhibitor herbicides).
[4690] Accordingly, in one embodiment, the increase of the level of
the respective fine chemical is used to protect plants against
herbicides destroying membranes due to the creation of free oxygen
radicals.
[4691] Examples of inhibitors or herbicides building up oxidative
stress are aryl triazion, e.g. sulfentrazone, carfentrazone, or
diphenylethers, e.g. acifluorfen, lactofen, or oxyfluorfen, or
N-Phenylphthalimide, e.g. flumiclorac or flumioxazin, substituted
ureas, e.g. fluometuron, tebuthiuron, or diuron, linuron, or
triazines, e.g. atrazine, prometryn, ametryn, metributzin,
prometon, simazine, or hexazinone, or uracils, e.g. bromacil or
terbacil.
[4692] Carotenoid inhibitors are e.g. Pyridines and Pyridazinones,
e.g. norflurazon, fluridone or dithiopyr. Thus, in one embodiment,
the present invention relates to the use of an increase of the
respective respective fine chemical according to the present
invention for the protection of plants against carotenoids
inhibitors as pyridines and pyridazinones.
[4693] [0418.0.0.10] to [0423.0.0.10]: see [0418.0.0.0] to
[0423.0.0.0]
[4694] [0424.0.10.10] Accordingly, the nucleic acid of the
invention or the nucleic acid molecule used in the method of the
invention, the polypeptide of the invention or the polypeptid used
in the method of the invention, the nucleic acid construct of the
invention, the organisms, the host cell, the microorganisms, the
plant, plant tissue, plant cell, or the part thereof of the
invention, the vector of the invention, the agonist identified with
the method of the invention, the nucleic acid molecule identified
with the method of the present invention, can be used for the
production of the respective fine chemical or of the respective
fine chemical and one or more other carotenoids, in particular
other xanthophylls, e.g. astaxanthin.
[4695] Accordingly, the nucleic acid of the invention, the nucleic
acid molecule used in the method of the invention, or the nucleic
acid molecule identified with the method of the present invention
or the complement sequences thereof, the polypeptide of the
invention or the polypeptide used in the method of the invention,
the nucleic acid construct of the invention, the organisms, the
host cell, the microorganisms, the plant, plant tissue, plant cell,
or the part thereof of the invention, the vector of the invention,
the antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the respective
fine chemical in a organism or part thereof, e.g. in a cell.
[4696] [0424.1.0.10] In a further embodiment the present invention
relates to the use of the antagonist of the present invention, the
plant of the present invention or a part thereof, the microorganism
or the host cell of the present invention or a part thereof for the
production a cosmetic composition or a pharmaceutical composition.
Such a composition has antioxidative activity, photoprotective
activity, tanning activity, can be used for the treating of high
levels of cholesterol and/or lipids, can be used to protect, treat
or heal the above mentioned diseases, e.g. retinal disorders,
hyperholsterolemia, hyperlipidemia, and ahterosclerosis, or can be
used for the cleaning, conditioning, and/or treating of the skin,
e.g. if combined with a pharmaceutically or cosmetically acceptable
carrier.
[4697] The xanthophylls can be also used as stabilizer of other
colours or oxygen sensitive compounds.
[4698] [0425.0.0.10] to [0434.0.0.10]: see [0425.0.0.0] to
[0434.0.0.0]
[0435.0.10.10] Example 3
In-Vivo and In-Vitro Mutagenesis
[4699] [0436.0.10.10] An in vivo mutagenesis of organisms such as
green algae (e.g. Spongiococcum sp, e.g. Spongiococcum exentricum,
Chlorella sp.), Saccharomyces, Mortierella, Escherichia and others
mentioned above, which are beneficial for the production of
xanthophylls can be carried out by passing a plasmid DNA (or
another vector DNA) containing the desired nucleic acid sequence or
nucleic acid sequences, e.g. the nucleic acid molecule of the
invention or the vector of the invention, through E. coli and other
microorganisms (for example Bacillus spp. or yeasts such as
Saccharomyces cerevisiae) which are not capable of maintaining the
integrity of its genetic information. Usual mutator strains have
mutations in the genes for the DNA repair system [for example
mutHLS, mutD, mutT and the like; for comparison, see Rupp, W. D.
(1996) DNA repair mechanisms in Escherichia coli and Salmonella,
pp. 2277-2294, ASM: Washington]. The skilled worker knows these
strains. The use of these strains is illustrated for example in
Greener, A. and Callahan, M. (1994) Strategies 7; 32-34.
[4700] In-vitro mutation methods such as increasing the spontaneous
mutation rates by chemical or physical treatment are well known to
the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[4701] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new
[4702] DNA synthesis to create a complementary full-length sequence
containing the desired mutation. Another site-directed mutagensis
method is the PCR mismatch primer mutagensis method also known to
the skilled person. Dpnl site-directed mutagensis is a further
known method as described for example in the Stratagene
Quickchange.TM. site-directed mutagenesis kit protocol. A huge
number of other methods are also known and used in common
practice.
[4703] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[4704] [0436.1.0.10] see [0436.1.0.0]
[0437.0.10.10] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpina
[4705] [0438.0.10.10] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology, VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[4706] [0439.0.10.10] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255f) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[4707] [0440.0.10.10] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002,) teaches a homolog recombination system dependent
on rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this
system for homologous recombination are vectors derived from the
Ylp series. Plasmid vectors derived for example from the 2p-Vector
are known by the skilled worker and used for the expression in
yeasts. Other preferred vectors are for example pART1, pCHY21 or
pEVP11 as they have been described by McLeod et al. (EMBO J. 1987,
6:729-736) and Hoffman et al. (Genes Dev. 5, 1991:561-571.) or
Russell et al. (J. Biol. Chem. 258, 1983: 143-149.). Other
beneficial yeast vectors are plasmids of the REP, REP-X, pYZ or RIP
series.
[4708] [0441.0.0.10] to [0443.0.0.10] see [0441.0.0.0] to
[0443.0.0.0]
[0444.0.10.10] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[4709] [0445.0.10.10] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[4710] [0446.0.0.10] to [0454.0.0.10]: see [0446.0.0.0] to
[0454.0.0.0]
[4711] [0455.0.10.10] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a fatty acid) can be determined by growing the
modified microorganisms or the modified plant under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the elevated production of
desired product (i.e. of the lipids or a fatty acid). These
analytical techniques are known to the skilled worker and comprise
spectroscopy, thin-layer chromatography, various types of staining
methods, enzymatic and microbiological methods and analytical
chromatography such as high-performance liquid chromatography (see,
for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2,
p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.,
(1987) "Applications of HPLC in Biochemistry" in: Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et
al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery
and purification", p. 469-714, VCH: Weinheim; Better, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[4712] In addition to the abovementioned processes, plant lipids
are extracted from plant material as described by Cahoon et al.
(1999) Proc. Natl. Acad. Sci. USA 96 (22): 12935-12940 and Browse
et al. (1986) Analytic Biochemistry 152:141-145. The qualitative
and quantitative analysis of lipids is described by Christie,
William W., Advances in Lipid Methodology, Ayr/Scotland: Oily Press
(Oily Press Lipid Library; 2); Christie, William W., Gas
Chromatography and Lipids. A Practical Guide--Ayr, Scotland: Oily
Press, 1989, Repr. 1992, IX, 307 pp. (Oily Press Lipid Library; 1);
"Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952)--16
(1977) under the title: Progress in the Chemistry of Fats and Other
Lipids CODEN.
[4713] [0456.0.0.10]: see [0456.0.0.0]
[0457.0.10.10] Example 9
Purification of the Xanthophylls and Determination of the
Carotenoids Content
[4714] [0458.0.10.10] Abbreviations: GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[4715] The unambiguous detection for the presence of Xanthophylls
can be obtained by analyzing recombinant organisms using analytical
standard methods: LC, LC-MSMS or TLC, as described The total
xanthophylls produced in the organism for example in yeasts used in
the inventive process can be analysed for example according to the
following procedure:
[4716] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods.
[4717] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[4718] A typical sample pretreatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography. E.g.:
[4719] For analysis, solvent delivery and aliquot removal can be
accomplished with a robotic system comprising a single injector
valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000
W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of
50% potassium hydroxide hydro-ethanolic solution (4 water:1
ethanol) can be added to each vial, followed by the addition of 3
ml of octanol. The saponification treatment can be conducted at
room temperature with vials maintained on an IKA HS 501 horizontal
shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours
at 250 movements/minute, followed by a stationary phase of
approximately one hour.
[4720] Following saponification, the supernatant can be diluted
with 0.10 ml of methanol. The addition of methanol cqan be
conducted under pressure to ensure sample homogeneity. Using a 0.25
ml syringe, a 0.1 ml aliquot can be removed and transferred to HPLC
vials for analysis.
[4721] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete
with a quaternary pump, vacuum degassing system, six-way injection
valve, temperature regulated autosampler, column oven and
Photodiode Array detector can be used [Agilent
[4722] Technologies available through Ultra Scientific Inc., 250
Smith Street, North Kingstown, R.I.]. The column can be a Waters
YMC30, 5-micron, 4.6.times.250 mm with a guard column of the same
material [Waters, 34 Maple Street, Milford, Mass.]. The solvents
for the mobile phase can be 81 methanol: 4 water: 15
tetrahydrofuran (THF) stabilized with 0.2% BHT
(2,6-di-tert-butyl-4-methylphenol). Injections were 20 l.
Separation can be isocratic at 30.degree. C. with a flow rate of
1.7 ml/minute. The peak responses can be measured by absorbance at
447 nm.
[4723] Carotenoid compositions can be determined for wild-type and
mutant samples selected from those identified in a screening
procedure. Petal samples can be stored in a -80.degree. C. freezer
until mutants were identified. Samples can be lyophilized, and the
dried tissue can be stored under argon at -80.degree. C. until
ready for analysis.
[4724] Extraction procedures can be performed under red light.
Dried petals can be ground to pass through a No. 40 sieve mesh
size. A ground sample can be accurately weighed and transferred
into a 100 ml red volumetric flask. To the sample, 500 microliters
I) of H.sub.2O can be added, and the mixture can be swirled for 1
minute. Thirty ml of extractant solvent (10 ml hexane+7 ml
acetone+6 ml absolute alcohol+7 ml toluene) can be added, and the
flask can be shaken at 160 rpm for 10 minutes.
[4725] For saponification, 2 ml of 40% methanolic KOH can be added
into the flask, which can be then swirled for one minute. The flask
can be placed in a 56.degree. C. H.sub.2O bath for 20 minutes. An
air condenser can be attached to prevent loss of solvent. The
sample can be cooled in the dark for one hour with the condenser
attached. After cooling, 30 ml of hexane can be added, and the
flask can be shaken at 160 rpm for 10 minutes.
[4726] The shaken sample can be diluted to volume (100 ml) with 10%
sodium sulfate solution and shaken vigorously for one minute. The
sample can be remained in the dark for at least 30 minutes. A 35 ml
aliquot can be removed from the approximately 50 ml upper phase,
and transferred to a sample cup. An additional 30 ml of hexane can
be added into the flask that can be then shaken at 160 rpm for 10
minutes. After approximately one hour, the upper phases can be
combined. For HPLC analysis, 10 ml aliquots can be dried under
nitrogen and stored under argon at -80.degree. C.
[4727] HPLC equipment comprised an Alliance 2690 equipped with a
refrigerated autosampler, column heater and a Waters Photodiode
Array 996 detector (Waters Corp., 34 Maple Street Milford, Mass.
01757). Separation can be obtained with a YMC30 column, 3 m,
2.0.times.150 mm with a guard column of the same material.
Standards can be obtained from ICC Indorespective fine chemicals
Somerville, N.J. 088876 and from DHI-Water & Environment,
DK-2970 Horsholm, Denmark. The dried mutant samples can be
resuspended in tetrahydrofuran and methanol to a total volume of
200 l and filtered, whereas the control can be not additionally
concentrated. Carotenoids can be separated using a gradient method.
Initial gradient conditions can be 90% methanol: 5% water: 5%
methyl tert-butyl ether at a flow rate of 0.4 milliliters per
minute (ml/min). From zero to 15 minutes, the mobile phase can be
changed from the initial conditions to 80 methanol: 5 water: 15
methyl tert-butyl ether, and from 15 to 60 minutes to 20 methanol:
5 water: 75 methyl tert-butyl ether. For the following 10 minutes,
the mobile phase can be returned to the initial conditions and the
column equilibrated for an additional 10 minutes. The column
temperature can be maintained at 27.degree. C. and the flow rate
was 0.4 ml/minute. Injections were 10 l. The majority of peak
responses can be measured at 450 nm and additional areas added from
286, 348, 400 and 472 nm extracted channels.
[4728] [0459.0.10.10] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the xanthophylls can be further purified with a
so-called RTHPLC. As eluent acetonitrile/water or
chloroform/acetonitrile mixtures can be used. If necessary, these
chromatography steps may be repeated, using identical or other
chromatography resins. The skilled worker is familiar with the
selection of suitable chromatography resin and the most effective
use for a particular molecule to be purified.
[4729] [0460.0.10.10] see [0460.0.0.0]
[0461.0.10.10] Example 10
Cloning SEQ ID NO: 10215 for the Expression in Plants
[4730] [0462.0.0.10] see [0462.0.0.0]
[4731] [0463.0.10.10] SEQ ID NO: 10215 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[4732] [0464.0.0.10] to [0466.0.0.10]: see [0464.0.0.0] to
[0466.0.0.0]
[4733] [0466.1.0.10] In case the Herculase enzyme can be used for
the amplification, the PCR amplification cycles were as follows: 1
cycle of 2-3 minutes at 94.degree. C., followed by 25-30 cycles of
in each case 30 seconds at 94.degree. C., 30 seconds at
55-60.degree. C. and 5-10 minutes at 72.degree. C., followed by 1
cycle of 10 minutes at 72.degree. C., then 4.degree. C.
[4734] [0467.0.10.10] The following primer sequences were selected
for the gene SEQ ID NO: 10215:
TABLE-US-00037 i) forward primer (SEQ ID NO: 10235) atgcgccctc
ttattttatc gatttt ii) reverse primer (SEQ ID NO: 10236) ttatggtgcg
ggtttaagaa acgtc
[4735] [0468.0.0.10] to [0479.0.0.10]: see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.10.10] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 10215
[4736] [0481.0.0.10] to [0513.0.0.10]: see [0481.0.0.0] to
[0513.0.0.0]
[4737] [0514.0.10.10] As an alternative, xanthophylls can be
detected as described in Deli, J. & Molnar, P. Paprika
carotenoids: Analysis, isolation, structure eucidation. Curr. Org.
Chem. 6, 1197-1219 (2004) or Fraser, P. D., Pinto, M. E., Holloway,
D. E. & Bramley, P. M. Technical advance: application of
high-performance liquid chromatography with photodiode array
detection to the metabolic profiling of plant isoprenoids. Plant J.
24, 551-558 (2000).
[4738] The results of the different plant analyses can be seen from
the table 1 which follows:
TABLE-US-00038 TABLE 1 ORF Metabolite Method Min Max YCL040W
Cryptoxanthin LC 1,36 1,51 b0986 Cryptoxanthin LC 1,33 1,40 b3684
Cryptoxanthin LC 1,35 1,67 b4401 Cryptoxanthin LC 1,48 1,60 b3926
Cryptoxanthin LC 1.33 1.33 b0851 Cryptoxanthin LC 1.57 1.76 b2211
Cryptoxanthin LC 1.05 1.26 b0050 Cryptoxanthin LC 1.42 1.84 YHR055C
Zeaxanthin LC 1,05 1,38 b2699 Zeaxanthin LC 1,25 1,48
[4739] [0515.0.0.10] to [0552.0.0.10]: see [0515.0.0.0] to
[0552.0.0.0] including [0530.1.0.0] to [0530.6.0.0] as well as
[0552.1.0.0] and [0552.2.0.0]
[4740] [0553.0.10.10] [4741] 1. A process for the production of
xanthopyhlls, which comprises (a) increasing or generating the
activity of one or more proteins as indicated in Table II, columns
5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108
or a functional equivalent thereof in a non-human organism, or in
one or more parts thereof; and (b) growing the organism under
conditions which permit the production of xanthopyhlls in said
organism. [4742] 2. A process for the production of xanthopyhlls,
comprising the increasing or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [4743] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108 or a fragment
thereof, which confers an increase in the amount of xanthopyhlls in
an organism or a part thereof; [4744] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108; [4745] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of xanthopyhlls in an
organism or a part thereof; [4746] d) nucleic acid molecule which
encodes a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
xanthopyhlls in an organism or a part thereof; [4747] e) nucleic
acid molecule which hybridizes with a nucleic acid molecule of (a)
to (c) under under stringent hybridisation conditions and
conferring an increase in the amount of xanthopyhlls in an organism
or a part thereof; [4748] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table III,
columns 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or
108 and conferring an increase in the amount of
xanthopyhllsrespective fine chemical in an organism or a part
thereof; [4749] g) nucleic acid molecule encoding a polypeptide
which is isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of xanthopyhlls in an
organism or a part thereof; [4750] h) nucleic acid molecule
encoding a polypeptide comprising a consensus as indicated in Table
IV, columns 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108 and conferring an increase in the amount of
xanthopyhllsrespective fine chemical in an organism or a part
thereof; and [4751] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of xanthopyhllsrespective fine chemical in an organism or a
part thereof. [4752] or comprising a sequence which is
complementary thereto. [4753] 3. The process of claim 1 or 2,
comprising recovering of the free or bound xanthopyhlls. [4754] 4.
The process of any one of claims 1 to 3, comprising the following
steps: [4755] (a) selecting an organism or a part thereof
expressing a polypeptide encoded by the nucleic acid molecule
characterized in claim 2; [4756] (b) mutagenizing the selected
organism or the part thereof; [4757] (c) comparing the activity or
the expression level of said polypeptide in the mutagenized
organism or the part thereof with the activity or the expression of
said polypeptide of the selected organisms or the part thereof;
[4758] (d) selecting the mutated organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
[4759] (e) optionally, growing and cultivating the organisms or the
parts thereof; and [4760] (f) recovering, and optionally isolating,
the free or bound xanthopyhlls produced by the selected mutated
organisms or parts thereof. [4761] 5. The process of any one of
claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. [4762] 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [4763] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 103 to
106 and/or 468 to 471 or lines 107 and/or 108 or a fragment
thereof, which confers an increase in the amount of xanthopyhlls in
an organism or a part thereof; [4764] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108; [4765] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of xanthopyhlls in an
organism or a part thereof; [4766] d) nucleic acid molecule which
encodes a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
xanthopyhlls in an organism or a part thereof; [4767] e) nucleic
acid molecule which hybridizes with a nucleic acid molecule of (a)
to (c) under under stringent hybridisation conditions and
conferring an increase in the amount of xanthopyhlls in an organism
or a part thereof; [4768] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table III,
columns 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or
108 and conferring an increase in the amount of
xanthopyhllsrespective fine chemical in an organism or a part
thereof; [4769] g) nucleic acid molecule encoding a polypeptide
which is isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of xanthopyhlls in an
organism or a part thereof; [4770] h) nucleic acid molecule
encoding a polypeptide comprising a consensus as indicated in Table
IV, columns 7, lines 103 to 106 and/or 468 to 471 or lines 107
and/or 108 and conferring an increase in the amount of
xanthopyhllsrespective fine chemical in an organism or a part
thereof; and [4771] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of xanthopyhllsrespective fine chemical in an organism or a
part thereof. [4772] whereby the nucleic acid molecule
distinguishes over the sequence as indicated in Table I A, columns
5 or 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108
by one or more nucleotides. [4773] 7. A nucleic acid construct
which confers the expression of the nucleic acid molecule of claim
6, comprising one or more regulatory elements. [4774] 8. A vector
comprising the nucleic acid molecule as claimed in claim 6 or the
nucleic acid construct of claim 7. [4775] 9. The vector as claimed
in claim 8, wherein the nucleic acid molecule is in operable
linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [4776] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [4777] 11. The host cell of claim 10, which is a
transgenic host cell. [4778] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [4779] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [4780] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table II A, columns 5
or 7, lines 103 to 106 and/or 468 to 471 or lines 107 and/or 108 by
one or more amino acids [4781] 15. An antibody, which binds
specifically to the polypeptide as claimed in claim 14. [4782] 16.
A plant tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [4783] 17. A method for screening for agonists
and antagonists of the activity of a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of xanthopyhlls in an organism or a part thereof comprising:
[4784] (a) contacting cells, tissues, plants or microorganisms
which express the a polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
xanthopyhlls in an organism or a part thereof with a candidate
compound or a sample comprising a plurality of compounds under
conditions which permit the expression the polypeptide; [4785] (b)
assaying the linoleic acid level or the polypeptide expression
level in the cell, tissue, plant or microorganism or the media the
cell, tissue, plant or microorganisms is cultured or maintained in;
and [4786] (c) identifying a agonist or antagonist by comparing the
measured xanthopyhlls level or polypeptide expression level with a
standard linoleic acid or polypeptide expression level measured in
the absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. [4787] 18. A process for
the identification of a compound conferring increased xanthopyhlls
production in a plant or microorganism, comprising the steps:
[4788] (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of xanthopyhlls in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
xanthopyhlls in an organism or a part thereof; [4789] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [4790] 19. A method for the identification of a
gene product conferring an increase in xanthopyhlls production in a
cell, comprising the following steps: [4791] (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in xanthopyhlls
after expression with the nucleic acid molecule of claim 6; [4792]
(b) identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; [4793] (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing xanthopyhlls;
[4794] (d) expressing the identified nucleic acid molecules in the
host cells; [4795] (e) assaying the xanthopyhlls level in the host
cells; and [4796] (f) identifying nucleic acid molecule and its
gene product which expression confers an increase in the
xanthopyhlls level in the host cell in the host cell after
expression compared to the wild type. [4797] 20. A method for the
identification of a gene product conferring an increase in
xanthopyhlls production in a cell, comprising the following steps:
(a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the xanthopyhllsamount or level
in an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing xanthopyhlls; (c) expressing the
identified nucleic acid molecules in the host cells; (d) assaying
the xanthopyhllslevel in the host cells; and (e) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the xanthopyhlls level in the host cell after
expression compared to the wild type. [4798] 21. A method for the
production of an agricultural composition comprising the steps of
the method of any one of claims 17 to 20 and formulating the
compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. [4799] 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [4800] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
xanthopyhlls after expression. [4801] 24. Use of the polypeptide of
claim 14 or the nucleic acid construct claim 7 or the gene product
identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of
xanthopyhlls levels in an organism.
[4802] 25. Cosmetic, pharmaceutical, food or feed composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20. [4803] 26. The method of any one of
claims 1 to 5, the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20, wherein the xanthylphyll is
beta-cryptoxanthin or zeaxanthin, resp. [4804] 27. Use of the
nucleic acid molecule of claim 6, the polypeptide of claim 14, the
nucleic acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20 for the protection of a plant against a oxidative stress. [4805]
28. Use of the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20 for the protection of a plant against a
oxidative stress causing or a carotenoid synthesis inhibiting
herbicide. [4806] 29. Use of the agonist identified according to
claim 17, the plant or plant tissue of claim 16, the harvested
material of claim 16, or the host cell of claim 10 to 12 for the
production of a cosmetic composition.
[4807] [0554.0.0.10] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[4808] [0000.0.0.11] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[4809] [0001.0.0.11] see [0001.0.0.0]
[4810] [0002.0.11.11] Carotenoids are red, yellow and orange
pigments that are widely distributed in nature. Although specific
carotenoids have been identified in photosynthetic centers in
plants, in bird feathers, in crustaceans and in marigold petals,
they are especially abundant in yellow-orange fruits and vegetables
and dark green, leafy vegetables. Of the more than 700 naturally
occurring carotenoids identified thus far, as many as 50 may be
absorbed and metabolized by the human body. To date, only 14
carotenoids have been identified in human serum.
[4811] In animals some carotenoids (particularly beta-carotene)
serve as dietary precursors to Vitamin A, and many of them may
function as fat-soluble antioxidants. In plants carotenes serve for
example as antioxidants to protect the highly reactive photosystems
and act as accessory photopigments. In vitro experiments have shown
that lycopene, alpha-carotene, zeaxanthin, lutein and cryptoxanthin
quench singlet oxygen and inhibit lipid peroxidation. The isolation
and identification of oxidized metabolites of lutein, zeaxanthin
and lycopene provide direct evidence of the antioxidant action of
these carotenoids.
[4812] Carotenoids are 40-carbon (C.sub.40) terpenoids generally
comprising eight isoprene (C.sub.5) units joined together. Linking
of the units is reversed at the center of the molecule.
"Ketocarotenoid" is a general term for carotenoid pigments that
contain a keto group in the ionene ring portion of the molecule,
whereas "hydroxycarotenoid" refers to carotenoid pigments that
contain a hydroxyl group in the ionene ring. Trivial names and
abbreviations will be used throughout this disclosure, with
IUPAC-recommended semi-systematic names usually being given in
parentheses after first mention of a trivial name. Owing to its
three chiral centers, there are 2.sup.3 or 8 stereoisomers of
lutein.
[4813] The principal natural stereoisomer of lutein and the form of
lutein in the plasma is (3R,3'R,6'R)-lutein, thus a preferred form
of the compound. Lutein is also known as xanthophyll (also, the
group name of the oxygen-containing carotenoids), vegetable lutein,
vegetable luteol and beta, epsilon-carotene-3,3'diol. The molecular
formula of lutein is C.sub.40H.sub.56O.sub.2 and its molecular
weight is 568.88 daltons. The chemical name of the principal
natural stereoisomer of lutein is
(3R,3'R,6'R)-beta,epsilon-carotene-3,3'-diol.
[4814] Lutein and zeaxanthin esters are hydrolyzed in the small
intestine via esterases and lipases. Lutein and zeaxanthin that are
derived from supplements or released from the matrices of foods,
are either solubilized in the lipid core of micelles (formed from
bile salts and dietary lipids) in the lumen of the small intestine,
or form clathrate complexes with conjugated bile salts. Micelles
and possibly clathrate complexes deliver lutein and zeaxanthin to
the enterocytes. Lutein and zeaxanthin are released from the
enterocytes into the lymphatics in the form of chylomicrons. They
are transported by the lymphatics to the general circulation via
the thoracic duct. In the circulation, lipoprotein lipase
hydrolyzes much of the triglycerides in the chylomicrons, resulting
in the formation of chylomicron remnants. Chylomicron remnants
retain apolipoproteins E and B48 on their surfaces and are mainly
taken up by the hepatocytes and to a smaller degree by other
tissues. Within hepatocytes, lutein and zeaxanthin are incorporated
into lipoproteins. Lutein and zeaxanthin appear to be released into
the blood mainly in the form of high-density lipoproteins (HDL)
and, to a lesser extent, in the form of very-low density
lipoprotein (VLDL). Lutein and zeaxanthin are transported in the
plasma predominantly in the form of HDL. Lutein and zeaxanthin are
mainly accumulated in the macula of the retina, where they bind to
the retinal protein tuberlin. Zeaxanthin is specifically
concentrated in the macula, especially in the fovea. Lutein is
distributed throughout the retina. Zeaxanthin found in plasma is
predominantly (3R,3'R)-zeaxanthin. Lutein appears to undergo some
metabolism in the retina to meso-zeaxanthin.
[4815] Carotenoids are synthesized from a five carbon atom
metabolic precursor, isopentenyl pyrophosphate (IPP). There are at
least two known biosynthetic pathways in the formation of IPP, the
universal isoprene unit. One pathway begins with mevalonic acid,
the first specific precursor of terpenoids, formed from acetyl-CoA
via HMG-CoA (3-hydroxy-3-methylglutaryl-CoA), that is itself
converted to isopentenyl pyrophosphate
[4816] (IPP). Later, condensation of two geranylgeranyl
pyrophosphate (GGPP) molecules with each other produces colorless
phytoene, which is the initial carotenoid. Studies have also shown
the existence of an alternative, mevalonate-independent pathway for
IPP formation that was characterized initially in several species
of eubacteria, a green alga, and in the plastids of higher plants.
The first reaction in this alternative pathway is the
transketolase-type condensation reaction of pyruvate and
D-glyceraldehylde-3-phosphate to yield
1-deoxy-D-xylulose-5-phosphate (DXP or DOXP) as an
intermediate.
[4817] Through a series of desaturation reactions, phytoene is
converted to phytofluene, .zeta.-carotene, neurosporene and finally
to lycopene. Subsequently, lycopene is converted by a cyclization
reaction to .beta.-carotene that contains two .beta.-ionene rings.
A keto-group and/or a hydroxyl group are introduced into each ring
of .beta.-carotene to thereby synthesize canthaxanthin, zeaxanthin,
astaxanthin. A hydroxylase enzyme has been shown to convert
canthaxanthin to astaxanthin. Similarly, a ketolase enzyme has been
shown to convert zeaxanthin to astaxanthin. The ketolase also
converts .beta.-carotene to canthaxanthin and the hydroxylase
converts .beta.-carotene to zeaxanthin. In many plants, lycopene is
a branch point in carotenoid biosynthesis. Thus, some of the
plant's lycopene is made into beta-carotene and zeaxanthin, and
sometimes zeaxanthin diglucoside, whereas remaining portions of
lycopene are formed into alpha-carotene and lutein
(3,3'-dihydroxy-.alpha.-carotene), another hydroxylated
compound.
[4818] Lutein and zeaxanthin exist in several forms. Lutein and
zeaxanthin also occur in plants in the form of mono- or diesters of
fatty acids. For example, lutein and zeaxanthin dipalmitates,
dimyristates and monomyristates are found in the petals of the
marigold flower (Tagetes erecta). Many of the marketed lutein
nutritional supplements contain lutein esters, with much smaller
amounts of zeaxanthin esters, which are derived from the dried
petals of marigold flowers. Lutein dipalmitate is found in the
plant Helenium autumnale L. Compositae. It is also known as
helenien and it is used in France for the treatment of visual
disorders. Zeaxanthin in its fatty acid ester forms, is the
principal carotenoid found in the plant Lycium chinese Mill. Lycium
chinese Mill, also known as Chinese boxthorn, is used in
traditional Chinese medicine for the treatment of a number of
disorders, including visual problems. Nutritional supplement forms
are comprised of these carotenoids either in their free
(non-esterified) forms or in the form of fatty acid esters.
[4819] Lutein and zeaxanthin exist in a matrix in foods. In the
case of the chicken egg yolk, the matrix is comprised of lipids
(cholesterol, phospholipid, triglycerides). The carotenoids are
dispersed in the matrix along with fat-soluble nutrients, including
vitamins A, D and E. In the case of plants, lutein and zeaxanthin
are associated with chloroplasts or chromoplasts.
[4820] Carotenoids absorb light in the 400-500 nm region of the
visible spectrum. This physical property imparts the characteristic
red/yellow colour of the pigments. A conjugated backbone composed
of isoprene units is usually inverted at the centre of the
molecule, imparting symmetry. Changes in geometrical configuration
about the double bonds result in the existence of many cis- and
trans-isomers. Hydroxylated, oxidized, hydrogenated or
ring-containing derivatives also exist. Hydrocarbon carotenoids are
classified as carotenes while those containing oxygen are known as
xanthophylls.
[4821] In animals, carotenoids are absorbed from the intestine with
the aid of dietary fat and incorporated into chylomicrons for
transport in the serum. The different structural features possessed
by carotenoids account for selective distribution in organ tissue,
biological activity and pro-vitamin A potency, or in vivo
conversion to vitamin A. Due to the hydrophobic character,
carotenoids are associated with lipid portions of human tissues,
cells, and membranes. In general, 80-85% of carotenoids are
distributed in adipose tissue, with smaller amounts found in the
liver, muscle, adrenal glands, and reproductive organs.
Approximately 1% circulate in the serum on high and low density
lipoproteins. Serum concentrations are fairly constant and slow to
change during periods of low intake. The estimated half-life was
estimated to be 11-14 days for lycopene, -carotene, -carotene,
lutein and zeaxanthin. Evidence for the existence of more than one
body pool has been published. The major serum carotenoids are
-carotene, -carotene, lutein, zeaxanthin, lycopene and
cryptoxanthin. Smaller amounts of polyenes such as phytoene and
phytofluene are also present.
[4822] Human serum levels reflect lifestyle choices and dietary
habits within and between cultures. Approximately only 15
carotenoids circulate in the blood, on HDL and LDL. Variations can
be attributed to different intakes, unequal abilities to absorb
certain carotenoids, and different rates of metabolism and tissue
uptake. Decreased serum levels occur with alcohol consumption, the
use of oral contraceptives, smoking and prolonged exposure to UV
light.
[4823] [0003.0.11.11] The established efficacy of lutein in
quenching singlet oxygen and intercepting deleterious free radicals
and reactive oxygen species can make it part of the diverse
antioxidant defense system in humans. Reactive oxygen species have
been implicated in the development of many diseases, including
ischemic heart disease, various cancers, cataracts and macular
degeneration. Because the conjugated polyene portion of
beta-carotene confers its antioxidant capability and all
carotenoids possess this structural feature, research efforts have
been directed at evaluating the efficacy of other carotenoids in
the prevention of free radical-mediated diseases. Indeed, in vitro
experiments have demonstrated that lycopene, alpha-carotene,
zeaxanthin, lutein and cryptoxanthin quench singlet oxygen and
inhibit lipid peroxidation. The isolation and identification of
oxidized metabolites of lutein, zeaxanthin and lycopene may provide
direct evidence of the antioxidant action of these carotenoids.
[4824] In addition to antioxidant capability, other biological
actions of carotenoids include the ability to enhance
immunocompetence and in vitro gap junction communication, reduce or
inhibited mutagenesis and inhibit cell transformations in
vitro.
[4825] Many epidemiological studies have established an inverse
correlation between dietary intake of yellow-orange fruit and dark
green, leafy vegetables and the incidence of various cancers,
especially those of the mouth, pharynx, larynx, esophagus, lung,
stomach, cervix and bladder. While a number of protective compounds
may be responsible for this observation, the co-incidence of
carotenoids in these foods has been noted. Because nutritionists
and medical professionals currently recognize the occurrence of a
large number of distinct carotenoids in food, interest in their
functions and biological impact on health is burgeoning.
[4826] Lutein exists in the retina. It functions to protect
photoreceptor cells from light-generated oxygen radicals, and thus
plays a key role in preventing advanced macular degeneration.
Lutein possesses chemopreventive activity, induces gap junction
communication between cells and inhibits lipid peroxidation in
vitro more effectively than beta-carotene, alpha-carotene and
lycopene. High levels of lutein in serum have been inversely
correlated with lung cancer.
[4827] In addition to lutein, zeaxanthin exists in the retina and
confers protection against macular degeneration. Zeaxanthin is also
prevalent in ovaries and adipocyte tissue. This xanthophyll does
not possess provitamin A activity.
[4828] Alcohol consumption has been shown to influence lipid
peroxidation. Anhydrolutein, an oxidative by-product of lutein and
zeaxanthin, was higher in plasma after alcohol ingestion, while
concentrations of these xanthophylls were reduced. Lutein and
zeaxanthin may therefore have protective effects against LDL
oxidation.
[4829] [0004.0.11.11] In plants, approximately 80-90% of the
carotenoids present in green, leafy vegetables such as broccoli,
kale, spinach and brussel sprouts are xanthophylls, whereas 10-20%
are carotenes. Conversely, yellow and orange vegetables including
carrots, sweet potatoes and squash contain predominantly carotenes.
Up to 60% of the xanthophylls and 15% of the carotenes in these
foods are destroyed during microwave cooking. Of the xanthophylls,
lutein appears to be the most stable.
[4830] Lutein occurs in mango, papaya, oranges, kiwi, peaches,
squash, peas, lima beans, green beans, broccoli, brussel sprouts,
cabbage, kale, lettuce, prunes, pumpkin, sweet potatoes and
honeydew melon. Commercial sources are obtained from the extraction
of marigold petals. Lutein does not possess provitamin A
activity.
[4831] Dietary sources of Zeaxanthin include peaches, squash,
apricots, oranges, papaya, prunes, pumpkin, mango, kale, kiwi,
lettuce, honeydew melon and yellow corn.
[4832] [0005.0.11.11] Some carotenoids occur particularly in a wide
variety of marine animals including fish such as salmonids and sea
bream, and crustaceans such as crab, lobster, and shrimp. Because
animals generally cannot biosynthesize carotenoids, they obtain
those carotenoids present in microorganisms or plants upon which
they feed.
[4833] Carotenoids, e.g. xanthophylls, in particular lutein,
supplied from biological sources, such as crustaceans, yeast, and
green alga is limited by low yield and costly extraction methods
when compared with that obtained by organic synthetic methods.
Synthetic methods are e.g. described in Hansgeorg Ernst, Pure Appl.
Chem., Vol. 74, No. 8, pp. 1369-1382, 2002. Usual synthetic
methods, however, produce by-products that can be considered
unacceptable. It is therefore desirable to find a relatively
inexpensive source of carotenoids, in particular lutein, to be used
as a feed supplement in aquaculture and as a valuable chemical for
other industrial uses and for diets. Sources of xanthophylls
include crustaceans such as a krill in the Antarctic Ocean,
cultured products of the yeast Phaffia, cultured products of a
green alga Haematococcus pluvialis, and products obtained by
organic synthetic methods. However, when crustaceans such as a
krill or the like are used, a great deal of work and expense are
required for the isolation of xanthophylls from contaminants such
as lipids and the like during the harvesting and extraction.
Moreover, in the case of the cultured product of the yeast Phaffia,
a great deal of expense is required for the gathering and
extraction of astaxanthin because the yeast has rigid cell walls
and produces xanthophylls only in a low yield. One approach to
increase the productivity of some xanthophylls' production in a
biological system is to use genetic engineering technology.
[4834] [0006.0.11.11] Carotenoids in higher plants; i.e.,
angiosperms, are found in plastids; i.e., chloroplasts and
chromoplasts. Plastids are intracellular storage bodies that differ
from vacuoles in being surrounded by a double membrane rather than
a single membrane. Plastids such as chloroplasts can also contain
their own DNA and ribosomes, can reproduce independently, and
synthesize some of their own proteins. Plastids thus share several
characteristics of mitochondria. In leaves, carotenoids are usually
present in the grana of chloroplasts where they provide a
photoprotective function. Beta-carotene and lutein are the
predominant carotenoids, with the epoxidized carotenoids
violaxanthin and neoxanthin being present in smaller amounts.
Carotenoids accumulate in developing chromoplasts of flower petals,
usually with the disappearance of chlorophyll. As in flower petals,
carotenoids appear in fruit chromoplasts as they develop from
chloroplasts. Most enzymes that take part in conversion of phytoene
to carotenes and xanthophylls are labile, membrane-associated
proteins that lose activity upon solubilization. In maize,
cartonoids were present in horny endosperm (74% to 86%), floury
endosperm (9%-23%) and in the germ and bran of the kernel.
[4835] [0007.0.11.11] At the present time only a few plants are
widely used for commercial coloured carotenoid production. However,
the productivity of coloured carotenoid synthesis in most of these
plants is relatively low and the resulting carotenoids are
expensively produced.
[4836] Dried marigold petals and marigold petal concentrates
obtained from so-called xanthophyll marigolds are used as feed
additives in the poultry industry to intensify the yellow color of
egg yolks and broiler skin. The pigmenting ability of marigold
petal meal resides largely in the carotenoid fraction known as the
xanthophylls, primarily lutein esters. The xanthophyll zeaxanthin,
also found in marigold petals, has been shown to be effective as a
broiler pigmenter, producing a highly acceptable yellow to
yellow-orange color. Of the xanthophylls, the pigments lutein and
zeaxanthin are the most abundant in commercially available
hybrids.
[4837] Carotenoids have been found in various higher plants in
storage organs and in flower petals. For example, marigold flower
petals accumulate large quantities of esterified lutein as their
predominant xanthophyll carotenoid (about 75 to more than 90
percent), with smaller amounts of esterified zeaxanthin. Besides
lutein and zeaxanthin, marigold flower petals also typically
exhibit a small accumulation of .beta.-carotene and epoxidized
xanthophylls, but do not produce or accumulate canthaxanthin or
astaxanthin because a 4-keto-.beta.-ionene ring-forming enzyme is
absent in naturally-occurring marigolds or their hybrids.
[4838] [0008.0.11.11] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce coloured carotenoids generally and,
with the use of recent advances in determining carotenoid
biosynthesis from .beta.-carotene to xanthophylls to control the
production of carotenoids. That type of production permits control
over quality, quantity, and selection of the most suitable and
efficient producer organisms. The latter is especially important
for commercial production economics and therefore availability to
consumers.
[4839] Methods of recombinant DNA technology have been used for
some years to improve the production of Xanthophylls in
microorganisms, in particular algae or in plants by amplifying
individual xanthophyll biosynthesis genes and investigating the
effect on xanthophyll production. It is for example reported, that
the five ketocarotenoids, e.g. the xanthophyll astaxanthin could be
produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. The Pds gene was said by
those workers to direct transcription and expression in
chloroplasts and/or chromoplast-containing tissues of plants. Those
results indicated that about 75 percent of the carotenoids found in
the flower of the transformed plant contained a keto group.
Further, in maize the phytonene synthase (Psy), Phytone desaturase
(Pds), and the -carotene desaturase were identified and it was
shown, that PSY activity is an important control point for the
regulation of the flux.
[4840] Genes suitable for conversion of microorganisms have also
been reported (U.S. Pat. No. 6,150,130 WO 99/61652). Two different
genes that can convert a carotenoid [3-ionene ring compound into
astaxanthin have been isolated from the green alga Haematococcus
pluvialis. Zeaxanthin or -carotene were also found in the marine
bacteria Agrobacterium aurantiacum, Alcaligenes PC-1, Erwinia
uredovora. An A. aurantiacum crtZ gene was introduced to an E. coli
transformant that accumulated all-trans-.beta.-carotene. The
transformant so formed produced zeaxanthin. A gene cluster encoding
the enzymes for a carotenoid biosynthesis pathway has been also
cloned from the purple photosynthetic bacterium Rhodobacter
capsulatus. A similar cluster for carotenoid biosynthesis from
ubiquitous precursors such as farnesyl pyrophosphate and geranyl
pyrophosphate has been cloned from the non-photosynthetic bacteria
Erwinia herbicola. Yet another carotenoid biosynthesis gene cluster
has been cloned from Erwinia uredovora. It is yet unknown and
unpredictable as to whether enzymes encoded by other organisms
behave similarly to that of A. aurantiacum in vitro or in vivo
after transformation into the cells of a higher plant.
[4841] [0009.0.11.11] Thus, it would be advantageous if an algae or
other microorganism were available which produce large amounts of
.beta.-carotene, beta-cryptoxanthin, lutein, zeaxanthin, or other
carotenoids. The invention discussed hereinafter relates in some
embodiments to such transformed prokaryotic or eukaryotic
microorganisms.
[4842] It would also be advantageous if a marigold or other plants
were available whose flowers produced large amounts of
.beta.-carotene, beta-cryptoxanthin, lutein, zeaxanthin, or other
carotenoids. The invention discussed hereinafter relates in some
embodiments to such transformed plants.
[4843] [0010.0.11.11] Therefore improving the quality of foodstuffs
and animal feeds is an important task of the food-and-feed
industry. This is necessary since, for example, as mentioned above
xanthophylls, which occur in plants and some microorganisms are
limited with regard to the supply of mammals. Especially
advantageous for the quality of foodstuffs and animal feeds is as
balanced as possible a carotenoids profile in the diet since a
great excess of some carotenoids above a specific concentration in
the food has only some positive effect. A further increase in
quality is only possible via addition of further carotenoids, which
are limiting.
[4844] [0011.0.11.11] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add one or a plurality of
carotenoids in a balanced manner to suit the organism.
[4845] [0012.0.11.11] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
regulators which participate in the biosynthesis of lutein, and
make it possible to produce them specifically on an industrial
scale without unwanted byproducts forming. In the selection of
genes for biosynthesis two characteristics above all are
particularly important. On the one hand, there is as ever a need
for improved processes for obtaining the highest possible contents
of carotenoids like lutein; on the other hand as less as possible
byproducts should be produced in the production process.
[4846] [0013.0.0.11] see [0013.0.0.0]
[4847] [0014.0.11.11] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is lutein. Accordingly, in the
present invention, the term "the fine chemical" as used herein
relates to a "lutein".
[4848] [0015.0.11.11] In one embodiment, the term "the respective
fine chemical" means lutein. Throughout the specification the term
"the respective fine chemical" or the term "lutein" means lutein in
its free form, its salts, ester, its mono- or diesters of fatty
acids, e.g. as lutein dipalmitates, dimyristates or monomyristates
or bound to proteins, e.g. lipoproteins or tuberlin, or bound to
other compounds.
[4849] Lutein exist in a matrix in foods. Thus, in one embodiment,
the fine chemical produced according to the process of the
invention is a matrix comprising inter alia lipids, in particular
cholesterol, phospholipid, and/or triglycerides, and lutein.
[4850] Thus in one embodiment, the fine chemical is a lutein ester.
In one particular embodiment, the fine chemical is a lutein ester
of a natural occurring, preferably in plants or microorganisms
occurring fatty acid. In a further embodiment, the fine chemical is
a lutein monoester. In a further embodiment, the fine chemical is a
lutein diester. In a further embodiment, the fine chemical is
lutein dipalmitates, dimyristates or monomyristates. In a further
embodiment, the fine chemical is a lutein comprising matrix. In a
further embodiment, the fine chemical is a lutein comprising
micelle, e.g. a micelle formed from bile salts or dietary lipids,
or a clathrate complex, e.g. with conjugated bile salts. In a
further embodiment, the fine chemical is lutein in the form of
chylomicrons. In a further embodiment, the fine chemical is lutein
in the form of chylomicron remnants. In a further embodiment, the
fine chemical is lutein incorporated into lipoproteins, e.g. HDL or
VLDL. In a further embodiment, the fine chemical is lutein bound to
tuberlin. In a further embodiment, the fine chemical is free
lutein, in particular (3R,3'R,6'R)-lutein.
[4851] [0016.0.11.11] Accordingly, the present invention relates to
a process comprising [4852] (a) increasing or generating the
activity of one or more b4401-, b2699- or YFR007W-protein(s) in a
non-human organism in one or more parts thereof; and [4853] (b)
growing the organism under conditions which permit the production
of the respective fine chemical, thus, of lutein in said
organism.
[4854] Accordingly, the present invention relates to a process
comprising [4855] (a) increasing or generating the activity of one
or more proteins having the activity of a protein indicated in
Table II, column 3, lines 109 to 111, resp., or having the sequence
of a polypeptide encoded by a nucleic acid molecule indicated in
Table I, column 5 or 7, lines 109 to 111, resp., in a non-human
organism in one or more parts thereof; and [4856] (b) growing the
organism under conditions which permit the production of the
respective fine chemical, thus, lutein in said organism.
[4857] [0016.1.0.11] -/-
[4858] [0017.0.0.11] to [0018.0.0.11]: see [0017.0.0.0] to
[0018.0.0.0]
[4859] [0019.0.11.11] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the respective fine chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table II, column 3, lines 109 to 111 or encoded by
nucleic acid molecule indicated in Table I, columns 5 or 7, lines
109 to 111.
[4860] [0020.0.11.11] Surprisingly it was found, that the
transgenic expression of the Saccharomyces cerevisiae protein
YFR007W, and/or the Escherichia coli K12 protein b2699, and/or
b4401, in particular as indicated in Table II, column 3, lines 109
to 111, resp., conferred in Arabidopsis thaliana an increase in the
"fine chemical" or "the fine respective chemical" content of the
transformed plants.
[4861] [0021.0.0.11] see [0021.0.0.0]
[4862] [0022.0.11.11] The sequence of b2699 from Escherichia coli
K12 has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as a DNA strand
exchange and recombination protein with protease and nuclease
activity. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein with DNA
recombination and DNA repair activity, a pheromone response
activity, a mating-type determination activity, a sex-specific
protein activity, a nucleotide binding activity and/or a protease
and nuclease activity, in particular a DNA strand exchange and
recombination protein with protease and nuclease activity, in
particular of the superfamily of the recombination protein recA, in
particular of a protein with protease and nuclease activity, in
particular DNA strand exchange and recombination protein with
protease and nuclease activity of the superfamily of recombination
protein rec A or its homologs, e.g. as shown herein in Table II or
as encoded by the nucleic acid molecule shown in table I, column 5
or 7, lines 110, for the production of the respective fine
chemical, meaning of lutein, preferably in free or bound or
derivative form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a protein with protease and nuclease activity, in
particular DNA strand exchange and recombination protein with
protease and nuclease activity of the superfamily of recombination
protein rec A or its homolog is increased or generated, e.g. from
E. coli or a homolog thereof.
[4863] The sequence of b4401 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a response regulator of the
OmpR family. Accordingly, in one embodiment, the process of the
present invention comprises the use of protein having a
transcriptional control activity, unspecified signal transduction
activity, two-component signal transduction system (E.g. response
regulator component) activity, transcriptional activator activity,
regulation of respiration activity, aerobic respiration activity,
and/or anaerobic respiration activity, in particular, having the
activity of a protein of the superfamily of the ompR protein,
preferably a protein having a response regulator of the OmpR family
activity, in particular of E. coli, or its homolog, e.g. as shown
herein in Table II or as encoded by the nucleic acid molecule shown
in table I, column 5 or 7, lines 111, for the production of the
respective fine chemical, i.e. lutein, preferably in free or bound
or derivative form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention said
activity, e.g the activity of response regulator of the OmpR family
is increased or generated, e.g. from E. coli or a homolog thereof.
The sequence of YFR007W from Saccharomyces cerevisiae has been
published in Jacq et al., Nature 387 (6632 Suppl), 75-78, 1997, and
Goffeau et al., Science 274 (5287), 546-547, 1996, and its activity
has not been annotated, yet. Accordingly, in one embodiment, the
process of the present invention comprises the use of YFR007W
protein or its homolog, e.g. as shown herein in Table II or as
encoded by the nucleic acid molecule shown in Table I, columns 5 or
7, lines 109, for the production of the respective fine chemical in
free or bound or derivative form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention said activity, e.g. the activity of YFR007W is increased
or generated, e.g. from Saccharomyces cerevisiae, or a homolog
thereof.
[4864] [0023.0.11.11] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[4865] In one embodiment, a homolog of any one of the polypeptides
indicated in Table II, column 3, line 109 is a homolog having the
same or a similar activity as described herein or annotated. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms. In one
embodiment, the homologue is a homolog with a sequence as indicated
in Table I or II, column 7, lines 109, resp. In one embodiment, the
homologue of one of the polypeptides indicated in Table II, column
3, line 109 is derived from an eukaryotic organism. In one
embodiment, the homologue is derived from Fungi. In one embodiment,
the homologue of a polypeptide indicated in Table II, column 3,
line 109 is derived from Ascomyceta. In one embodiment, the
homologue of a polypeptide indicated in Table II, column 3, line
109 is derived from Saccharomycotina. In one embodiment, the
homologue of a polypeptide indicated in Table II, column 3, line
109 is derived from Saccharomycetes. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, line 109 is a
homologue being derived from Saccharomycetales. In one embodiment,
the homologue of a polypeptide indicated in Table II, column 3,
line 109 is a homologue having the same or a similar activity being
derived from Saccharomycetaceae. In one embodiment, the homologue
of a polypeptide indicated in Table II, column 3, line 109 is a
homologue having the same or a similar activity, in particular an
increase of activity confers an increase in the content of lutein
in a organisms or part thereof, being derived from
Saccharomycetes.
[4866] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 111 is a homolog
having the same or a similar activity as described herein or
annotated. In particular an increase of activity confers an
increase in the content of the respective fine chemical. In one
embodiment, the homolog is a homolog with a sequence as indicated
in Table I or II, column 7, line 111, resp. In one embodiment, the
homolog of one of the polypeptides indicated in Table II, column 3,
line 111 is derived from an bacteria. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 111
is derived from Proteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 111 is a homolog
having the same or a similar activity being derived from
Gammaproteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 111 is derived
from Enterobacteriales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 111 is a homolog
being derived from Enterobacteriaceae. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 111
is a homolog having the same or a similar activity, in particular
an increase of activity confers an increase in the content of
lutein in a organisms or part thereof being derived from
Escherichia.
[4867] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 110 is a homolog
having the same or a similar activity as described herein or
annotated. In particular an increase of activity confers an
increase in the content of the respective fine chemical in the
organisms. In one embodiment, the homolog is a homolog with a
sequence as indicated in Table I or II, column 7, line 110, resp.
In one embodiment, the homolog of one of the polypeptides indicated
in Table II, column 3, line 110 is derived from an bacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 110 is derived from Proteobacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 110 is a homolog having the same or a similar
activity being derived from Gammaproteobacteria. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, line
110 is derived from Enterobacteriales. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 110
is a homolog being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 110 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the lutein in the organisms or part thereof,
being derived from Escherichia.
[4868] [0023.1.0.11] Homologs of the polypeptides indicated in
Table II, column 3, lines 109 to 111 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 109 to 111, respectively or may be the polypeptides
indicated in Table II, column 7, lines 109 to 111 having a lutein
content and/or amount increasing activity. Homologs of the
polypeptides indicated in Table II, column 3, lines 109 to 111 may
be the polypeptides encoded by the nucleic acid molecules indicated
in Table I, column 7, lines 109 to 111 or may be the polypeptides
indicated in Table II, column 7, lines 109 to 111 having a lutein
content and/or amount increasing activity.
[4869] [0024.0.0.11] see [0024.0.0.0]
[4870] [0025.0.11.11] In accordance with the invention, a protein
or polypeptide has the "activity of an protein of the invention",
e.g. the activity of a protein indicated in Table II, column 3,
lines 109 to 111 if its de novo activity, or its increased
expression directly or indirectly leads to an increased the
respective fine chemical, in particular lutein, preferably free
lutein level, in the organism or a part thereof, preferably in a
cell of said organism. In a preferred embodiment, the protein or
polypeptide has the above-mentioned additional activities of a
protein indicated in Table II, column 3, lines 109 to 111.
Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of any one of the proteins indicated in Table II, column
3, lines 109 to 111, or which has at least 10% of the original
enzymatic activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to any one of the
proteins indicated in Table II, column 3, line 109 of Saccharomyces
cerevisiae and/or any one of the proteins indicated in Table II,
column 3, line 110 or line 111 of E. coli K12.
[4871] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4, lines 109 to 111, and is expressed in an organism, which
is evolutionary distant to the origin organism. For example origin
and expressing organism are derived from different families,
orders, classes or phylums whereas origin and the organism
indicated in Table I, column 4, lines 109 to 111 are derived from
the same families, orders, classes or phylums.
[4872] [0025.1.0.11] see [0025.1.0.0]
[4873] [0026.0.0.11] to [0033.0.0.11]: see [0026.0.0.0] to
[0033.0.0.0]
[4874] [0034.0.11.11] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of an protein having the activity of a
protein as indicated in Table II, column 3, lines 109 to 111 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 109 to 111 or its homologs, e.g. as indicated in
Table I, column 7, lines 109 to 111, its biochemical or genetic
causes. It therefore shows the increased amount of the respective
fine chemical.
[4875] [0035.0.0.11] to [0044.0.0.11]: see [0035.0.0.0] to
[0044.0.0.0]
[4876] [0045.0.11.11] In one embodiment, the activity of the
Escherichia coli K12 protein b4401 or its homologs, e.g. a protein
of a transcriptional control activity, a unspecified signal
transduction activity, a two-component signal transduction system
(e.g. response regulator component activity), a transcriptional
activator activity, a regulation of respiration activity, an
aerobic respiration activity, and/or an anaerobic respiration
activity, e.g. of a response regulator, in particular of a response
regulator of the OmpR family, e.g. as indicated in Table II,
columns 5 or 7, line 111, is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of free lutein, between 25% and 42% or more is conferred.
[4877] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2699 or its homologs, e.g. a DNA recombination
and DNA repair activity, a pheromone response activity, a
mating-type determination activity, a sex-specific protein
activity, a nucleotide binding activity and/or a protease and
nuclease activity, in particular a DNA strand exchange and
recombination protein with protease and nuclease activity, in
particular of the superfamily of the recombination protein recA,
e.g. as indicated in Table II, columns 5 or 7, line 110, is
increased, preferably, in one embodiment the increase of the
respective fine chemical, preferably of free lutein between 31% and
58% or more is conferred.
[4878] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YFR007w or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 107, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of free lutein between 33% and 78% or more is
conferred.
[4879] [0046.0.11.11] In one embodiment, the activity of the
Escherichia coli K12 protein b4401 or its homologs e.g. a protein
of a transcriptional control activity, a unspecified signal
transduction activity, a two-component signal transduction system
(e.g. response regulator component activity), a transcriptional
activator activity, a regulation of respiration activity, an
aerobic respiration activity, and/or an anaerobic respiration
activity, e.g. of a response regulator, in particular of a response
regulator of the OmpR family, e.g. as indicated in Table II,
columns 5 or 7, line 111, is increased, preferably an increase of
the respective fine chemical and of further carotenoids, preferably
xanthophylls, in particular zeaxanthin, is conferred.
[4880] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2699 or its homologs e.g. a DNA recombination and
DNA repair activity, a pheromone response activity, a mating-type
determination activity, a sex-specific protein activity, a
nucleotide binding activity and/or a protease and nuclease
activity, in particular a DNA strand exchange and recombination
protein with protease and nuclease activity, in particular of the
superfamily of the recombination protein recA, e.g. as indicated in
Table II, columns 5 or 7, line 110, is increased, preferably an
increase of the respective fine chemical and of further
carotenoids, preferably xanthophylls, e.g. zeaxanthin, is
conferred.
[4881] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YFR007W or its homologs or its homolog, e.g. as
indicated in Table I, columns 5 or 7, line 109, is increased,
preferably an increase of the respective fine chemical and of
further carotenoids, preferably xanthophylls, e.g. zeaxanthin is
conferred.
[4882] [0047.0.0.11] to [0048.0.0.11]: see [0047.0.0.0] to
[0048.0.0.0]
[4883] [0049.0.11.11] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
lutein preferably has the structure of the polypeptide described
herein. In a particular embodiment, the polypeptides used in the
process of the present invention or the polypeptide of the present
invention comprises the sequence of a consensus sequence as
indicated in Table IV, column 7, lines 109 to 111, respectively, or
of a polypeptide as indicated in Table II, columns 5 or 7, lines
109 to 111, or of a functional homologue thereof as described
herein, or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 109 to 111 or its herein described
functional homologues and has the herein mentioned activity
conferring an increase in the lutein level.
[4884] [0050.0.11.11] -/-
[4885] [0051.0.11.11] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the fine
chemical free or bound, e.g compositions comprising lutein and (a)
fatty acid(s), dietary oil(s), such as corn oil, and/or
triglycerides, in particular medium-chain (e.g. C.sub.4 to
C.sub.18-, in particular C.sub.6 to C.sub.14-) triglycerides,
lipoproteins, e.g. HDL and/or VLDL, micelles, clathrate complexes,
e.g. conjugated with bile salts, chylomicrons, chylomicron
remnants, tuberlin and/or other carotenoids, e.g. xanthophylls, in
particular zeaxanthin. Depending on the choice of the organism used
for the process according to the present invention, for example a
microorganism or a plant, compositions or mixtures of various
carotenoids and luteincan be produced.
[4886] [0052.0.0.11] see [0052.0.0.0]
[4887] [0053.0.11.11] In one embodiment, the process of the present
invention comprises one or more of the following steps [4888] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3 or 5,
lines 109 to 111 or its homologs, e.g. as indicated in Table II,
columns 7, lines 109 to 111, activity having herein-mentioned the
respective fine chemical increasing activity; [4889] b) stabilizing
a mRNA conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3
or 5, lines 109 to 111 or its homologs activity, e.g. as indicated
in Table II, columns 7, lines 109 to 111, or of a mRNA encoding the
polypeptide of the present invention having herein-mentioned the
respective fine chemical increasing activity; [4890] c) increasing
the specific activity of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3 or 5, lines 109 to 111 or its homologs
activity, e.g. as indicated in Table II, columns 7, lines 109 to
111, or decreasing the inhibitory regulation of the polypeptide of
the invention; [4891] d) generating or increasing the expression of
an endogenous or artificial transcription factor mediating the
expression of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention having herein-mentioned the
respective fine chemical-increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3
or 5, lines 109 to 111 or its homologs activity, e.g. as indicated
in Table II, columns 7, lines 109 to 111; [4892] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned the respective fine chemical-increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3 or 5, lines 109 to 111 or line 109 and/or 111
or its homologs activity, e.g. as indicated in Table II, columns 7,
lines 109 to 111, by adding one or more exogenous inducing factors
to the organism or parts thereof; [4893] f) expressing a transgenic
gene encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned the respective fine chemical-increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3 or 5, lines 109 to 111 or its homologs
activity, e.g. as indicated in Table II, columns 7, lines 109 to
111, and/or [4894] g) increasing the copy number of a gene
conferring the increased expression of a nucleic acid molecule
encoding a polypeptide encoded by the nucleic acid molecule of the
invention or the polypeptide of the invention having
herein-mentioned the respective fine chemical-increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3 or 5, lines 109 to 111 or its homologs, e.g.
as indicated in Table II, columns 7, lines 109 to 111, activity.
[4895] h) Increasing the expression of the endogenous gene encoding
the polypeptide of the invention, e.g. a polypeptide having an
activity of a protein as indicated in Table II, column 3 or 5,
lines 109 to 111 or its homologs activity, e.g. as indicated in
Table II, columns 7, lines 109 to 111, by adding positive
expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; and/or
[4896] i) Modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced respective fine chemical
production. [4897] j) selecting of organisms with especially high
activity of the proteins of the invention from natural or from
mutagenized resources and breeding them into the target organisms,
e.g. the elite crops.
[4898] [0054.0.11.11] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table II, columns 5, lines
109 to 111, or its homologs activity, e.g. as indicated in Table
II, columns 7, lines 109 to 111.
[4899] [0055.0.0.11] to [0067.0.0.11]: see [0055.0.0.0] to
[0067.0.0.0]
[4900] [0068.0.11.11] The mutation is introduced in such a way that
the production of the lutein is not adversely affected.
[4901] [0069.0.0.11] see [0069.0.0.0]
[4902] [0070.0.11.11] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolites composition in the organism, e.g. an advantageous
composition of carotenoids comprising a higher content of (from a
viewpoint of nutritional physiology limited) carotenoids, e.g.
xanthophylls, in particular lutein.
[4903] It can also be advantageous to increase the level of a
metabolic precursor of lutein in the organism or part thereof, e.g.
of phytoene, lycopene, alpha-carotene.
[4904] It can also be advantageous owing to the introduction of a
gene or a plurality of genes conferring the expression of a
inhibitory nucleic acid molecule, e.g. for a gene k.o., e.g.
[4905] a iRNA or a antisense nucleic acid, to decrease the level of
production of neoxanthin or one or more precursor thereof, e.g.
vialastaxanthin, zeaxanthin, and/or beta-carotene as this might
increase the level of lycopene to be provided for the production of
lutein according to method of present invention.
[4906] [0071.0.0.11] see [0071.0.0.0]
[4907] [0072.0.11.11] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to lutein are further carotenoids, e.g. carotenes or
xanthophylls, in particular ketocarotenoids, or hydrocarotenoids,
e.g. beta-cryptoxanthin, zeaxanthin, astaxanthin, lycopene,
alpha-carotene, or beta-carentene, or compounds for which lutein is
a precursor compound or medium-chain (e.g C.sub.4 to C.sub.18-, in
particular C.sub.6 to C.sub.14-) triglycerides, lipoproteins, e.g.
HDL and/or VLDL, micelles, clathrate complexes, e.g. conjugated
with bile salts, chylomicrons, chylomicron remnants, and/or
tuberlin.
[4908] [0073.0.11.11] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[4909] i) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [4910] j) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table II, columns 5 or 7, lines 109 to 111, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the respective fine chemical in an organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [4911] k) growing an organism,
preferably a microorganism, a non-human animal, a plant or animal
cell, a plant or animal tissue or a plant under conditions which
permit the production of the respective fine chemical in the
organism, preferably the microorganism, the plant cell, the plant
tissue or the plant; and [4912] l) if desired, recovering,
optionally isolating, the free and/or bound the respective fine
chemical and, optionally further free and/or bound carotenoids, in
particular ketocarotenoids, or hydrocarotenoids, e.g.
beta-cryptoxanthin, zeaxanthin, astaxanthin, lycopene,
alpha-carotene, or beta-carotene, synthesized by the organism, the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue or the plant.
[4913] [0074.0.11.11] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the respective fine chemical or the free and
bound the respective fine chemical but as option it is also
possible to produce, recover and, if desired isolate, other free
or/and bound carotenoids, in particular ketocarotenoids, or
hydrocarotenoids, e.g. beta-cryptoxanthin, zeaxanthin, astaxanthin,
lycopene, alpha-carotene, or beta-carotene.
[4914] [0075.0.0.11] to [0077.0.0.11]: see [0075.0.0.0] to
[0077.0.0.0]
[4915] [0078.0.11.11] The organism such as microorganisms or plants
or the recovered, and if desired isolated, the respective fine
chemical can then be processed further directly into foodstuffs or
animal feeds or for other applications, for example according to
the disclosures made in the following US applications: U.S. Pat.
No. 6,380,442: Process for the isolation of mixed carotenoids from
plants, U.S. Pat. No. 6,376,722: Lutein to zeaxanthin isomerization
process and product, U.S. Pat. No. 6,362,221: Compositions
containing natural lycopene and natural tocopherol, U.S. Pat. No.
6,329,557: Purification of xanthophylls from marigold extracts that
contain high levels of chlorophylls, U.S. Pat. No. 6,328,995:
Stable vitamin and/or carotenoid products in powder form and
process for their production, U.S. Pat. No. 6,313,169: Lutein
esters having high bioavailability, U.S. Pat. No. 6,309,677:
Multi-carotenoid product, U.S. Pat. No. 299.912: Preparation for
administration to animals and feeding method thereof, U.S. Pat. No.
6,299,896: Multi-vitamin and mineral supplement, U.S. Pat. No.
6,296,877: Stable, aqueous dispersions and stable,
water-dispersible dry xanthophyll powder, their production and use,
U.S. Pat. No. 6,291,533: Dietary supplements for each specific
blood type, U.S. Pat. No. 6,287,615: Use of solubilized carotenoid
preparations for coloring food preparations, U.S. Pat. No.
6,271,408: Process for making metabolites of lycopene, U.S. Pat.
No. 6,262,284: Process for extraction and purification of lutein,
zeaxanthin and rare carotenoids from marigold flowers and plants
U.S. Pat. No. 6,261,622: Water-dispersible carotenoid pigment
preparation, U.S. Pat. No. 6,261,598: Carotenoid formulations,
comprising a mixture of B-carotens, lycopene and lutein U.S. Pat.
No. 6,248,378: Enhanced food products, U.S. Pat. No. 6,248,374:
Stabilized food additive, U.S. Pat. No. 6,241,987: Dietary
supplement containing saw palmetto, pumpkin seed, and nettle root,
U.S. Pat. No. 6,254,898: Nutraceutical composition for protection
against solar radiation. Pharmaceutical and other compositions
comprising lutein are e.g. described in the following US
applications: U.S. Pat. No. 6,383,523: Pharmaceutical compositions
and methods for managing skin conditions, U.S. Pat. No. 6,368,621:
Preparation in particular for use as a medication and/or food
supplement, U.S. Pat. No. 6,361,800: Multi-vitamin and mineral
supplement, U.S. Pat. No. 6,348,200: Cosmetic composition, U.S.
Pat. No. 6,329,432: Mesozeaxanthin formulations for treatment of
retinal disorders, U.S. Pat. No. 6,310,090: Process and product for
enhancing immune response in companion animals using a combination
of antioxidants, U.S. Pat. No. 6,303,586: Supportive therapy for
diabetes, hyperglycemia and hypoglycemia, U.S. Pat. No. 6,296,880:
Pharmaceutical compositions and methods for managing skin
conditions, U.S. Pat. No. 6,271,246: Pharmaceutical compositions
for managing scalp conditions, U.S. Pat. No. 6,262,109: Methods of
preventing and/or treating high serum levels of cholesterol and/or
lipids, U.S. Pat. No. 6,258,855: Method of retarding and
ameliorating carpal tunnel syndrome, U.S. Pat. No. 6,248,363: Solid
carriers for improved delivery of active ingredients in
pharmaceutical compositions.
[4916] The cited literature describes some preferred embodiments
without being meant to be limiting. The fermentation broth,
fermentation products, plants or plant products can be purified as
described in above mentioned applications and other methods known
to the person skilled in the art, e.g. as described in Methods in
Enzymology: Carotenoids, Part A: Chemistry, Separation,
Quantitation and Antioxidation, by John N Abelson or Part B,
Metabolism, Genetics, and Biochemistry, or described herein below.
Products of these different work-up procedures are
lutein-comprising compositions, which still comprise fermentation
broth, plant particles and cell components in different amounts,
advantageously in the range of from 0 to 99% by weight, preferably
below 80% by weight, especially preferably between below 50% by
weight.
[4917] [0079.0.0.11] to [0084.0.0.11]: see [0079.0.0.0] to
[0084.0.0.0]
[4918] [0084.1.11.11] The invention also contemplates embodiments
in which the respective fine chemical, is present in the flowers of
the flowering plant chosen as the host (for example,
marigolds).
[4919] In one embodiment, preferred flowering plants include, but
are not limited to: Amaryllidaceae (Allium, Narcissus); Apocynaceae
(Catharanthus); Asteraceae, alternatively Compositae (Aster,
Calendula, Callistephus, Cichorium, Coreopsis, Dahlia,
Dendranthema, Gazania, Gerbera, Helianthus, Helichrysum, Lactuca,
Rudbeckia, Tagetes, Zinnia); Balsaminaceae (Impatiens); Begoniaceae
(Begonia); Caryophyllaceae (Dianthus); Chenopodiaceae (Beta,
Spinacia); Cucurbitaceae (Citrullus, Curcurbita, Cucumis);
Cruciferae (Alyssum, Brassica, Erysimum, Matthiola, Raphanus);
Gentinaceae (Eustoma); Geraniaceae (Pelargonium); Graminae,
alternatively Poaceae, (Avena, Horedum, Oryza, Panicum, Pennisetum,
Poa, Saccharum, Secale, Sorghum, Triticum, Zea); Euphorbiaceae
(Poinsettia); Labiatae (Salvia); Leguminosae (Glycine, Lathyrus,
Medicago, Phaseolus, Pisum); Liliaceae (Cilium); Lobeliaceae
(Lobelia); Malvaceae (Abelmoschus, Gossypium, MaIva);
Plumbaginaceae (Limonium); Polemoniaceae (Phlox); Primulaceae
(Cyclamen); Ranunculaceae (Aconitum, Anemone, Aquilegia, Caltha,
Delphinium, Ranunculus); Rosaceae (Rosa); Rubiaceae (Pentas);
Scrophulariaceae (Angelonia, Antirrhinum, Torenia); Solanaceae
(Capsicum, Lycopersicon, Nicotiana, Petunia, Solanum); Umbelliferae
(Apium, Daucus, Pastinaca); Verbenaceae (Verbena, Lantana);
Violaceae (Viola).
[4920] [0085.0.11.11] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [4921] a) a nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 109 to 111, or a
derivative thereof, or [4922] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 109 to
111, or a derivative thereof, or [4923] c) (a) and (b) is/are not
present in its/their natural genetic environment or has/have been
modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[4924] [0086.0.0.11] to [0087.0.0.11]: see [0086.0.0.0] to
[0087.0.0.0]
[4925] [0088.0.11.11] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned
essential vitamins, e.g. carotenoids, and the general amount of
lutein as source in feed. Further, this is also important for the
production of cosmetic compostions since, for example, the
antioxidant level of plant extraction is dependent on the amount of
th abovementioned of xanthophylls and/or the amount of carotenoids
as antioxidants.
[4926] [0088.1.0.11] see [0088.1.0.0]
[4927] [0089.0.0.11] to [0094.0.0.11]: see [0089.0.0.0] to
[0094.0.0.0]
[4928] [0095.0.11.11] It may be advantageous to increase the pool
of said carotenoids in the transgenic organisms by the process
according to the invention in order to isolate high amounts of the
pure respective fine chemical.
[4929] [0096.0.11.11] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical in the organism,
is useful to increase the production of the respective fine
chemical. It has been reported, that the inhibition of Lyopene
production increases the amount of other xanthophylls in the cell.
Further it may be, that the inhibition of enzymes using
beta-carotene as substrate increases the amount of said chemicals
in a cell. For example, in one embodiment, it can be advantageous
to inhibit the production of neoxanthin, if a high amount of lutein
is desired.
[4930] [0097.0.11.11] Lutein mono- or diesters of fatty acids, e.g.
as lutein dipalmitates, dimyristates or monomyristates or
associated with lipids or proteins, e.g. lipoproteins or tuberlin,
as well as other modification of lutein are known to a person
skilled in the art.
[4931] In may also be advantageous to increase the content of the
bound respective fine chemical, e.g. of modification of lutein, in
particular mono- or diesters of fatty acids or lipids or protein
associated derivatives.
[4932] [0098.0.11.11] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated. The production of further carotenoids, e.g.
carotenes or xanthophylls, in particular ketocarotenoids or
hydrocarotenoids, e.g. zeaxanthin, beta-cryptoxanthin, astaxanthin,
lycopene, alpha-carotene, or beta-carotene, or mixtures thereof or
mixtures of other carotenoids by the process according to the
invention is advantageous.
[4933] [0099.0.11.11] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods
[4934] [0100.0.11.11] Transgenic plants which comprise lutein or in
one embodiment comprising lutein and advantageously further
carotenoids such as xanthophylls, e.g. zeaxanthin, and/or mono- or
diesters of fatty acids, triglycerides, lipids or protein
associated derivatives, and/or dietary oils, like e.g. plant oils,
e.g. corn oil, synthesized in the process according to the
invention can advantageously be marketed directly without there
being any need for the carotenoids synthesized to be isolated.
Plants for the process according to the invention are listed as
meaning intact plants and all plant parts, plant organs or plant
parts such as leaf, stem, seeds, root, tubers, anthers, fibers,
root hairs, stalks, embryos, calli, cotelydons, petioles, harvested
material, plant tissue, reproductive tissue and cell cultures which
are derived from the actual transgenic plant and/or can be used for
bringing about the transgenic plant. In this context, the seed
comprises all parts of the seed such as the seed coats, epidermal
cells, seed cells, endosperm or embryonic tissue.
[4935] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, in the form of their oils, fats,
lipids, as extracts, e.g. ether, alcohol, or other organic solvents
or water containing extract and/or in the form of their mono- or
diesters of fatty acids or lipids or protein associated derivatives
or as free lutein. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts, e.g. the plant seeds
or the plant flowers. To increase the efficiency of extraction it
is beneficial to clean, to temper and if necessary to hull and to
flake the plant material.E.g., oils, fats, and/or lipids comprising
lutein can be obtained by what is known by processes known to the
skilled person e.g. known as cold beating or cold pressing without
applying heat. To allow for greater ease of disruption of the plant
parts, e.g. the seeds, they can previously be comminuted, steamed
or roasted. However, it is known that lutein is heat instable.
Thus, to maximize the availability of the carotenoids to be
extracted, the samples can be steamed only lightly and are
preferably not being roasted. Seeds, which have been pretreated in
this manner can subsequently be pressed or extracted with solvents
such as warm hexane. The solvent is subsequently removed. In the
case of microorganisms, the latter are, after harvesting, for
example extracted directly without further processing steps or
else, after disruption, extracted via various methods with which
the skilled worker is familiar. Thereafter, the resulting products
can be processed further, i.e. degummed and/or refined. In this
process, substances such as the plant mucilages and suspended
matter can be first removed. What is known as desliming can be
affected enzymatically or, for example, chemico-physically by
addition of acid such as phosphoric acid. However, in one
embodiment, lutein is extracted from photosynthetic tissues or
cells. In another embodiment, lutein is extracted from flowers.
[4936] Because carotenoids in microorganisms are localized
intracellular, their recovery essentials comes down to the
isolation of the biomass. Well-established approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Of the residual
hydrocarbon, adsorbed on the cells, has to be removed. Solvent
extraction or treatment with surfactants have been suggested for
this purpose. However, it can be advantageous to avoid this
treatment as it can result in cells devoid of most carotenoids,
e.g. lutein.
[4937] [0101.0.11.11] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[4938] [0102.0.11.11] Lutein can for example be detected
advantageously via HPLC, LC or GC separation methods. The
unambiguous detection for the presence of lutein containing
products can be obtained by analyzing recombinant organisms using
analytical standard methods: LC, LC-MS, MS or TLC). The material to
be analyzed can be disrupted by sonication, grinding in a glass
mill, liquid nitrogen and grinding, cooking, or via other
applicable methods.
[4939] However, as mentioned herein, it is in one embodiment
advantageous to provide an organism or part thereof produced
according to the process of the invention as food source for
lutein. To maximize the availability of the carotenoids in the
food, the food should be eaten raw or steamed lightly.
[4940] Carotenoids such as lutein and zeaxanthin are fat-soluble
substances and as such require the presence of dietary fat for the
proper absorption through the digestive tract. Consequently in one
embodiment, the organism or the part thereof or the lutein isolated
or a composition comprising the isolated lutein, is combined with
dietary fat or a dietary fat is added thereto.
[4941] [0103.0.11.11] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [4942] a) nucleic acid molecule encoding, preferably
at least the mature form, of the polypeptide having a sequence as
indicated in Table II, columns 5 or 7, lines 109 to 111, or a
fragment thereof, which confers an increase in the amount of the
respective fine chemical in an organism or a part thereof; [4943]
b) nucleic acid molecule comprising, preferably at least the mature
form, of a nucleic acid molecule having a sequence as indicated in
Table I, columns 5 or 7, lines 109 to 111, [4944] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [4945] d) nucleic acid molecule encoding a polypeptide
which has at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [4946] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridization conditions and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [4947] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [4948] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [4949]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers pairs having a
sequence as indicated in Table III, columns 7, lines 109 to 111,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [4950] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from an
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [4951] j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence having a sequences as
indicated in Table IV, columns 7, lines 109 to 111, and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [4952] k) nucleic acid molecule
comprising one or more of the nucleic acid molecule encoding the
amino acid sequence of a polypeptide encoding a domain of the
polypeptide indicated in Table II, columns 5 or 7, lines 109 to
111, and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; and [4953] l)
nucleic acid molecule which is obtainable by screening a suitable
library under stringent conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[4954] [0103.1.11.11.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 109 to 111,
by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence shown in indicated in Table I A, columns 5 or 7, lines
109 to 111. In one embodiment, the nucleic acid molecule used in
the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table I A,
columns 5 or 7, lines 109 to 111. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II A, columns 5 or 7, lines 109 to 111.
[4955] [0103.2.11.11.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, lines 109 to 111,
by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence shown in indicated in Table I B, columns 5 or 7, lines
109 to 111. In one embodiment, the nucleic acid molecule used in
the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table I B,
columns 5 or 7, lines 109 to 111. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II B, columns 5 or 7, lines 109 to 111.
[4956] [0104.0.11.11] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence indicated in
Table I, columns 5 or 7, lines 109 to 111 by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of the sequence
indicated in Table I, columns 5 or 7, lines 109 to 111. In one
embodiment, the nucleic acid molecule of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to the
sequence indicated in Table I, columns 5 or 7, lines 109 to 111. In
another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II, columns 5 or 7,
lines 109 to 111.
[4957] [0105.0.0.11] to [0107.0.0.11]: see [0105.0.0.0] to
[0107.0.0.0]
[4958] [0108.0.11.11] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 109 to 111, nucleic
acid molecules which are derived from an amino acid sequences as
indicated in Table II, columns 5 or 7, lines 109 to 111, or from
polypeptides comprising the consensus sequence as indicated in
Table IV, columns 7, lines 109 to 111, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of a polypeptide as indicated in Table II, column 3, 5 or
7, lines 109 to 111, e.g. conferring the increase of the respective
fine chemical, after increasing its expression or activity, are
advantageously increased in the process according to the
invention.
[4959] [0109.0.0.11] to [0111.0.0.11]: see [0109.0.0.0] to
[0111.0.0.0]
[4960] [0112.0.11.11] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table II, column 3, lines 109 to
111, or having the sequence of a polypeptide as indicated in Table
II, columns 5 or 7, lines 109 to 111, and conferring an increase in
the lutein level.
[4961] [0113.0.0.11] to [0120.0.0.11]: see [0113.0.0.0] to
[0120.0.0.0]
[4962] [0120.1.11.11]: Production strains which are also
advantageously selected in the process according to the invention
are microorganisms selected from the group green algae, like
Spongioccoccum exentricum, Chlorella sorokiniana (pyrenoidosa,
7-11-05), or form the group of fungi like fungi belonging to the
Daccrymycetaceae family, or non-photosynthetic bacteria, like
methylotrophs, flavobacteria, actinomycetes, like streptomyces ch
restomyceticus, Mycobacteria like Mycobacterim phlei, Rhodobacter
capsulatus, or Brevibacterium linens, Dunaliella spp., Phaffia
rhodozyma, Phycomyces sp., Rhodotorula spp.
[4963] [0121.0.11.11] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 109 to 111 or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a lutein level increase after increasing
the activity of the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 109 to 111.
[4964] [0122.0.0.11] to [0127.0.0.11]: see [0122.0.0.0] to
[0127.0.0.0]
[4965] [0128.0.11.11] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, columns 7,
lines 109 to 111, by means of polymerase chain reaction can be
generated on the basis of a sequence shown herein, for example the
sequence as indicated in Table I, columns 5 or 7, lines 109 to 111,
resp., or the sequences derived from a sequences as indicated in
Table II, columns 5 or 7, lines 109 to 111, resp.
[4966] [0129.0.11.11] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved regions are those, which show a
very little variation in the amino acid in one particular position
of several homologs from different origin. The consensus sequences
indicated in table IV, columns 7, lines 109 to 111 are derived from
said alignments.
[4967] [0130.0.11.11] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of the
respective fine chemical after increasing the expression or
activity of a protein comprising said fragment.
[4968] [0131.0.0.11] to [0138.0.0.11]: see [0131.0.0.0] to
[0138.0.0.0]
[4969] [0139.0.11.11] Polypeptides having above-mentioned activity,
i.e. conferring the increase of the respective fine chemical,
derived from other organisms, can be encoded by other DNA sequences
which hybridise to a sequences indicated in Table I, columns 5 or
7, lines 109 to 111, preferably of Table I B, columns 5 or 7, lines
109 to 111 for lutein under relaxed hybridization conditions and
which code on expression for peptides having the respective fine
chemical-increasing activity.
[4970] [0140.0.0.11] to [0146.0.0.11]: see [0140.0.0.0] to
[0146.0.0.0]
[4971] [0147.0.11.11] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 109 to 111, preferably of Table I B,
columns 5 or 7, lines 109 to 111 is one which is sufficiently
complementary to one of said nucleotide sequences such that it can
hybridise to one of said nucleotide sequences, thereby forming a
stable duplex. Preferably, the hybridisation is performed under
stringent hybridization conditions. However, a complement of one of
the herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[4972] [0148.0.11.11] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines
109 to 111, preferably of Table I B, columns 5 or 7, lines 109 to
111 or a portion thereof and preferably has above mentioned
activity, in particular having a lutein increasing activity after
increasing the activity or an activity of a product of a gene
encoding said sequences or their homologs.
[4973] [0149.0.11.11] The nucleic acid molecule of the invention or
used in the process of the invention comprises a nucleotide
sequence which hybridizes, preferably hybridizes under stringent
conditions as defined herein, to one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 109 to 111, preferably
of Table I B, columns 5 or 7, lines 109 to 111 or a portion thereof
and encodes a protein having above-mentioned activity, e.g.
conferring a of lutein-increase, resp., and optionally, the
activity of a protein indicated in Table II, column 5, lines 109 to
111, preferably of Table II B, columns 5 or 7, lines 109 to
111.
[4974] [00149.1.11.11] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table I, columns 5 or 7, lines 109 to 111,
preferably of Table I B, columns 5 or 7, lines 109 to 111 has
further one or more of the activities annotated or known for the a
protein as indicated in Table II, column 3, lines 109 to 111,
preferably of Table II B, columns 3, lines 109 to 111.
[4975] [0150.0.11.11] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table I, columns 5 or 7, lines 109 to
111, preferably of Table I B, columns 5 or 7, lines 109 to 111 for
example, a fragment which can be used as a probe or primer or a
fragment encoding a biologically active portion of the polypeptide
of the present invention or of a polypeptide used in the process of
the present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of lutein, if its activity is increased. The
nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table I, columns
5 or 7, lines 109 to 111, an anti-sense sequence of one of the
sequences, e.g., as indicated in Table I, columns 5 or 7, lines 109
to 111, or naturally occurring mutants thereof. Primers based on a
nucleotide of invention can be used in PCR reactions to clone
homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table III, column 7, lines 109 to 111 will result in a fragment of
a polynucleotide sequence as indicated in Table I, columns 5 or 7,
lines 109 to 111 or its gene product. Preferred is Table II B,
columns 7, lines 109 to 111.
[4976] [0151.0.0.11]: see [0151.0.0.0]
[4977] [0152.0.11.11] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 109 to 111
such that the protein or portion thereof maintains the ability to
participate in the respective fine chemical production, in
particular a lutein-increasing activity as mentioned above or as
described in the examples in plants or microorganisms is
comprised.
[4978] [0153.0.11.11] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 109
to 111 such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table II, columns 5 or 7, lines 109 to 111 has for
example an activity of a polypeptide indicated in Table II, column
3, lines 109 to 111.
[4979] [0154.0.11.11] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 109 to 111
and has above-mentioned activity, e.g. conferring preferably the
increase of the respective fine chemical.
[4980] [0155.0.0.11] to [0156.0.0.11]: see [0155.0.0.0] to
[0156.0.0.0] [0157.0.11.11] The invention further relates to
nucleic acid molecules that differ from one of the nucleotide
sequences as indicated in Table I, columns 5 or 7, lines 109 to 111
(and portions thereof) due to degeneracy of the genetic code and
thus encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
polypeptides comprising the sequence as indicated in Table IV,
columns 5 or 7, lines 109 to 111 or as polypeptides depicted in
Table II, columns 5 or 7, lines 109 to 111 or the functional
homologues. Advantageously, the nucleic acid molecule of the
invention comprises, or in an other embodiment has, a nucleotide
sequence encoding a protein comprising, or in an other embodiment
having, an amino acid sequence of a consensus sequences as
indicated in Table IV, columns 5 or 7, lines 109 to 111 or of the
polypeptide as indicated in Table II, columns 5 or 7, lines 109 to
111, resp., or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
IV, columns 5 or 7, lines 109 to 111 or of a polypeptide as
indicated in Table II, columns 5 or 7, lines 109 to 111 or the
functional homologues. However, in a preferred embodiment, the
nucleic acid molecule of the present invention does not consist of
a sequence as indicated in Table I, columns 5 or 7, lines 109 to
111, preferably as indicated in Table I A, columns 5 or 7, lines
109 to 111. Preferably the nucleic acid molecule of the invention
is a functional homologue or identical to a nucleic acid molecule
indicated in Table I B, columns 5 or 7, lines 109 to 111.
[4981] [0158.0.0.11] to [0160.0.0.11]: see [0158.0.0.0] to
[0160.0.0.0]
[4982] [0161.0.11.11] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 109 to 111.
The nucleic acid molecule is preferably at least 20, 30, 50, 100,
250 or more nucleotides in length.
[4983] [0162.0.0.11] see [0162.0.0.0]
[4984] [0163.0.11.11] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 109 to 111
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the increase of
the amount of the respective fine chemical in a organism or a part
thereof, e.g. a tissue, a cell, or a compartment of a cell, after
increasing the expression or activity thereof or the activity of a
protein of the invention or used in the process of the
invention.
[4985] [0164.0.0.11] see [0164.0.0.0]
[4986] [0165.0.11.11] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 109 to 111, resp.,
[4987] [0166.0.0.11] to [0167.0.0.11]: see [0166.0.0.0] to
[0167.0.0.0]
[4988] [0168.0.11.11] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 109 to 111, preferably of Table II B, column 7, lines 109 to
111, resp., yet retain said activity described herein. The nucleic
acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table II, columns 5 or 7, lines 109 to 111, preferably
of Table II B, column 7, lines 109 to 111, resp., and is capable of
participation in the increase of production of the respective fine
chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table II,
columns 5 or 7, lines 109 to 111, preferably of Table II B, column
7, lines 109 to 111, resp., more preferably at least about 70%
identical to one of the sequences as indicated in Table II, columns
5 or 7, lines 109 to 111, preferably of Table II B, column 7, lines
109 to 111 resp., even more preferably at least about 80%, 90%, 95%
homologous to a sequence as indicated in Table II, columns 5 or 7,
lines 109 to 111, preferably of Table II B, column 7, lines 109 to
111, resp., and most preferably at least about 96%, 97%, 98%, or
99% identical to the sequence as indicated in Table II, columns 5
or 7, lines 109 to 111.
[4989] [0169.0.0.11] to [0172.0.0.11]: see [0169.0.0.0] to
[0172.0.0.0]
[4990] [0173.0.11.11] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 11430 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 11430 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[4991] [0174.0.0.11]: see [0174.0.0.0]
[4992] [0175.0.11.11] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 11431 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 11431 by the above program algorithm with the
above parameter set, has a 80% homology.
[4993] [0176.0.11.11] Functional equivalents derived from one of
the polypeptides as indicated in Table II, columns 5 or 7, lines
109 to 111, resp., according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 109 to 111, resp., according to the invention
and are distinguished by essentially the same properties as a
polypeptide as indicated in Table II, columns 5 or 7, lines 109 to
111, resp.
[4994] [0177.0.11.11] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 109 to
111, preferably of Table I B, column 7, lines 109 to 111, resp.,
according to the invention by substitution, insertion or deletion
have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%,
60%, 65% or 70% by preference at least 80%, especially preferably
at least 85% or 90%, 91%, 92%, 93% or 94%, very especially
preferably at least 95%, 97%, 98% or 99% homology with one of the
polypeptides as indicated in Table II, columns 5 or 7, lines 109 to
111, resp., according to the invention and encode polypeptides
having essentially the same properties as a polypeptide as
indicated in Table II, columns 5 or 7, lines 109 to 111, preferably
of Table II B, column 7, lines 109 to 111, resp.
[4995] [0178.0.0.11] see [0178.0.0.0]
[4996] [0179.0.11.11] A nucleic acid molecule encoding an
homologous to a protein sequence as indicated in Table II, columns
5 or 7, lines 109 to 111, preferably of Table II B, column 7, lines
109 to 111, resp., can be created by introducing one or more
nucleotide substitutions, additions or deletions into a nucleotide
sequence of the nucleic acid molecule of the present invention, in
particular as indicated in Table I, columns 5 or 7, lines 109 to
111, resp., such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences of a
sequences as indicated in Table I, columns 5 or 7, lines 109 to
111, resp., by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis.
[4997] [0180.0.0.11] to [0183.0.0.11]: see [0180.0.0.0] to
[0183.0.0.0]
[4998] [0184.0.11.11] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table I, columns 5 or 7,
lines 109 to 111, preferably of Table I B, column 7, lines 109 to
111, resp., or of the nucleic acid sequences derived from a
sequences as indicated in Table II, columns 5 or 7, lines 109 to
111, preferably of Table II B, column 7, lines 109 to 111, resp.,
comprise also allelic variants with at least approximately 30%,
35%, 40% or 45% homology, by preference at least approximately 50%,
60% or 70%, more preferably at least approximately 90%, 91%, 92%,
93%, 94% or 95% and even more preferably at least approximately
96%, 97%, 98%, 99% or more homology with one of the nucleotide
sequences shown or the abovementioned derived nucleic acid
sequences or their homologues, derivatives or analogues or parts of
these. Allelic variants encompass in particular functional variants
which can be obtained by deletion, insertion or substitution of
nucleotides from the sequences shown, preferably from a sequence as
indicated in Table I, columns 5 or 7, lines 109 to 111, resp., or
from the derived nucleic acid sequences, the intention being,
however, that the enzyme activity or the biological activity of the
resulting proteins synthesized is advantageously retained or
increased.
[4999] [0185.0.11.11] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table I, columns 5 or 7, lines 109 to 111, preferably of Table I B,
column 7, lines 109 to 111, resp. In one embodiment, it is
preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table I, columns 5 or 7, lines 109 to 111, preferably
of Table I B, column 7, lines 109 to 111, resp. In one embodiment,
the nucleic acid molecule comprises less than 500, 400, 300, 200,
100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a further
embodiment, the nucleic acid molecule comprises less than 30, 20 or
10 further nucleotides. In one embodiment, a nucleic acid molecule
used in the process of the invention is identical to a sequences as
indicated in Table I, columns 5 or 7, lines 109 to 111, preferably
of Table I B, column 7, lines 109 to 111, resp.
[5000] [0186.0.11.11] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 109 to 111, preferably of Table II B, column 7, lines
109 to 111, resp. In one embodiment, the nucleic acid molecule
encodes less than 150, 130, 100, 80, 60, 50, 40 or 30 further amino
acids. In a further embodiment, the encoded polypeptide comprises
less than 20, 15, 10, 9, 8, 7, 6 or 5 further amino acids. In one
embodiment, the encoded polypeptide used in the process of the
invention is identical to the sequences as indicated in Table II,
columns 5 or 7, lines 109 to 111, preferably of Table II B, column
7, lines 109 to 111, resp.
[5001] [0187.0.11.11] In one embodiment, a nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II, columns 5 or 7,
lines 109 to 111, preferably of Table II B, column 7, lines 109 to
111, resp., comprises less than 100 further nucleotides. In a
further embodiment, said nucleic acid molecule comprises less than
30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence
encoding a sequence as indicated in Table II, columns 5 or 7, lines
109 to 111, preferably of Table II B, column 7, lines 109 to 111,
resp.
[5002] [0188.0.11.11] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 109 to 111, resp., and is expressed under
identical conditions. In one embodiment, the polypeptide of the
invention is a homolog consisting of or comprising the sequence as
indicated in Table II B, columns 7, lines 109 to 111.
[5003] [0189.0.11.11] Homologues of a sequences as indicated in
Table I, columns 5 or 7, lines 109 to 111, resp., or of a derived
sequences as indicated in Table II, columns 5 or 7, lines 109 to
111, resp., also mean truncated sequences, cDNA, single-stranded
DNA or RNA of the coding and noncoding DNA sequence. Homologues of
said sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[5004] [0190.0.0.11] to [0203.0.0.11]: see [0190.0.0.0] to
[0203.0.0.0]
[5005] [0204.0.11.11] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [5006] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, columns 5 or 7, lines 109 to
111, preferably of Table II B, column 7, lines 109 to 111, resp.;
or a fragment thereof conferring an increase in the amount of the
respective fine chemical, in an organism or a part thereof; [5007]
b) nucleic acid molecule comprising, preferably at least the mature
form, of a nucleic acid molecule as indicated in Table I, columns 5
or 7, lines 109 to 111, preferably of Table I B, column 7, lines
109 to 111, resp., or a fragment thereof conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [5008] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [5009] d) nucleic
acid molecule encoding a polypeptide whose sequence has at least
50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [5010] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [5011] f) nucleic acid molecule encoding a polypeptide,
the polypeptide being derived by substituting, deleting and/or
adding one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[5012] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [5013] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table III, columns 5 or 7, lines 109 to 111 and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [5014] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [5015] j) nucleic acid molecule which encodes a
polypeptide comprising a consensus sequence as indicated in Table
IV, columns 5 or 7, lines 109 to 111 and conferring an increase in
the amount of the respective fine chemical, in an organism or a
part thereof; [5016] k) nucleic acid molecule encoding the amino
acid sequence of a polypeptide encoding a domaine of a polypeptide
as indicated in Table II, columns 5 or 7, lines 109 to 111,
preferably of Table II B, column 7, lines 109 to 111, resp., and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; and [5017] l) nucleic
acid molecule which is obtainable by screening a suitable nucleic
acid library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment of at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (h) or of a nucleic acid molecule as
indicated in Table I, columns 5 or 7, lines 109 to 111, preferably
of Table I B, column 7, lines 109 to 111, resp., or a nucleic acid
molecule encoding, preferably at least the mature form of, a
polypeptide as indicated in Table II, columns 5 or 7, lines 109 to
111, preferably of Table II B, column 7, lines 109 to 111, resp.,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which encompasses a
sequence which is complementary thereto; whereby, preferably, the
nucleic acid molecule according to (a) to (l) distinguishes over
the sequence indicated in Table IA or I B, columns 5 or 7, lines
109 to 111, by one or more nucleotides. In one embodiment, the
nucleic acid molecule does not consist of the sequence shown and
indicated in Table I A or I B, columns 5 or 7, lines 109 to 111. In
one embodiment, the nucleic acid molecule is less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence indicated in
Table I A or I B, columns 5 or 7, lines 109 to 111. In another
embodiment, the nucleic acid molecule does not encode a polypeptide
of a sequence indicated in Table II A or II B, columns 5 or 7,
lines 109 to 111. In an other embodiment, the nucleic acid molecule
of the present invention is at least 30%, 40%, 50%, or 60%
identical and less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I A or I B, columns 5 or
7, lines 109 to 111. In a further embodiment the nucleic acid
molecule does not encode a polypeptide sequence as indicated in
Table II A or II B, columns 5 or 7, lines 109 to 111. Accordingly,
in one embodiment, the nucleic acid molecule of the differs at
least in one or more residues from a nucleic acid molecule
indicated in Table I A or I B, columns 5 or 7, lines 109 to 111.
Accordingly, in one embodiment, the nucleic acid molecule of the
present invention encodes a polypeptide, which differs at least in
one or more amino acids from a polypeptide indicated in Table II A
or I B, columns 5 or 7, lines 109 to 111. In another embodiment, a
nucleic acid molecule indicated in Table I A or I B, columns 5 or
7, lines 109 to 111 does not encode a protein of a sequence
indicated in Table II A or II B, columns 5 or 7, lines 109 to 111.
Accordingly, in one embodiment, the protein encoded by a sequences
of a nucleic acid according to (a) to (l) does not consist of a
sequence as indicated in Table II A or II B, columns 5 or 7, lines
109 to 111. In a further embodiment, the protein of the present
invention is at least 30%, 40%, 50%, or 60% identical to a protein
sequence indicated in Table II A or II B, columns 5 or 7, lines 109
to 111 and less than 100%, preferably less than 99.999%, 99.99% or
99.9%, more preferably less than 99%, 98%, 97%, 96% or 95%
identical to a sequence as indicated in Table I A or II B, columns
5 or 7, lines 109 to 111.
[5018] [0205.0.0.11] to [0206.0.0.11]: see [0205.0.0.0] to
[0206.0.0.0]
[5019] [0207.0.11.11] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the e.g. the carotenoid
metabolism, e.g. the xanthophyll metabolism, e.g. the zeaxanthin
metabolism, the amino acid metabolism, of glycolysis, of the
tricarboxylic acid metabolism, oxidative stress protection or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[5020] [0208.0.0.11] to [0226.0.0.11]: see [0208.0.0.0] to
[0226.0.0.0]
[5021] [0227.0.11.11] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[5022] In addition to a sequence indicated in Table I, columns 5 or
7, lines 109 to 111 or its derivatives, it is advantageous to
express and/or mutate further genes in the organisms. It can be
especially advantageous, if additionally at least one further gene
of the lutein biosynthetic pathway, e.g. of the DOXP pathway of
isoprenoids biosynthesis, is expressed in the organisms such as
plants or microorganisms. It is also possible that the regulation
of the natural genes has been modified advantageously so that the
gene and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the amino acids desired since, for example, feedback
regulations no longer exist to the same extent or not at all. In
addition it might be advantageously to combine one or more of the
sequences indicated in Table I, columns 5 or 7, lines 109 to 111,
resp., with genes which generally support or enhances the growth or
yield of the target organisms, for example genes which lead to
faster growth rate of microorganisms or genes which produces
stress-, pathogen, or herbicide resistant plants.
[5023] [0227.1.0.11] In addition it might be also advantageously to
combine one or more of the sequences indicated in Table I, columns
5 or 7, lines 109 to 111, resp., with genes which modify plant
architecture of flower development, in the way, that the plant
either produces more flowers, or produces flowers with more sepals
in order to increase the respective fine chemical production
capacity.
[5024] [0228.0.11.11] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the carotenoids
metabolism, in particular in synthesis of zeaxanthin, e.g. as
described in Burrr B J. Carotenoids and gege expression. Nutrition
2000, 31; 16(7-8):577-8; Delagado-Vargas F, Natural pigments:
carotenoids, anthocyanins, and betalains--characteristics,
biosynthesis, processing, and stability. Crit Rev Food Sci Nutr
2000; 40(3):173-289.
[5025] [0229.0.11.11] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the carotenoids biosynthetic
pathway, such as -Lycopene cyclase, -lycopene cyclase,
beta-carotene hydroxylase, and/or -carotene hydroxylase. These
genes may lead to an increased synthesis of the essential
carotenoids, in particular lutein.
[5026] [0230.0.0.11] see [230.0.0.0]
[5027] [0231.0.11.11] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a lutein degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[5028] [0232.0.0.11] to [0276.0.0.11]: see [0232.0.0.0] to
[0276.0.0.0]
[5029] [0277.0.11.11] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously. The fine chemical and other carotenoids produced by
this process can be obtained by harvesting the organisms, either
from the crop in which they grow, or from the field. This can be
done via pressing or extraction of the plant parts.
[5030] [0278.0.0.11] to [0282.0.0.11]: see [0278.0.0.0] to
[0282.0.0.0]
[5031] [0283.0.11.11] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table II,
column 3, lines 109 to 111, resp., or, in an other embodiment, with
an antibody against a polypeptide as indicated in
[5032] Table II, columns 5 or 7, lines 109 to 111, resp., which can
be produced by standard techniques utilizing the polypeptid of the
present invention or fragment thereof, i.e., the polypeptide of
this invention. Preferred are monoclonal antibodies.
[5033] [0284.0.0.11] see [0284.0.0.0]
[5034] [0285.0.11.11] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 109 to 111, resp., or as encoded by a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 109 to 111, resp., or functional homologues thereof.
[5035] [0286.0.11.11] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, columns 7, lines 109 to 111 and in one another
embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table IV, columns 7, lines 109 to 111 whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid.
[5036] [0287.0.0.11] to [0290.0.0.11]: see [0287.0.0.0] to
[0290.0.0.0]
[5037] [0291.0.11.11] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[5038] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 109 to 111, resp., by one or more amino
acids. In one embodiment, polypeptide distinguishes from a sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 109 to 111,
resp., by more than 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, even more preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 109 to 111,
resp., by not more than 80% or 70% of the amino acids, preferably
not more than 60% or 50%, more preferred not more than 40% or 30%,
even more preferred not more than 20% or 10%. In an other
embodiment, said polypeptide of the invention does not consist of a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
109 to 111.
[5039] [0292.0.0.11] see [0292.0.0.0]
[5040] [0293.0.11.11] In one embodiment, the invention relates to
polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or by a nucleic acid molecule used in the process of the
invention. In one embodiment, the polypeptide of the invention or
used in the process of the invention has a sequence which
distinguishes from a sequence as indicated in Table II A or II B,
columns 5 or 7, lines 109 to 111, resp., by one or more amino
acids. In an other embodiment, said polypeptide of the invention or
used in the process of the invention does not consist of the
sequence as indicated in Table II A or II B, columns 5 or 7, lines
109 to 111, resp., In a further embodiment, said polypeptide of the
present invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by a nucleic acid molecules as indicated in
Table I A or I B, columns 5 or 7, lines 109 to 111, resp.
[5041] [0294.0.11.11] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 109 to 111, resp., which
distinguishes over a sequence as indicated in Table II A or II B,
columns 5 or 7, lines 109 to 111, resp., by one or more amino
acids, preferably by more than 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, even more
preferred are more than 40, 50, or 60 amino acids but even more
preferred by less than 70% of the amino acids, more preferred by
less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[5042] [0295.0.0.11] to [0297.0.0.11]: see [0295.0.0.0] to
[0297.0.0.0]
[5043] [00297.1.11.11] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table II, columns 3, 5 or 7, lines 109 to 111,
resp.
[5044] [0298.0.11.11] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table II, columns 5 or 7, lines 109 to 111, resp.
The portion of the protein is preferably a biologically active
portion as described herein. Preferably, the polypeptide used in
the process of the invention has an amino acid sequence identical
to a sequence as indicated in Table II, columns 5 or 7, lines 109
to 111, resp.
[5045] [0299.0.11.11] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
109 to 111, resp., The preferred polypeptide of the present
invention preferably possesses at least one of the activities
according to the invention and described herein. A preferred
polypeptide of the present invention includes an amino acid
sequence encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions, to a nucleotide
sequence as indicated in Table I, columns 5 or 7, lines 109 to 111,
resp., or which is homologous thereto, as defined above.
[5046] [0300.0.11.11] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 109 to 111, resp., in amino acid sequence due
to natural variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprise an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence of a sequence as indicated in Table II A or II
B, columns 5 or 7, lines 109 to 111, resp.
[5047] [0301.0.0.11] see [0301.0.0.0]
[5048] [0302.0.11.11] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table II, columns 5 or 7, lines 109 to 111, resp., or the amino
acid sequence of a protein homologous thereto, which include fewer
amino acids than a full length polypeptide of the present invention
or used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[5049] [0303.0.0.11] see [0303.0.0.0]
[5050] [0304.0.11.11] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 109 to 111 but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[5051] [0305.0.0.11] to [0308.0.0.11]: see [0305.0.0.0] to
[0308.0.0.0]
[5052] [0309.0.11.11] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table II,
columns 5 or 7, lines 109 to 111, resp., refers to a polypeptide
having an amino acid sequence corresponding to the polypeptide of
the invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table II, columns 5 or 7, lines 109 to 111, resp.,
refers to a polypeptide having an amino acid sequence corresponding
to a protein which is not substantially homologous a polypeptide of
the invention, preferably which is not substantially homologous to
a polypeptide as indicated in Table II, columns 5 or 7, lines 109
to 111, resp., e.g., a protein which does not confer the activity
described herein or annotated or known for as indicated in Table
II, column 3, lines 109 to 111, resp., and which is derived from
the same or a different organism. In one embodiment, a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table II, columns 5 or 7, lines 109 to 111, resp.,
does not confer an increase of the respective fine chemical in an
organism or part thereof.
[5053] [0310.0.0.11] to [0334.0.0.11]: see [0310.0.0.0] to
[0334.0.0.0]
[5054] [0335.0.11.11] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table I, columns 5 or
7, lines 109 to 111, resp., and/or homologs thereof. As described
inter alia in WO 99/32619, dsRNAi approaches are clearly superior
to traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived there from),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table I,
columns 5 or 7, lines 109 to 111, resp., and/or homologs thereof.
In a double-stranded RNA molecule for reducing the expression of an
protein encoded by a nucleic acid sequence sequences as indicated
in Table I, columns 5 or 7, lines 109 to 111, resp., and/or
homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[5055] [0336.0.0.11] to [0342.0.0.11]: see [0336.0.0.0] to
[0342.0.0.0]
[5056] [0343.0.11.11] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 109 to 111,
resp., or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table I, columns 5 or 7, lines 109 to 111, resp., or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[5057] [0344.0.0.11] to [0361.0.0.11]: see [0344.0.0.0] to
[0361.0.0.0]
[5058] [0362.0.11.11] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, columns 5 or 7, lines 109 to 111, resp., e.g. encoding a
polypeptide having protein activity, as indicated in Table II,
columns 3, lines 109 to 111, resp., Due to the above mentioned
activity the respective fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. Transgenic for a polypeptide having an activity of a
polypeptide as indicated in Table II, columns 5 or 7, lines 109 to
111, resp., means herein that due to modulation or manipulation of
the genome, an activity as annotated for a polypeptide as indicated
in Table II, column 3, lines 109 to 111, e.g. having a sequence as
indicated in Table II, columns 5 or 7, lines 109 to 111, resp., is
increased in a cell or an organism or a part thereof. Examples are
described above in context with the process of the invention
[5059] [0363.0.0.11] to [0373.0.0.11]: see [0363.0.0.0] to
[0373.0.0.0]
[5060] [0374.0.11.11] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. Carotenoids, in particular
the respective fine chemical, produced in the process according to
the invention may, however, also be isolated from the plant in the
form of their free carotenoids, in particular the free respective
fine chemical, or bound in or to compounds or moieties, e.g. to
mono- or diester of fatty acids, triglycerides or associated with
lipoproteins or lipids. The respective fine chemical produced by
this process can be harvested by harvesting the organisms either
from the culture in which they grow or from the field. This can be
done via expressing, grinding and/or extraction, salt precipitation
and/or ion-exchange chromatography or other chromatographic methods
of the plant parts, preferably the plant seeds, plant fruits, plant
tubers and the like.
[5061] [0375.0.0.11] to [0376.0.0.11]: see [0375.0.0.0] to
[0376.0.0.0]
[5062] [0377.0.11.11] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced carotenoids, in particular lutein, comprising composition
is isolated. In one embodiment, the produced respective fine
chemical, preferably free lutein, is isolated.
[5063] [0378.0.11.11] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the respective
fine chemical produced in the process can be isolated. The
resulting lutein comprising composition can, if appropriate,
subsequently be further purified, if desired mixed with other
active ingredients such as vitamins, amino acids, carbohydrates,
antibiotics and the like, and, if appropriate, formulated.
[5064] [0379.0.11.11] In one embodiment, the isolated carotenoids
are a mixture comprising the respective fine chemical. In one
embodiment, the carotenoids are a mixture of the respective fine
chemical with other carotenoids, e.g. zeaxanthin.
[5065] [0380.0.11.11] The respective fine chemical obtained in the
process are suitable as starting material for the synthesis of
further products of value. For example, they can be used in
combination with each other or alone for the production of
pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the carotenoids containing,
in particular lutein containing composition produced or the
respective fine chemical produced if desired and formulating the
product with a pharmaceutical or cosmetically acceptable carrier or
formulating the product in a form acceptable for an application in
agriculture. A further embodiment according to the invention is the
use of the carotenoids, in particular of lutein, produced in the
process or of the transgenic organisms in animal feeds, foodstuffs,
medicines, food supplements, cosmetics or pharmaceuticals.
[5066] [0381.0.0.11] to [0382.0.0.11]: see [0381.0.0.0] to
[0382.0.0.0]
[5067] [0383.0.11.11] ./.
[5068] [0384.0.0.11] see [0384.0.0.0]
[5069] [0385.0.11.11] The fermentation broths obtained in this way,
containing in particular lutein in mixtures with other carotenoids,
in particular with other xanthophylls, e.g. with zeaxanthin, or
containing microorganisms or parts of microorganisms, like plastids
or cytoplasm, containing lutein in mixtures with other carotenoids,
in particular with other xanthophylls, e.g. with zeaxanthin,
normally have a dry matter content of from 1 to 70% by weight,
preferably 7.5 to 25% by weight. Sugar-limited fermentation is
additionally advantageous, e.g. at the end, for example over at
least 30% of the fermentation time. This means that the
concentration of utilizable sugar in the fermentation medium is
kept at, or reduced to, 0 to 10 g/l, preferably to 0 to 3 g/l
during this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed or isolated
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation, coagulation/flocculation
or a combination of these methods, from the fermentation broth or
left completely in it.
[5070] The fermentation broth can then be thickened or concentrated
by known methods, such as, for example, with the aid of a rotary
evaporator, thin-film evaporator, falling film evaporator, by
reverse osmosis or by nanofiltration. This concentrated
fermentation broth can then be worked up by freeze-drying, spray
drying, spray granulation or by other processes.
[5071] As carotenoids are synthesized in the plastids and are often
localized in membranes or plastids, in one embodiment it is
advantageous to avoid a leaching of the cells when the biomass is
isolated entirely or partly by separation methods, such as, for
example, centrifugation, filtration, decantation,
coagulation/flocculation or a combination of these methods, from
the fermentation broth. The dry biomass can directly be added to
animal feed, provided the carotenoids concentration is sufficiently
high and no toxic compounds are present. In view of the instability
of carotenoids, conditions for drying, e.g. spray or flash-drying,
can be mild and can be avoiding oxidation and cis/trans
isomerization. For example antioxidants, e.g. BHT, ethoxyquin or
other, can be added. In case the carotenoids concentration in the
biomass is to dilute, solvent extraction can be used for their
isolation, e.g. with alcohols, ether or other organic solvents,
e.g. with methanol, ethanol, aceton, alcoholic potassium hydroxide,
glycerol-phenol, liquefied phenol or for example with acids or
bases, like trichloroacetatic acid or potassium hydroxide. A wide
range of advantageous methods and techniques for the isolation of
carotenoids, in particular of lutein, can be found in the state of
the art. In case phenol is used it can for example be removed with
ether and water extraction and the dry eluate comprises a mixture
of the carotenoids of the biomass. Extraction of carotenoids, in
particular lutein, are described inter alia in U.S. Pat. No.
6,387,370, U.S. Pat. No. 6,380,442, U.S. Pat. No. 6,329,557,U.S.
Pat. No. 6,262,284 and literature cited therein.
[5072] [0386.0.11.11] Accordingly, it is possible to purify the
carotenoids, in particular lutein produced according to the
invention further. For this purpose, the product-containing
composition, e.g. a total or partial lipid extraction fraction
using organic solvents, e.g. as described above, is subjected for
example to a saponification to remove triglycerides, partition
between e.g. hexane/methanol (separation of non-polar epiphase from
more polar hypophasic derivates) and separation via e.g. an open
column chromatography or HPLC in which case the desired product or
the impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use.
[5073] [0387.0.0.11] to [0392.0.0.11]: see [0387.0.0.0] to
[0392.0.0.0]
[5074] [0393.0.11.11] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps:
(a) contacting e.g. hybridising, the nucleic acid molecules of a
sample, e.g. cells, tissues, plants or microorganisms or a nucleic
acid library, which can contain a candidate gene encoding a gene
product conferring an increase in the respective fine chemical
after expression, with the nucleic acid molecule of the present
invention; (b) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table I, columns 5 or 7, lines
109 to 111, preferably in Table I B, columns 5 or 7, lines 109 to
111, resp., and, optionally, isolating the full length cDNA clone
or complete genomic clone; (c) introducing the candidate nucleic
acid molecules in host cells, preferably in a plant cell or a
microorganism, appropriate for producing the respective fine
chemical; (d) expressing the identified nucleic acid molecules in
the host cells; (e) assaying the respective fine chemical level in
the host cells; and (f) identifying the nucleic acid molecule and
its gene product which expression confers an increase in the
respective fine chemical level in the host cell after expression
compared to the wild type.
[5075] [0394.0.0.11] to [0398.0.0.11]: see [0394.0.0.0] to
[0398.0.0.0]
[5076] [0399.0.11.11] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increased the respective fine chemical production in a
plant or microorganism, comprising the steps:
(a) culturing a cell or tissue or microorganism or maintaining a
plant expressing the polypeptide according to the invention or a
nucleic acid molecule encoding said polypeptide and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and the polypeptide of the present invention or used
in the process of the invention; and (b) identifying if the
compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout
system.
[5077] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in respective fine chemical
production.
[5078] [00399.1.11.11] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 109 to 111 or a homolog thereof, e.g.
comparing the phenotype of nearly identical organisms with low and
high activity of a protein as indicated in Table II, columns 5 or
7, lines 109 to 111 after incubation with the drug.
[5079] [0400.0.0.11] to [0416.0.0.11]: see [0400.0.0.0] to
[0416.0.0.0]
[5080] [0417.0.11.11] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the lutein production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect an organism such as a
microorganism or a plant against inhibitors, which block the
carotenoid synthesis, in particular the respective fine chemical
synthesis in said organism. Examples of inhibitors or herbicides
blocking the synthesis of carotenoids in organism such as
microorganism or plants are for example classified in two groups.
The first group consists of inhibitors that cause the accumulation
of early intermediates in the pathway, particularly the colorless
phytene, e.g. diphenylamine. Other inhibitors preferentially block
late reactions in the pathway, notably the cyclization of lycopene.
Inhibitors are e.g. nicotine, 2-(4-chlorophenylthio)-triethylamine
and other substituted amines as well as nitrogenous heterocyclic
bases, e.g. imidazole.
[5081] As lutein can protect organisms against damages of oxidative
stress, especially singlet oxygens, a increased level of the
respective fine chemical can protect plants against herbicides
which cause the toxic build-up of oxidative compounds, e.g. singlet
oxygen. For example, inhibition of the protoporphorineogen oxidase
(Protox), an enzyme important in the synthesis of chlorophyll and
heme biosynthesis results in the loss of chlorophyll and
carotenoids and in leaky membranes; the membrane destruction is due
to creation of free oxygen radicals (which is also reported for
other classic photosynthetic inhibitor herbicides).
[5082] Accordingly, in one embodiment, the increase of the level of
the respective fine chemical is used to protect plants against
herbicides destroying membranes due to the creation of free oxygen
radicals.
[5083] Examples of inhibitors or herbicides building up oxidative
stress are aryl triazion, e.g. sulfentrazone, carfentrazone, or
diphenylethers, e.g. acifluorfen, lactofen, or oxyfluorfen, or
N-Phenylphthalimide, e.g. flumiclorac or flumioxazin, substituted
ureas, e.g. fluometuron, tebuthiuron, or diuron, linuron, or
triazines, e.g. atrazine, prometryn, ametryn, metributzin,
prometon, simazine, or hexazinone, or uracils, e.g. bromacil or
terbacil.
[5084] Carotenoid inhibitors are e.g. Pyridines and Pyridazinones,
e.g. norflurazon, fluridone or dithiopyr. Thus, in one embodiment,
the present invention relates to the use of an increase of the
respective fine chemical according to the present invention for the
protection of plants against carotenoids inhibitors as pyridines
and pyridazinones.
[5085] [0418.0.0.11] to [0423.0.0.11]: see [0418.0.0.0] to
[0423.0.0.0]
[5086] [0424.0.11.11] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other carotenoids, in
particular other xanthophylls, e.g. zeaxanthin.
[5087] Accordingly, the nucleic acid of the invention, or the
nucleic acid molecule identified with the method of the present
invention or the complement sequences thereof, the polypeptide of
the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the respective fine chemical in a organism or part
thereof, e.g. in a cell.
[5088] [0424.1.11.11] In a further embodiment the present invention
relates to the use of the the plant of the present invention or a
part thereof, the microorganism or the host cell of the present
invention or a part thereof for the production a cosmetic
composition or a pharmaceutical composition. Such a composition has
antioxidative activity, photoprotective activity, tanning activity,
can be used for the treating of high levels of cholesterol and/or
lipids, can be used to protect, treat or heal the above mentioned
diseases, e.g. retinal disorders, hyperholsterolemia,
hyperlipidemia, arteriosclerosis, Acquired Immunodeficiency
Syndrome (AIDS), Age-related macular degeneration,
[5089] Angina pectoris, Asthma, Cataracts, Cervical cancer,
Cervical dysplasia, Chlamydial infection, Heart disease, Laryngeal
cancer (cancer of the larynx), Lung cancer, Male and female
infertility, Osteoarthritis, Photosensitivity, Pneumonia, Prostate
cancer, Rheumatoid arthritis, Skin cancer, Vaginal candidiasis or
can be used for the cleaning, conditioning, and/or treating of the
skin, e.g. if combined with a pharmaceutically or cosmetically
acceptable carrier.
[5090] The xanthophylls can be also used as stabilizer of other
colours or oxygen sensitive compounds.
[5091] [0425.0.0.11] to [0435.0.0.11]: see [0425.0.0.0] to
[0435.0.0.0]
[5092] [0436.0.11.11] An in vivo mutagenesis of organisms such as
green algae (e.g. Spongiococcum sp, e.g. Spongiococcum exentricum,
Chlorella sp.), Saccharomyces, Mortierella, Escherichia and others
mentioned above, which are beneficial for the production of lutein
can be carried out by passing a plasmid DNA (or another vector DNA)
containing the desired nucleic acid sequence or nucleic acid
sequences, e.g. the nucleic acid molecule of the invention or the
vector of the invention, through E. coli and other microorganisms
(for example Bacillus spp. or yeasts such as Saccharomyces
cerevisiae) which are not capable of maintaining the integrity of
its genetic information. Usual mutator strains have mutations in
the genes for the DNA repair system [for example mutHLS, mutD, mutT
and the like; for comparison, see Rupp, W. D. (1996) DNA repair
mechanisms in Escherichia coli and Salmonella, pp. 2277-2294, ASM:
Washington]. The skilled worker knows these strains. The use of
these strains is illustrated for example in Greener, A. and
Callahan, M. (1994) Strategies 7; 32-34. In-vitro mutation methods
such as increasing the spontaneous mutation rates by chemical or
physical treatment are well known to the skilled person. Mutagens
like 5-bromo-uracil, N-methyl-N-nitro-N-nitrosoguanidine (=NTG),
ethyl methanesulfonate (=EMS), hydroxylamine and/or nitrous acid
are widly used as chemical agents for random in-vitro mutagensis.
The most common physical method for mutagensis is the treatment
with UV irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[5093] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[5094] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.11.11] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpina
[5095] [0438.0.11.11] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker.
[5096] An easy method to isolate such shuttle vectors is disclosed
by Soni R. and Murray J. A. H. [Nucleic Acid Research, vol. 20 no.
21, 1992: 5852]: If necessary such shuttle vectors can be
constructed easily using standard vectors for E. coli (Sambrook, J.
et al., (1989), "Molecular Cloning: A Laboratory Manual", Cold
Spring Harbor Laboratory Press or Ausubel, F. M. et al. (1994)
"Current Protocols in Molecular Biology", John Wiley & Sons)
and/or the aforementioned vectors, which have a replication origin
for, and suitable marker from, Escherichia coli, Saccharomyces
cerevisiae or Mortierella alpina added. Such replication origins
are preferably taken from endogenous plasmids, which have been
isolated from species used in the inventive process. Genes, which
are used in particular as transformation markers for these species
are genes for kanamycin resistance (such as those which originate
from the Tn5 or Tn-903 transposon) or for chloramphenicol
resistance (Winnacker, E. L. (1987) "From Genes to
Clones--Introduction to Gene Technology, VCH, Weinheim) or for
other antibiotic resistance genes such as for G418, gentamycin,
neomycin, hygromycin or tetracycline resistance.
[5097] [0439.0.11.11] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255f) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[5098] [0440.0.11.11] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002,) teaches a homolog recombination system dependent
on rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this
system for homologous recombination are vectors derived from the
Ylp series. Plasmid vectors derived for example from the
2.mu.-Vector are known by the skilled worker and used for the
expression in yeasts. Other preferred vectors are for example
pART1, pCHY21 or pEVP11 as they have been described by McLeod et
al. (EMBO J. 1987, 6:729-736) and Hoffman et al. (Genes Dev. 5,
1991: :561-571.) or Russell et al. (J. Biol. Chem. 258, 1983:
143-149.). Other beneficial yeast vectors are plasmids of the REP,
REP-X, pYZ or RIP series.
[5099] [0441.0.0.11] to [0443.0.0.11]: see [0441.0.0.0] to
[0443.0.0.0]
[0444.0.11.11] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[5100] [0445.0.11.11] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[5101] [0446.0.0.11] to [0454.0.0.11]: see [0446.0.0.0] to
[0454.0.0.0]
[5102] [0455.0.11.11] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a fatty acid) can be determined by growing the
modified microorganisms or the modified plant under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the elevated production of
desired product (i.e. of the lipids or a fatty acid). These
analytical techniques are known to the skilled worker and comprise
spectroscopy, thin-layer chromatography, various types of staining
methods, enzymatic and microbiological methods and analytical
chromatography such as high-performance liquid chromatography (see,
for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2,
p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.,
(1987) "Applications of HPLC in Biochemistry" in: Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et
al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery
and purification", p. 469-714, VCH: Weinheim; Better, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[5103] In addition to the abovementioned processes, plant lipids
are extracted from plant material as described by Cahoon et al.
(1999) Proc. Natl. Acad. Sci. USA 96 (22): 12935-12940 and Browse
et al. (1986) Analytic Biochemistry 152:141-145. The qualitative
and quantitative analysis of lipids is described by Christie,
William W., Advances in Lipid Methodology, Ayr/Scotland: Oily Press
(Oily Press Lipid Library; 2); Christie, William W., Gas
Chromatography and Lipids. A Practical Guide--Ayr, Scotland: Oily
Press, 1989, Repr. 1992, IX, 307 pp. (Oily Press Lipid Library; 1);
"Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952)--16
(1977) under the title: Progress in the Chemistry of Fats and Other
Lipids CODEN.
[5104] [0456.0.0.11]: see [0456.0.0.0]
[0457.0.11.11] Example 9
Purification of the Lutein and Determination of the Carotenoids
Content
[5105] [0458.0.11.11] Abbreviations: GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[5106] The unambiguous detection for the presence of lutein can be
obtained by analyzing recombinant organisms using analytical
standard methods: LC, LC-MSMS or TLC, as described The total lutein
produced in the organism for example in yeasts used in the
inventive process can be analysed for example according to the
following procedure:
[5107] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods.
[5108] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[5109] A typical sample pretreatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography.
[5110] For analysis, solvent delivery and aliquot removal can be
accomplished with a robotic system comprising a single injector
valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000
W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of
50% potassium hydroxide hydro-ethanolic solution (4 water:1
ethanol) can be added to each vial, followed by the addition of 3
ml of octanol. The saponification treatment can be conducted at
room temperature with vials maintained on an IKA HS 501 horizontal
shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours
at 250 movements/minute, followed by a stationary phase of
approximately one hour.
[5111] Following saponification, the supernatant can be diluted
with 0.11 ml of methanol. The addition of methanol cqan be
conducted under pressure to ensure sample homogeneity. Using a 0.25
ml syringe, a 0.1 ml aliquot can be removed and transferred to HPLC
vials for analysis.
[5112] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete
with a quaternary pump, vacuum degassing system, six-way injection
valve, temperature regulated autosampler, column oven and
Photodiode Array detector can be used [Agilent Technologies
available through Ultra Scientific Inc., 250 Smith Street, North
Kingstown, R.I.]. The column can be a Waters YMC30, 5-micron,
4.6.times.250 mm with a guard column of the same material [Waters,
34 Maple Street, Milford, Mass.]. The solvents for the mobile phase
can be 81 methanol: 4 water: 15 tetrahydrofuran (THF) stabilized
with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injections were
20 l. Separation can be isocratic at 30.degree. C. with a flow rate
of 1.7 ml/minute. The peak responses can be measured by absorbance
at 447 nm.
[5113] Carotenoid compositions can be determined for wild-type and
mutant samples selected from those identified in a screening
procedure. Petal samples can be stored in a -80.degree. C. freezer
until mutants were identified. Samples can be lyophilized, and the
dried tissue can be stored under argon at -80.degree. C. until
ready for analysis.
[5114] Extraction procedures can be performed under red light.
Dried petals can be ground to pass through a No. 40 sieve mesh
size. A ground sample can be accurately weighed and transferred
into a 100 ml red volumetric flask. To the sample, 500 microliters
I) of H.sub.2O can be added, and the mixture can be swirled for 1
minute. Thirty ml of extractant solvent (10 ml hexane+7 ml
acetone+6 ml absolute alcohol+7 ml toluene) can be added, and the
flask can be shaken at 160 rpm for 10 minutes.
[5115] For saponification, 2 ml of 40% methanolic KOH can be added
into the flask, which can be then swirled for one minute. The flask
can be placed in a 56.degree. C. H.sub.2O bath for 20 minutes. An
air condenser can be attached to prevent loss of solvent. The
sample can be cooled in the dark for one hour with the condenser
attached. After cooling, 30 ml of hexane can be added, and the
flask can be shaken at 160 rpm for 10 minutes.
[5116] The shaken sample can be diluted to volume (100 ml) with 10%
sodium sulfate solution and shaken vigorously for one minute. The
sample can be remained in the dark for at least 30 minutes. A 35 ml
aliquot can be removed from the approximately 50 ml upper phase,
and transferred to a sample cup. An additional 30 ml of hexane can
be added into the flask that can be then shaken at 160 rpm for 10
minutes. After approximately one hour, the upper phases can be
combined. For HPLC analysis, 10 ml aliquots can be dried under
nitrogen and stored under argon at -80.degree. C.
[5117] HPLC equipment comprised an Alliance 2690 equipped with a
refrigerated autosampler, column heater and a Waters Photodiode
Array 996 detector (Waters Corp., 34 Maple Street Milford, Mass.
01757). Separation can be obtained with a YMC30 column, 3 m,
2.0.times.150 mm with a guard column of the same material.
Standards can be obtained from ICC Indorespective fine chemicals
Somerville, N.J. 088876 and from DHI-Water & Environment,
DK-2970 Horsholm, Denmark.
[5118] The dried mutant samples can be resuspended in
tetrahydrofuran and methanol to a total volume of 200 l and
filtered, whereas the control can be not additionally concentrated.
Carotenoids can be separated using a gradient method. Initial
gradient conditions can be 90% methanol: 5% water: 5% methyl
tert-butyl ether at a flow rate of 0.4 milliliters per minute
(ml/min). From zero to 15 minutes, the mobile phase can be changed
from the initial conditions to 80 methanol: 5 water: 15 methyl
tert-butyl ether, and from 15 to 60 minutes to 20 methanol: 5
water: 75 methyl tert-butyl ether. For the following 10 minutes,
the mobile phase can be returned to the initial conditions and the
column equilibrated for an additional 10 minutes. The column
temperature can be maintained at 27.degree. C. and the flow rate
was 0.4 ml/minute. Injections were 10 The majority of peak
responses can be measured at 450 nm and additional areas added from
286, 348, 400 and 472 nm extracted channels.
[5119] Methods for the extraction are further described in the
following patent applications and in the literature cited therein:
U.S. Pat. No. 5,854,015; WO 99/20587, U.S. Pat. No. 6,380,442, U.S.
Pat. Nos. 6,362,221, 6,329,557, 6,262,284,
[5120] [0459.0.11.11] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the lutein can be further purified with a so-called
RTHPLC. As eluent acetonitrile/water or chloroform/acetonitrile
mixtures can be used.
[5121] If necessary, these chromatography steps may be repeated,
using identical or other chromatography resins. The skilled worker
is familiar with the selection of suitable chromatography resin and
the most effective use for a particular molecule to be
purified.
[5122] [0460.0.11.11] see [0460.0.0.0]
[0461.0.11.11] Example 10
Cloning SEQ ID NO: 11430 for the Expression in Plants
[5123] [0462.0.0.11] see [0462.0.0.0]
[5124] [0463.0.11.11] SEQ ID NO: 11430 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[5125] [0464.0.0.11] to [0466.0.0.11]: see [0464.0.0.0] to
[0466.0.0.0]
[5126] [0466.1.11.11] In case the Herculase enzyme can be used for
the amplification, the PCR amplification cycles were as follows: 1
cycle of 2-3 minutes at 94.degree. C., followed by 25-30 cycles of
in each case 30 seconds at 94.degree. C., 30 seconds at
55-60.degree. C. and 5-10 minutes at 72.degree. C., followed by 1
cycle of 10 minutes at 72.degree. C., then 4.degree. C.
[5127] [0467.0.11.11] The following primer sequences were selected
for the gene SEQ ID NO: 11430:
TABLE-US-00039 i) forward primer (SEQ ID NO: 11760) atgcagaccc
cgcacattct t ii) reverse primer (SEQ ID NO: 11761) ttaatcttcc
agatcaccgc agaa
[5128] [0468.0.0.11] to [0479.0.0.11]: see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.11.11] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 11430
[5129] [0481.0.0.11] to [0513.0.0.11]: see [0481.0.0.0] to
[0513.0.0.0]
[5130] [0514.0.11.11] As an alternative, lutein can be detected as
described in Deli, J. & Molnar, P. Paprika carotenoids:
Analysis, isolation, structure eucidation. Curr. Org. Chem. 6,
1197-1219 (2004) or Fraser, P. D., Pinto, M. E., Holloway, D. E.
& Bramley, P. M. Technical advance: application of
high-performance liquid chromatography with photodiode array
detection to the metabolic profiling of plant isoprenoids. Plant J.
24, 551-558 (2000).
[5131] The results of the different plant analyses can be seen from
the table 1 which follows:
TABLE-US-00040 TABLE 1 ORF Metabolite Method Min Max b4401 Lutein
LC 1,25 1,42 b2699 Lutein LC 1,31 1,58 YFR007W Lutein LC 1,33
1,78
[5132] [0515.0.0.11] to [0552.0.0.11]: see [0515.0.0.0] to
[0552.0.0.0]
[5133] [0553.0.11.11] We claim: [5134] 1. A process for the
production of lutein, which comprises [5135] (a) increasing or
generating the activity of a protein as indicated in Table II,
columns 5 or 7, lines 109 to 111 or a functional equivalent thereof
in a non-human organism, or in one or more parts thereof; and
[5136] (b) growing the organism under conditions which permit the
production of lutein in said organism. [5137] 2. A process for the
production of lutein, comprising the increasing or generating in an
organism or a part thereof the expression of at least one nucleic
acid molecule comprising a nucleic acid molecule selected from the
group consisting of: [5138] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 109 to
111 or a fragment thereof, which confers an increase in the amount
of lutein in an organism or a part thereof; [5139] b) nucleic acid
molecule comprising of a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 109 to 111; [5140] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of lutein in an organism or a part thereof; [5141] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of lutein in an organism or a part
thereof; [5142] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of lutein in an organism or a part thereof; [5143] f) nucleic acid
molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table III, columns 5 or 7, lines 109 to 111 and conferring an
increase in the amount of lutein in an organism or a part thereof;
[5144] g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of lutein in an
organism or a part thereof; [5145] h) nucleic acid molecule
encoding a polypeptide comprising a consensus as indicated in Table
IV, columns 5 or 7, lines 109 to 111 and conferring an increase in
the amount of lutein in an organism or a part thereof; and [5146]
i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of lutein in an
organism or a part thereof. [5147] or comprising a sequence which
is complementary thereto. [5148] 3. The process of claim 1 or 2,
comprising recovering of the free or bound lutein. [5149] 4. The
process of any one of claims 1 to 3, comprising the following
steps: [5150] (a) selecting an organism or a part thereof
expressing a polypeptide encoded by the nucleic acid molecule
characterized in claim 2; [5151] (b) mutagenizing the selected
organism or the part thereof; [5152] (c) comparing the activity or
the expression level of said polypeptide in the mutagenized
organism or the part thereof with the activity or the expression of
said polypeptide of the selected organisms or the part thereof;
[5153] (d) selecting the mutated organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
[5154] (e) optionally, growing and cultivating the organisms or the
parts thereof; and [5155] (f) recovering, and optionally isolating,
the free or bound lutein produced by the selected mutated organisms
or parts thereof. [5156] 5. The process of any one of claims 1 to
4, wherein the activity of said protein or the expression of said
nucleic acid molecule is increased or generated transiently or
stably. [5157] 6. An isolated nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [5158]
a) nucleic acid molecule encoding of a polypeptide as indicated in
Table II, columns 5 or 7, lines 109 to 111 or a fragment thereof,
which confers an increase in the amount of lutein in an organism or
a part thereof; [5159] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 109 to 111; [5160] c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as a result of the degeneracy of the
genetic code and conferring an increase in the amount of lutein in
an organism or a part thereof; [5161] d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of lutein in an organism or a part thereof; [5162] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under under stringent hybridisation conditions and
conferring an increase in the amount of lutein in an organism or a
part thereof; [5163] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, columns 5 or 7,
lines 109 to 111 and conferring an increase in the amount of lutein
in an organism or a part thereof; [5164] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
lutein in an organism or a part thereof; [5165] h) nucleic acid
molecule encoding a polypeptide comprising a consensus as indicated
in Table IV, columns 5 or 7, lines 109 to 111 and conferring an
increase in the amount of lutein in an organism or a part thereof;
and [5166] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of lutein in an organism or a part thereof. [5167] whereby
the nucleic acid molecule distinguishes over the sequence as
indicated in Table I A, columns 5 or 7, lines 109 to 111 by one or
more nucleotides. [5168] 7. A nucleic acid construct which confers
the expression of the nucleic acid molecule of claim 6, comprising
one or more regulatory elements. [5169] 8. A vector comprising the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7. [5170] 9. The vector as claimed in claim 8,
wherein the nucleic acid molecule is in operable linkage with
regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic, host. [5171] 10. A
host cell, which has been transformed stably or transiently with
the vector as claimed in claim 8 or 9 or the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 2 to 5. [5172] 11.
The host cell of claim 10, which is a transgenic host cell. [5173]
12. The host cell of claim 10 or 11, which is a plant cell, an
animal cell, a microorganism, or a yeast cell, a fungus cell, a
prokaryotic cell, an eukaryotic cell or an archaebacterium. [5174]
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. [5175] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table II A, columns 5 or 7, lines 109 to 111 by one or
more amino acids [5176] 15. An antibody, which binds specifically
to the polypeptide as claimed in claim 14. [5177] 16. A plant
tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [5178] 17. A method for screening for agonists
and antagonists of the activity of a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of lutein in an organism or a part thereof comprising:
[5179] (a) contacting cells, tissues, plants or microorganisms
which express the a polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of lutein
in an organism or a part thereof with a candidate compound or a
sample comprising a plurality of compounds under conditions which
permit the expression the polypeptide; [5180] (b) assaying the
lutein level or the polypeptide expression level in the cell,
tissue, plant or microorganism or the media the cell, tissue, plant
or microorganisms is cultured or maintained in; and [5181] (c)
identifying a agonist or antagonist by comparing the measured
lutein level or polypeptide expression level with a standard lutein
or polypeptide expression level measured in the absence of said
candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. [5182] 18. A process for the
identification of a compound conferring increased lutein production
in a plant or microorganism, comprising the steps: [5183] (a)
culturing a plant cell or tissue or microorganism or maintaining a
plant expressing the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of lutein
in an organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of lutein in an organism or a
part thereof; [5184] (b) identifying if the compound is an
effective agonist by detecting the presence or absence or increase
of a signal produced by said readout system. [5185] 19. A method
for the identification of a gene product conferring an increase in
lutein production in a cell, comprising the following steps: [5186]
(a) contacting the nucleic acid molecules of a sample, which can
contain a candidate gene encoding a gene product conferring an
increase in lutein after expression with the nucleic acid molecule
of claim 6; [5187] (b) identifying the nucleic acid molecules,
which hybridise under relaxed stringent conditions with the nucleic
acid molecule of claim 6; [5188] (c) introducing the candidate
nucleic acid molecules in host cells appropriate for producing
lutein; [5189] (d) expressing the identified nucleic acid molecules
in the host cells; [5190] (e) assaying the lutein level in the host
cells; and [5191] (f) identifying nucleic acid molecule and its
gene product which expression confers an increase in the lutein
level in the host cell in the host cell after expression compared
to the wild type. [5192] 20. A method for the identification of a
gene product conferring an increase in lutein production in a cell,
comprising the following steps: [5193] (a) identifying in a data
bank nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the luteinamount or level in an organism or a part thereof after
expression, and which are at least 20% homolog to the nucleic acid
molecule of claim 6; [5194] (b) introducing the candidate nucleic
acid molecules in host cells appropriate for producing lutein;
[5195] (c) expressing the identified nucleic acid molecules in the
host cells; [5196] (d) assaying the luteinlevel in the host cells;
and [5197] (e) identifying nucleic acid molecule and its gene
product which expression confers an increase in the lutein level in
the host cell after expression compared to the wild type. [5198]
21. A method for the production of an agricultural composition
comprising the steps of the method of any one of claims 17 to 20
and formulating the compound identified in any one of claims 17 to
20 in a form acceptable for an application in agriculture. [5199]
22. A composition comprising the nucleic acid molecule of claim 6,
the polypeptide of claim 14, the nucleic acid construct of claim 7,
the vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [5200] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
lutein after expression. [5201] 24. Use of the polypeptide of claim
14 or the nucleic acid construct claim 7 or the gene product
identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of lutein
levels in an organism. [5202] 25. Cosmetical, pharmaceutical, food
or feed composition comprising the nucleic acid molecule of claim
6, the polypeptide of claim 14, the nucleic acid construct of claim
7, the vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. [5203] 26. Use of the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the harvested material of
claim 16, the host cell of claim 10 to 12 or the gene product
identified according to the method of claim 19 or 20 for the
protection of a plant against a oxidative stress.
[5204] 27. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the protection of a plant against
a oxidative stress causing or a carotenoid synthesis inhibiting
herbicide. [5205] 28. Use of the agonist identified according to
claim 17, the plant or plant tissue of claim 16, the harvested
material of claim 16, or the host cell of claim 10 to 12 for the
production of a cosmetic composition.
[5206] [0554.0.0.11] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[5207] [0001.0.0.12] for the disclosure of this paragraph see
[0001.0.0.0].
[5208] [0002.0.12.12] Sterols are a class of essential, natural
compounds required by all eukaryotes to complete their life cycle.
In animals, cholesterol is typically the major sterol while in
fungi it is ergosterol. Plants produce a class of sterols called
phytosterols. Phytosterols are natural components of many
vegetables and grains. The term "phytosterols" covers plant sterols
and plant stanols. Plant sterols are naturally occurring substances
present in the diet as minor components of vegetable oils. The
structures of these plant sterols are similar to that of
cholesterol with an extra methyl or ethyl group and a double bond
in the side chain. Saturated plant sterols, referred to as stanols,
have no double bond in the ring structure.
[5209] Phytosterols (including plant sterols and stanols) are
natural components of plant foods, especially plant oils, seeds and
nuts, cereals and legumes specially of edible vegetable oils such
as sunflower seed oil and, as such are natural constituents of the
human diet. The most common phytosterols are beta-sitosterol,
campesterol, and stigmasterol. Beta-sitosterol is found in high
amounts in nuts.
[5210] [0003.0.12.12] A high concentration of cholesterol in serum,
i.e., hypercholesterolemia, is a wellknown risk factor for coronary
heart disease (CHD). Blood cholesterol levels can be decreased by
following diets, which are low in saturated fat, high in
polyunsaturated fat and low in cholesterol. Although considerable
achievements have been made in terms of knowledge and education,
consumers still find it difficult to follow healthy eating
advice.
[5211] Both plant sterols and plant stanols are effective in
lowering plasma total and low density lipoprotein (LDL) cholesterol
and this occurs by inhibiting the absorption of cholesterol from
the small intestine. The plasma cholesterol-lowering properties of
plant sterols have been known since the 1950s (Pollak, Circulation,
7, 702-706.1953). They have been used as cholesterol-lowering
agents, first in a free form (Pollak and Kritchevsky, Sitosterol.
In: Monographs on Aherosclerosis. Clarkson T B, Kritchevsky D,
Pollak O J, eds. New York, Basel, Karger 1981; 1-219) and recently
mainly as esterified phytosterols (Katan et al., Mayo Clin Proc
2003; 78: 965-978).
[5212] The consumption of plant sterols and plant stanols lowers
blood cholesterol levels by inhibiting the absorption of dietary
and endogenously-produced cholesterol from the small intestine and
the plant sterols/stanols are only very poorly absorbed themselves.
This inhibition is related to the similarity in physico-chemical
properties of plant sterols and stanols and cholesterol. Plant
sterols and plant stanols appear to be without hazard to health,
having been shown without adverse effects in a large number of
human studies. They show no evidence of toxicity even at high dose
levels and gastro-intestinal absorption is low.
[5213] [0004.0.12.12] The most abundant sterols of vascular plants
are campesterol, sitosterol and stigmasterol, all of which contain
a double bond between the carbon atoms at positions 5 and 6 and are
classified as delta-5 sterols.
[5214] Exemplary naturally occurring delta-5 plant sterols
isofucosterol, sitosterol, stigmasterol, campesterol, cholesterol,
and di hydrobrassicasterol. Exemplary naturally occurring
non-delta-5 plant sterols are cycloartenol, 24-methylene
cycloartenol, cycloeucalenol, and obtusifoliol.
[5215] The ratio of delta-5 to non-delta-5 sterols in plants can be
an important factor relating to insect pest resistance. Insect
pests are unable to synthesize de novo the steroid nucleus and
depend upon external sources of sterols in their food source for
production of necessary steroid compounds. In particular, insect
pests require an external source of delta-5 sterols. By way of
example, externally provided delta-5 sterols are necessary for the
production of ecdysteroids, hormones that control reproduction and
development. See, e.g., Costet et al., Proc. Natl. Acad. Sci. USA,
84:643 (1987) and Corio-Costet et al., Archives of Insect Biochem.
Physiol., 11:47 (1989).
[5216] [0005.0.12.12] US 20020148006 and WO 98/45457 describes the
modulation of phytosterol compositions to confer resistance to
insects, nematodes, fungi and/or environmental stresses, and/or to
improve the nutritional value of plants by using a DNA sequence
encoding a first enzyme; which binds a first sterol and is
preferably selected from the group consisting of
S-adenosyl-L-methionine-.sub.24(25)-sterol methyl transferase, a
C-4 demethylase, a cycloeucalenol to obtusifoliol-isomerase, a
14-demethylase, a .sub.8 to .sub.7-isomerase, a
.sub.7-C-S-desaturase and a 24,25-reductase, and produces a second
sterol and a 3' non-translated region which causes polyadenylation
at the 3' end of the RNA.
[5217] WO 93/16187 discloses new plants containing in its genome
one or more genes involved in the early stages of phytosterol
biosynthesis, preferably the genes encode mevalonate kinase.
[5218] U.S. Pat. No. 5,306,862, U.S. Pat. No. 5,589,619, U.S. Pat.
No. 5,365,017, U.S. Pat. No. 5,349,126 and US 20030150008 describe
a method of increasing sterol (and squalene) accumulation in a
plant based on an increased HMG-CoA reductase activity to increase
the pest resistance of transgenic plants.
[5219] WO 97/48793 discloses a C-14 sterol reductase polypeptide
for the genetic manipulation of a plant sterol biosynthetic
pathway.
[5220] US 20040172680 disclose the use of a gene expressing a SMT1
(sterol methyltransferase) to increase the level of sterols in the
seeds of plants. A DNA sequence encoding sterol methyltransferase
isolated from Zea mays is disclosed in WO 00/08190. Bouvier-Nav et
al in Eur. J. Biochem. 256, 88-96 (1988) describes two families of
sterol methyl transf erases (SMTs), The first (SMT1) applying to
cycloartenol and the second (SMT2) to 24-methylene lophenol.
Schaller et al (Plant Physiology (1998) 118: 461-169) describes the
over-expression of SMT2 from Arabidopsis in tobacco resulting in a
change in the ratio of 24-methyl cholesterol to sitosterol in the
tobacco leaf.
[5221] U.S. Pat. No. 6,723,837 and US 20040199940 disclose nucleic
acid molecules encoding proteins and fragments of proteins
associated with sterol and phytosterol metabolism as well as cells,
that have been manipulated to contain increased levels or
overexpress at least one sterol or phytosterol compound. The
protein or fragment is selected from the group consisting of a
HES1, HMGCoA reductase, squalene synthase, cycloartenol synthase,
SMTI, SMTII and UPC, preferably from member of the KES1/HES1/OSH1
family of oxysterol-binding (OSBP) proteins comprising an
oxysterol-binding protein consensus sequence--E(K, Q) xSH (H, R)
PPx (S, T, A, C, F)A. One class of proteins, oxysterol-binding
proteins, have been reported in humans and yeast (Jiang et al.,
Yeast 10: 341-353 (1994), the entirety of which is herein
incorporated by reference). These proteins have been reported to
modulate ergosterol levels in yeast (Jiang et al., Yeast 10:
341-353 (1994)). In particular, Jiang et al., reported three genes
KES1, HES1 and OSH1, which encode proteins containing an
oxysterol-binding region.
[5222] [0006.0.12.12] Transgenic plants having altered sterol
profiles could be instrumental in establishing a dietary approach
to cholesterol management and cardiovascular disease prevention.
The altered phytosterol profile further leads to pest
resistance.
[5223] [0007.0.12.12] Although people consume plant sterols every
day in their normal diet, the amount is not great enough to have a
significant blood cholesterol lowering effect. The intake of
phytosterols varies among different populations according to the
food products being consumed, but the average daily Western diet is
reported to contain 150-300 mg of these sterols (de Vries et al., J
Food Comp Anal 1997; 19: 115-141; Bjorkhem et al. Inborn errors in
bile acid biosynthesis and storage of sterols other than
cholesterol. In: The Metabolic and Molecular Bases of Inherited
Disease. Scriver C S, Beaudet A L, Sly W S, Valle D, eds. New York,
McGraw-Hill 2001; 2961-2988). In order to achieve a
cholesterol-lowering benefit, approximately 1 g/day of plant
sterols need to be consumed (Hendriks et al., European Journal of
Clinical Nutrition, 53, 319-327.1999).
[5224] [0008.0.12.12] Phytosterols are found naturally in plant
foods at low levels. The enrichment of foods such as margarines
with plant sterols and stanols is one of the recent developments in
functional foods to enhance the cholesterol-lowering ability of
traditional food products. Incorporation of additional phytosterols
into the diet may be an effective way of lowering total and LDL
cholesterol levels. The non-esterified phytosterols can be used as
novel food ingredients in: [5225] (a) bakery products and cereals
(eg, breakfast cereals, breakfast bars); [5226] (b) dairy products
such as low and reduced fat liquid milk, low and reduced fat
yoghurt and yoghurt products, and dairy based desserts; [5227] (c)
non-carbonated soft drinks like low and reduced fat soy beverages
and low and reduced fat soy-based yoghurts; [5228] (d) meat
products or edible fats and oils (eg, mayonnaise, spice sauces,
salad dressings); [5229] (e) margarine; and [5230] table spreads or
dietary fats.
[5231] [0009.0.12.12] When edible oils undergo normal refining,
plant sterols are partially extracted. It is estimated that 2500
tonnes of vegetable oil needs to be refined to produce 1 tonne of
plant sterols. Plant stanols are obtained by hydrogenation of the
plant sterols.
[5232] [0010.0.12.12] Another source of plant sterols is tall oil,
derived from the process of paper production from wood and
approximately 2500 tonnes of pine is required to produce 1 tonne of
plant sterols. Tall oil also contains a higher proportion of plant
stanols (primarily b-sitostanol) than do vegetable oils.
[5233] [0011.0.12.12] As described above, phytosterols are used in
a lot of different applications, for example in cosmetics,
pharmaceuticals and in feed and food. Therefore improving the
quality of foodstuffs and animal feeds is an important task of the
food-and-feed industry. Especially advantageous for the quality of
foodstuffs and animal feeds is as balanced as possible a sterol
profile in the diet.
[5234] [0012.0.12.12] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes which
participate in the biosynthesis of phytosterols and make it
possible to produce them specifically on an industrial scale
without unwanted byproducts forming. In the selection of genes for
or regulators of biosynthesis two characteristics above all are
particularly important. On the one hand, there is as ever a need
for improved processes for obtaining the highest possible contents
of phytosterols; on the other hand as less as possible by products
should be produced in the production process.
[5235] [0013.0.0.12] for the disclosure of this paragraph see
[0013.0.0.0] above.
[5236] [0014.0.12.12] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is/are phytosterols. The term
"phytosterols" covers plant sterols and plant stanols. Accordingly,
in the present invention, the term "the fine chemical" as used
herein relates to "phytosterols". Further, the term "the fine
chemicals" as used herein also relates to fine chemicals comprising
plant sterols and plant stanols, preferably beta-sitosterol,
campesterol, and/or stigmasterol.
[5237] [0015.0.12.12] In one embodiment, the term "the fine
chemical" means phytosterols, plant sterols and plant stanols.
Throughout the specification the term "the fine chemical" means
phytosterols and ester, thioester or sterols in free form or bound
to other compounds. For the purpose of this description, the term
sterol/stanol refers both to free sterols/stanols and conjugated
sterols/stanols, for example, where the 3-hydroxy group is
esterified by a fatty acid chain or phenolic acid to give a
steryl/stanyl ester. As used herein, the term "phytosterol"
includes all phytosterols without limitation, for example:
sitosterol, campesterol, stigmasterol, brassicasterol, desmosterol,
chalinosterol, poriferasterol, clionasterol, the corresponding
stanols and all natural or synthesized forms and derivatives
thereof, including isomers. It is to be understood that
modifications to the phytosterols i.e. to include side chains also
falls within the purview of this invention. All those derivates
forms are summarized as "conjugates". In an preferred embodiment,
the term "the fine chemical" or the term "phytosterol" or the term
"the respective fine chemical" means at least one chemical compound
plant sterols and plant stanols selected from the group
"beta-sitosterol, sitostanol, stigmasterol, brassicasterol,
campestanol, isofucosterol and campesterol", preferred
"beta-sitosterol, campesterol, and/or stigmasterol", most preferred
"beta-sitosterol and/or campesterol". Also preferably, are esters
of sterols/stanols with C10-24 fatty acids.
[5238] Increased phytosterol content normally means an increased
total phytosterol content. However, an increased phytosterol
content also means, in particular, a modified content of the
above-described compounds ("beta-sitosterol, sitostanol,
stigmasterol, brassicasterol, campestanol, isofucosterol and
campesterol") with phytosterol activity, without the need for an
inevitable increase in the total phytosterol content.
[5239] [0016.0.12.12] Accordingly, the present invention relates to
a process for the production of phytosterol comprising [5240] (a)
increasing or generating the activity of one or more [5241]
YER156C, YKR057W, YOR084W, b0019, b0421, b2699, b0050, b0161 and/or
b4129 protein(s) and/or [5242] YER156C, YER173W, YKR057W, YOR044W,
YPR172W, b2699, b3256, b0161, b0464, b1896, b2341, b2478 and/or
b2822 protein(s) in a non-human organism in one or more parts
thereof and [5243] (b) growing the organism under conditions which
permit the production of the fine chemical, thus, phytosterol,
plant sterol and stanol, preferably beta-sitosterol, sitostanol,
stigmasterol, brassicasterol, campestanol, isofucosterol and
campesterol, more preferred beta-sitosterol, campesterol, and/or
stigmasterol, most preferred beta-sitosterol and/or campesterol or
fine chemicals comprising phytosterol, in said organism.
[5244] Accordingly, the present invention relates to a process for
the production of phytosterol comprising [5245] (a) increasing or
generating the activity of one or more proteins having the activity
of a protein indicated in Table II, column 3, lines 112 to 124
and/or lines 483 to 491 or having the sequence of a polypeptide
encoded by a nucleic acid molecule indicated in Table I, column 5
or 7, lines 112 to 124 and/or lines 483 to 491, in a non-human
organism in one or more parts thereof and [5246] (b) growing the
organism under conditions which permit the production of the fine
chemical, thus, phytosterol, in particular beta-sitosterol and/or
campesterol.
[5247] [0016.1.12.12] Accordingly, the term "the fine chemical"
means in one embodiment "beta-sitosterol" in relation to all
sequences listed in Table I to IV, lines 112 to 117 and/or 483 to
485 or homologs thereof and means in one embodiment "campesterol"
in relation to all sequences listed in Tables I to IV, lines 118 to
124 and/or 486 to 491 or homologs thereof.
[5248] Accordingly, in one embodiment the term "the fine chemical"
means "beta-sitosterol" and "campesterol" in relation to all
sequences listed in Table I to IV, lines 113 or 120 and 120; in one
embodiment the term "the fine chemical" means "beta-sitosterol" and
"campesterol" in relation to all sequences listed in Table I to IV,
lines 114 or 122; in one embodiment the term "the fine chemical"
means "beta-sitosterol" and "campesterol" in relation to all
sequences listed in Table I to IV, lines 115 or 124, in one
embodiment the term "the fine chemical" means "beta-sitosterol" and
"campesterol" in relation to all sequences listed in Table I to IV,
lines 484 or 486.
[5249] Accordingly, the term "the fine chemical" can mean
"beta-sitosterol" and "campesterol", owing to circumstances and the
context. In order to illustrate that the meaning of the term "the
fine chemical" means ""beta-sitosterol" and "campesterol" the term
"the respective fine chemical" is also used.
[5250] [0017.0.0.12] and [0018.0.0.12] for the disclosure of the
paragraphs [0017.0.0.12] and [0018.0.0.12] see paragraphs
[0017.0.0.0] and [0018.0.0.0] above.
[5251] [0019.0.12.12] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the respective fine chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the fine respective chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein indicated in Table II, column 3, lines
112 to 124 and/or lines 483 to 491 or encoded by nucleic acid
molecule indicated in Table I, columns 5 or 7, lines 112 to 124
and/or lines 483 to 491.
[5252] [0020.0.12.12] Surprisingly it was found, that the
transgenic expression of the Saccaromyces cerevisiae protein
YER156C, YKR057W and/or YOR084W and/or the Escherichia coli K12
protein b0019, b0421, b2699, b0050, b0161 and/or b4129 in
[5253] Arabidopsis thaliana conferred an increase in the
beta-sitosterol content of the transformed plants.
[5254] Surprisingly it was found, that the transgenic expression of
the Saccaromyces cerevisiae protein YER156C, YER173W, YKR057W,
YOR044W and/or YPR172W and/or the Escherichia coli K12 protein
b2699, b3256, b0161, b0464, b1896, b2341, b2478 and/or b2822 in
Arabidopsis thaliana conferred an increase in campesterol content
of the transformed plants.
[5255] Accordingly, it was surprisingly found, that the transgenic
expression of the Saccaromyces cerevisiae protein YER156C in
Arabidopsis thaliana conferred an increase in beta-sitosterol
and/or campesterol (or the respective fine chemical) content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of beta-sitosterol; in one
embodiment, said protein or its homologs are used for the
production of campesterol; in one embodiment, said protein or its
homologs are used for the production of beta-sitosterol and
campesterol.
[5256] Accordingly, it was surprisingly found, that the transgenic
expression of the Saccaromyces cerevisiae protein YKR057W in
Arabidopsis thaliana conferred an increase in beta-sitosterol
and/or campesterol (or the respective fine chemical) content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of beta-sitosterol; in one
embodiment, said protein or its homologs are used for the
production of campesterol; in one embodiment, said protein or its
homologs are used for the production of beta-sitosterol and
campesterol.
[5257] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein b2699 in Arabidopsis
thaliana conferred an increase in beta-sitosterol and/or
campesterol (or the respective fine chemical) content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of beta-sitosterol; in one
embodiment, said protein or its homologs are used for the
production of campesterol; in one embodiment, said protein or its
homologs are used for the production of beta-sitosterol and
campesterol.
[5258] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein b0161 in Arabidopsis
thaliana conferred an increase in beta-sitosterol and/or
campesterol (or the respective fine chemical) content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of beta-sitosterol; in one
embodiment, said protein or its homologs are used for the
production of campesterol; in one embodiment, said protein or its
homologs are used for the production of beta-sitosterol and
campesterol.
[5259] [0021.0.0.12] for the disclosure of this paragraph see
[0021.0.0.0] above.
[5260] [0022.0.12.12] The sequence of YER156C from Saccharomyces
cerevisiae has been published in Dietrich, et al. (Nature 387 (6632
Suppl), 78-81 (1997)), and its activity has not been characterized
yet. It seems to have an activity similar to Arabidopsis thaliana
hypothetical protein F2K15.180. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
with the activity of a YER156C from Saccharomyces cerevisiae or of
a Arabidopsis thaliana hypothetical protein F2K15.180 or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of phytosterol, e.g. beta-sitosterol and/or
campesterol and/or conjugates, preferably in free or bound form in
an organism or a part thereof, as mentioned. In one further
embodiment the YER156C protein expression is increased together
with the increase of another gene of the phytosterol biosynthesis
pathway, preferably with a gene encoding a protein being involved
in the production of phytosterol from the intermediates like
squalene and squalene epoxide or cycloartenol. In one embodiment,
in the process of the present invention said activity, e.g. of a
YER156C protein is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof. The sequence of YER173W from
Saccharomyces cerevisiae has been published in Dietrich et al.
(Nature 387 (6632 Suppl), 78-81, 1997), and Goffeau et al. (Science
274 (5287), 546-547, 1996), and its activity is being defined as an
"Checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints". Accordingly, in one embodiment,
the process of the present invention comprises the use of a YER173W
activity from Saccharomyces cerevisiae or its homolog, e.g. as
shown herein, for the production of phytosterol, meaning of
campesterol and/or conjugates, in particular for increasing the
amount of campesterol and/or conjugates, preferably campesterol in
free or bound form in an organism or a part thereof, as mentioned.
In one further embodiment the YER173W protein expression is
increased together with the increase of another gene of the
phytosterol biosynthesis pathway, preferably with a gene encoding a
protein being involved in the production of phytosterol from the
intermediates like squalene and squalene epoxide or cycloartenol.
In one embodiment, in the process of the present invention the
activity of a YER173W protein is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[5261] The sequence of YKR057W from Saccharomyces cerevisiae has
been published in Dujon et al., Nature 369 (6479), 371-378, 1994
and Goffeau et al., Science 274 (5287), 546-547, 1996 and its
activity is being defined as a ribosomal protein, similar to S21
ribosomal proteins, involved in ribosome biogenesis and
translation. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein involved in the
ribosome biogenesis and translation, in particular of the
superfamiliy of the ribosomal protein, preferably having a S21
ribosomal protein activity or its homolog, e.g. as shown herein,
for the production of the respective fine chemical, meaning of
phytosterol, e.g. beta-sitosterol and/or campesterol and/or
conjugates, preferably in free or bound form in an organism or a
part thereof, as mentioned. In one further embodiment the YKR057W
protein expression is increased together with the increase of
another gene of the phytosterol biosynthesis pathway, preferably
with a gene encoding a protein being involved in the production of
phytosterol from the intermediates like squalene and squalene
epoxide or cycloartenol. In one embodiment, in the process of the
present invention said activity, e.g. of a protein of the yeast
ribosomal protein superfamily or preferably having a activity
involved in ribosome biogenesis and translation is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[5262] The sequence of YOR044W from Saccharomyces cerevisiae has
been published in Dujon et al., Nature 387 (6632 Suppl), 98-102
(1997), and its activity has not been characterized yet.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein with the activity of a
YOR044w from Saccharomyces cerevisiae or of a Saccharomyces
cerevisiae probable membrane protein YOR044w or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, meaning of phytosterol, e.g. campesterol and/or
conjugates, preferably in free or bound form in an organism or a
part thereof, as mentioned. In one further embodiment the YOR044W
protein expression is increased together with the increase of
another gene of the phytosterol biosynthesis pathway, preferably
with a gene encoding a protein being involved in the production of
phytosterol from the intermediates like squalene and squalene
epoxide or cycloartenol. In one embodiment, in the process of the
present invention said activity, e.g. of a YOR044W protein is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof. The sequence of YOR084W from Saccharomyces
cerevisiae has been published in Dujon, Nature 387 (6632 Suppl),
98-102, 1997, and Goffeau, Science 274 (5287), 546-547, 1996, and
its activity is being defined as a putative lipase of the
peroxisomal matrix. Accordingly, in one embodiment, the process of
the present invention comprises the use of a putative lipase of the
peroxisomal matrix protein or its homolog, e.g. as shown herein,
for the production of the respective fine chemical, meaning of
phytosterol, e.g. beta-sitosterol and/or conjugates, preferably in
free or bound form in an organism or a part thereof, as mentioned.
In one further embodiment the YOR084W protein expression is
increased together with the increase of another gene of the
phytosterol biosynthesis pathway, preferably with a gene encoding a
protein being involved in the production of phytosterol from the
intermediates like squalene and squalene epoxide or cycloartenol.
In one embodiment, in the process of the present invention said
activity, e.g. of a a putative lipase of the peroxisomal matrix
protein is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof. The sequence of YPR172W from
Saccharomyces cerevisiae has been published in Bussey et al.,
Nature 387 (6632 Suppl), 103-105 (1997), and its activity has not
been characterized yet. Accordingly, in one embodiment, the process
of the present invention comprises the use of a protein with the
activity of a YPR172W from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, meaning of phytosterol, e.g. campesterol and/or
conjugates, preferably in free or bound form in an organism or a
part thereof, as mentioned. In one further embodiment the YPR172W
protein expression is increased together with the increase of
another gene of the phytosterol biosynthesis pathway, preferably
with a gene encoding a protein being involved in the production of
phytosterol from the intermediates like squalene and squalene
epoxide or cycloartenol. In one embodiment, in the process of the
present invention said activity, e.g. of a YPR172W protein is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[5263] The sequence of b0019 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as protein for the transport of
small molecules, preferably cations, meaning a Na+/H+ antiporter
protein. In a more preferred embodiment the protein has the
activity of a Na+/H+ antiporter, responsive to stress, especially
to high salinity and pH. Accordingly, in one embodiment, the
process of the present invention comprises the use of a transport
protein, in particular of the superfamiliy of Na+/H+-exchanging
protein nhaA, preferably having a Na+/H+ antiporter activity from
E. coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of phytosterol, e.g.
beta-sitosterol and/or conjugates, preferably in free or bound form
in an organism or a part thereof, as mentioned. In one further
embodiment the b0019 protein expression is increased together with
the increase of another gene of the phytosterol biosynthesis
pathway, preferably with a gene encoding a protein being involved
in the production of phytosterol from the intermediates like
squalene and squalene epoxide or cycloartenol. In one embodiment,
in the process of the present invention said activity, e.g. of a
transport protein, in particular of the superfamiliy of
Na+/H+-exchanging protein nhaA, preferably having a Na+/H+
antiporter activity from E. coli is increased or generated, e.g.
from E. coli or a homolog thereof.
[5264] The sequence of b0421 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as geranyltranstransferase
(=farnesyldiphosphate synthase). Accordingly, in one embodiment,
the process of the present invention comprises the use of a
geranyltranstransferase (=farnesyldiphosphate synthase), in
particular of the superfamiliy of dimethylallyltrans-transferase,
geranyltranstransferase from E. coli or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of phytosterol, e.g. beta-sitosterol and/or conjugates, preferably
in free or bound form in an organism or a part thereof, as
mentioned. In one further embodiment the b0421 protein expression
is increased together with the increase of another gene of the
phytosterol biosynthesis pathway, preferably with a gene encoding a
protein being involved in the production of phytosterol from the
intermediates like squalene and squalene epoxide or cycloartenol.
In one embodiment, in the process of the present invention said
activity, e.g. of a geranyltranstransferase (=farnesyldiphosphate
synthase), in particular of the superfamiliy of
dimethylallyltrans-transferase, geranyltranstransferase is
increased or generated, e.g. from E. coli or a homolog thereof.
[5265] The sequence of b2699 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a DNA strand exchange and
recombination protein with protease and nuclease activity of the
superfamily of the recombination protein recA. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein with a DNA recombination and DNA repair activity, a
pheromone response activity, a mating-type determination activity,
a sex-specific protein activity, a nucleotide binding activity
and/or a protease and nuclease activity, in particular a DNA strand
exchange and recombination protein with protease and nuclease
activity, in particular of the superfamily of the recombination
protein recA from E. coli or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, meaning of
phytosterol, in particular for increasing the amount of
beta-sitosterol and/or campesterol and/or conjugates, preferably in
free or bound form in an organism or a part thereof, as mentioned.
In one further embodiment the b2699 protein expression is increased
together with the increase of another gene of the phytosterol
biosynthesis pathway, preferably with a gene encoding a protein
being involved in the production of phytosterol from the
intermediates like squalene and squalene epoxide or cycloartenol.
In one embodiment, in the process of the present invention said
activity, e.g. the activity of a protease and nuclease activity, in
particular a DNA strand exchange and recombination protein with
protease and nuclease activity, in particular of the superfamily of
the recombination protein recA is increased or generated, e.g. from
E. coli or a homolog thereof.
[5266] The sequence of b3256 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as acetyl CoA carboxylase, biotin
carboxylase subunit. Accordingly, in one embodiment, the process of
the present invention comprises the use of a acetyl CoA
carboxylase, in particular of the superfamiliy of biotin
carboxylase from E. coli or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, meaning of
phytosterol, in particular for increasing the amount of campesterol
and/or conjugates, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one further embodiment the
b3256 protein expression is increased together with the increase of
another gene of the phytosterol biosynthesis pathway, preferably
with a gene encoding a protein being involved in the production of
phytosterol from the intermediates like squalene and squalene
epoxide or cycloartenol. In one embodiment, in the process of the
present invention said activity, e.g. the activity of a acetyl CoA
carboxylase, in particular of the superfamiliy of biotin
carboxylase is increased or generated, e.g. from E. coli or a
homolog thereof.
[5267] The sequence of b0050 (Accession number NP.sub.--414592)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a conserved protein potentially involved in protein
protein interaction. Accordingly, in one embodiment, the process of
the present invention comprises the use of said protein from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of phytosterol, e.g. beta-sitosterol
and/or conjugates, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one further embodiment the
b0050 protein expression is increased together with the increase of
another gene of the phytosterol biosynthesis pathway, preferably
with a gene encoding a protein being involved in the production of
phytosterol from the intermediates like squalene and squalene
epoxide or cycloartenol. In one embodiment, in the process of the
present invention the activity of said protein is increased or
generated, e.g. from E. coli or a homolog thereof.
[5268] The sequence of b0161 (Accession number NP.sub.--414703)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a periplasmic serine protease (heat shock protein).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a periplasmic serine protease from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of phytosterol, e.g. beta-sitosterol
and/or campesterol and/or conjugates, preferably in free or bound
form in an organism or a part thereof, as mentioned. In one further
embodiment the b0161 protein expression is increased together with
the increase of another gene of the phytosterol biosynthesis
pathway, preferably with a gene encoding a protein being involved
in the production of phytosterol from the intermediates like
squalene and squalene epoxide or cycloartenol. In one embodiment,
in the process of the present invention the activity of a
periplasmic serine protease is increased or generated, e.g. from E.
coli or a homolog thereof.
[5269] The sequence of b4129 (Accession number NP.sub.--418553)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a lysine tRNA synthetase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a lysine tRNA synthetase protein from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of phytosterol, e.g. beta-sitosterol and/or conjugates,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one further embodiment the b4129 protein
expression is increased together with the increase of another gene
of the phytosterol biosynthesis pathway, preferably with a gene
encoding a protein being involved in the production of phytosterol
from the intermediates like squalene and squalene epoxide or
cycloartenol. In one embodiment, in the process of the present
invention the activity of a lysine tRNA synthetase protein is
increased or generated, e.g. from E. coli or a homolog thereof.
[5270] The sequence of b0464 (Accession number NP.sub.--414997)
from Escherichia coli K12 has been published in Blattner, Science
277(5331), 1453-1474, 1997, and its activity is being defined as
a''transcriptional repressor for multidrug efflux pump (TetR/AcrR
family)". Accordingly, in one embodiment, the process of the
present invention comprises the use of a "transcriptional repressor
for multidrug efflux pump (TetR/AcrR family)" from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of phytosterol, in particular for increasing the
amount of campesterol and/or conjugates, preferably in free or
bound form in an organism or a part thereof, as mentioned. In one
further embodiment the b0464 protein expression is increased
together with the increase of another gene of the phytosterol
biosynthesis pathway, preferably with a gene encoding a protein
being involved in the production of phytosterol from the
intermediates like squalene and squalene epoxide or cycloartenol.
In one embodiment, in the process of the present invention the
activity of a "transcriptional repressor for multidrug efflux pump
(TetR/AcrR family)" is increased or generated, e.g. from E. coli or
a homolog thereof.
[5271] The sequence of b1896 (Accession number NP.sub.--416410)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277(5331), 1453-1474, 1997, and its activity is being
defined as a trehalose-6-phosphate synthase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a trehalose-6-phosphate synthase from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of phytosterol, in particular for increasing the amount of
campesterol and/or conjugates, preferably in free or bound form in
an organism or a part thereof, as mentioned. In one further
embodiment the b1896 protein expression is increased together with
the increase of another gene of the phytosterol biosynthesis
pathway, preferably with a gene encoding a protein being involved
in the production of phytosterol from the intermediates like
squalene and squalene epoxide or cycloartenol. In one embodiment,
in the process of the present invention the activity of a
trehalose-6-phosphate synthase is increased or generated, e.g. from
E. coli or a homolog thereof.
[5272] The sequence of b2341 (Accession number NP.sub.--416410)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277(5331), 1453-1474, 1997, and its activity is being
defined as a "bifunctional anaerobic fatty acid oxidation complex
protein: enoyl-CoA hydratase/epimerase/isomerase (N-terminal);
3-hydroxyacyl-CoA dehydrogenase (C-terminal)". Accordingly, in one
embodiment, the process of the present invention comprises the use
of a bifunctional anaerobic fatty acid oxidation complex protein:
enoyl-CoA hydratase/epimerase/isomerase (N-terminal);
3-hydroxyacyl-CoA dehydrogenase (C-terminal) from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of phytosterol, in particular for increasing the
amount of campesterol and/or conjugates, preferably in free or
bound form in an organism or a part thereof, as mentioned. In one
further embodiment the b2341 protein expression is increased
together with the increase of another gene of the phytosterol
biosynthesis pathway, preferably with a gene encoding a protein
being involved in the production of phytosterol from the
intermediates like squalene and squalene epoxide or cycloartenol.
In one embodiment, in the process of the present invention the
activity of a bifunctional anaerobic fatty acid oxidation complex
protein: enoyl-CoA hydratase/epimerase/isomerase (N-terminal);
3-hydroxyacyl-CoA dehydrogenase (C-terminal) is increased or
generated, e.g. from E. coli or a homolog thereof.
[5273] The sequence of b2478 (Accession number NP.sub.--416973)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277(5331), 1453-1474, 1997, and its activity is being
defined as a "dihydrodipicolinate synthase". Accordingly, in one
embodiment, the process of the present invention comprises the use
of a dihydrodipicolinate synthase from E. coli or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
of phytosterol, in particular for increasing the amount of
campesterol and/or conjugates, preferably in free or bound form in
an organism or a part thereof, as mentioned. In one further
embodiment the b2478 protein expression is increased together with
the increase of another gene of the phytosterol biosynthesis
pathway, preferably with a gene encoding a protein being involved
in the production of phytosterol from the intermediates like
squalene and squalene epoxide or cycloartenol. In one embodiment,
in the process of the present invention the activity of a
dihydrodipicolinate synthase is increased or generated, e.g. from
E. coli or a homolog thereof.
[5274] The sequence of b2822 (Accession number NP.sub.--417299)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a DNA helicase, ATP-dependent dsDNA/ssDNA exonuclease V
subunit, ssDNA endonuclease. Accordingly, in one embodiment, the
process of the present invention comprises the use of a DNA
helicase, ATP-dependent dsDNA/ssDNA exonuclease V subunit, ssDNA
endonuclease protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
phytosterol, in particular for increasing the amount of campesterol
and/or conjugates, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one further embodiment the
b2822 protein expression is increased together with the increase of
another gene of the phytosterol biosynthesis pathway, preferably
with a gene encoding a protein being involved in the production of
phytosterol from the intermediates like squalene and squalene
epoxide or cycloartenol. In one embodiment, in the process of the
present invention the activity of a DNA helicase, ATP-dependent
dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease protein is
increased or generated, e.g. from E. coli or a homolog thereof.
[5275] [0023.0.12.12] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[5276] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 112, 113 or 114,
resp. is a homolog having the same or a similar activity, resp. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of beta-sitosterol. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
line 112, 113 or 114, resp. In one embodiment, the homolog of one
of the polypeptides indicated in Table II, column 3, line 112, 113
or 114 resp., is derived from an eukaryotic. In one embodiment, the
homolog is derived from Fungi. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 112, 113 or 114,
resp., is derived from Ascomycota. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, line 112, 113 or
114 resp., is derived from Saccharomycotina. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 112,
113 or 114, resp., is derived from Saccharomycetes. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 112, 113 or 114, resp., is a homolog being derived
from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 112, 113 or 114,
resp., is a homolog having the same or a similar activity being
derived from Saccharomycetaceae. In one embodiment, the homolog of
a polypeptide indicated in Table II, column 3, line 112, 113 or 114
resp., is a homolog having the same or a similar activity being
derived from Saccharomyces.
[5277] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 118 to 122 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms, preferably of
phytosterol, preferably of campesterol. In one embodiment, the
homolog is a homolog with a sequence as indicated in Table I or II,
column 7, lines 118 to 122. In one embodiment, the homolog of one
of the polypeptides indicated in Table II, column 3, lines 118 to
122, is derived from an eukaryotic. In one embodiment, the homolog
is derived from Fungi. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 118 to 122, is
derived from Ascomycota. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 118 to 122, is
derived from Saccharomycotina. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 118 to 122, is
derived from Saccharomycetes. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 118 to 122, is a
homolog being derived from Saccharomycetales. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
118 to 122, is a homolog having the same or a similar activity
being derived from Saccharomycetaceae. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 118
to 122, is a homolog having the same or a similar activity being
derived from Saccharomyces.
[5278] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 115, 116 or 117
and 483 to 485 is a homolog having the same or a similar activity.
In particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of phytosterol, more preferably beta-sitosterol. In one
embodiment, the homolog is a homolog with a sequence as indicated
in Table I or II, column 7, lines 115, 116 or 117 and 483 to 485,
resp. In one embodiment, the homolog of one of the polypeptides
indicated in Table II, column 3, 115, 116 or 117 and 483 to 485 is
derived from a bacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 115, 116 or 117
and 483 to 485 is derived from Proteobacteria. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
115, 116 or 117 and 483 to 485 is a homolog having the same or a
similar activity being derived from Gammaproteobacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 115, 116 or 117 and 483 to 485 is derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 115, 116 or 117 and 483 to
485 is a homolog being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 115, 116 or 117 and 483 to 485 is a homolog having
the same or a similar activity and being derived from
Escherichia.
[5279] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 123 or 124 and
486 to 491 is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of phytosterol, more preferably of campesterol. In one
embodiment, the homolog is a homolog with a sequence as indicated
in Table I or II, column 7, lines 123 or 124 and 486 to 491, resp.
In one embodiment, the homolog of one of the polypeptides indicated
in Table II, column 3, lines 123 or 124 and 486 to 491 is derived
from an bacteria. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 123 or 124 and 486 to 491 is
derived from Proteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 123 or 124 and
486 to 491 is a homolog having the same or a similar activity being
derived from Gammaproteobacteria. In one embodiment, the homolog of
a polypeptide indicated in Table II, column 3, lines 123 or 124 and
486 to 491 is derived from Enterobacteriales. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
123 or 124 and 486 to 491 is a homolog being derived from
Enterobacteriaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 123 or 124 and 486 to 491 is
a homolog having the same or a similar activity and being derived
from Escherichia.
[5280] [0023.1.12.12] Homologs of the polypeptide indicated in
Table II, column 3, lines 112 to 124 and/or lines 483 to 491 may be
the polypeptides encoded by the nucleic acid molecules indicated in
Table I, column 7, lines 112 to 124 and/or lines 483 to 491, resp.,
or may be the polypeptides indicated in Table II, column 7, lines
112 to 124 and/or lines 483 to 491, resp. Homologs of the
polypeptides indicated in Table II, column 3, lines 112 to 124
and/or lines 483 to 491 may be the polypeptides encoded by the
nucleic acid molecules indicated in Table I, column 7, lines 112 to
124 and/or lines 483 to 491, resp., or may be the polypeptides
indicated in Table II, column 7, lines 112 to 124 and/or lines 483
to 491.
[5281] Homologs of the polypeptides indicated in Table II, column
3, lines 112 to 117 and 483 to 485 may be the polypeptides encoded
by the nucleic acid molecules indicated in Table I, column 7, lines
112 to 117 and 483 to 485, respectively or may be the polypeptides
indicated in Table II, column 7, lines 112 to 117 and 483 to 485,
having a beta-sitosterol content- and/or amount-increasing
activity. Homologs of the polypeptides indicated in Table II,
column 3, lines 112 to 117 and 483 to 485 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 112 to 117 and 483 to 485 or may be the polypeptides
indicated in Table II, column 7, lines 112 to 117 and 483 to 485
having a beta-sitosterol content- and/or amount-increasing
activity.
[5282] Homologs of the polypeptides indicated in Table II, column
3, lines 118 to 124 and 486 to 491 may be the polypeptides encoded
by the nucleic acid molecules indicated in Table I, column 7, 118
to 124 and 486 to 491, respectively or may be the polypeptides
indicated in Table II, column 7, 118 to 124 and 486 to 491, having
a campesterol content- and/or amount-increasing activity. Homologs
of the polypeptides indicated in Table II, column 3, 118 to 124 and
486 to 491 may be the polypeptides encoded by the nucleic acid
molecules indicated in Table I, column 7, 118 to 124 and 486 to 491
or may be the polypeptides indicated in Table II, column 7, lines
118 to 124 and 486 to 491 having a campesterol content- and/or
amount-increasing activity.
[5283] [0024.0.0.12] for the disclosure of this paragraph see
[0024.0.0.0] above.
[5284] [0025.0.12.12] In accordance with the invention, a protein
or polypeptide has the "activity of an protein of the invention",
e.g. the activity of a protein indicated in Table II, column 3,
lines 112 to 117 and/or lines 483 to 485 and/or lines 118 to 124
and/or lines 486 to 491 resp., if its de novo activity, or its
increased expression directly or indirectly leads to an increased
phytosterol level, in particular to a increased beta-sitosterol
and/or campesterol, resp., in the organism or a part thereof,
preferably in a cell of said organism. In a preferred embodiment,
the protein or polypeptide has the above-mentioned additional
activities of a protein indicated in Table II, column 3, lines 112
to 117 and/or lines 483 to 485 and/or lines 118 to 124 and/or lines
486 to 491. Throughout the specification the activity or preferably
the biological activity of such a protein or polypeptide or an
nucleic acid molecule or sequence encoding such protein or
polypeptide is identical or similar if it still has the biological
or enzymatic activity of any one of the proteins indicated in Table
II, column 3, lines 112 to 117 and/or lines 483 to 485 and/or lines
118 to 124 and/or lines 486 to 491 resp. or which has at least 10%
of the original enzymatic activity, preferably 20%, particularly
preferably 30%, most particularly preferably 40% in comparison to
any one of the proteins indicated in Table II, column 3, lines 112,
113, 114, 118, 119, 120, 121 or 122 of Saccharomyces cerevisiae
and/or any one of the proteins indicated in Table II, column 3,
lines 115, 116, 117, 123 or 124 or 483 to 491 of E. coli K12.
[5285] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[5286] [0025.1.0.12] and [0025.2.0.12] for the disclosure of the
paragraphs [0025.1.0.12] and [0025.2.0.12] see [0025.1.0.0] and
[0025.2.0.0] above.
[5287] [0026.0.0.12] to [0033.0.0.12] for the disclosure of the
paragraphs [0026.0.0.12] to [0033.0.0.12] see paragraphs
[0026.0.0.0] to [0033.0.0.0] above.
[5288] [0034.0.12.12] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of an protein having the activity of a
protein as indicated in Table II, column 3, lines 112 to 117 and/or
lines 483 to 485 and/or lines 118 to 124 and/or lines 486 to 491
resp., or being encoded by a nucleic acid molecule as indicated in
Table I, column 5, lines 112 to 117 and/or lines 483 to 485 and/or
lines 118 to 124 and/or lines 486 to 491 resp., or its homologs,
e.g. as indicated in Table I, column 7, lines 112 to 117 and/or
lines 483 to 485 and/or lines 118 to 124 and/or lines 486 to 491
resp. its biochemical or genetic causes and therefore shows the
increased amount of the respective fine chemical.
[5289] [0035.0.0.12] to [0038.0.0.12] and [0039.0.5.12] for the
disclosure of the paragraphs [0035.0.0.12] to [0038.0.0.12] and
[0039.0.5.12] see paragraphs [0035.0.0.0] to [0039.0.0.0]
above.
[5290] [0040.0.0.12] to [0044.0.0.12] for the disclosure of the
paragraphs [0040.0.0.12] to [0044.0.0.12] see paragraphs
[0035.0.0.0] and [0044.0.0.0] above.
[5291] [0045.0.12.12.] In one embodiment, the activity of the
Saccharomyces cerevisiae protein YER156C or its homologs, e.g. an
activity of a Arabidopsis thaliana hypothetical protein F2K15.180,
e.g. as indicated in Table I, columns 5 or 7, lines 114 or 122, is
increased conferring an increase of the respective fine chemical,
preferably of beta-sitosterol between 18% and 39% or more or an
increase of the respective fine chemical, preferably of campesterol
between 17% and 95% or more or an increase of the respective fine
chemical, preferably of beta-sitosterol and campesterol between 17%
and 95%, preferably between 18% and 39% or more.
[5292] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER173W or its homologs, e.g. an activity of a
checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints, e.g. as indicated in Table I,
columns 5 or 7, line 121, is increased conferring an increase of
the respective fine chemical, preferably of the campesterol between
23% and 51% or more.
[5293] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YKR057W or its homologs, e.g. an activity of
ribosomal protein, similar to S21 ribosomal proteins, involved in
ribosome biogenesis and translation, e.g. as indicated in Table I,
columns 5 or 7, lines 113 or 120, is increased conferring an
increase of the respective fine chemical, preferably of
beta-sitosterol between 14% and 32% or more or an increase of the
respective fine chemical, preferably of campesterol between 20% and
39% or more or an increase of the respective fine chemical,
preferably of beta-sitosterol and campesterol between 14% and 39%,
preferably between 20% and 32% or more.
[5294] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR044W or its homologs, e.g. an activity of a
probable membrane protein YOR044w, e.g. as indicated in Table I,
columns 5 or 7, line 119, is increased conferring an increase of
the respective fine chemical, preferably of the campesterol between
28% and 57% or more.
[5295] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR084W or its homologs, e.g. an activity of a
putative lipase of the peroxisomal matrix, e.g. as indicated in
Table I, columns 5 or 7, line 112, is increased conferring an
increase of the respective fine chemical, preferably of the
beta-sitosterol between 100% and 259% or more.
[5296] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YPR172W or its homologs, e.g. an activity of a
YPR172W protein, e.g. as indicated in Table I, columns 5 or 7, line
118, is increased conferring an increase of the respective fine
chemical, preferably of the campesterol between 17% and 62% or
more.
[5297] In one embodiment the activity of the Escherichia coli K12
protein b0019 or its homologs, e.g. having a Na+/H+ antiporter
activity, in particular, of the superfamiliy of Na+/H+-exchanging
protein nhaA, e.g. as indicated in Table I, columns 5 or 7, line
117, is increased conferring an increase of the respective fine
chemical, preferably of the beta-sitosterol between 20% and 47% or
more.
[5298] In one embodiment, the activity of the Escherichia coli K12
protein b0421 or its homologs, e.g. with a geranyltranstransferase
(=farnesyldiphosphate synthase) activity, in particular, of the
superfamiliy of dimethylallyltrans-transferase,
geranyltranstransferase, e.g. as indicated in Table I, columns 5 or
7, line 116, is increased conferring an increase of the respective
fine chemical, preferably of beta-sitosterol between 13% and 52% or
more.
[5299] In one embodiment, the activity of the Escherichia coli K12
protein b2699 or its homologs, e.g. a protein with a DNA strand
exchange and recombination protein with protesase and
nuclease-activity, e.g. as indicated in Table I, columns 5 or 7,
lines 115 or 124, is increased conferring an increase of the
respective fine chemical, preferably of preferably of
beta-sitosterol between 14% and 35% or more or an increase of the
respective fine chemical, preferably of campesterol between 17% and
62% or more or an increase of the respective fine chemical,
preferably of beta-sitosterol and campesterol between 14% and 62%,
preferably between 17% and 35% or more.
[5300] In one embodiment, the activity of the Escherichia coli K12
protein b3256 or its homologs, e.g. a protein with acetyl CoA
carboxylase, biotin carboxylase subunit activity, in particular, of
the superfamiliy of biotin carboxylase, e.g. as indicated in Table
I, columns 5 or 7, line 123, is increased conferring an increase of
the respective fine chemical, preferably of campesterol between 20%
and 22% or more.
[5301] In case the activity of the Escherichia coli K12 protein
b0050 or its homologs e.g. a conserved protein potentially involved
in protein protein interaction e.g. as indicated in Table II,
columns 5 or 7, line 483, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
preferably of the beta-sitosterol between 15% and 24% or more is
conferred.
[5302] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0161 or its homologs e.g. a periplasmic serine
protease e.g. as indicated in Table II, columns 5 or 7, line 484 or
486, is increased, preferably of beta-sitosterol between 12% and
26% or more or an increase of the respective fine chemical,
preferably of campesterol between 17% and 42% or more or an
increase of the respective fine chemical, preferably of
beta-sitosterol and campesterol between 12% and 42%, preferably
between 17% and 26% or more.
[5303] In case the activity of the Escherichia coli K12 protein
b4129 or its homologs e.g. a lysine tRNA synthetase e.g. as
indicated in Table II, columns 5 or 7, line 485, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of the beta-sitosterol between 21% and 23% or more is
conferred.
[5304] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0464 or its homologs, e.g. the activity of a
transcriptional repressor for multidrug efflux pump (TetR/AcrR
family) protein as indicated in Table II, columns 5 or 7, line 487,
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of campesterol between 16% and 32% or
more is conferred.
[5305] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs, e.g. the activity of a
trehalose-6-phosphate synthase as indicated in Table II, columns 5
or 7, line 488, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of campesterol between
18% and 59% or more is conferred.
[5306] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2341 or its homologs, e.g. the activity of a
bifunctional anaerobic fatty acid oxidation complex protein:
enoyl-CoA hydratase/epimerase/isomerase (N-terminal);
3-hydroxyacyl-CoA dehydrogenase (C-terminal) as indicated in Table
II, columns 5 or 7, line 489, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
campesterol between 18% and 48% or more is conferred.
[5307] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2478 or its homologs, e.g. the activity of a
dihydrodipicolinate synthase as indicated in Table II, columns 5 or
7, line 490, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of campesterol between
17% and 37% or more is conferred.
[5308] In case the activity of the Escherichia coli K12 protein
b2822 or its homologs e.g. a DNA helicase, ATP-dependent
dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease e.g. as
indicated in Table II, columns 5 or 7, line 491, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of campesterol between 21% and 32% or more is
conferred.
[5309] [0046.0.12.12] In one embodiment, the activity of the
Saccharomyces cerevisiae protein YER156C or its homologs, e.g. an
activity of a Arabidopsis thaliana hypothetical protein F2K15.180,
e.g. as indicated in Table I, columns 5 or 7, lines 114 or 122
confers an increase of the respective fine chemical and of further
phytosterol activity-having compounds or their precursors.
[5310] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER173W or its homologs, e.g. an activity of a
checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints, e.g. as indicated in Table I,
columns 5 or 7, line 121, confers an increase of the respective
fine chemical and of further phytosterol activity-having compounds
or their precursors.
[5311] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YKR057W or its homologs, e.g. an activity of
ribosomal protein, similar to S21 ribosomal proteins, involved in
ribosome biogenesis and translation, e.g. as indicated in Table I,
columns 5 or 7, lines 113 or 120, confers an increase of the
respective fine chemical and of further phytosterol activity-having
compounds or their precursors. In one embodiment, the activity of
the Saccharomyces cerevisiae protein YOR044W or its homologs, e.g.
an activity of a probable membrane protein YOR044w, e.g. as
indicated in Table I, columns 5 or 7, line 119, confers an increase
of the respective fine chemical and of further phytosterol
activity-having compounds or their precursors. In one embodiment,
the activity of the Saccharomyces cerevisiae protein YOR084W or its
homologs, e.g. an activity of a putative lipase of the peroxisomal
matrix, e.g. as indicated in Table I, columns 5 or 7, line 112
confers an increase of the respective fine chemical and of further
phytosterol activity-having compounds or their precursors. In one
embodiment, the activity of the Saccharomyces cerevisiae protein
YPR172W or its homologs, e.g. an activity of a YPR172W protein,
e.g. as indicated in Table I, columns 5 or 7, line 118, confers an
increase of the respective fine chemical and of further phytosterol
activity-having compounds or their precursors.
[5312] In one embodiment the activity of the Escherichia coli K12
protein b0019 or its homologs, e.g. having a Na+/H+ antiporter
activity, in particular, of the superfamiliy of Na+/H+-exchanging
protein nhaA, e.g. as indicated in Table I, columns 5 or 7, line
117 confers an increase of the respective fine chemical and of
further phytosterol activity-having compounds or their
precursors.
[5313] In one embodiment, the activity of the Escherichia coli K12
protein b0421 or its homologs, e.g. with a geranyltranstransferase
(=farnesyldiphosphate synthase) activity, in particular, of the
superfamiliy of dimethylallyltrans-transferase,
geranyltranstransferase, e.g. as indicated in Table I, columns 5 or
7, line 116 confers an increase of the respective fine chemical and
of further phytosterol activity-having compounds or their
precursors.
[5314] In one embodiment, the activity of the Escherichia coli K12
protein b2699 or its homologs, e.g. a protein with a DNA strand
exchange and recombination protein with protesase and
nuclease-activity, e.g. as indicated in Table I, columns 5 or 7,
lines 115 or 124 confers an increase of the respective fine
chemical and of further phytosterol activity-having compounds or
their precursors.
[5315] In one embodiment, the activity of the Escherichia coli K12
protein b3256 or its homologs, e.g. a protein with acetyl CoA
carboxylase, biotin carboxylase subunit activity, in particular, of
the superfamiliy of biotin carboxylase, e.g. as indicated in Table
I, columns 5 or 7, line 123, confers an increase of the respective
fine chemical and of further phytosterol activity-having compounds
or their precursors.
[5316] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0050 or its homologs e.g. a conserved protein
potentially involved in protein protein interaction is increased,
preferably an increase of the fine chemical and of further
phytosterol activity-having compounds or their precursor is
conferred.
[5317] In case the activity of the Escherichia coli K12 protein
b0161 or its homologs e.g. a periplasmic serine protease is
increased, preferably, in one embodiment an increase of one or more
fine chemical, preferably an increase of one or more the fine
chemical and of further phytosterol activity-having compounds or
their precursor is conferred. In one embodiment, in case the
activity of the Escherichia coli K12 protein b4129 or its homologs
e.g. a lysine tRNA synthetase is increased, preferably, in one
embodiment an increase of the fine chemical, preferably an increase
of the fine chemical and of further phytosterol activity-having
compounds or their precursor is conferred. In one embodiment, in
case the activity of the Escherichia coli K12 protein b0464 or its
homologs e.g. a transcriptional repressor for multidrug efflux pump
(TetR/AcrR family) is increased, preferably, in one embodiment an
increase of the fine chemical, preferably an increase of the fine
chemical and of further phytosterol activity-having compounds or
their precursor is conferred.
[5318] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs e.g. a trehalose-6-phosphate
synthase is increased, preferably, in one embodiment an increase of
the fine chemical, preferably an increase of the fine chemical and
of further phytosterol activity-having compounds or their precursor
is conferred.
[5319] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2341 or its homologs e.g. a bifunctional
anaerobic fatty acid oxidation complex protein: enoyl-CoA
hydratase/epimerase/isomerase (N-terminal); 3-hydroxyacyl-CoA
dehydrogenase (C-terminal) is increased, preferably, in one
embodiment an increase of the fine chemical, preferably an increase
of the fine chemical and of further phytosterol activity-having
compounds or their precursor is conferred.
[5320] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2478 or its homologs e.g. a dihydrodipicolinate
synthase is increased, preferably, in one embodiment an increase of
the fine chemical, preferably an increase of the fine chemical and
of further phytosterol activity-having compounds or their precursor
is conferred.
[5321] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2822 or its homologs e.g. a DNA helicase,
ATP-dependent dsDNA/ssDNA exonuclease V subunit, ssDNA endonuclease
is increased, preferably, in one embodiment an increase of the fine
chemical, preferably an increase of the fine chemical and of
further phytosterol activity-having compounds or their precursor is
conferred.
[5322] [0047.0.0.12] to [0048.0.0.12] for the disclosure of the
paragraphs [0047.0.0.12] and [0048.0.0.12] see paragraphs
[0047.0.0.0] and [0048.0.0.0] above.
[5323] [0049.0.7.7] A protein having an activity conferring an
increase in the amount or level of the beta-sitosterol preferably
has the structure of the polypeptide described herein. In a
particular embodiment, the polypeptides used in the process of the
present invention or the polypeptide of the present invention
comprises the sequence of a consensus sequence as shown in Table
IV, column 7, lines 112 to 117 and 483 to 485 and/or the sequence
of a consensus sequence as indicated in Table IV, columns 5 or 7,
lines 112 to 117 and 483 to 485 and/or the sequence of a
polypeptide as indicated in Table II, columns 5 or 7, lines 112 to
117 and 483 to 485 or of a functional homologue thereof as
described herein, or of a polypeptide encoded by the nucleic acid
molecule characterized herein or the nucleic acid molecule
according to the invention, for example by a nucleic acid molecule
as indicated in Table I, columns 5 or 7, lines 112 to 117 and 483
to 485 or its herein described functional homologues and has the
herein mentioned activity conferring an increase in the
beta-sitosterol level.
[5324] A protein having an activity conferring an increase in the
amount or level of the campesterol preferably has the structure of
the polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence as shown in Table IV, column 7, lines 118 to 124
and 486 to 491 and/or the sequence of a consensus sequence as
indicated in Table IV, columns 5 or 7, lines 118 to 124 and 486 to
491 and/or the sequence of a polypeptide as indicated in Table II,
columns 5 or 7, lines 118 to 124 and 486 to 491 or of a functional
homologue thereof as described herein, or of a polypeptide encoded
by the nucleic acid molecule characterized herein or the nucleic
acid molecule according to the invention, for example by a nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines 118 to
124 and 486 to 491 or its herein described functional homologues
and has the herein mentioned activity conferring an increase in the
campesterol level.
[5325] [0050.0.12.12] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of phytosterols or their ester.
[5326] [0051.0.5.12] and [0052.0.0.12] for the disclosure of the
paragraphs [0051.0.5.12] and [0052.0.0.12] see paragraphs
[0051.0.0.0] and [0052.0.0.0] above.
[5327] [0053.0.12.12] In one embodiment, the process of the present
invention comprises one or more of the following steps [5328] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 112
to 117 or lines 483 to 485 or lines 118 to 124 or lines 486 to 491
or its homologs, e.g. as indicated in Table II, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 or lines 118 to 124 or lines
486 to 491, activity having herein-mentioned the respective fine
chemical-increasing activity; [5329] b) stabilizing a mRNA
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 112 to 117 or lines 483 to 485 or lines 118 to 124 or lines
486 to 491 or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 or lines 118
to 124 or lines 486 to 491, or of a mRNA encoding the polypeptide
of the present invention having herein-mentioned the respective
fine chemical-increasing activity; [5330] c) increasing the
specific activity of a protein conferring the increased expression
of a protein encoded by the nucleic acid molecule of the invention
or of the polypeptide of the present invention having
herein-mentioned the respective fine chemical-increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 112 to 117 or lines 483 to 485 or
lines 118 to 124 or lines 486 to 491 or its homologs activity, e.g.
as indicated in Table II, columns 5 or 7, lines 112 to 117 or lines
483 to 485 or lines 118 to 124 or lines 486 to 491, or decreasing
the inhibitory regulation of the polypeptide of the invention;
[5331] d) generating or increasing the expression of an endogenous
or artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned the respective fine
chemical-increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 112
to 117 or lines 483 to 485 or lines 118 to 124 or lines 486 to 491
or its homologs activity, e.g. as indicated in Table II, columns 5
or 7, lines 112 to 117 or lines 483 to 485 or lines 118 to 124 or
lines 486 to 491; [5332] e) stimulating activity of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the present invention or a polypeptide of
the present invention having herein-mentioned the respective fine
chemical-increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 112
to 117 or lines 483 to 485 or lines 118 to 124 or lines 486 to 491
or its homologs activity, e.g. as indicated in Table II, columns 5
or 7, lines 112 to 117 or lines 483 to 485 or lines 118 to 124 or
lines 486 to 491, by adding one or more exogenous inducing factors
to the organism or parts thereof; [5333] f) expressing a transgenic
gene encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned the respective fine chemical-increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 112 to 117 or lines 483 to 485 or
lines 118 to 124 or lines 486 to 491 or its homologs activity, e.g.
as indicated in Table II, columns 5 or 7, lines 112 to 117 or lines
483 to 485 or lines 118 to 124 or lines 486 to 491, and/or [5334]
g) increasing the copy number of a gene conferring the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or the
polypeptide of the invention having herein-mentioned the respective
fine chemical-increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 112
to 117 or lines 483 to 485 or lines 118 to 124 or lines 486 to 491
or its homologs, e.g. as indicated in Table II, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 or lines 118 to 124 or lines
486 to 491, activity. [5335] h) Increasing the expression of the
endogenous gene encoding the polypeptide of the invention, e.g. a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 112 to 117 or lines 483 to 485 or lines 118 to
124 or lines 486 to 491 or its homologs activity, e.g. as indicated
in Table II, columns 5 or 7, lines 112 to 117 or lines 483 to 485
or lines 118 to 124 or lines 486 to 491, by adding positive
expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; [5336] i)
Modulating growth conditions of an organism in such a manner, that
the expression or activity of the gene encoding the protein of the
invention or the protein itself is enhanced for example
microorganisms or plants can be grown for example under a higher
temperature regime leading to an enhanced expression of heat shock
proteins, which can lead an enhanced fine chemical production
and/or [5337] j) selecting of organisms with especially high
activity of the proteins of the invention from natural or from
mutagenized resources and breeding them into the target organisms,
eg the elite crops.
[5338] [0054.0.12.12] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table II, column 5, lines 112
to 117 or lines 483 to 485 or lines 118 to 124 or lines 486 to 491
resp., or its homologs activity, e.g. as indicated in Table II,
column 5, lines 112 to 117 or lines 483 to 485 or lines 118 to 124
or lines 486 to 491 resp.
[5339] [0055.0.0.12] to [0067.0.0.12] for the disclosure of the
paragraphs [0055.0.0.12] to [0067.0.0.12] see paragraphs
[0055.0.0.0] to [0067.0.0.0] above.
[5340] [0068.0.12.12] The mutation is introduced in such a way that
the production of the phytosterol(s) is not adversely affected.
[5341] [0069.0.0.12] for the disclosure of this paragraph see
paragraphs [0069.0.0.0] above.
[5342] [0070.0.12.12] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding the
YER156C, YKR057W, YOR084W, b0019, b0421, b2699, b0050, b0161 and/or
b4129 protein or of the YER156C, YER173W, YKR057W, YOR044W,
YPR172W, b2699, b3256, b0161, b0464, b1896, b2341, b2478 and/or
b2822 protein into an organism alone or in combination with other
genes, it is possible not only to increase the biosynthetic flux
towards the end product, but also to increase, modify or create de
novo an advantageous, preferably novel metabolites composition in
the organism, e.g. an advantageous fatty acid composition
comprising a higher content of (from a viewpoint of nutritional
physiology limited) phytosterol(s) etc.
[5343] [0071.0.5.12] for the disclosure of this paragraph see
[0071.0.0.0] above.
[5344] [0072.0.12.12] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to phytosterols further sterols, stanols or squalene,
squalene epoxide or cycloartenol.
[5345] [0073.0.12.12] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[5346] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [5347] b) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table II, columns 5 or 7, lines 112 to 117 or lines
483 to 485 or lines 118 to 124 or lines 486 to 491, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the respective fine chemical in an organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [5348] c) growing an organism,
preferably a microorganism, a non-human animal, a plant or animal
cell, a plant or animal tissue or a plant under conditions which
permit the production of the respective fine chemical in the
organism, preferably the microorganism, the plant cell, the plant
tissue or the plant; and [5349] d) if desired, revovering,
optionally isolating, the free and/or bound the respective fine
chemical and, optionally further free and/or bound phytosterol or
its conjugate synthesised by the organism, the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[5350] [0074.0.12.12] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the respective fine chemical or the free and
bound respective fine chemical but as option it is also possible to
produce, recover and, if desired isolate, other free or/and bound
of phytosterol(s) or its/their conjugates.
[5351] [0075.0.0.12] to [0077.0.0.12] for the disclosure of the
paragraphs [0075.0.0.12] to [0077.0.0.12] see paragraphs
[0075.0.0.0] to [0077.0.0.0] above.
[5352] [0078.0.12.12] The organism such as microorganisms or plants
or the recovered, and if desired isolated, respective fine chemical
can then be processed further directly into foodstuffs or animal
feeds or for other applications, for example according to the
disclosures made in:
US 20040101829, which disclose a methods for treating
hyperlipidemia and to reduce Low Density Lipoprotein ("LDL") levels
in a subject, US 20040047971, which disclose the preparation of a
fat composition containing sterol esters characterised by direct
interesterification of sterol with triglyceride, U.S. Pat. No.
5,965,449, which describes phytosterol-based compositions useful in
preventing and treating cardiovascular disease and other disorders,
U.S. Pat. No. 5,523,087, which is for a pharmaceutical composition
containing beta-sitosterol for the treatment of diabetic male
sexual dysfunction; U.S. Pat. No. 5,747,464, which discloses a
composition for inhibiting absorption of fat and cholesterol from
the gut comprising beta.-sitosterol bound irreversibly to pectin,
U.S. Pat. No. 4,588,717, which describes a vitamin supplement which
comprises a fatty acid ester of a phytosterol, U.S. Pat. No.
5,270,041, which teaches the use of small amounts of sterols, their
fatty acid esters and glucosides for the treatment of tumours, U.S.
Pat. No. 6,087,353, which comprises methods of making a composition
suitable for incorporation into foods, beverages, pharmaceuticals,
nutraceuticals and the like which comprises condensing a suitable
aliphatic acid with a phytosterol to form a phytosterol ester and
subsequently hydrogenating the phytosterol ester to form a
hydrogenated phytosterol ester, which are expressly incorporated
herein by reference.
[5353] The fermentation broth, fermentation products, plants or
plant products can be treated with water and a mixture of organic
solvents (hexane and acetone) in order to extract the phytosterols.
Crude phytosterols are obtained from the organic phase by removal
of the solvents, complexation of the sterols in the extract with
calcium chloride in methanol, separation of the sterol-complexes by
centrifugation, dissociation of the complexes by heating in water
and removal of the water. The crude phytosterols can be further
purified by crystallisation from isopropanol. According to an other
production process the tall oil soap is first subjected to
fractional distillation which removes volatile compounds. The
resulting residue (tall oil pitch) containing sterols in esterified
form is treated with alkali to liberate these sterols. After
neutralisation, the material is subjected to a two-stage
distillation process. The distillate is then dissolved in
methanol/methylethylketone solvent and the sterols crystallising
from this solution are obtained by filtration, washed with solvent
and dried. U.S. Pat. No. 4,420,427 teaches the preparation of
sterols from vegetable oil sludge using solvents such as methanol.
Alternatively, phytosterols may be obtained from tall oil pitch or
soap, by-products of the forestry practise as described in
PCT/CA95/00555, incorporated herein by reference. The extraction
and crystallization may be performed by other methods known to the
person skilled in the art and described herein below.
[5354] To form a phytosterol ester in accordance with the U.S. Pat.
No. 6,087,353, the selected phytosterol and aliphatic acid or its
ester with volatile alcohol are mixed together under reaction
conditions to permit condensation of the phytosterol with the
aliphatic acid to produce an ester. A most preferred method of
preparing these esters which is widely used in the edible fat and
oil industry is described in U.S. Pat. No. 5,502,045 (which is
incorporated herein by reference). The stanol and/or sterol esters
with the desired fatty acid composition can also be produced by
direct, preferably catalytic esterification methods, e.g. U.S. Pat.
No. 5,892,068, between free fatty acids or fatty acid blends of the
composition and the stanol and/or sterol. In addition, stanol
and/or sterol esters can also be produced by enzymatic
esterification e.g. as outlined in EP 195 311 (which are
incorporated herein by reference).
[5355] Products of these different work-up procedures are
phytosterols and/or esters and/or conjugates or compositions which
still comprise fermentation broth, plant particles and cell
components in different amounts, advantageously in the range of
from 0 to 99% by weight, preferably below 80% by weight, especially
preferably between below 50% by weight.
[5356] [0079.0.0.12] to [0084.0.0.12] for the disclosure of the
paragraphs [0079.0.0.12] to [0084.0.0.12] see paragraphs
[0079.0.0.0] to [0084.0.0.0] above.
[5357] [0085.0.12.12] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [5358] a) a nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 112 to 117 or lines 483
to 485 or lines 118 to 124 or lines 486 to 491, resp. or a
derivative thereof, or [5359] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 112 to
117 or lines 483 to 485 or lines 118 to 124 or lines 486 to 491,
resp. or a derivative thereof, or [5360] c) (a) and (b) is/are not
present in its/their natural genetic environment or has/have been
modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[5361] [0086.0.0.12] and [0087.0.0.12] for the disclosure of the
paragraphs [0086.0.0.12] and [0087.0.0.12] see paragraphs
[0086.0.0.0] and [0087.0.0.0] above.
[5362] [0088.0.12.12] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose phytosterol
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for poultry is dependent on the abovementioned phytosterol and the
general amount of phytosterol as source in feed and/or food.
Further, this is also important since, for example a balanced
content of different phytosterols induces stress resistance to
plants. After the YER156C, YKR057W, YOR084W, b0019, b0421, b2699,
b0050, b0161 and/or b4129 and/or YER156C, YER173W, YKR057W,
YOR044W, YPR172W, b2699, b3256, b0161, b0464, b1896, b2341, b2478
and/or b2822 protein activity has been increased or generated, or
after the expression of nucleic acid molecule or polypeptide
according to the invention has been generated or increased, the
transgenic plant generated thus is grown on or in a nutrient medium
or else in the soil and subsequently harvested.
[5363] [0088.1.0.12], [0089.0.0.12] and [0090.0.0.12] for the
disclosure of the paragraphs [0088.1.0.12], [0089.0.0.12] and
[0090.0.0.12] see paragraphs [0088.1.0.0],
[5364] [0089.0.0.0] and [0090.0.0.0] above.
[5365] [0091.0.12.12] Thus, the content of plant components and
preferably also further impurities is as low as possible, and the
abovementioned phytosterols are obtained in as pure form as
possible. In these applications, the content of plant components
advantageously amounts to less than 10%, preferably 1%, more
preferably 0.1%, very especially preferably 0.01% or less.
[5366] [0092.0.0.12] to [0094.0.0.12] for the disclosure of the
paragraphs [0092.0.0.12] to [0094.0.0.12] see paragraphs
[0092.0.0.0] to [0094.0.0.0] above.
[5367] [0095.0.12.12] It may be advantageous to increase the pool
of said phytosterols in the transgenic organisms by the process
according to the invention in order to isolate high amounts of the
pure respective fine chemical.
[5368] [0096.0.12.12] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example
beta-sitosterol or campesterol in the organism, is useful to
increase the production of the respective fine chemical.
[5369] [0097.0.0.12] for the disclosure of this paragraph see
paragraph [0097.0.0.0] above.
[5370] [0098.0.12.12] In a preferred embodiment, the respective
fine chemical (beta-sitosterol or campesterol) is produced in
accordance with the invention and, if desired, is isolated. The
production of further phytosterols or conjugates or mixtures
thereof or mixtures with other compounds by the process according
to the invention is advantageous.
[5371] [0099.0.12.12] In the case of the fermentation of
microorganisms, the abovementioned phytosterol (preferably
beta-sitosterol and/or campesterol) accumulate in the medium and/or
the cells. If microorganisms are used in the process according to
the invention, the fermentation broth can be processed after the
cultivation. Depending on the requirement, all or some of the
biomass can be removed from the fermentation broth by separation
methods such as, for example, centrifugation, filtration, decanting
or a combination of these methods, or else the biomass can be left
in the fermentation broth. The fermentation broth can subsequently
be reduced, or concentrated, with the aid of known methods such as,
for example, rotary evaporator, thin-layer evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. Afterwards
advantageously further compounds for formulation can be added such
as corn starch or silicates. This concentrated fermentation broth
advantageously together with compounds for the formulation can
subsequently be processed by lyophilization, spray drying, and
spray granulation or by other methods. Preferably the respective
fine chemical or the phytosterol (preferably beta-sitosterol and/or
campesterol) comprising compositions are isolated from the
organisms, such as the microorganisms or plants or the culture
medium in or on which the organisms have been grown, or from the
organism and the culture medium, in the known manner, for example
via extraction, distillation, crystallization, chromatography or a
combination of these methods. These purification methods can be
used alone or in combination with the aforementioned methods such
as the separation and/or concentration methods.
[5372] [0100.0.12.12] Transgenic plants which comprise the
phytosterol (preferably beta-sitosterol and/or campesterol)
synthesized in the process according to the invention can
advantageously be marketed directly without there being any need
for the phytosterols (preferably beta-sitosterol and/or
campesterol) (oils, lipids or fatty acids synthesized to be
isolated). Plants for the process according to the invention are
listed as meaning intact plants and all plant parts, plant organs
or plant parts such as leaf, stem, seeds, root, tubers, anthers,
fibers, root hairs, stalks, embryos, calli, cotelydons, petioles,
harvested material, plant tissue, reproductive tissue and cell
cultures which are derived from the actual transgenic plant and/or
can be used for bringing about the transgenic plant. In this
context, the seed comprises all parts of the seed such as the seed
coats, epidermal cells, seed cells, endosperm or embryonic tissue.
However, the respective fine chemical produced in the process
according to the invention can also be isolated from the organisms,
advantageously plants, in the form of their oils, fats, lipids,
esters and/or as extracts, e.g. ether, alcohol, or other organic
solvents or water containing extract and/or free phytosterol(s).
The respective fine chemical produced by this process can be
obtained by harvesting the organisms, either from the crop in which
they grow, or from the field. This can be done via pressing or
extraction of the plant parts, preferably the plant seeds. To
increase the efficiency of extraction it is beneficial to clean, to
temper and if necessary to hull and to flake the plant material
especially the seeds. In this context, the oils, fats, lipids,
esters and/or free phytosterols can be obtained by what is known as
cold beating or cold pressing without applying heat. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they are previously comminuted, steamed or roasted. The
seeds, which have been pretreated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. In this manner, more than 96% of the compounds produced
in the process can be isolated. Thereafter, the resulting products
are processed further, i.e. degummed and/or refined. In this
process, substances such as the plant mucilages and suspended
matter are first removed. What is known as desliming can be
affected enzymatically or, for example, chemico-physically by
addition of acid such as phosphoric acid.
[5373] Plant sterols (phytosterols) are by-products of traditional
vegetable oil refining. The source may be commonly a blend of crude
edible oils, consisting of soy bean oil or of other edible oils,
e.g. corn, rapeseed, olive and palm oil in varying proportions.
Hemp may also be a source of new oilseed, oil and food ingredients
as well as Sea buckthorn (hippophae rhamnoides). The crude oil,
which is obtained by pressing or solvent extraction, may undergoes
a series of refining processes to remove solvents, lecithins, free
fatty acids, color bodies, off-odors and off-flavors. In one of
these steps, the oil may be subjected to steam distillation at
reduced pressure (deodorisation) and the resulting distillate
contains the phytosterol fraction. From this fraction, fatty acids,
lecithins and other compounds are removed by fractional
distillation, ethanolysis/transesterification, distillation and
crystallisation from a heptane solution, and the phytosterols are
further purified by recrystallisation using food grade materials
and good manufacturing practices. The extraction and purification
steps are standard methods and similar to the procedures used
traditionally by the food industry for the production of plant
sterols. Phytosterol esters may be produced from the sterols using
food grade vegetable oil-derived fatty acids or triglycerides and
applying standard methods for esterification or transesterification
commonly used in the fats and oils industry.
[5374] Phytosterol in microorganisms may be localized
intracellularly, therefor their recovery essentials comes down to
the isolation of the biomass. Well-establisthed approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Determination of
tocopherols in cells has been described by Tan and Tsumura 1989,
see also Biotechnology of Vitamins, Pigments and Growth Factors,
Edited by Erik J. Vandamme, London, 1989, p. 96 to 103. Many
further methods to determine the tocopherol content are known to
the person skilled in the art.
[5375] [0101.0.12.12] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michel, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[5376] [0102.0.12.12] Phytosterols can for example be analyzed
advantageously via HPLC or GC separation methods and detected by MS
oder MSMS methods. The unambiguous detection for the presence of
beta-sitosterol and/or campesterol containing products can be
obtained by analyzing recombinant organisms using analytical
standard methods: GC, GC-MS or TLC, as described on several
occasions by Christie and the references therein (1997, in:
Advances on Lipid Methodology, Fourth Edition: Christie, Oily
Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfah ren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). The
material to be analyzed can be disrupted by sonication, grinding in
a glass mill, liquid nitrogen and grinding, cooking, or via other
applicable methods; see also Biotechnology of Vitamins, Pigments
and Growth Factors, Edited by Erik J. Vandamme, London, 1989, p. 96
to 103.
[5377] For the sterol isolation and analysis the German standard
method F III (1) may be used. The method comprises: saponification
of fat, isolation of unsaponifiable matter using an aliminium oxide
column, separation of sterol fraction by preparative TLC and
determination of the composition of sterols as trimetysilyl ethers
by GLC.
[5378] [0103.0.12.12] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [5379] a) nucleic acid molecule encoding, preferably
at least the mature form, of the polypeptide having a sequence as
indicated in Table II, columns 5 or 7, lines 112 to 117 or lines
483 to 485 or lines 118 to 124 or lines 486 to 491, resp. or a
fragment thereof, which confers an increase in the amount of the
respective fine chemical in an organism or a part thereof; [5380]
b) nucleic acid molecule comprising, preferably at least the mature
form, of a nucleic acid molecule having a sequence as indicated in
Table I, columns 5 or 7, lines 112 to 117 or lines 483 to 485 or
lines 118 to 124 or lines 486 to 491, resp. [5381] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [5382] d) nucleic acid molecule encoding a polypeptide
which has at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [5383] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [5384] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [5385] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [5386]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers pairs having a
sequence as indicated in Table III, column 7, lines 112 to 117 or
lines 483 to 485 or lines 118 to 124 or lines 486 to 491, resp. and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [5387] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from an
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [5388] j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence having a sequences as
indicated in Table IV, column 7, lines 112 to 117 or lines 483 to
485 or lines 118 to 124 or lines 486 to 491, resp., and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [5389] k) nucleic acid molecule
comprising one or more of the nucleic acid molecule encoding the
amino acid sequence of a polypeptide encoding a domain of the
polypeptide indicated in Table II, columns 5 or 7, lines 112 to 117
or lines 483 to 485 or lines 118 to 124 or lines 486 to 491, resp.
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; and [5390] l) nucleic
acid molecule which is obtainable by screening a suitable library
under stringent conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k), preferably to
(a) to (c), or with a fragment of at least 15 nt, preferably 20 nt,
30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k), preferably to (a) to (c), and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which comprises a
sequence which is complementary thereto.
[5391] [00103.1.0.12.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491, by
one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence shown in indicated in Table I A, columns 5 or 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491: In one embodiment, the nucleic acid molecule used in the
process of the invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence indicated in Table I A, columns 5 or
7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491. In another embodiment, the nucleic acid molecule
does not encode a polypeptide of a sequence indicated in Table II
A, columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491.
[5392] [00103.2.0.12.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, lines 112 to 117
or lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491, by
one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence shown in indicated in Table I B, columns 5 or 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491. In one embodiment, the nucleic acid molecule used in the
process of the invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence indicated in Table I B, columns 5 or
7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491. In another embodiment, the nucleic acid molecule
does not encode a polypeptide of a sequence indicated in Table II
B, columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491.
[5393] [0104.0.12.12] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence indicated in
Table I, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp., by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of the sequence
indicated in Table I, columns 5 or 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, resp., In one
embodiment, the nucleic acid molecule of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to the
sequence indicated in Table I, columns 5 or 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp.
In another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp.
[5394] [0105.0.0.12] to [0107.0.0.12] for the disclosure of the
paragraphs [0105.0.0.12] to [0107.0.0.12] see paragraphs
[0105.0.0.0] and [0107.0.0.0] above.
[5395] [0108.0.12.12] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, resp., nucleic
acid molecules which are derived from an amino acid sequences as
indicated in Table II, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp., or
from polypeptides comprising the consensus sequence as indicated in
Table IV, column 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491 resp. or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of an activity of a polypeptide as indicated in Table II,
column 3, 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp., or conferring an increase of
the respective fine chemical, meaning phytosterol, in particular,
beta-sitosterol and/or campesterol after increasing its expression
or activity are advantageously increased in the process according
to the invention.
[5396] [0109.0.0.12] for the disclosure of this paragraph see
[0109.0.0.0] above.
[5397] [0110.0.12.12] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide used in the
method of the invention or used in the process of the invention,
e.g. of a protein as shown in Table II, columns 5 or 7, lines 112
to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491 or being encoded by a nucleic acid molecule indicated in Table
I, columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491 or of its homologs, e.g. as
indicated in Table II, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491 can be
determined from generally accessible databases.
[5398] [0111.0.0.12] for the disclosure of this paragraph see
[0111.0.0.0] above.
[5399] [0112.0.12.12] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table II, column 3, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
4914 resp., or having the sequence of a polypeptide as indicated in
Table II, columns 5 and 7 lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491 resp., and conferring
an increase in the level of phytosterols, preferably
beta-sitosterol and/or campesterol.
[5400] [0113.0.0.12] to [0120.0.0.12] for the disclosure of the
paragraphs [0113.0.0.12] to [0120.0.0.12] see paragraphs
[0113.0.0.0] and [0120.0.0.0] above.
[5401] [0121.0.12.12] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp., or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring an increase in the level of
beta-sitosterol after increasing the activity of the polypeptide
sequences indicated in Table II, columns 5 or 7, lines 112 to 117
and/or lines 483 to 485, or conferring increase in the level of
campesterol after increasing the activity of the polypeptide
sequences indicated in Table II, columns 5 or 7, lines 118 to 124
and/or lines 486 to 491.
[5402] [0122.0.0.12] to [0127.0.0.12] for the disclosure of the
paragraphs [0122.0.0.12] to [0127.0.0.12] see paragraphs
[0122.0.0.0] and [0127.0.0.0] above.
[5403] [0128.0.12.12] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, column 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp. by means of polymerase chain reaction can
be generated on the basis of a sequence shown herein, for example
the sequence as indicated in Table I, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp. or the sequences derived from a sequence as indicated in
Table II, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp.
[5404] [0129.0.12.12] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved regions are those, which show a
very little variation in the amino acid in one particular position
of several homologs from different origin. The consensus sequences
indicated in Table IV, column 7, lines 112 to 117 or lines 483 to
485 and/or lines 118 to 124 or lines 486 to 491, resp. are derived
from said alignments.
[5405] [0130.0.12.12] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of
phytosterol, preferably beta-sitosterol and/or campesterol resp
after increasing the expression or activity of the protein
comprising said fragment.
[5406] [0131.0.0.12] to [0138.0.0.12] for the disclosure of the
paragraphs [0131.0.0.12] to [0138.0.0.12] see paragraphs
[0131.0.0.0] to [0138.0.0.0] above.
[5407] [0139.0.12.12] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical increase, derived from
other organisms, can be encoded by other DNA sequences which
hybridize to a sequences indicated in Table I, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, preferably of Table I B, columns 5 or 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491 under relaxed hybridization conditions and which code on
expression for peptides having the phytosterol, preferably
beta-sitosterol and/or campesterol increasing activity.
[5408] [0140.0.0.12] to [0146.0.0.12] for the disclosure of the
paragraphs [0140.0.0.12] to [0146.0.0.12] see paragraphs
[0140.0.0.0] to [0146.0.0.0] above.
[5409] [0147.0.12.12] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491, preferably of Table I B,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491 resp., is one which is sufficiently
complementary to one of said nucleotide sequences such that it can
hybridize to one of said nucleotide sequences, thereby forming a
stable duplex. Preferably, the hybridisation is performed under
stringent hybridization conditions. However, a complement of one of
the herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[5410] [0148.0.12.12] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491, preferably of Table I B, columns 5 or 7, lines 112 to 117
or lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491
resp. or a portion thereof and preferably has above mentioned
activity, in particular having a phytosterol, in particular, of
beta-sitosterol and/or campesterol increasing activity after
increasing the activity or an activity of a product of a gene
encoding said sequences or their homologs.
[5411] [0149.0.12.12] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, preferably of Table I B, columns 5 or 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491 resp. or a portion thereof and encodes a protein having
above-mentioned activity, e.g. conferring increase of phytosterol,
in particular, of beta-sitosterol and/or campesterol resp., and
optionally, the activity of a protein indicated in Table II, column
5, lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491.
[5412] [00149.1.12.12] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table I, columns 5 or 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
preferably of Table I B, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491 resp., has
further one or more of the activities annotated or known for the a
protein as indicated in Table II, column 3, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491.
[5413] [0150.0.12.12] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table I, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, preferably of Table I B, columns 5 or 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491 resp.
for example a fragment which can be used as a probe or primer or a
fragment encoding a biologically active portion of the polypeptide
of the present invention or of a polypeptide used in the process of
the present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of phytosterol, in particular, of
beta-sitosterol and/or campesterol resp., if its activity is
increased. The nucleotide sequences determined from the cloning of
the present protein-according-to-the-invention-encoding gene allows
for the generation of probes and primers designed for use in
identifying and/or cloning its homologues in other cell types and
organisms. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, 15 preferably about 20 or 25, more
preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one of the sequences set forth, e.g., as indicated in
Table I, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp. an anti-sense
sequence of one of the sequences, e.g., as indicated in Table I,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp. or naturally occurring
mutants thereof. Primers based on a nucleotide of invention can be
used in PCR reactions to clone homologues of the polypeptide of the
invention or of the polypeptide used in the process of the
invention, e.g. as the primers described in the examples of the
present invention, e.g. as shown in the examples. A PCR with the
primer pairs indicated in Table III, column 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp., will result in a fragment of a polynucleotide sequence as
indicated in Table I, columns 5 or 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, resp., or its
gene product.
[5414] [0151.0.0.12] for the disclosure of this paragraph see
paragraph [0151.0.0.0] above.
[5415] [0152.0.12.12] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 112 to 117
or lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp., such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular an activity increasing the level of phytosterol, in
particular, of beta-sitosterol and/or campesterol, resp., as
mentioned above or as described in the examples in plants or
microorganisms is comprised.
[5416] [0153.0.12.12] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 112
to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491 resp., such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table II, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp., has
for example an activity of a polypeptide as indicated in Table II,
column 3, lines 112 to 117 or lines 483 to 485 and/or lines 118 to
124 or lines 486 to 491, resp.
[5417] [0154.0.12.12] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 112 to 117
or lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp., and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[5418] [0155.0.0.12] and [0156.0.0.12] for the disclosure of the
paragraphs [0155.0.0.12] and [0156.0.0.12] see paragraphs
[0155.0.0.0] and [0156.0.0.0] above.
[5419] [0157.0.12.12] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences as
indicated in Table I, columns 5 or 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, resp., (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides encoded by the sequence as indicated in Table I,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp., or of the polypeptide as
indicated in Table II, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp., or
the functional homologues. Advantageously, the nucleic acid
molecule of the invention comprises, or in an other embodiment has,
a nucleotide sequence encoding a protein comprising, or in an other
embodiment having, an amino acid sequence of a consensus sequences
as indicated in Table IV, column 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, resp., or of
the polypeptide as indicated in Table II, columns 5 or 7, lines 112
to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp., or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
IV, column 7, lines 112 to 117 or lines 483 to 485 and/or lines 118
to 124 or lines 486 to 491, resp., or of a polypeptide as indicated
in Table II, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp., or the
functional homologues. However, in a preferred embodiment, the
nucleic acid molecule of the present invention does not consist of
a sequence as indicated in Table I, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, preferably as indicated in Table I A, columns 5 or 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491, resp. Preferably the nucleic acid molecule of the invention
is a functional homologue or identical to a nucleic acid molecule
indicated in Table I B, column 7, lines 112 to 117 or lines 483 to
485 and/or lines 118 to 124 or lines 486 to 491.
[5420] [0158.0.0.12] to [0160.0.0.12] for the disclosure of the
paragraphs [0158.0.0.12] to [0160.0.0.12] see paragraphs
[0158.0.0.0] to [0160.0.0.0] above.
[5421] [0161.0.12.12] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 112 to 117
or lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[5422] [0162.0.0.12] for the disclosure of this paragraph see
paragraph [0162.0.0.0] above.
[5423] [0163.0.12.12] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp.,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[5424] [0164.0.0.12] for the disclosure of this paragraph see
paragraph [0164.0.0.0] above.
[5425] [0165.0.12.12] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, resp.
[5426] [0166.0.0.12] and [0167.0.0.12] for the disclosure of the
paragraphs [0166.0.0.12] and [0167.0.0.12] see paragraphs
[0166.0.0.0] and [0167.0.0.0] above.
[5427] [0168.0.12.12] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp., yet retain said activity described herein.
The nucleic acid molecule can comprise a nucleotide sequence
encoding a polypeptide, wherein the polypeptide comprises an amino
acid sequence at least about 50% identical to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 112 to 117
or lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp., and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table II, columns 5 or 7, lines 112 to 117
or lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp. more preferably at least about 70% identical to one of the
sequences as indicated in Table II, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp. even more preferably at least about 80%, 90%, 95%
homologous to a sequence as indicated in Table II, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp. and most preferably at least about 96%,
97%, 98%, or 99% identical to the sequence as indicated in Table
II, columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491. Accordingly, the invention
relates to nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table II,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, preferably of Table II B, column 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491 yet retain said activity described herein. The
nucleic acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table II, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, preferably
of Table II B, column 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491 and is capable of
participation in the increase of production of the respective fine
chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table II,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, preferably of Table II B, column 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, more preferably at least about 70% identical to
one of the sequences as indicated in Table II, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, preferably of Table II B, column 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table II, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, preferably of Table II B, column 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, and most preferably at least about 96%, 97%, 98%, or 99%
identical to the sequence as indicated in Table II, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, preferably of Table II B, column 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491.
[5428] [0169.0.0.12] to [0174.0.0.12] for the disclosure of the
paragraphs [0169.0.0.12] to [0174.0.0.12] see paragraphs
[0169.0.0.0] to [0174.0.0.0] above.
[5429] [0175.0.12.12] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 12136 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 12136 by the above program algorithm with the
above parameter set, has a 80% homology.
[5430] [0176.0.12.12] Functional equivalents derived from one of
the polypeptides as indicated in Table II, columns 5 or 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491, resp., according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp., according to the invention
and are distinguished by essentially the same properties as a
polypeptide as indicated in Table II, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp.
[5431] [0177.0.12.12] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp., according to the invention by substitution, insertion
or deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at
least 55%, 60%, 65% or 70% by preference at least 80%, especially
preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very
especially preferably at least 95%, 97%, 98% or 99% homology with
one of the polypeptides as indicated in Table II, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp., according to the invention and encode
polypeptides having essentially the same properties as a
polypeptide as indicated in Table II, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp.
[5432] [0178.0.0.12] for the disclosure of this paragraph see
[0178.0.0.0] above.
[5433] [0179.0.12.12] A nucleic acid molecule encoding an
homologous to a protein sequence as indicated in Table II, columns
5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to
124 or lines 486 to 491, preferably of Table II B, column 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491, resp., can be created by introducing one or more nucleotide
substitutions, additions or deletions into a nucleotide sequence of
the nucleic acid molecule of the present invention, in particular
as indicated in Table I, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp., such
that one or more amino acid substitutions, additions or deletions
are introduced into the encoded protein. Mutations can be
introduced into the encoding sequences of a sequence as indicated
in Table I, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp., by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[5434] [0180.0.0.12] to [0183.0.0.12] for the disclosure of the
paragraphs [0180.0.0.12] to [0183.0.0.12] see paragraphs
[0180.0.0.0] to [0183.0.0.0] above.
[5435] [0184.0.12.12] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table I, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, preferably of Table I B, column 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp., or of the nucleic acid sequences derived from a
sequences as indicated in Table II, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, preferably of Table II B, column 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp.
comprise also allelic variants with at least approximately 30%,
35%, 40% or 45% homology, by preference at least approximately 50%,
60% or 70%, more preferably at least approximately 90%, 91%, 92%,
93%, 94% or 95% and even more preferably at least approximately
96%, 97%, 98%, 99% or more homology with one of the nucleotide
sequences shown or the abovementioned derived nucleic acid
sequences or their homologues, derivatives or analogues or parts of
these. Allelic variants encompass in particular functional variants
which can be obtained by deletion, insertion or substitution of
nucleotides from the sequences shown, preferably from a sequence as
indicated in Table I, columns 5 or 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, resp., or from
the derived nucleic acid sequences, the intention being, however,
that the enzyme activity or the biological activity of the
resulting proteins synthesized is advantageously retained or
increased.
[5436] [0185.0.12.12] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table I, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, preferably of Table I
B, column 7, lines 112 to 117 or lines 483 to 485 and/or lines 118
to 124 or lines 486 to 491, resp. In one embodiment, it is
preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table I, columns 5 or 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, preferably of
Table I B, column 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491, resp. In one embodiment, the
nucleic acid molecule comprises less than 500, 400, 300, 200, 100,
90, 80, 70, 60, 50 or 40 further nucleotides. In a further
embodiment, the nucleic acid molecule comprises less than 30, 20 or
10 further nucleotides. In one embodiment, a nucleic acid molecule
used in the process of the invention is identical to a sequences as
indicated in Table I, columns 5 or 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, preferably of
Table I B, column 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491, resp.
[5437] [0186.0.12.12] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to
124 or lines 486 to 491, preferably of Table II B, column 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491, resp. In one embodiment, the nucleic acid molecule encodes
less than 150, 130, 100, 80, 60, 50, 40 or 30 further amino acids.
In a further embodiment, the encoded polypeptide comprises less
than 20, 15, 10, 9, 8, 7, 6 or 5 further amino acids. In one
embodiment, the encoded polypeptide used in the process of the
invention is identical to the sequences as indicated in Table II,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, preferably of Table II B, column 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp.
[5438] [0187.0.12.12] In one embodiment, a nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, preferably of Table II B, column 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp., comprises less than 100 further nucleotides. In a
further embodiment, said nucleic acid molecule comprises less than
30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence of
the amino sequences indicated in Table II, columns 5 or 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491, preferably of Table II B, column 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp.
[5439] [0188.0.12.12] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491, resp., and is expressed under
identical conditions. In one embodiment, the polypeptide of the
invention is a homolog consisting of or comprising the sequence as
indicated in Table II B, columns 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491.
[5440] [0189.0.12.12] Homologues of a sequences as indicated in
Table I, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp., or of a derived
sequences as indicated in Table II, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp., also mean truncated sequences, cDNA, single-stranded
DNA or RNA of the coding and noncoding DNA sequence. Homologues of
said sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[5441] [0190.0.0.12] to [0203.0.0.12] for the disclosure of the
paragraphs [0190.0.0.12] to [0203.0.0.12] see paragraphs
[0190.0.0.0] to [0203.0.0.0] above.
[5442] [0204.0.12.12] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [5443] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, preferably of Table II B, column 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp.; or a
fragment thereof conferring an increase in the amount of the
respective fine chemical, i.e. phytosterol, in particular, of
beta-sitosterol (lines 112 to 117 and/or lines 483 to 485) and/or
campesterol (lines 118 to 124 and/or lines 486 to 491) resp., in an
organism or a part thereof [5444] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 112 to 117
or lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
preferably of Table I B, column 7, lines 112 to 117 or lines 483 to
485 and/or lines 118 to 124 or lines 486 to 491, resp., or a
fragment thereof conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [5445]
c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [5446] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [5447] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[5448] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[5449] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [5450] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table III, column 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, resp., and
conferring an increase in the amount of the respective fine
chemical, i.e. phytosterol, in particular, of beta-sitosterol
(lines 112 to 117) and/or campesterol (lines 118 to 124) resp., in
an organism or a part thereof; [5451] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from a expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [5452] j) nucleic acid molecule which encodes a
polypeptide comprising a consensus sequence as indicated in Table
IV, column 7, lines 112 to 117 or lines 483 to 485 and/or lines 118
to 124 or lines 486 to 491, resp., and conferring an increase in
the amount of the respective fine chemical, i.e. phytosterol, in
particular, of beta-sitosterol (lines 112 to 117 and/or lines 483
to 485) and/or campesterol (lines 118 to 124 and/or lines 486 to
491) resp., in an organism or a part thereof; [5453] k) nucleic
acid molecule encoding the amino acid sequence of a polypeptide
encoding a domaine of a polypeptide as indicated in Table II,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, preferably of Table II B, column 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp., and conferring an increase in the amount
of the respective fine chemical, i.e. phytosterol, in particular,
of beta-sitosterol (lines 112 to 117 and/or lines 483 to 485)
and/or campesterol (lines 118 to 124 and/or lines 486 to 491)
resp., in an organism or a part thereof; and [5454] l) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment of at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (h) or of a nucleic acid molecule as
indicated in Table I, columns 5 or 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, preferably of
Table I B, column 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491, resp., or a nucleic acid
molecule encoding, preferably at least the mature form of, a
polypeptide as indicated in Table II, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, preferably of Table II B, column 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp. and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which encompasses a
sequence which is complementary thereto; whereby, preferably, the
nucleic acid molecule according to (a) to (l) distinguishes over a
sequence as indicated in Table IA, columns 5 or 7, lines 112 to 117
or lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp., by one or more nucleotides. In one embodiment, the nucleic
acid molecule of the invention does not consist of the sequence as
indicated in Table IA, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp., In
an other embodiment, the nucleic acid molecule of the present
invention is at least 30% identical and less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to a sequence as indicated in Table
IA, columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491, resp. In a further embodiment
the nucleic acid molecule does not encode a polypeptide sequence as
indicated in Table IIA, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp.
Accordingly, in one embodiment, the nucleic acid molecule of the
present invention encodes in one embodiment a polypeptide which
differs at least in one or more amino acids from a polypeptide
indicated in Table IIA, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp., does
not encode a protein of a sequence as indicated in Table IIA,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp. Accordingly, in one
embodiment, the protein encoded by a sequences of a nucleic acid
according to (a) to (l) does not consist of a sequence as indicated
in Table IIA, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp. In a further
embodiment, the protein of the present invention is at least 30%
identical to a protein sequence indicated in Table IIA, columns 5
or 7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124
or lines 486 to 491, resp., and less than 100%, preferably less
than 99.999%, 99.99% or 99.9%, more preferably less than 99%, 985,
97%, 96% or 95% identical to a sequence as indicated in Table IIA,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp.
[5455] [0205.0.0.12] and [0206.0.0.12] for the disclosure of the
paragraphs [0205.0.0.12] and [0206.0.0.12] see paragraphs
[0205.0.0.0] and [0206.0.0.0] above.
[5456] [0207.0.12.12] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the sterol metabolism, the squalen
metabolism, the amino acid metabolism, of glycolysis, of the
tricarboxylic acid metabolism or their combinations. As described
herein, regulator sequences or factors can have a positive effect
on preferably the gene expression of the genes introduced, thus
increasing it. Thus, an enhancement of the regulator elements may
advantageously take place at the transcriptional level by using
strong transcription signals such as promoters and/or enhancers. In
addition, however, an enhancement of translation is also possible,
for example by increasing mRNA stability or by inserting a
translation enhancer sequence.
[5457] [0208.0.0.12] to [0226.0.0.12] for the disclosure of the
paragraphs [0208.0.0.12] to [0226.0.0.12] see paragraphs
[0208.0.0.0] to [0226.0.0.0] above.
[5458] [0227.0.12.12] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[5459] In addition to a sequence indicated in Table I, columns 5 or
7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491 resp., or its derivatives, it is advantageous
additionally to express and/or mutate further genes in the
organisms. Especially advantageously, additionally at least one
further gene of the sterol biosynthetic pathway such as for a
phytosterol precursor, for example squalene epoxide is expressed in
the organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine one or more of the sequences indicated in
Table I, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp., with genes
which generally support or enhances to growth or yield of the
target organisms, for example genes which lead to faster growth
rate of microorganisms or genes which produces stress-, pathogen,
or herbicide resistant plants.
[5460] [0228.0.12.12] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the
sterol/phytosterol metabolism, in particular in synthesis of of
beta-sitosterol and/or campesterol.
[5461] [0229.0.12.12] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the phytosterol biosynthetic
pathway, such as the enzymes catalyzing the production of acetyl
CoA HMGCoA, mevalonate, mevalonate 5 phosphate, mevalonate
5-pyrophosphate, isopentyl diphosphate, 5-pyrophosphatemevalonate,
isopentyl pyrophosphate (PIP), dimethylallyl pyrophosphate (DMAPP),
PIP+DMAPP, geranyl pyrophosphate+IPP, farnesyl pyrophosphate, 2
farnesyl pyrophosphate, squalene (squalene synthase) and squalene
epoxide, or cycloartenol synthase controling the cyclization of
squalene epoxide, S-adenosyl-L-methionine:sterol C-24 methyl
transferase (EC 2.1.1.41) (SMT1) catalyzing the transfer of a
methyl group from a cofactor, SMT2 catalyzing the second methyl
transfer reaction, sterol C-14 demethylase catalyzing the
demethylation at C-14, removing the methyl group and creating a
double bond. These genes can lead to an increased synthesis of the
essential phytosterol, in particular, of beta-sitosterol and/or
campesterol resp.
[5462] [0230.0.0.12] for the disclosure of this paragraph see
paragraph [0230.0.0.0] above.
[5463] [0231.0.12.12] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a protein degrading phytosterol, in
particular, of beta-sitosterol and/or campesterol, resp., is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[5464] [0232.0.0.5] to [0276.0.0.5] for the disclosure of the
paragraphs [0232.0.0.5] to [0276.0.0.5] see paragraphs [0232.0.0.0]
to [0276.0.0.0] above.
[5465] [0277.0.12.12] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
is familiar. For example, via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously. The respective fine chemical produced by this process
can be obtained by harvesting the organisms, either from the crop
in which they grow, or from the field. This can be done via
pressing or extraction of the plant parts.
[5466] [0278.0.0.12] to [0282.0.0.12] for the disclosure of the
paragraphs [0278.0.0.12] to [0282.0.0.12] see paragraphs
[0278.0.0.0] to [0282.0.0.0] above.
[5467] [0283.0.12.12] Moreover, a native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table II,
column 3, lines 112 to 117 or lines 483 to 485 and/or lines 118 to
124 or lines 486 to 491, resp., or an antibody against a
polypeptide as indicated in Table II, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp. which can be produced by standard techniques utilizing
the polypeptid of the present invention or fragment thereof, i.e.,
the polypeptide of this invention. Preferred are monoclonal
antibodies.
[5468] [0284.0.0.12] for the disclosure of this paragraph see
[0284.0.0.0] above.
[5469] [0285.0.12.12] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491, resp., or as encoded by a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp., or functional homologues thereof.
[5470] [0286.0.12.12] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491, resp. In another embodiment,
the present invention relates to a polypeptide comprising or
consisting of a consensus sequence as indicated in Table IV, column
7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp., whereby 20 or less, preferably 15 or 10,
preferably 9, 8, 7, or 6, more preferred 5 or 4, even more
preferred 3, even more preferred 2, even more preferred 1, most
preferred 0 of the amino acids positions indicated can be replaced
by any amino acid or, in an further embodiment, can be replaced
and/or absent. In one embodiment, the present invention relates to
the method of the present invention comprising a polypeptide or to
a polypeptide comprising more than one consensus sequences (of an
individual line) as indicated in Table IV, column 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491.
[5471] [0287.0.0.12] to [0290.0.0.12] for the disclosure of the
paragraphs [0287.0.0.12] to [0290.0.0.12] see paragraphs
[0287.0.0.0] to [0290.0.0.0] above.
[5472] [0291.0.12.12] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[5473] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp., by one or more amino acids.
In one embodiment, polypeptide distinguishes from a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp., by more than 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp., by not more than 80% or 70% of the amino acids, preferably
not more than 60% or 50%, more preferred not more than 40% or 30%,
even more preferred not more than 20% or 10%. In an other
embodiment, said polypeptide of the invention does not consist of a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491, resp.
[5474] [0292.0.0.12] for the disclosure of this paragraph see
[0292.0.0.0] above.
[5475] [0293.0.12.12] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, said polypeptide
is having a sequence which distinguishes from a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp., by one or more amino acids. In an other embodiment, the
polypeptide of the invention does not consist of the sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp.
In a further embodiment, the polypeptide of the present invention
is less than 100%, 99.999%, 99.99%, 99.9% or 99% identical.
[5476] [0294.0.12.12] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table IIA or IIB, column 3, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp.,
which distinguishes over a sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491, resp., by one or more amino
acids, preferably by more than 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, evenmore
preferred are more than 40, 50, or 60 amino acids but even more
preferred by less than 70% of the amino acids, more preferred by
less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[5477] [0295.0.0.12] to [0297.0.0.12] for the disclosure of the
paragraphs [0295.0.0.12] to [0297.0.0.12] see paragraphs
[0295.0.0.0] to [0297.0.0.0] above.
[5478] [0297.1.12.12] Non polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table II, columns 3, 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp.
[5479] [0298.0.12.12] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence, which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 112 to 117
or lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp. The portion of the protein is preferably a biologically
active portion as described herein. Preferably, the polypeptide
used in the process of the invention has an amino acid sequence
identical to a sequence as indicated in Table II, columns 5 or 7,
lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp.
[5480] [0299.0.12.12] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491, resp. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table I, lines 112 to 117 or lines 483 to 485 and/or lines 118 to
124 or lines 486 to 491, resp., or which is homologous thereto, as
defined above.
[5481] [0300.0.12.12] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp., in amino acid sequence due
to natural variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprise an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence of a sequence as indicated in Table II, columns
5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to
124 or lines 486 to 491, resp.
[5482] [0301.0.0.12] for the disclosure of this paragraph see
[0301.0.0.0] above.
[5483] [0302.0.12.12] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., the amino acid sequence as indicated in
Table II, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp., or the amino
acid sequence of a protein homologous thereto, which include fewer
amino acids than a full length polypeptide of the present invention
or used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[5484] [0303.0.0.12] for the disclosure of this paragraph see
[0303.0.0.0] above.
[5485] [0304.0.12.12] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 112 to 117 or lines 483 to 485 and/or lines 118
to 124 or lines 486 to 491, resp., but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[5486] [0305.0.0.12], [0306.0.0.12] and [0306.1.0.12] for the
disclosure of the paragraphs [0305.0.0.12], [0306.0.0.12] and
[0306.1.0.12] see paragraphs [0305.0.0.0],
[5487] [0306.0.0.0] and [0306.1.0.0] above.
[5488] [0307.0.0.12] and [0308.0.0.12] for the disclosure of the
paragraphs [0307.0.0.12] and [0308.0.0.12] see paragraphs
[0307.0.0.0 and [0308.0.0.0] above.
[5489] [0309.0.12.12] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table II,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp., refers to a polypeptide
having an amino acid sequence corresponding to the polypeptide of
the invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table II, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp.,
refers to a polypeptide having an amino acid sequence corresponding
to a protein which is not substantially homologous a polypeptide of
the invention, preferably which is not substantially homologous to
a polypeptide as indicated in Table II, columns 5 or 7, lines 112
to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp., e.g., a protein which does not confer the activity
described herein or annotated or known for as indicated in Table
II, column 3, lines 112 to 117 or lines 483 to 485 and/or lines 118
to 124 or lines 486 to 491, resp., and which is derived from the
same or a different organism. In one embodiment, a "non-polypeptide
of the invention" or "other polypeptide" not being indicated in
Table II, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp., does not confer
an increase of the respective fine chemical in an organism or part
therof.
[5490] [0310.0.0.12] to [0334.0.0.12] for the disclosure of the
paragraphs [0310.0.0.12] to [0334.0.0.12] see paragraphs
[0310.0.0.0] to [0334.0.0.0] above.
[5491] [0335.0.12.12] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table I, columns 5 or
7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp., and/or homologs thereof. As described
inter alia in WO 99/32619, dsRNAi approaches are clearly superior
to traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table I,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp., and/or homologs thereof. In
a double-stranded RNA molecule for reducing the expression of an
protein encoded by a nucleic acid sequence sequences as indicated
in Table I, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp., and/or homologs
thereof, one of the two RNA strands is essentially identical to at
least part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[5492] [0336.0.0.12] to [0342.0.0.12] for the disclosure of the
paragraphs [0336.0.0.12] to [0342.0.0.12] see paragraphs
[0336.0.0.0] to [0342.0.0.0] above.
[5493] [0343.0.12.12] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 112 to 117
or lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491,
resp., or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table I, columns 5 or 7, lines 112 to 117 or lines 483
to 485 and/or lines 118 to 124 or lines 486 to 491, resp., or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[5494] [0344.0.0.12] to [0361.0.0.12] for the disclosure of the
paragraphs [0344.0.0.12] to [0361.0.0.12] see paragraphs
[0344.0.0.0] to [0361.0.0.0] above.
[5495] [0362.0.12.12] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, columns 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491, resp. e.g. encoding a
polypeptide having protein activity, as indicated in Table II,
column 3, lines lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp. Due to the above mentioned
activity the respective fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. Transgenic for a polypeptide having an activity of a
polypeptide as indicated in Table II, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp., means herein that due to modulation or manipulation of
the genome, an activity as annotated for a polypeptide as indicated
in Table II, column 3, lines 112 to 117 or lines 483 to 485 and/or
lines 118 to 124 or lines 486 to 491, resp. e.g. having a sequence
as indicated in Table II, columns 5 or 7, lines 112 to 117 or lines
483 to 485 and/or lines 118 to 124 or lines 486 to 491, resp., is
increased in a cell or an organism or a part thereof. Examples are
described above in context with the process of the invention
[5496] [0363.0.0.12] for the disclosure of this paragraph see
paragraph [0363.0.0.0] above.
[5497] [0364.0.12.12] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a polypeptide of the invention as indicated in Table
II, column 3, lines 112 to 117 or lines 483 to 485 and/or lines 118
to 124 or lines 486 to 491, resp., with the corresponding
protein-encoding sequence as indicated in
[5498] Table I, column 3, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 4914, resp., --becomes a
transgenic expression cassette when it is modified by non-natural,
synthetic "artificial" methods such as, for example,
mutagenization. Such methods have been described (U.S. Pat. No.
5,565,350; WO 00/15815; also see above).
[5499] [0365.0.0.12] to [0373.0.0.12] for the disclosure of the
paragraphs [0365.0.0.12], to [0373.0.0.12] see paragraphs
[0365.0.0.0] to [0373.0.0.0] above.
[5500] [0374.0.12.12] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. Phytosterol, in particular,
of beta-sitosterol and/or campesterol resp., produced in the
process according to the invention may, however, also be isolated
from the plant in the form of their free form or bound in or to
compounds or moieties. Phytosterol, in particular, of
beta-sitosterol and/or campesterol resp., produced by this process
can be harvested by harvesting the organisms either from the
culture in which they grow or from the field. This can be done via
expressing, grinding and/or extraction, salt precipitation and/or
ion-exchange chromatography or other chromatographic methods of the
plant parts, preferably the plant seeds, plant fruits, plant tubers
and the like.
[5501] [0375.0.0.12] and [0376.0.0.12] for the disclosure of the
paragraphs [0375.0.0.12] and [0376.0.0.12] see paragraphs
[0375.0.0.0] and [0376.0.0.0] above.
[5502] [0377.0.12.12] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced phytosterol, in particular, of beta-sitosterol and/or
campesterol comprising composition or the produced the respective
fine chemical is isolated.
[5503] [0378.0.12.12] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the phytosterol,
in particular, of beta-sitosterol and/or campesterol, resp.,
produced in the process can be isolated. The resulting phytosterol,
in particular, of beta-sitosterol and/or campesterol resp., can, if
appropriate, subsequently be further purified, if desired mixed
with other active ingredients such as vitamins, amino acids,
carbohydrates, antibiotics and the like, and, if appropriate,
formulated.
[5504] [0379.0.12.12] In one embodiment, the phytosterol, in
particular, of beta-sitosterol and/or campesterol resp., is a
mixture comprising of one or more the respective fine chemicals. In
one embodiment, the respective fine chemical means here
phytosterol, in particular, of beta-sitosterol and/or campesterol.
In one embodiment, phytosterol means here a mixture of the
respective fine chemicals.
[5505] [0380.0.12.12] The phytosterol, in particular, the
beta-sitosterol and/or campesterol resp., obtained in the process
are suitable as starting material for the synthesis of further
products of value. For example, they can be used in combination
with each other or alone for the production of pharmaceuticals,
foodstuffs, animal feeds or cosmetics. Accordingly, the present
invention relates a method for the production of pharmaceuticals,
food stuff, animal feeds, nutrients or cosmetics comprising the
steps of the process according to the invention, including the
isolation of the phytosterol comprising composition produced or the
respective fine chemical produced if desired and formulating the
product with a pharmaceutical acceptable carrier or formulating the
product in a form acceptable for an application in agriculture. A
further embodiment according to the invention is the use of the
phytosterol resp., produced in the process or of the transgenic
organisms in animal feeds, foodstuffs, medicines, food supplements,
cosmetics or pharmaceuticals.
[5506] [0381.0.0.12] and [0382.0.0.12] for the disclosure of the
paragraphs [0381.0.0.12] and [0382.0.0.12] see paragraphs
[0381.0.0.0] and [0382.0.0.0] above.
[5507] [0383.0.12.12] For preparing sterol compound-containing fine
chemicals, in particular the respective fine chemical of the
invention, it is possible to use as source organic compounds such
as, for example, acetyl CoA HMGCoA, mevalonate, mevalonate 5
phosphate, mevalonate 5-pyrophosphate, isopentyl diphosphate,
5-pyrophosphatemevalonate, isopentyl pyrophosphate (PIP),
dimethylallyl pyrophosphate (DMAPP), PIP+DMAPP, geranyl
pyrophosphate+IPP, farnesyl pyrophosphate, 2 farnesyl
pyrophosphate, squalene and squalene epoxide, cycloartenol and for
preparing esters comprising the phytosterols of the invention oils,
fats and/or lipids comprising fatty acids such as fatty acids
having a carbon back bone between C.sub.10- to C.sub.16-carbon
atoms and/or small organic acids such acetic acid, propionic acid
or butanoic acid as precursor compounds.
[5508] [0384.0.0.12] for the disclosure of this paragraph see
paragraph [0384.0.0.0] above.
[5509] [0385.0.12.12] The fermentation broths obtained in this way,
containing in particular phytosterol, in particular, of
beta-sitosterol and/or campesterol resp., in mixtures with other
compounds, in particular with other sterols or vitamins, e.g. with
carotenoids, e.g. with astaxanthin, or fatty acids or containing
microorganisms or parts of microorganisms, like plastids,
containing phytosterol, in particular, of beta-sitosterol and/or
campesterol resp., in mixtures with other compounds, e.g. with
vitamins, normally have a dry matter content of from 7.5 to 25% by
weight. Sugar-limited fermentation is additionally advantageous,
e.g. at the end, for example over at least 30% of the fermentation
time. This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 3 g/l during
this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed or isolated
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation, coagulation/flocculation
or a combination of these methods, from the fermentation broth or
left completely in it. The fermentation broth can then be thickened
or concentrated by known methods, such as, for example, with the
aid of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[5510] As phytosterol is often localized in membranes or plastids,
in one embodiment it is advantageous to avoid a leaching of the
cells when the biomass is isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth. The dry biomass can directly
be added to animal feed; provided the vitamin E concentration is
sufficiently high and no toxic compounds are present.
[5511] [0386.0.12.12] Accordingly, it is possible to further purify
the produced phytosterol, in particular, of beta-sitosterol and/or
campesterol resp. For this purpose, the product-containing
composition, e.g. a total or partial lipid extraction fraction
using organic solvents, e.g. as described above, is subjected for
example to a saponification to remove triglycerides, partition
between e.g. hexane/methanol (separation of non-polar epiphase from
more polar hypophasic derivates) and separation via e.g. an open
column chromatography, preparative thin layer chromatography or
HPLC in which case the desired product or the impurities are
retained wholly or partly on the chromatography resin (see e.g.
Kaluzny et al., J Lipid Res 1985; 26: 135-140). These
chromatography steps can be repeated if necessary, using the same
or different chromatography resins. The skilled worker is familiar
with the choice of suitable chromatography resins and their most
effective use.
[5512] [0387.0.0.12] to [0392.0.0.12] for the disclosure of the
paragraphs [0387.0.0.12] to [0392.0.0.12] see paragraphs
[0387.0.0.0] to [0392.0.0.0] above.
[5513] [0393.0.12.12] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [5514] (g) contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [5515] (h) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table I, columns 5 or 7, lines
112 to 117 or lines 483 to 485 and/or lines 118 to 124 or lines 486
to 491, preferably in Table I B, clumns 5 or 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491 resp.,
and, optionally, isolating the full length cDNA clone or complete
genomic clone; [5516] (i) introducing the candidate nucleic acid
molecules in host cells, preferably in a plant cell or a
microorganism, appropriate for producing the respective fine
chemical; [5517] (j) expressing the identified nucleic acid
molecules in the host cells; [5518] (k) assaying the the respective
fine chemical level in the host cells; and [5519] (l) identifying
the nucleic acid molecule and its gene product which expression
confers an increase in the respective fine chemical level in the
host cell after expression compared to the wild type.
[5520] [0394.0.0.12] to [0399.0.0.12] for the disclosure of the
paragraphs [0394.0.0.12] to [0399.0.0.12] see paragraphs
[0394.0.0.0] to [0399.0.0.0] above.
[5521] [0399.1.12.12] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp., or a homolog thereof, e.g.
comparing the phenotyp of nearly identical organisms with low and
high activity of a protein as indicated in Table II, columns 5 or
7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to 124 or
lines 486 to 491, resp., after incubation with the drug.
[5522] [0400.0.0.12] to [0416.0.0.12] for the disclosure of the
paragraphs [0400.0.0.12] to [0416.0.0.12] see paragraphs
[0400.0.0.0] to [0416.0.0.0] above.
[5523] [0417.0.12.12] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the phytosterol production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect an organism such as a
microorganism or a plant against inhibitors, which block the
phytosterol, in particular the respective fine chemical, synthesis
in said organism. Examples of inhibitors or herbicides blocking the
phytosterol synthesis in organism such as microorganism or plants
are for example compounds which inhibit the cytochrom P450 such as
Tetcyclasis, triazoles like Paclobutrazol or Epoxiconazol,
pyridines like Obtusifoliol, demethylases inhibitors, or compounds
like Mevilonin, which inhibits the HMG-CoA reductase.
[5524] [0418.0.0.12] to [0423.0.0.12] for the disclosure of the
paragraphs [0418.0.0.12] to [0423.0.0.12] see paragraphs
[0418.0.0.0] to [0423.0.0.0] above.
[5525] [0424.0.12.12] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other sterols,
phytosterols, carotenoids, vitamins or fatty acids. Accordingly,
the nucleic acid of the invention, or the nucleic acid molecule
identified with the method of the present invention or the
complement sequences thereof, the polypeptide of the invention, the
nucleic acid construct of the invention, the organisms, the host
cell, the microorganisms, the plant, plant tissue, plant cell, or
the part thereof of the invention, the vector of the invention, the
antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the respective
fine chemical in a organism or part thereof, e.g. in a cell.
[5526] [0425.0.0.12] to [0430.0.0.12] for the disclosure of the
paragraphs [0425.0.0.12] to [0430.0.0.12] see paragraphs
[0425.0.0.0] to [0430.0.0.0] above.
[0431.0.12.12] Example 1
Cloning SEQ ID NO: 12135 in Escherichia coli
[5527] [0432.0.12.12] SEQ ID NO: 12135 was cloned into the plasmids
pBR322 (Sutcliffe, J. G. (1979) Proc. Natl Acad. Sci. USA, 75:
3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol. 134:
1141-1156); plasmids of the pBS series (pBSSK+, pBSSK- and others;
Stratagene, LaJolla, USA) or cosmids such as SuperCos1 (Stratagene,
LaJolla, USA) or Lorist6 (Gibson, T. J. Rosenthal, A., and
Waterson, R. H. (1987) Gene 53: 283-286) for expression in E. coli
using known, well-established procedures (see, for example,
Sambrook, J. et al. (1989) "Molecular Cloning: A Laboratory
Manual". Cold Spring Harbor Laboratory Press or Ausubel, F. M. et
al. (1994) "Current Protocols in Molecular Biology", John Wiley
& Sons).
[5528] [0433.0.0.12] and [0434.0.0.12] for the disclosure of the
paragraphs [0433.0.0.12] and [0434.0.0.12] see paragraphs
[0433.0.0.0] and [0434.0.0.0] above.
[0435.0.12.12] Example 3
In-Vivo and In-Vitro Mutagenesis
[5529] [0436.0.12.12] An in vivo mutagenesis of organisms such as
green algae (e.g. Spongiococcum sp, e.g. Spongiococcum exentricum,
Chlorella sp., Haematococcus, Phaedactylum tricornatum, Volvox or
Dunaliella), Synchocytic spec. PLL 6803, Physocmetrella patens,
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of phytosterol can be
carried out by passing a plasmid DNA (or another vector DNA)
containing the desired nucleic acid sequences, e.g. the nucleic
acid molecule of the invention or the vector of the invention, or
nucleic acid sequences through E. coli and other microorganisms
(for example Bacillus spp. or yeasts such as Saccharomyces
cerevisiae) which are not capable of maintaining the integrity of
its genetic information. Usual mutator strains have mutations in
the genes for the DNA repair system [for example mutHLS, mutD, mutT
and the like; for comparison, see Rupp, W. D. (1996) DNA repair
mechanisms in Escherichia coli and Salmonella, pp. 2277-2294, ASM:
Washington]. The skilled worker knows these strains. The use of
these strains is illustrated for example in Greener, A. and
Callahan, M. (1994) Strategies 7; 32-34.
[5530] In-vitro mutation methods such as increasing the spontaneous
mutation rates by chemical or physical treatment are well known to
the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[5531] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[5532] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[5533] [0437.0.5.12] to [0440.0.5.12] and [0441.0.0.12] for the
disclosure of the paragraphs [0437.0.5.12] to [0440.0.5.12] and
[0441.0.0.12] see paragraphs [0437.0.5.5] to [0444.0.5.5] and
[0441.0.0.0] above.
[5534] [0442.0.5.12], [0443.0.0.12], [0444.0.5.12] and
[0445.0.5.12] for the disclosure of the paragraphs [0442.0.5.12],
[0443.0.0.12], [0444.0.5.12] and [0445.0.5.12] see paragraphs
[0442.0.5.5], [0443.0.0.0], [0444.0.5.5] and [0445.0.5.5]
above.
[5535] [0446.0.0.12] to [0450.0.0.12] and [0451.0.5.12] for the
disclosure of the paragraphs [0446.0.0.12] to [0450.0.0.12] and
[0451.0.5.12] see paragraphs [0446.0.0.0] to [0450.0.0.0] and
[0451.0.5.5] above.
[5536] [0452.0.0.12] to [0454.0.0.12], [0455.0.5.12] and
[0456.0.0.12] for the disclosure of the paragraphs [0452.0.0.12] to
[0454.0.0.12], [0455.0.5.12] and [0456.0.0.12] see
[5537] [0452.0.0.0] to [0454.0.0.0], [0455.0.5.5] and [0456.0.0.0]
above.
[0457.0.12.12] Example 9
Purification of the Phytosterol
[5538] [0458.0.12.12] One example is the analysis of phytosterol:
the content of the phytosterols of the invention can be
determinated by gas chromatography with flame ionisation detection
(GC-FID; column SAC-5, 30 m.times.0.25 mm, 0.25 .mu.m, samples not
silylated) using standards for these phytosterols. Another method
is the detection by gas chromatography-mass spectrometry (GC-MS)
using the same type of column as indicated above.
[5539] For the analysis of the concentrations of sterols by gas
chromatography mass spectrometry a Hewlett-Packard (HP) 5890 gas
chromatograph equipped with an NB-54 fused-silica capillary column
(15 mx0.20 mm I.D.; Nordion, Helsinki, Finland) and interfaced with
an HP 5970A mass spectrometry detector operating in electron impact
mode (70 eV) can be used. The column oven is programmed from
230.degree. C. to 285.degree. C. at 10.degree. C./min and injector
and detector should be at 285.degree. C. The lipids from the
samples (200 .mu.l) are extracted with chloroform/methanol (2:1)
and transesterified with sodium methoxide. The released free
sterols are trimethylsilylated as described previously (Gylling et
al. J. Lipid Res 40: 593-600, 1999) and quantified by single ion
monitoring technique using m/z 129 (cholesterol, campesterol and
.beta.-sitosterol), m/z 215 ([3-sitostanol), m/z 343 (desmosterol),
m/z 255 (lathosterol) and m/z 217 (5-.alpha.-cholestane, internal
standard) as selected ions (Vaskonen, Dissertation, Biomedicum
Helsinki, Jun. 19, 2002).
[5540] [0459.0.12.12] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the phytosterol, in particular, of beta-sitosterol
and/or campesteroll, can be further purified with a so-called
RTHPLC. As eluent acetonitrile/water or chloroform/acetonitrile
mixtures can be used. If necessary, these chromatography steps may
be repeated, using identical or other chromatography resins. The
skilled worker is familiar with the selection of suitable
chromatography resin and the most effective use for a particular
molecule to be purified.
[5541] [0460.0.0.12] for the disclosure of this paragraph see
[0460.0.0.0] above.
[0461.0.12.12] Example 10
Cloning SEQ ID NO: 12135 for the Expression in Plants
[5542] [0462.0.0.12] for the disclosure of this paragraph see
[0462.0.0.0] above.
[5543] [0463.0.12.12] SEQ ID NO: 12135 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[5544] [0464.0.0.12] to [0466.0.0.12] for the disclosure of the
paragraphs [0464.0.0.12] to [0466.0.0.12] see paragraphs
[0464.0.0.0] to [0466.0.0.0] above.
[5545] [0467.0.12.12] The following primer sequences were selected
for the gene SEQ ID NO:
[5546] 12135:
TABLE-US-00041 i) forward primer (SEQ ID NO: 12137)
ATGGAACAGAACAGGTTCAAGAAAG ii) reverse primer (SEQ ID NO: 12138)
TTACAGTTTTTGTTTAGTCGTTTTAAC
[5547] [0468.0.0.12] to [0479.0.0.12] for the disclosure of the
paragraphs [0468.0.0.12] to [0479.0.0.12] see paragraphs
[0468.0.0.0] to [0479.0.0.0] above.
[0480.0.12.12] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 12135
[5548] [0481.0.0.12] to [0513.0.0.12] for the disclosure of the
paragraphs [0481.0.0.12] to [0513.0.0.12] see paragraphs
[0482.0.0.0] to [0513.0.0.0] above.
[5549] [0514.0.12.12] As an alternative, phytosterols can be
detected via HPLC, e.g. reversed-phase HPLC, as described by
Heftmann, E. and Hunter, I. R. (J Chromatogr 1979; 165: 283-299).
As separating principles of HPLC and GC are complementary,
preparative reversed-phase HPLC followed by GC-MS analysis of the
obtained sterol fractions is a preferred method to analyze sterols
from natural products (Bianchini, J.-P. et al.; J Chromatogr 1985;
329: 231-246).
[5550] The results of the different plant analyses can be seen from
the table, which follows:
TABLE-US-00042 TABLE 1 ORF Metabolite Method Min Max YER156C
beta-Sitosterol GC 1.18 1.39 YER156C Campesterol GC 1.17 1.95
YER173W Campesterol GC 1.23 1.51 YKR057W beta-Sitosterol GC 1.14
1.32 YKR057W Campesterol GC 1.20 1.39 YOR044W Campesterol GC 1.28
1.57 YOR084W beta-Sitosterol GC 3.59 3.59 YPR172W Campesterol GC
1.17 1.62 b0019 beta-Sitosterol GC 1.20 1.47 b0421 beta-Sitosterol
GC 1.13 1.52 b2699 beta-Sitosterol GC 1.14 1.35 b2699 Campesterol
GC 1.17 1.62 b3256 Campesterol GC 1.20 1.22 b0050 beta-Sitosterol
GC 1,15 1,24 b0161 beta-Sitosterol GC 1,12 1,26 b0161 Campesterol
GC 1,17 1,42 b0464 Campesterol GC 1,16 1,32 b1896 Campesterol GC
1,18 1,59 b2341 Campesterol GC 1,18 1,48 b2478 Campesterol GC 1,17
1,37 b2822 Campesterol GC 1,21 1,32 b4129 beta-Sitosterol GC 1,21
1,23
[5551] [0515.0.12.12] Column 2 shows the phytosterol analyzed.
Columns 4 and 5 shows the ratio of the analyzed phytosterol between
the transgenic plants and the wild type; Increase of the
metabolites: Max: maximal x-fold (normalised to wild type)-Min:
minimal x-fold (normalised to wild type). Decrease of the
metabolites: Max: maximal x-fold (normalised to wild type) (minimal
decrease), Min: minimal x-fold (normalised to wild type) (maximal
decrease). Column 3 indicates the analytical method.
[5552] [0516.0.0.12] for the disclosure of this paragraph see
[0516.0.0.0] above.
[0517.0.12.12] Example 14a
Engineering Ryegrass Plants by Over-Expressing YOR084W from
Saccharomyces cerevisiae or Other Nucleic Acids of the Present
Invention
[5553] [0518.0.0.12] to [0524.0.0.12] for the disclosure of the
paragraphs [0518.0.0.12] to [0524.0.0.12] see paragraphs
[0518.0.0.0] to [0524.0.0.0] above.
[0525.0.12.12] Example 14b
Engineering Soybean Plants by Over-Expressing YOR084W from
Saccharomyces cerevisiae or Other Nucleic Acids of the Present
Invention
[5554] [0526.0.0.12] to [0529.0.0.12] for the disclosure of the
paragraphs [0526.0.0.12] to [0529.0.0.12] see paragraphs
[0526.0.0.0] to [0529.0.0.0] above.
[0530.0.12.12] Example 14c
Engineering Corn Plants by Over-Expressing YOR084W from
Saccharomyces cerevisiae or Other Nucleic Acids of the Present
Invention
[5555] [0530.1.0.12] to [0530.6.0.12] for the disclosure of the
paragraphs [0530.1.0.12] to [0530.6.0.12] see paragraphs
[0530.1.0.0] to [0530.6.0.0] above.
[5556] [0531.0.0.12] to [0533.0.0.12] for the disclosure of the
paragraphs [0531.0.0.12] to [0533.0.0.12] see paragraphs
[0531.0.0.0] to [0533.0.0.0] above.
[0534.0.12.12] Example 14d
Engineering Wheat Plants by Over-Expressing YOR084W from
Saccharomyces cerevisiae or Other Nucleic Acids of the Present
Invention
[5557] [0535.0.0.12] to [0537.0.0.12] for the disclosure of the
paragraphs [0535.0.0.12] to [0537.0.0.12] see paragraphs
[0535.0.0.0] to [0537.0.0.0] above.
[0538.0.12.12] Example 14e
Engineering Rapeseed/Canola Plants by Over-Expressing YOR084W from
Saccharomyces cerevisiae or Other Nucleic Acids of the Present
Invention
[5558] [0539.0.0.12] to [0542.0.0.12] for the disclosure of the
paragraphs [0539.0.0.12] to [0542.0.0.12] see paragraphs
[0539.0.0.0] to [0542.0.0.0] above.
[0543.0.12.12] Example 14f
Engineering Alfalfa Plants by Over-Expressing YOR084W from
Saccharomyces cerevisiae or Other Nucleic Acids of the Present
Invention.
[5559] [0544.0.0.12] to [0547.0.0.12] for the disclosure of the
paragraphs [0544.0.0.12] to [0547.0.0.12] see paragraphs
[0544.0.0.0] to [0547.0.0.0] above.
[0548.0.12.12] Example 14 g
Engineering Alfalfa Plants by Over-Expressing YOR084W from
Saccharomyces cerevisiae or Other Nucleic Acids of the Present
Invention
[5560] [0549.0.0.12] to [0552.0.0.12] for the disclosure of the
paragraphs [0549.0.0.12] to [0552.0.0.12] see paragraphs
[0549.0.0.0] to [0552.0.0.0] above.
[0552.1.12.12] Example 15
Metabolite Profiling Info from Zea mays
[5561] Zea mays plants were engineered, grown and analyzed as
described in Example 14c.
[5562] The results of the different Zea mays plants analysed can be
seen from Table 2 which follows:
TABLE-US-00043 TABLE 2 ORF_NAME Metabolite Min Max YKR057W
Campesterol 1.29 1.47 YPR172W Campesterol 1.23 1.30
[5563] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in Campesterol in
genetically modified corn plants expressing the Saccharomyces
cerevisiae nucleic acid sequence YKR057W or YPR172W resp.
[5564] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YKR057W or its homologs, e.g. "an activity of
ribosomal protein, similar to S21 ribosomal proteins, involved in
ribosome biogenesis and translation", is increased in corn plants,
preferably, an increase of the fine chemical Campesterol between
29% and 47% is conferred.
[5565] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YPR172W or its homologs, its activity has not
been characterized yet, is increased in corn plants, preferably, an
increase of the fine chemical Campesterol between 23% and 30% is
conferred.
[5566] [0552.2.0.12] for the disclosure of this paragraph see
[0552.2.0.0] above.
[5567] [0553.0.12.12] [5568] 1. A process for the production of
phytosterol, in particular, of beta-sitosterol and/or campesterol,
which comprises [5569] (a) increasing or generating the activity of
a YER156C, YKR057W, YOR084W, b0019, b0421, b2699, b0050, b0161
and/or b4129 protein and/or YER156C, YER173W, YKR057W, YOR044W,
YPR172W, b2699, b3256, b0161, b0464, b1896, b2341, b2478 and/or
b2822 protein in a non-human organism, or in one or more parts
thereof; and [5570] (b) growing the organism under conditions which
permit the production of phytosterol, in particular, of
beta-sitosterol and/or campesterol in said organism. [5571] 2. A
process for the production of phytosterol, in particular, of
beta-sitosterol and/or campesterol comprising the increasing or
generating in an organism or a part thereof the expression of at
least one nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [5572] a) nucleic acid
molecule encoding of the polypeptide as indicated in Table II,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491 resp., or a fragment thereof, which
confers an increase in the amount of phytosterol, in particular, of
beta-sitosterol and/or campesterol in an organism or a part
thereof; [5573] b) nucleic acid molecule comprising of the nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines 112 to
117 or lines 483 to 485 and/or lines 118 to 124 or lines 486 to
491, resp; [5574] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of phytosterol, in
particular, of beta-sitosterol and/or campesterol in an organism or
a part thereof; [5575] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of phytosterol,
in particular, of beta-sitosterol and/or campesterol in an organism
or a part thereof; [5576] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to [5577] (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of phytosterol, in particular, of beta-sitosterol and/or
campesterol in an organism or a part thereof; [5578] f) nucleic
acid molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers as shown in table III,
column 7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to
124 or lines 486 to 491 resp., and conferring an increase in the
amount of phytosterol, in particular, of beta-sitosterol and/or
campesterol in an organism or a part thereof; [5579] g) nucleic
acid molecule encoding a polypeptide which is isolated with the aid
of monoclonal antibodies against a polypeptide encoded by one of
the nucleic acid molecules of (a) to (f) and conferring an increase
in the amount of phytosterol, in particular, of beta-sitosterol
and/or campesterol in an organism or a part thereof; [5580] h)
nucleic acid molecule encoding a polypeptide comprising the
consensus sequence shown in table IV, column 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491 resp.,
and conferring an increase in the amount of phytosterol, in
particular, of beta-sitosterol and/or campesterol in an organism or
a part thereof; and [5581] i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of phytosterol, in particular, of beta-sitosterol and/or
campesterol in an organism or a part thereof. or comprising a
sequence which is complementary thereto. [5582] 3. The process of
claim 1 or 2, comprising recovering of the free or bound
phytosterol, in particular, of beta-sitosterol and/or campesterol.
[5583] 4. The process of any one of claims 1 to 3, comprising the
following steps: [5584] (a) selecting an organism or a part thereof
expressing a polypeptide encoded by the nucleic acid molecule
characterized in claim 2; [5585] (b) mutagenizing the selected
organism or the part thereof; [5586] (c) comparing the activity or
the expression level of said polypeptide in the mutagenized
organism or the part thereof with the activity or the expression of
said polypeptide of the selected organisms or the part thereof;
[5587] (d) selecting the mutated organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
[5588] (e) optionally, growing and cultivating the organisms or the
parts thereof; and [5589] (f) recovering, and optionally isolating,
the free or bound phytosterol, in particular, of beta-sitosterol
and/or campesterol produced by the selected mutated organisms or
parts thereof. [5590] 5. The process of any one of claims 1 to 4,
wherein the activity of said protein or the expression of said
nucleic acid molecule is increased or generated transiently or
stably. [5591] 6. An isolated nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [5592]
a) nucleic acid molecule encoding of the polypeptide as indicated
in Table II, column 5 or 7, lines 112 to 117 or lines 483 to 485
and/or lines 118 to 124 or lines 486 to 491 resp. or a fragment
thereof, which confers an increase in the amount of phytosterol, in
particular, of beta-sitosterol and/or campesterol in an organism or
a part thereof; [5593] b) nucleic acid molecule comprising of the
nucleic acid molecule shown in
[5594] Table I, columns 5 or 7, lines 112 to 117 or lines 483 to
485 and/or lines 118 to 124 or lines 486 to 491, resp.; [5595] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of phytosterol, in particular,
of beta-sitosterol and/or campesterol in an organism or a part
thereof; [5596] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of phytosterol,
in particular, of beta-sitosterol and/or campesterol in an organism
or a part thereof; [5597] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to [5598] (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of phytosterol, in particular, of beta-sitosterol and/or
campesterol in an organism or a part thereof; [5599] f) nucleic
acid molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers as shown in table III,
column 7, lines 112 to 117 or lines 483 to 485 and/or lines 118 to
124 or lines 486 to 491 resp., and conferring an increase in the
amount of phytosterol, in particular, of beta-sitosterol and/or
campesterol in an organism or a part thereof; [5600] g) nucleic
acid molecule encoding a polypeptide which is isolated with the aid
of monoclonal antibodies against a polypeptide encoded by one of
the nucleic acid molecules of (a) to (f) and conferring an increase
in the amount of phytosterol, in particular, of beta-sitosterol
and/or campesterol in an organism or a part thereof; [5601] h)
nucleic acid molecule encoding a polypeptide comprising the
consensus sequence shown in table IV, column 7, lines 112 to 117 or
lines 483 to 485 and/or lines 118 to 124 or lines 486 to 491 resp.,
and conferring an increase in the amount of phytosterol, in
particular, of beta-sitosterol and/or campesterol in an organism or
a part thereof; and [5602] i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of phytosterol, in particular, of beta-sitosterol and/or
campesterol in an organism or a part thereof, whereby the nucleic
acid molecule distinguishes over the sequence as shown in Table IA,
columns 5 or 7, lines 112 to 117 or lines 483 to 485 and/or lines
118 to 124 or lines 486 to 491, resp., by one or more nucleotides.
[5603] 7. A nucleic acid construct which confers the expression of
the nucleic acid molecule of claim 6, comprising one or more
regulatory elements. [5604] 8. A vector comprising the nucleic acid
molecule as claimed in claim 6 or the nucleic acid construct of
claim 7. [5605] 9. he vector as claimed in claim 8, wherein the
nucleic acid molecule is in operable linkage with regulatory
sequences for the expression in a prokaryotic or eukaryotic, or in
a prokaryotic and eukaryotic, host. [5606] 10. A host cell, which
has been transformed stably or transiently with the vector as
claimed in claim 8 or 9 or the nucleic acid molecule as claimed in
claim 6 or the nucleic acid construct of claim 7 or produced as
described in claim any one of claims 2 to 5. [5607] 11. The host
cell of claim 10, which is a transgenic host cell. [5608] 12. The
host cell of claim 10 or 11, which is a plant cell, an animal cell,
a microorganism, or a yeast cell, a fungus cell, a prokaryotic
cell, an eukaryotic cell or an archaebacterium. [5609] 13. A
process for producing a polypeptide, wherein the polypeptide is
expressed in a host cell as claimed in any one of claims 10 to 12.
[5610] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over the sequence as
indicated in Table IIA, columns 5 or 7, I A by one or more amino
acids [5611] 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. [5612] 16. A plant tissue,
propagation material, harvested material or a plant comprising the
host cell as claimed in claim 12 which is plant cell or an
Agrobacterium. [5613] 17. A method for screening for agonists and
antagonists of the activity of a polypeptide encoded by the nucleic
acid molecule of claim 6 conferring an increase in the amount of
phytosterol, in particular, of beta-sitosterol and/or campesterol
in an organism or a part thereof comprising: [5614] (a) contacting
cells, tissues, plants or microorganisms which express the a
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of phytosterol, in particular,
of beta-sitosterol and/or campesterol in an organism or a part
thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; [5615] (b) assaying the phytosterol, in
particular, of beta-sitosterol and/or campesterol level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [5616] (c)
identifying a agonist or antagonist by comparing the measured
phytosterol, in particular, of beta-sitosterol and/or campesterol
level or polypeptide expression level with a standard phytosterol,
in particular, of beta-sitosterol and/or campesterol or polypeptide
expression level measured in the absence of said candidate compound
or a sample comprising said plurality of compounds, whereby an
increased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an agonist and
a decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[5617] 18. A process for the identification of a compound
conferring increased phytosterol, in particular, of beta-sitosterol
and/or campesterol production in a plant or microorganism,
comprising the steps: [5618] (a) culturing a plant cell or tissue
or microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of phytosterol, in particular, of
beta-sitosterol and/or campesterol in an organism or a part thereof
and a readout system capable of interacting with the polypeptide
under suitable conditions which permit the interaction of the
polypeptide with dais readout system in the presence of a compound
or a sample comprising a plurality of compounds and capable of
providing a detectable signal in response to the binding of a
compound to said polypeptide under conditions which permit the
expression of said readout system and of the polypeptide encoded by
the nucleic acid molecule of claim 6 conferring an increase in the
amount of phytosterol, in particular, of beta-sitosterol and/or
campesterol in an organism or a part thereof; [5619] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [5620] 19. A method for the identification of a
gene product conferring an increase in phytosterol, in particular,
of beta-sitosterol and/or campesterol production in a cell,
comprising the following steps: [5621] (a) contacting the nucleic
acid molecules of a sample, which can contain a candidate gene
encoding a gene product conferring an increase in phytosterol, in
particular, of beta-sitosterol and/or campesterol after expression
with the nucleic acid molecule of claim 6; [5622] (b) identifying
the nucleic acid molecules, which hybridise under relaxed stringent
conditions with the nucleic acid molecule of claim 6; [5623] (c)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing phytosterol, in particular, of
beta-sitosterol and/or campesterol; [5624] (d) expressing the
identified nucleic acid molecules in the host cells; [5625] (e)
assaying the phytosterol, in particular, of beta-sitosterol and/or
campesterol level in the host cells; and [5626] (f) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the phytosterol, in particular, of beta-sitosterol
and/or campesterol level in the host cell in the host cell after
expression compared to the wild type. [5627] 20. A method for the
identification of a gene product conferring an increase in
phytosterol, in particular, of beta-sitosterol and/or campesterol
production in a cell, comprising the following steps: [5628] (a)
identifying in a data bank nucleic acid molecules of an organism;
which can contain a candidate gene encoding a gene product
conferring an increase in the phytosterol, in particular, of
beta-sitosterol and/or campesterol amount or level in an organism
or a part thereof after expression, and which are at least 20%
homolog to the nucleic acid molecule of claim 6; [5629] (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing phytosterol, in particular, of
beta-sitosterol and/or campesterol; [5630] (c) expressing the
identified nucleic acid molecules in the host cells; [5631] (d)
assaying the phytosterol, in particular, of beta-sitosterol and/or
campesterol level in the host cells; and [5632] (e) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the phytosterol, in particular, of beta-sitosterol
and/or campesterol level in the host cell after expression compared
to the wild type. [5633] 21. A method for the production of an
agricultural composition comprising the steps of the method of any
one of claims 17 to 20 and formulating the compound identified in
any one of claims 17 to 20 in a form acceptable for an application
in agriculture. [5634] 22. A composition comprising the nucleic
acid molecule of claim 6, the polypeptide of claim 14, the nucleic
acid construct of claim 7, the vector of any one of claim 8 or 9,
an antagonist or agonist identified according to claim 17, the
compound of claim 18, the gene product of claim 19 or 20, the
antibody of claim 15, and optionally an agricultural acceptable
carrier. [5635] 23. Use of the nucleic acid molecule as claimed in
claim 6 for the identification of a nucleic acid molecule
conferring an increase of phytosterol, in particular, of
beta-sitosterol and/or campesterol after expression. [5636] 24. Use
of the polypeptide of claim 14 or the nucleic acid construct claim
7 or the gene product identified according to the method of claim
19 or 20 for identifying compounds capable of conferring a
modulation of phytosterol, in particular, of beta-sitosterol and/or
campesterol levels in an organism. [5637] 25. Food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. [5638] 26. Use of the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the host cell of claim 10 to
12 or the gene product identified according to the method of claim
19 or 20 for the protection of a plant against a beta-sitosterol
and/or campesterol synthesis inhibiting herbicide.
[5639] [0554.0.0.12] Abstract: see [0554.0.0.0]:
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[5640] [0000.0.0.13] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and corresponding embodiments as described herein
as follows.
[5641] [0001.0.0.13]: see [0001.0.0.0]
[5642] [0002.0.13.13] Fatty acids and triglycerides have numerous
applications in the food and feed industry, in cosmetics and in the
drug sector. Depending on whether they are free saturated or
unsaturated fatty acids or triglycerides with an increased content
of saturated or unsaturated fatty acids, they are suitable for the
most varied applications; thus, for example, polyunsaturated fatty
acids (=PUFAs) are added to infant formula to increase its
nutritional value. The various fatty acids and triglycerides are
mainly obtained from microorganisms such as fungi or from
oil-producing plants including phytoplankton and algae, such as
soybean, oilseed rape, sunflower and others, where they are usually
obtained in the form of their triacylglycerides.
[5643] [0003.0.13.13] Straight- or normal-chain (even-numbered),
monoenoic components, i.e. with one double bond, make up a high
proportion of the total fatty acids in most natural lipids.
Normally the double bond is of the cis- or Z-configuration,
although some fatty acids with trans- or E-double bonds are known.
The most abundant monoenoic fatty acids in animal and plant tissues
are straight-chain compounds with 16 or 18 carbon atoms, but
analogous fatty acids with 10 to 36 carbon atoms have been found in
nature in esterified form. Very long-chain (20:1 upwards)
cis-monoenoic fatty acids have relatively high melting points, but
the more common C.sub.18 monoenes tend to be liquid at room
temperature. Triacylglycerols (or oils and fats) containing high
proportions of monoenoic fatty acids are usually liquid at ambient
temperature. Analogous fatty acids with trans double bonds are
normally higher melting. Very-long-chain monoenoic fatty acids of
the (n-9) family occur in a variety of natural sources, often
accompanied by analogous fatty acids of the (n-7) family),
especially in animal tissues. For example, monoenes from 20:1 to
26:1 are normal constituents of animal sphingolipids. Odd-numbered
very-long chain monoenes (23:1 upwards) from brain belong to (n-8)
and (n-10) families, presumably because they are formed by
chain-elongation of 9-17:1 (17:1(n-8)) and 9-19:1 (19:1(n-10)),
respectively (see below). An even wider range of chain-lengths is
found in monoenes from plant waxes and sponge lipids.
[5644] In animals and yeasts, stearoyl-CoA is converted directly to
oleoyl-CoA by a concerted removal of hydrogen atoms from carbons 9
and 10 (D-stereochemistry in each instance). Subsequently, oleate
can be chain elongated by two carbon atoms to give longer-chain
fatty acids of the (n-9) family, while palmitoleate is the
precursor of the (n-7) family of fatty acids. Certain bacteria
produce mono-unsaturated fatty acids by an anaerobic mechanism that
involves the fatty acid synthetase. During the fourth cycle of
chain elongation, a branch point occurs in fatty acid synthesis
following the dehydrase step. Chain elongation can proceed as
normal, or an isomerase can convert the trans-2-decanoyl-ACP too
cis-3-decanoyl-ACP. The latter is not a substrate for the enoyl-ACP
reductase, but it can be further elongated with eventual formation
of a cis-11-18:1 fatty acid. For many years, this was thought to be
the major pathway for biosynthesis of unsaturated fatty acids in
bacteria, but it is now recognised that it is restricted to a few
proteobacteria, such as E. coli. Aerobic mechanisms certainly
exist, but other mechanisms and enzymes have yet to be adequately
characterized for most bacterial species.
[5645] [0004.0.13.13] Principally microorganisms such as
Mortierella or oil producing plants such as soybean, rapeseed or
sunflower or algae such as Crytocodinium or Phaeodactylum are a
common source for oils containing fatty acids, where they are
usually obtained in the form of their triacyl glycerides.
Alternatively, they are obtained advantageously from animals, such
as fish. The free fatty acids are prepared advantageously by
hydrolysis with a strong base such as potassium or sodium
hydroxide.
[5646] [0005.0.13.13] Erucic acid is found in high amounts in seed
oils of the Cruciferae such as Naturtium, rape, mustard, Lunaria,
and of Tropaeolaceae. Other species haven porposed as source of
erucic acid as Crambe (Crambe abyssinica) and meadowfoam
(Limnanthes alba).
[5647] [0006.0.13.13] 11-cis-Eicosenoic acid (gadoleic acid) is a
common if minor constituent of animal tissues and fish oils, often
accompanied by the 13-isomer. It is also found in rapeseed oil and
seed oils of related species. cis-5-20:1 can amount to 67% of the
total fatty acids in meadowfoam oil.
[5648] Similarly, erucic acid (13-22:1) occurs naturally in fish
oils and in small amounts in the phospholipids of animal tissues
(often with some 15-22:1), but it is probably best known as the
major component (up to 66%) of the total fatty acids in rapeseed
oil. 5-22:1 is present in meadowfoam oil.
[5649] [0007.0.13.13] cis-Monoenoic acids obviously have desirable
physical properties for membranes lipids, and they are now
recognised by nutritionists as being beneficial in the human
diet.
[5650] [0008.0.13.13] The exception is erucic acid as there is
evidence from studies with laboratory rats that it may adversely
affect the metabolism of the heart. Erucic acid is used in the
manufacture of industrial oils, e.g. for production of polyethylene
film.
[5651] [0009.0.13.13] As described above, cis-Monoenoic acids are
used in a lot of different applications, for example in cosmetics,
pharmaceuticals and in feed and food.
[5652] [0010.0.13.13] Therefore improving the productivity of such
cis-Monoenoic acids and improving the quality of foodstuffs and
animal feeds is an important task of the different industries.
[5653] [0011.0.13.13] To ensure a high productivity of certain
cis-Monoenoic acids in plants or microorganism, it is necessary to
manipulate the natural biosynthesis of fatty acids in said
organism.
[5654] [0012.0.13.13] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
regulators which participate in the biosynthesis of cis-Monoenoic
acids and make it possible to produce certain cis-Monoenoic acids
specifically on an industrial scale without unwanted byproducts
forming. In the selection of genes for biosynthesis two
characteristics above all are particularly important. On the one
hand, there is as ever a need for improved processes for obtaining
the highest possible contents of cis-Monoenoic acids on the other
hand as less as possible byproducts should be produced in the
production process.
[5655] [0013.0.0.13] see [0013.0.0.0]
[5656] [0014.0.13.13] It was found that the overexpression of the
nucleic acid molecule characterized herein confers an increase in
the content of Eicosenic acid (20:1) in plants. Accordingly, in a
first embodiment, the invention relates to a process for the
production of Eicosenic acid. In Arabidopsis thaliana, Eicosenic
acid is predominately found in the stereoisomeric form 20:1 delta
9c (gadoleic acid). Accordingly, in a further embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is gadoleic acid or
tryglycerides, lipids, oils or fats containing gadoleic acid.
Accordingly, in the present invention, the term "the fine chemical"
as used herein relates to "gadoleic acid and/or tryglycerides,
lipids, oils and/or fats containing gadoleic acid". Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising gadoleic acid and/or triglycerides, lipids,
oils and/or fats containing gadoleic acid.
[5657] Advantageously, the increase of the Eicosenic acid (20:1
fatty acid) content, e.g. the gadoleic acid content, in a
composition produced according to the process of the invention
results in an incrase in the melting point of the composition, in
particular if the composition is a fatty composition as a oil or a
wax. As other monoenoic fatty acids, eicosenic acid, in particular
gadoleic acid, may be used as anti-foaming agent in detergents or
as an anti-blocking agent in the production of platics or used
preservation agent, flouering agent, plastic softener, formulation
agent, flotation agent, wetting agent emulsifer agend and/or
lubricating agents, as e.g. erucic acid, arachinic acid, pelagonic
acid, brassylic acid or erucic acid amidses. Furthermore,
monounsaturated fatty acids as eicosenic acid, in particular
gadolenic acid, are cholesterol lowering when they replace
significant levels of saturated fatty acids in the diet. Some
studies have found that diets high in monounsaturated fatty acids
compared with polyunsaturated fatty acids decrease LDL cholsterol
while maintaining HDL cholesterol levels. However, other studies
suggested that the effect of consuming polyunsaturated fat and
monounsaturated fat is similar and results in a decrease in both
LDL and HDL cholesterol (see e.g. homepage of the Instiute of
Shorting and Edible Oils for references).
[5658] [0015.0.13.13] In one embodiment, the term "the respective
fine chemical" means gadoleic acid and/or tryglycerides, lipids,
oils and/or fats containing gadoleic acid. Throughout the
specification the term "the respective fine chemical" means
gadoleic acid and/or tryglycerides, lipids, oils and/or fats
containing gadoleic acid, gadoleic acid and its salts, ester,
thioester or gadoleic acid in free form or bound to other compounds
such as triglycerides, glycolipids, phospholipids etc. In a
preferred embodiment, the term "the respective fine chemical" means
gadoleic acid, in free form or its salts or bound to triglycerides.
Triglycerides, lipids, oils, fats or lipid mixture thereof shall
mean any triglyceride, lipid, oil and/or fat containing any bound
or free gadoleic acid for example sphingolipids, phosphoglycerides,
lipids, glycolipids such as glycosphingolipids, phospholipids such
as phosphatidylethanolamine, phosphatidylcholine,
phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or
diphosphatidylglycerol, or as monoacylglyceride, diacylglyceride or
triacylglyceride or other fatty acid esters such as acetyl-Coenzym
A thioester, which contain further saturated or unsaturated fatty
acids in the fatty acid molecule.
[5659] [0016.0.13.13] Accordingly, the present invention relates to
a process comprising [5660] (a) increasing or generating the
activity of one or more of YDR513W proteins in a non-human organism
in one or more parts thereof and [5661] (b) growing the organism
under conditions which permit the production of the respective fine
chemical, thus, gadoleic acid or the respective fine chemical
comprising gadoleic acid, in said organism.
[5662] Accordingly, the present invention relates to a process
comprising [5663] (a) increasing or generating the activity of one
or more proteins having the activity of a protein indicated in
Table II, column 3, line 125 or having the sequence of a
polypeptide encoded by a nucleic acid molecule indicated in Table
I, column 5 or 7, lines 125, in a non-human organism in one or more
parts thereof; and [5664] (b) growing the organism under conditions
which permit the production of the respective fine chemical, thus,
gadoleic acid in said organism.
[5665] [0017.0.0.13] to [0018.0.0.13]: see [0017.0.0.0] to
[0018.0.0.0]
[5666] [0019.0.13.13] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the respective fine chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of an YDR513W protein.
[5667] [0020.0.13.13] Surprisingly it was found, that the
transgenic expression of the Saccaromyces cerevisiae protein
YDR513W in Arabidopsis thaliana conferred an increase in the
gadoleic acid content of the transformed plants.
[5668] [0021.0.0.13] see [0021.0.0.0]
[5669] [0022.0.13.13] The sequence of YDR513W from Saccharomyces
cerevisiae has been published in Jacq et al., Nature 387 (6632
Suppl), 75-78 (1997) and in Goffeau et al., Science 274 (5287),
546-547, 1996 and its cellular activity has characterized as
glutathione reductase. Accordingly, in one embodiment, the process
of the present invention comprises the use of glutathione reductase
or a protein of the glutaredoxin superfamily for the production of
gadoleic acid. Accordingly, in one embodiment, the process of the
present invention comprises the use of YDR513W, from Saccharomyces
cerevisiae, e.g. as indicated herein in Table II, line 125, columns
3 or 5, or its homologue, e.g. as shown herein in Table II, line
125, column 7, for the production of the respective fine chemical,
meaning of gadoleic acid and/or tryglycerides, lipids, oils and/or
fats containing gadoleic acid, in particular for increasing the
amount of gadoleic acid and/or tryglycerides, lipids, oils and/or
fats containing gadoleic acid, preferably gadoleic acid in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a glutathione reductase is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[5670] [0023.0.13.13] In one embodiment, the homolog of the YDR513W
is a homolog having said activity and being derived from an
eukaryotic. In one embodiment, the homolog of the YDR513W is a
homolog having said activity and being derived from Fungi. In one
embodiment, the homolog of the YDR513W is a homolog having said
activity and being derived from Ascomyceta. In one embodiment, the
homolog of the YDR513W is a homolog having said activity and being
derived from Saccharomycotina. In one embodiment, the homolog of
the YDR513W is a homolog having said activity and being derived
from Saccharomycetes. Iln one embodiment, the homolog of the
YDR513W is a homolog having said activity and being derived from
Saccharomycetales. In one embodiment, the homolog of the YDR513W is
a homolog having said activity and being derived from
Saccharomycetaceae. In one embodiment, the homolog of the YDR513W
is a homolog having said activity and being derived from
Saccharomycetes.
[5671] [0023.1.13.13] Homologs of the polypeptides indicated in
Table II, column 3, lines 125 may be the polypeptides encoded by
the nucleic acid molecules indicated in Table I, column 7, lines
125, respectively or may be the polypeptides indicated in Table II,
column 7, line 125 having a gadoleic acid content and/or amount
increasing activity.
[5672] [0024.0.0.13] see [0024.0.0.0]
[5673] [0025.0.13.13] In accordance with the invention, a protein
or polypeptide has the "activity of an YDR513W protein" if its de
novo activity, or its increased expression directly or indirectly
leads to an increased level of the respective fine chemical, in
particular gadoleic acid and/or tryglycerides, lipids, oils and/or
fats containing gadoleic acid in the organism or a part thereof,
preferably in a cell of said organism. In one embodiment, the
protein has the above mentioned activities of a protein as
indicated in Table II, line 125, columns 3 or 5. Throughout the
specification the activity or preferably the biological activity of
such a protein or polypeptide or an nucleic acid molecule or
sequence encoding such protein or polypeptide is identical or
similar if it still has the biological or enzymatic activity
protein as indicated in Table II, line 125, columns 3 or 5, or
which has at least 10% of the original enzymatic activity,
preferably 20%, particularly preferably 30%, most particularly
preferably 40% in comparison to an, YDR513W protein of
Saccharomyces cerevisiae
[5674] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organsim indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[5675] [0025.1.0.13] see [0025.1.0.0]
[5676] [0026.0.0.13] to [0033.0.0.13]: see [0026.0.0.0] to
[0033.0.0.0]
[5677] [0034.0.13.13] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention, e.g. by or in the expression
level or activity of an protein having the activity of a protein as
indicated in Table II, column 3, line 125 or being encoded by a
nucleic acid molecule indicated in Table I, column 5, line 125 or
its homologs, e.g. as indicated in Table I, column 7, line 125 its
biochemical or genetical causes and the increased amount of the
respective fine chemical.
[5678] [0035.0.0.13] to [0044.0.0.13]: see [0035.0.0.0] to
[0044.0.0.0]
[5679] [0045.0.13.13] In case the activity of the Saccharomyces
cerevisiae protein YDR513W or its homologs, e.g. a "glutaredoxin
(thioltransferase, glutathione reductase)" is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of gadoleic acid between 46% and 53% or more
is conferred.
[5680] [0046.0.13.13] In one embodiment the activity of the
Saccharomyces cerevisiae protein YDR513W or its homologs, e.g. a
"glutaredoxin (thioltransferase, glutathione reductase)", e.g. as
indicated in Table II, columns 5 or 7. resp., line 125 is
increased, preferably, is increased of the respective fine
chemical; preferably an increase of the respective fine chemical
and of tryglycerides, lipids, oils and/or fats containing gadoleic
acid is conferred.
[5681] [0047.0.0.13] to [0048.0.0.13]: see [0047.0.0.0] to
[0048.0.0.0]
[5682] [0049.0.13.13] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical has
the structure of the polypeptide described herein. In a particular
embodiment, the polypeptides used in the process of the present
invention or the polypeptide of the present invention comprises the
sequence of a consensus sequence as indicated in Table IV, column
7, line 125, respectively, or of a polypeptide as indicated in
Table II, columns 5, line 125, or of a functional homologue thereof
as described herein, e.g as indicated in Table II, columns 7, line
125, or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, line 125, or its herein described
functional homologues and has the herein mentioned activity
conferring an increase in the gadoleic acid level.
[5683] [0050.0.13.13] For the purposes of the present invention,
the term "gadoleic acid" also encompasses the corresponding salts,
such as, for example, the potassium or sodium salts of gadoleic
acid or the salts of gadoleic acid with amines such as
diethylamine.
[5684] [0051.0.13.13] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g monoenoic fatty acid compositions.
Depending on the choice of the organism used for the process
according to the present invention, for example a microorganism or
a plant, compositions or mixtures of various monoenoic fatty acid
can be produced.
[5685] [0052.0.0.13]: see [0052.0.0.0]
[5686] [0053.0.13.13] In one embodiment, the process of the present
invention comprises one or more of the following steps [5687] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention, e.g. of a polypeptide having an
YDR513W protein activity e.g. of a protein as indicated in Table
II, column 5, line 125 or being encoded by a nucleic acid molecule
indicated in Table II, column 5, line 125 or its homologs activity
having herein-mentioned gadoleic acid increasing activity, e.g. as
indicated in Table II, column 7, line 125; [5688] b) stabilizing a
mRNA conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention, e.g. of a polypeptide
having a YDR513W protein a protein as indicated in Table II, column
5, line 125 or being encoded by a nucleic acid molecule indicated
in Table I, column 5, line 125 or its homologs activity or of a
mRNA encoding the polypeptide of the present invention having
herein-mentioned gadoleic acid increasing activity, e.g. as
indicated in Table II, column 7, line 125; [5689] c) increasing the
specific activity of a protein conferring the increased expression
of a protein encoded by the nucleic acid molecule of the invention
or of the polypeptide of the present invention having
herein-mentioned gadoleic acid increasing activity, e.g. of a
polypeptide having a YDR513W protein activity, e.g of a protein as
indicated in Table II, column 5, line 125 or being encoded by a
nucleic acid molecule indicated in Table I, column 5, line 125 or
its homologs activity, e.g. as indicated in Table II, column 7,
line 125, or decreasing the inhibiitory regulation of the
polypeptide of the invention; [5690] d) generating or increasing
the expression of an endogenous or artificial transcription factor
mediating the expression of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptide of the invention having
herein-mentioned gadoleic acid increasing activity, e.g. of a
polypeptide having the YDR513W protein activity, e.g. of a protein
as indicated in Table II, column 5, line 125 or being encoded by a
nucleic acid molecule indicated in Table I, column 5, line 125, or
of their homologs, e.g. as indicated in Table I or II, column 7,
line 125; [5691] e) stimulating activity of a protein conferring
the increased expression of a protein encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention having herein-mentioned gadoleic acid increasing
activity, e.g. of a polypeptide having the YDR513W protein
activity, e.g. of a protein as indicated in Table II, column 5,
line 125 or being encoded by a nucleic acid molecule indicated in
Table I, column 5, line 125, or of their homologs, e.g. as
indicated in Table I or II, column 7, line 125, by adding one or
more exogenous inducing factors to the organisms or parts thereof;
[5692] f) expressing a transgenic gene encoding a protein
conferring the increased expression of a polypeptide encoded by the
nucleic acid molecule of the present invention or a polypeptide of
the present invention, having herein-mentioned gadoleic acid
increasing activity, e.g. of a polypeptide having the YDR513W
protein acitiviy, e.g. of a protein as indicated in Table II,
column 5, line 125 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, line 125, or of their homologs,
e.g. as indicated in Table I or II, column 7, line 125, [5693] g)
increasing the copy number of a gene conferring the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or the
polypeptide of the invention having herein-mentioned gadoleic acid
increasing activity, e.g. of a polypeptide having the YDR513W
protein activity, e.g. of a protein as indicated in Table II,
column 5, line 125 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, line 125, or of their homologs,
e.g. as indicated in Table I or II, column 7, line 125; [5694] h)
Increasing the expression of the endogenous gene encoding the
polypeptide of the invention, e.g. a polypeptide having the YDR513W
protein activity, e.g. of a protein as indicated in Table II,
column 5, line 125 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, line 125, or of their homologs,
e.g. as indicated in Table I or II, column 7, line 125, by adding
positive expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; [5695]
and/or [5696] i) Modulating growth conditions of an organism in
such a manner, that the expression or activity of the gene encoding
the protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production. [5697] j) selecting of organisms with especially high
activity of the proteins of the invention from natural or from
mutagenized resources and breeding them into the target organisms,
eg the elite crops.
[5698] [0054.0.13.13] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of gadoleic acid after
increasing the expression or activity of the encoded polypeptide or
having the activity of a polypeptide having an YDR513W protein,
e.g. of a protein as indicated in Table II, column 5, line 125 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, line 125, or of their homologs, e.g. as indicated in
Table I or II, column 7, line 125.
[5699] [0055.0.0.13] to [0067.0.0.13]: see [0055.0.0.0] to
[0067.0.0.0]
[5700] [0068.0.13.13] The mutation is introduced in such a way that
the production of the fatty acids is not adversely affected.
[5701] [0069.0.0.13]: see [0069.0.0.0]
[5702] [0070.0.13.13] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding the
YDR513W protein, e.g. of a protein as indicated in Table II, column
5, line 125 or being encoded by a nucleic acid molecule indicated
in Table I, column 5, line 125 or of its homologs, e.g. as
indicated in Table II, column 7, line 125, into an organism alone
or in combination with other genes, it is possible not only to
increase the biosynthetic flux towards the end product, but also to
increase, modify or create de novo an advantageous, preferably
novel metabolites composition in the organism, e.g. an advantageous
fatty acid composition comprising a higher content of (from a
viewpoint of nutritional physiology limited) fatty acids, like
palmitate, palmitoleate, oleate and/or linoleate. In one
embodiment, the expression of a protein as indicated in Table II,
column 5, line 125 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, line 125 or of its homologs, e.g.
as indicated in Table II, column 7, line 125 conferring an increase
of the eicosenic acid, in particular of gadoleic acid, is combined
with a low level of or an decrease of the production of erucic
acid. In one embodiment, the expression of a protein as indicated
in Table II, column 5, line 125 or being encoded by a nucleic acid
molecule indicated in Table I, column 5, line 125 or of its
homologs, e.g. as indicated in Table II, column 7, line 125
conferring an increase of eicosenic acid, in particular of gadoleic
acid, is combined with a low level of or a decrease of the
production of glucosinolate, preferably in the oil extraction press
cake. However, erucic acid can be biosynthesised from gadoleic acid
and a high level of erucic acid is advantageous in specific
embodiments, e.g. as described above, e.g. for the production of
fats and oils for industrial uses, e.g. for detergents and cleaning
agents through cosmetics to dye additives, lubricating agents and
hydraulic oils. In particular, a high content of erucic acid is
regarded a breeding goal in classic as well as in modern plant
breeding, since it is used as an anti-foaming agent, an anti
blocking agent, a preservation agent, flavouring agent, platic
softener, formulation agent, flotation agent, wetting agent,
emulsifier, or lubricating agent, e.g. as or together with its
derivatives arachinic acid, pelagonic acid, brassylic acid and/or
erucic acid amides. Thus, in an advantageous embodiment, the
gadoleic acid content is increased according to the process of the
present invention in an organism with a high erucic acid content or
together with enzymes or regulators conferring an erucic acid
increase, in particular with enzymes or regulators conferring the
formation or elongation of erucic acid from gadoleic acid.
[5703] [0071.0.0.13]: see [0071.0.0.0]
[5704] [0072.0.13.13] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to gadoleic acid, triglycerides, lipids, oils and/or fats
containing gadoleic acid compounds such as palmitate, palmitoleate,
oleate and/or linoleic acid.
[5705] [0073.0.13.13] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[5706] m) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [5707] n) increasing the YDR513W
protein activity or of a polypeptide being encoded by the nucleic
acid molecule of the present invention and described below, e.g. of
a protein as indicated in Table II, column 5, line 125 or being
encoded by a nucleic acid molecule indicated in Table I, column 5,
line 125 or of its homologs, e.g. as indicated in Table II, column
7, line 125, and conferring an increase of the respective fine
chemical in the organism, preferably in a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant, [5708] o) growing a organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant under conditions which permit the
production of the respective fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and [5709] p) if desired, revovering, optionally
isolating, the free and/or bound the respective fine chemical and,
optionally further free and/or bound fatty acids synthetized by the
organism, the microorganism, the non-human animal, the plant or
animal cell, the plant or animal tissue or the plant.
[5710] [0074.0.13.13] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the respective fine chemical or the free and
bound the respective fine chemical but as option it is also
possible to produce, recover and, if desired isolate, other free
or/and bound fatty acids, in particular gadoleic acid.
[5711] [0075.0.0.13] to [0077.0.0.13]: see [0075.0.0.0] to
[0077.0.0.0]
[5712] [0078.0.13.13] The organism such as microorganisms or plants
or the recovered, and if desired isolated, fine chemical can then
be processed further directly into foodstuffs or animal feeds or
for other applications, for example according to the disclosures
made in EP-A-0 568 608, EP-A-568 606, WO 2004/007732, WO 02/057465,
WO 01/02591, WO 2004/071467 or US 20020156254, which are expressly
incorporated herein by reference. The fermentation broth,
fermentation products, plants or plant products can be purified in
the customary manner by hydrolysis with strong bases, extraction
and crystallization or via thin layer chromatography and other
methods known to the person skilled in the art and described herein
below. Products of these different work-up procedures are fatty
acids or fatty acid compositions which still comprise fermentation
broth, plant particles and cell components in different amounts,
advantageously in the range of from 0 to 99% by weight, preferably
below 80% by weight, especially preferably between below 50% by
weight.
[5713] [0079.0.0.13] to [0084.0.0.13]: see [0079.0.0.0] to
[0084.0.0.0]
[5714] [0084.1.13.13] It was found that the level of eicosenic acid
is high in the following plants seeds with decreasing level:
Selenia grandis, Teesdalia nudicaulis (L.) R. Br., Teesdalia
nudicaulis (L.) R. Br., Cardiospermum canescens, Lesouerella
fendleri, Leavenworthia torulosa, Leavenworthia torulosa,
Cardiospermum grandiflorum, Cardiospermum halicacabum, Paullinia
elegans, Cupania anacardioides, Koelreuteria elegans, Koelreuteria
apiculata, Lobularia maritima, Iberis odorata, Alyssum maritimum,
Cardiospermum corindum, Biscutella laevigata, Biscutella
auriculata, Lobularia maritima, Dithyrea californica, Dithyrea
wislizenii, Thysanocarpus radians, Cheirantus maritimus, Cordia
verbenacea DC. , Conringia orientalis (L.) Dumort. , Tropaeolum
majus, Conringia orientalis (L.) Dumort. , Tropaeolum maius,
Arachis hypogaea, Neslia paniculata, Malcolmia maritima, Malcolmia
flexuosa, Arachis peruviana, Sapindus mukorossi, Conringia
orientalis, Arachis fastigiata, Arabidopsis thaliana, Arabidopsis
thaliana Schur, Sapindus mukorossi, Azima tetracantha, Tropaeolum
majus, Tropaeolum majus, Brassica repanda (WilId.) DC. , Calepina
irregularis, Arabidopsis thaliana (L.) Hevnh. Columbia, Crambe
orientalis, Nerisvrenia camporum, Crambe maritima L., Delphinium
ajacis, Tropaeolum minus L., Cardamine impatiens, Brassica oleracea
L. ssp. robertiana (Gay) Rouy et Foug., Delphinium spp., Camelina
rumelica, Juniperus chinensis, Capsella rubella, Capsella rubella,
Cordia moxa, Sapindus emardnatus, Lepidium cuneiforme, Caulanthus
inflatus, Malcolmia chia, Camelina sativa, Cardamine bellidifolia
L., Atalaya hemiglauca, Aesculus assamica, Leonurus sibiricus,
Capsella grandiflora, Capsella grandiflora, Sinapis arvensis L.,
Erysimum perovskianum, Camelina microcarpa, Schleichera trijuga,
Schleichera trijuga, Brassica iuncea, Tropaeolum maius, Schleichera
trijuga, Cardamine hirsuta, Cardamine amare, Lepidium lasiocarpum,
Stanleyella texana, Sapindus mukorossi, Goldbachia laevigata D. C.,
Descurainia bourgaeana Webb., Crambe maritima, Consolida
orientalis, Tropaeolum maius, Tropaeolum maius, Lunaria rediviva,
Tristellateia australasica, Lepidium perfoliatum, Camelina sativa,
Camelina sativa, Lepidium apetalum, Sapindus saponaria, Neslia
paniculata, Sapindus saponaria, Brassica sp., Brassica juncea,
Coronopus didymus, Fezia pterocarpa pitard, Arabis Qlabra, Eruca
sativa, Descurainia pinnata var. pinnata, Hornunda petraea,
Descurainia sophia, Phoenix dactvlifera L., Descurainia sophia,
Thlaspi alpinum, Capsella bursa-pastoris, Capsella bursa-pastoris,
Capsella spp., Capsella spp., Descurainia sophia, Capsella spp.,
Capsella spp., Tropaeolum minus L., Capsella bursa-pastoris,
Brassica campestris, Lunaria rediviva, Capsella spp., Descurainia
sophia, Capsella spp., Capsella bursa-pastoris, Clitoria
rubiginosa, Arabis laevigata, Thlaspi alpestre L., Camelina sativa,
Crambe cordifolia, Capsella bursa-pastoris, Savignya parviflora,
Brassica campestris, Lepidium sativum, Arabis virginica, Sophia
ochroleuca, Sapindus emardnatus, Brassica cossoneana (Boiss. et
Reuter) Maire, Parrya menziesii, Diplotaxis tenuifolia. For
example, it was found that the following content of eicosenic acid
is comprised in the oil of seeds: 58.50, GLC-Area-% , Selenia
grandis, 56.10, GLC-Area-%, Teesdalia nudicaulis (L.) R. Br. ,
56.00, GLC-Area-% , Teesdalia nudicaulis (L.) R. Br. , 55.70, GLC
area % , Cardiospermum canescens, 54.50, GLC-Area-%, Lespuerella
fendleri, 53.00, GLC-Area-% , Leavenworthia torulosa, 53.00,
GLC-Area-%, Leavenworthia torulosa, 52.60, GLC-Area-% ,
Cardiospermum grandiflorum, 49.10, GLC-Area-% , Cardiospermum
halicacabum, 48.70, GLC-Area-% , Paullinia elegans, 46.00,
GLC-Area-% , Cupania anacardioides, 45.30, GLC-Area-%, Koelreuteria
elegans, 44.60, GLC area % , Koelreuteria apiculata, 42.00,
GLC-Area-%, Lobularia maritima, 41.90, GLC-Area-% , Iberis odorata,
41.80, GLC-Area-%, Alyssum maritimum, 41.60, GLC-Area-% ,
Cardiospermum corindum, 40.00, GLC-Area-% , Biscutella laevigata,
36.10, GLC-area-% , Biscutella auriculata, 35.60, GLC-area-% ,
Lobularia maritima. A further list of plants with high gadoleic
acid can be found on webpage of the Agricultural Research service
(www.ars-grin.gov/cgi-bin/duke/chemical.pl?gadoleicacid) showing
plant species with the highest amount of gadoleic acid. The content
of fatty acids in the oil of plants and plants which have a high
level of distinct fatty acids can also be identified on the webpage
of the Institute of Chemistry and Physics of Lipids,
http://www.bagkf.de/sofa/.
[5715] Thus, in on embodiment, the process of the present invention
is performed in a plant of the above lists of identified in above
webpages, preferably in a plant with a high amount of eicosenic
acid, in particular with high gadoleic level and/or together with
further genes isolated from a plant with a high eicosenic acid
level, in particular a high gadoleic level. In a preferred
embodiment, the process of the present invention is performed in a
plant identified in above webpages with a low amount of erucic
acid, in particular with high gadoleic level and/or together with
further genes isolated from a plant with a high eicosenic acid
level, in particular a high gadoleic level. In another embodiment,
the process of the present invention is performed in a plant
identified in above webpages, preferably in a plant with a high
amount of erucic acid, in particular with high gadoleic level
and/or together with further genes isolated from a plant with a
high eicosenic acid level, in particular a high gadoleic level.
[5716] [0085.0.13.13] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [5717] a) a nucleic acid sequence as
shown in table I, line 125, columns 5 or 7 or a derivative thereof,
or [5718] b) a genetic regulatory element, for example a promoter,
which is functionally linked to the nucleic acid sequence as shown
table I, line 125, columns 5 or 7 or a derivative thereof, or
[5719] c) (a) and (b) is/are not present in its/their natural
genetic environment or has/have been modified by means of genetic
manipulation methods, it being possible for the modification to be,
by way of example, a substitution, addition, deletion, inversion or
insertion of one or more nucleotide. "Natural genetic environment"
means the natural chromosomal locus in the organism of origin or
the presence in a genomic library. In the case of a genomic
library, the natural, genetic environment of the nucleic acid
sequence is preferably at least partially still preserved. The
environment flanks the nucleic acid sequence at least on one side
and has a sequence length of at least 50 bp, preferably at least
500 bp, particularly preferably at least 1000 bp, very particularly
preferably at least 5000 bp.
[5720] [0086.0.0.13] to [0087.0.0.13]: see [0086.0.0.0] to
[0087.0.0.0]
[5721] [0088.0.13.13] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose fatty acid
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for poultry is dependent on the abovementioned essential fatty
acids and the general amount of fatty acids as energy source in
feed. After the YDR513W protein activity, e.g. of a protein as
indicated in Table II, column 5, line 125 or being encoded by a
nucleic acid molecule indicated in Table I, column 5, line 125 or
of its homologs, e.g. as indicated in Table II, column 7, line 125,
has been increased or generated, or after the expression of nucleic
acid molecule or polypeptide according to the invention has been
generated or increased, the transgenic plant generated thus is
grown on or in a nutrient medium or else in the soil and
subsequently harvested.
[5722] [0088.1.0.13]: see [0088.1.0.0]
[5723] [0089.0.0.13] to [0094.0.0.13]: see [0089.0.0.0] to
[0094.0.0.0]
[5724] [0095.0.13.13] It may be advantageous to increase the pool
of free fatty acids in the transgenic organisms by the process
according to the invention in order to isolate high amounts of the
pure fine chemical.
[5725] [0096.0.13.13] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid for example a fatty acid
transporter protein or a compound, which functions as a sink for
the desired fatty acid for example for linoleic or linolenic acid
in the organism is useful to increase the production of the
respective fine chemical (see Bao and Ohlrogge, Plant Physiol. 1999
August; 120 (4): 1057-1062). Such fatty acid transporter protein
may serve as a link between the location of fatty acid synthesis
and the socalled sink tissue, in which fatty acids, triglycerides,
oils and fats are stored.
[5726] [0097.0.0.13]: see [0097.0.0.0]
[5727] [0098.0.13.13] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated. The production of further fatty acids such as
palmitic acid, palmitoleic acid, oleic acid, linoleic acid and/or
linolenic acidmixtures thereof or mixtures of other fatty acids
and/or low in the level of erucic acid and/or of glucosinolate
level by the process according to the invention is advantageous,
e.g. for the production of compositions used in food and feed. In
another embodiment, the production of gadoleic acid is combined
with the production of erucic acid.
[5728] [0099.0.13.13] In the case of the fermentation of
microorganisms, the abovementioned fatty acids may accumulate in
the medium and/or the cells. If microorganisms are used in the
process according to the invention, the fermentation broth can be
processed after the cultivation. Depending on the requirement, all
or some of the biomass can be removed from the fermentation broth
by separation methods such as, for example, centrifugation,
filtration, decanting or a combination of these methods, or else
the biomass can be left in the fermentation broth. The fermentation
broth can subsequently be reduced, or concentrated, with the aid of
known methods such as, for example, rotary evaporator, thin-layer
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. Afterwards advantageously further compounds for
formulation can be added such as corn starch or silicates. This
concentrated fermentation broth advantageously together with
compounds for the formulation can subsequently be processed by
lyophilization, spray drying, spray granulation or by other
methods. Preferably the fatty acids or the fatty acid compositions
are isolated from the organisms, such as the microorganisms or
plants or the culture medium in or on which the organisms have been
grown, or from the organism and the culture medium, in the known
manner, for example via extraction, distillation, crystallization,
chromatography or a combination of these methods. These
purification methods can be used alone or in combination with the
aforementioned methods such as the separation and/or concentration
methods.
[5729] [0100.0.13.13] Transgenic plants which comprise the fatty
acids such as saturated or polyunsaturated fatty acids synthesized
in the process according to the invention can advantageously be
marketed directly without there being any need for the oils, lipids
or fatty acids synthesized to be isolated. Plants for the process
according to the invention are listed as meaning intact plants and
all plant parts, plant organs or plant parts such as leaf, stem,
seeds, root, tubers, anthers, fibers, root hairs, stalks, embryos,
calli, cotelydons, petioles, harvested material, plant tissue,
reproductive tissue and cell cultures which are derived from the
actual transgenic plant and/or can be used for bringing about the
transgenic plant. In this context, the seed comprises all parts of
the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue. However, the respective fine
chemical produced in the process according to the invention can
also be isolated from the organisms, advantageously plants, in the
form of their oils, fats, lipids and/or free fatty acids. Fatty
acids produced by this process can be obtained by harvesting the
organisms, either from the crop in which they grow, or from the
field. This can be done via pressing or extraction of the plant
parts, preferably the plant seeds. To increase the efficiency of
oil extraction it is beneficial to clean, to temper and if
necessary to hull and to flake the plant material especially the
seeds. In this context, the oils, fats, lipids and/or free fatty
acids can be obtained by what is known as cold beating or cold
pressing without applying heat. To allow for greater ease of
disruption of the plant parts, specifically the seeds, they are
previously comminuted, steamed or roasted. The seeds, which have
been pretreated in this manner can subsequently be pressed or
extracted with solvents such as warm hexane. The solvent is
subsequently removed. In the case of microorganisms, the latter
are, after harvesting, for example extracted directly without
further processing steps or else, after disruption, extracted via
various methods with which the skilled worker is familiar. In this
manner, more than 96% of the compounds produced in the process can
be isolated. Thereafter, the resulting products are processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter are first removed.
What is known as desliming can be affected enzymatically or, for
example, chemico-physically by addition of acid such as phosphoric
acid. Thereafter otionally, the free fatty acids are removed by
treatment with a base like alkali, for example aqueous KOH or NaOH,
or acid hydrolysis, advantageously in the presence of an alcohol
such as methanol or ethanol, or via enzymatic cleavage, and
isolated via, for example, phase separation and subsequent
acidification via, for example, H.sub.2SO.sub.4. The fatty acids
can also be liberated directly without the above-described
processing step. If desired the resulting product can be washed
thoroughly with water to remove traces of soap and the alkali
remaining in the product and then dried. To remove the pigment
remaining in the product, the products can be subjected to
bleaching, for example using filler's earth or active charcoal. At
the end, the product can be deodorized, for example using steam
distillation under vacuum. These chemically pure fatty acids or
fatty acid compositions are advantageous for applications in the
food industry sector, the cosmetic sector and especially the
pharmacological industry sector.
[5730] [0101.0.13.13] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[5731] [0102.0.13.13] Fatty acids can for excample be detected
advantageously via GC separation methods. The unambiguous detection
for the presence of fatty acid products can be obtained by
analyzing recombinant organisms using analytical standard methods:
GC, GC-MS or TLC, as described on several occasions by Christie and
the references therein (1997, in: Advances on Lipid Methodology,
Fourth Edition: Christie, Oily Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). One
example is the analysis of fatty acids via FAME and GC-MS or TLC
(abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer chromatography.
The material to be analyzed can be disrupted by sonication,
grinding in a glass mill, liquid nitrogen and grinding or via other
applicable methods. After disruption, the material must be
centrifuged. The sediment is resuspended in distilled water, heated
for 10 minutes at 100.degree. C., cooled on ice and recentrifuged,
followed by extraction for one hour at 90.degree. C. in 0.5 M
sulfuric acid in methanol with 2% dimethoxypropane, which leads to
hydrolyzed oil and lipid compounds, which give transmethylated
lipids. These fatty acid methyl esters are extracted in petroleum
ether and finally subjected to a GC analysis using a capillary
column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 .mu.m, 0.32
mm) at a temperature gradient of between 170.degree. C. and
240.degree. C. for 20 minutes and 5 minutes at 240.degree. C. The
identity of the resulting fatty acid methyl esters must be defined
using standards which are available from commercial sources (i.e.
Sigma).
[5732] [0103.0.13.13] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [5733] a) nucleic acid molecule encoding, preferably
at least the mature form, of the polypeptide shown in table II,
line 125, columns 5 or 7 or a fragment thereof, which confers an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [5734] b) nucleic acid molecule
comprising, preferably at least the mature form, of the nucleic
acid molecule shown in table I, line 125, columns 5 or 7; [5735] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [5736] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[5737] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [5738]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [5739] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [5740] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers shown in
table III, line 125, column 7 and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [5741] i) nucleic acid molecule encoding a polypeptide
which is isolated, e.g. from an expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (h), preferably to (a) to (c), and
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [5742] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence shown in table IV, line 125, column 7 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [5743] k) nucleic acid molecule
comprising one or more of the nucleic acid molecule encoding the
amino acid sequence of a polypeptide encoding a domain of the
polypeptide shown in table II, line 125, columns 5 or 7 and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; and [5744] l) nucleic
acid molecule which is obtainable by screening a suitable library
under stringent conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k), preferably to
(a) to (c), or with a fragment of at least 15 nt, preferably 20 nt,
30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k), preferably to (a) to (c), and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which comprises a
sequence which is complementary thereto.
[5745] [0103.1.13.13.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, line 125, by one or
more nucleotides. In one embodiment, the nucleic acid molecule used
in the process of the invention does not consist of the sequence
shown in indicated in Table I A, columns 5 or 7, line 125. In one
embodiment, the nucleic acid molecule used in the process of the
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I A, columns 5 or 7,
line 125. In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table II A, columns
5 or 7, line 125.
[5746] [0103.2.13.13.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, line 125, by one
or more nucleotides. In one embodiment, the nucleic acid molecule
used in the process of the invention does not consist of the
sequence shown in indicated in Table I B, columns 5 or 7, line 125.
In one embodiment, the nucleic acid molecule used in the process of
the invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I B, columns 5 or 7,
line 125. In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table II B, columns
5 or 7, line 125.
[5747] [0104.0.13.13] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence shown in table
I, line 125, columns 5 or 7 by one or more nucleotides or does not
consist of the sequence shown in table I, line 125, columns 5 or 7.
In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence shown in table I, line 125, columns 5 or
7. In another embodiment, the nucleic acid molecule does not encode
a polypeptide of the sequence shown in table II, line 125, columns
5 or 7.
[5748] [0105.0.0.13] to [0107.0.0.13]: see [0105.0.0.0] to
[0107.0.0.0]
[5749] [0108.0.13.13] Nucleic acid molecules with the sequence
shown in table I, line 125, columns 5 or 7, nucleic acid molecules
which are derived from the amino acid sequences shown in table II,
line 125, columns 5 or 7 or from polypeptides comprising the
consensus sequence shown in table IV, line 125, column 7, or their
derivatives or homologues encoding polypeptides with the enzymatic
or biological activity of an YDR513W protein or conferring of
eicosenic acid, in particular of gadoleic acid increase after
increasing its expression or activity are advantageously increased
in the process according to the invention.
[5750] [0109.0.0.13]: see [0109.0.0.0]
[5751] [0110.0.13.13] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with YDR513W protein activity, e.g. of a
protein as indicated in Table II, column 5, line 125 or being
encoded by a nucleic acid molecule indicated in Table I, column 5,
line 125 or of its homologs, e.g. as indicated in Table II, column
7, line 125, can be determined from generally accessible
databases.
[5752] [0111.0.0.13] see [0111.0.0.0]
[5753] [0112.0.13.13] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with YDR513W
protein activity and conferring gadoleic acid increase, e.g. a
protein as indicated in Table II, column 5, line 125 or being
encoded by a nucleic acid molecule indicated in Table I, column 5,
line 125, or of their homologs, e.g. as indicated in Table I or II,
column 7, line 125.
[5754] [0113.0.0.13] to [0120.0.0.13]: see [0113.0.0.0] to
[0120.0.0.0]
[5755] [0121.0.13.13] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences shown in table II, line
125, columns 5 or 7 or the functional homologues thereof as
described herein, preferably conferring above-mentioned activity,
i.e. conferring a respective fine chemical increase after
increasing its activity, e.g. having the activity of an YDR513W
protein, e.g. of a protein as indicated in Table II, column 5, line
125 or being encoded by a nucleic acid molecule indicated in Table
I, column 5, line 125 or of its homologs, e.g. as indicated in
Table II, column 7, line 125.
[5756] [0122.0.0.13] to [0127.0.0.13]: see [0122.0.0.0] to
[0127.0.0.0]
[5757] [0128.0.13.13] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs shown in table III, line 125,
column 7, by means of polymerase chain reaction can be generated on
the basis of a sequence shown herein, for example the sequence
shown in table I, line 125, columns 5 or 7 or the sequences derived
from table II, line 125, columns 5 or 7.
[5758] [0129.0.13.13] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence shown in table IV, line
125, column 7 is derived from said alignments.
[5759] [0130.0.13.13] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of the
respective fine chemical, in particular of gadoleic acid after
increasing the expression or activity or having an YDR513W activity
or further functional homologs of the polypeptide of the invention,
e.g. homologs from a protein as indicated in Table II, column 5,
line 125 or being encoded by a nucleic acid molecule indicated in
Table I, column 5, line 125 or of its homologs, e.g. as indicated
in Table II, column 7, line 125, from other organisms.
[5760] [0131.0.0.13] to [0138.0.0.13]: see [0131.0.0.0] to
[0138.0.0.0]
[5761] [0139.0.13.13] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical increase, derived from
other organisms, can be encoded by other DNA sequences which
hybridize to the sequences shown in table I, columns 5 or 7, line
125, preferably of Table I B, columns 5 or 7, line 125 under
relaxed hybridization conditions and which code on expression for
peptides having the fine chemical-increasing activity.
[5762] [0140.0.0.13] to [0146.0.0.13]: see [0140.0.0.0] to
[0146.0.0.0]
[5763] [0147.0.13.13] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences shown in Table I,
columns 5 or 7, line 125, preferably of Table I B, columns 5 or 7,
line 125 is one which is sufficiently complementary to one of the
nucleotide sequences shown in table I, line 125, columns 5 or 7
such that it can hybridize to one of the nucleotide sequences shown
in table I, line 125, columns 5 or 7, preferably of Table I B,
columns 5 or 7, line 125, thereby forming a stable duplex.
Preferably, the hybridisation is performed under stringent
hybridization conditions. However, a complement of one of the
herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[5764] [0148.0.13.13] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence shown in table I, line 125, columns 5 or 7,
preferably of Table I B, columns 5 or 7, line 125 or a functional
portion thereof and preferably has above mentioned activity, in
particular having a the fine chemical, in particular gadoleic
acid-increasing activity after increasing the activity or an
activity of an YDR513W gene product, e.g. a gene encoding a protein
as indicated in Table II, column 5, line 125, preferably of Table
II B, column 5, line 125 or comprising or expressing a nucleic acid
molecule indicated in Table I, column 5, line 125, or of their
homologs, e.g. as indicated in Table I or II, column 7, line
125.
[5765] [0149.0.13.13] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences shown in table I, line 125, columns 5 or
7, preferably of Table I B, columns 5 or 7, line 125 or a portion
thereof and encodes a protein having above-mentioned activity, e.g.
conferring an increase of the fine chemical, and optionally, the
activity of YDR513W, e.g. of a protein as indicated in Table II,
column 5, line 125, preferably of Table II B, column 5, line 125 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, line 125, or of their homologs, e.g. as indicated in
Table I or II, column 7, line 125.
[5766] [00149.1.0.13] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, line 125, preferably of Table I B, columns 5 or 7,
line 125 has further one or more of the activities annotated or
known for the a protein as indicated in Table II, column 3, line
125, preferably of Table II B, column 3, line 125.
[5767] [0150.0.13.13] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences shown in table I, line 125, columns 5 or 7,
preferably of Table I B, columns 5 or 7, line 125 for example a
fragment which can be used as a probe or primer or a fragment
encoding a biologically active portion of the polypeptide of the
present invention or of a polypeptide used in the process of the
present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of gadoleic acid if its activity is
increased. The nucleotide sequences determined from the cloning of
the present protein-according-to-the-invention-encoding gene allows
for the generation of probes and primers designed for use in
identifying and/or cloning its homologues in other cell types and
organisms. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, 15 preferably about 20 or 25, more
preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one of the sequences set forth, e.g., in table I, line
125, columns 5 or 7, an anti-sense sequence of one of the
sequences, e.g., set forth in table I, line 125, columns 5 or 7, or
naturally occurring mutants thereof. Primers based on a nucleotide
of invention can be used in PCR reactions to clone homologues of
the polypeptide of the invention or of the polypeptide used in the
process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primers pairs shown in table III, line 125, column 7
will result in a fragment of YDR513W gene product, e.g. of a gene
encoding of a protein as indicated in Table II, column 5, line 125
or expressing a nucleic acid molecule indicated in Table I, column
5, line 125 or of its homologs, e.g. as indicated in Table II,
column 7, line 125. Preferred is Table II B, column 7, line
125.
[5768] [0151.0.0.13] see [0151.0.0.0]
[5769] [0152.0.13.13] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to the amino acid
sequence shown in table II, line 125, columns 5 or 7 such that the
protein or portion thereof maintains the ability to participate in
the respective fine chemical production, in particular a gadoleic
acid increasing the activity as mentioned above or as described in
the examples in plants or microorganisms is comprised.
[5770] [0153.0.13.13] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence shown in table II, line 125, columns 5 or 7 such that
the protein or portion thereof is able to participate in the
increase of the respective fine chemical production. In one
embodiment, a protein or portion thereof as indicated in Table II,
columns 5 or 7, line 125 has for example an activity of a
polypeptide indicated in Table II, column 3, line 125.
[5771] [0154.0.13.13] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence of table II, line 125, columns 5 or 7 and having
above-mentioned activity, e.g. conferring preferably the increase
of the respective fine chemical.
[5772] [0155.0.0.13] to [0156.0.0.13]: see [0155.0.0.0] to
[0156.0.0.0]
[5773] [0157.0.13.13] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences shown in
table I, line 125, columns 5 or 7 (and portions thereof) due to
degeneracy of the genetic code and thus encode a polypeptide of the
present invention, in particular a polypeptide having above
mentioned activity, e.g. conferring an increase in the respective
fine chemical in a organism, e.g. as that polypeptides encoded by
the sequence shown in table II, line 125, columns 5 or 7 or the
functional homologues. Advantageously, the nucleic acid molecule of
the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, an amino acid sequence shown in table II, line
125, columns 5 or 7 or the functional homologues. In a still
further embodiment, the nucleic acid molecule of the invention
encodes a full length protein which is substantially homologous to
an amino acid sequence shown in table II, line 125, columns 5 or 7
or the functional homologues. However, in a preferred embodiment,
the nucleic acid molecule of the present invention does not consist
of a sequence as indicated in Table I, columns 5 or 7, line 125,
preferably as indicated in Table I A, columns 5 or 7, line 125.
Preferably the nucleic acid molecule of the invention is a
functional homologue or identical to a nucleic acid molecule
indicated in Table I B, columns 5 or 7, line 125.
[5774] [0158.0.0.13] to [0160.0.0.13]: see [0158.0.0.0] to
[0160.0.0.0]
[5775] [0161.0.13.13] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence shown in table I, line 125, columns 5 or 7. The nucleic
acid molecule is preferably at least 20, 30, 50, 100, 250 or more
nucleotides in length.
[5776] [0162.0.0.13] see [0162.0.0.0]
[5777] [0163.0.13.13] Preferably, a nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
shown in table I, line 125, columns 5 or 7 corresponds to a
naturally-occurring nucleic acid molecule of the invention. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein). Preferably, the nucleic
acid molecule encodes a natural protein having above-mentioned
activity, e.g. conferring the respective fine chemical increase
after increasing the expression or activity thereof or the activity
of a protein of the invention or used in the process of the
invention.
[5778] [0164.0.0.13] see [0164.0.0.0]
[5779] [0165.0.13.13] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. shown in
table I, line 125, columns 5 or 7.
[5780] [0166.0.0.13] to [0167.0.0.13]: see [0166.0.0.0] to
[0167.0.0.0]
[5781] [0168.0.13.13] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in the sequences shown in table II, line 125, columns 5
or 7, preferably of Table II B, column 7, lines 125 yet retain said
activity described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence shown in table II, line 125, columns 5 or 7,
preferably of Table II B, column 7, line 125 and is capable of
participation in the increase of production of the respective fine
chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to the sequence shown in table II, line
125, columns 5 or 7, preferably of Table II B, column 7, line 125,
more preferably at least about 70% identical to one of the
sequences shown in table II, line 125, columns 5 or 7, preferably
of Table II B, column 7, line 125, even more preferably at least
about 80%, 90%, 95% homologous to the sequence shown in table II,
line 125, columns 5 or 7, and most preferably at least about 96%,
97%, 98%, or 99% identical to the sequence shown in table II, line
125, columns 5 or 7, preferably of Table II B, column 7, line
125.
[5782] [0169.0.0.13] to [0172.0.0.13]: see [0169.0.0.0] to
[0172.0.0.0]
[5783] [0173.0.13.13] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 13791 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 13791 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[5784] [0174.0.0.13] see [0174.0.0.0]
[5785] [0175.0.13.13] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 13792 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 13792 by the above program algorithm with the
above parameter set, has a 80% homology.
[5786] [0176.0.13.13] Functional equivalents derived from one of a
polypeptides as shown in table II, line 125, columns 5 or 7
according to the invention by substitution, insertion or deletion
have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%,
60%, 65% or 70% by preference at least 80%, especially preferably
at least 85% or 90%, 91%, 92%, 93% or 94%, very especially
preferably at least 95%, 97%, 98% or 99% homology with one of a
polypeptides as shown in table II, line 125, columns 5 or 7
according to the invention and are distinguished by essentially the
same properties as a polypeptide as shown in table II, line 125,
columns 5 or 7.
[5787] [0177.0.13.13] Functional equivalents derived from a nucleic
acid sequence as shown in table I, line 125, columns 5 or 7,
preferably of Table I B, column 7, line 125 according to the
invention by substitution, insertion or deletion have at least 30%,
35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by
preference at least 80%, especially preferably at least 85% or 90%,
91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%,
98% or 99% homology with one of a polypeptides as shown in table
II, line 125, columns 5 or 7, preferably of Table II B, column 7,
line 125 according to the invention and encode polypeptides having
essentially the same properties as a polypeptide as shown in table
II, line 125, columns 5 or 7, preferably of Table II B, column 7,
line 125.
[5788] [0178.0.0.13]: see [0178.0.0.0]
[5789] [0179.0.13.13] A nucleic acid molecule encoding an
homologous to a protein sequence of table II, line 125, columns 5
or 7, preferably of Table II B, column 7, line 125 can be created
by introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of the nucleic acid molecule
of the present invention, in particular of table I, line 125,
columns 5 or 7 such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences of table I,
line 125, columns 5 or 7 by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
[5790] [0180.0.0.13] to [0183.0.0.13]: see [0180.0.0.0] to
[0183.0.0.0]
[5791] [0184.0.13.13] Homologues of the nucleic acid sequences
used, with a sequence shown in table I, line 125, columns 5 or 7,
preferably of Table I B, column 7, lines 125, comprise also allelic
variants with at least approximately 30%, 35%, 40% or 45% homology,
by preference at least approximately 50%, 60% or 70%, more
preferably at least approximately 90%, 91%, 92%, 93%, 94% or 95%
and even more preferably at least approximately 96%, 97%, 98%, 99%
or more homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from a
sequences shown, preferably from table I, line 125, columns 5 or 7,
or from the derived nucleic acid sequences, the intention being,
however, that the enzyme activity or the biological activity of the
resulting proteins synthesized is advantageously retained or
increased.
[5792] [0185.0.13.13] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more of thesequences shown in any
of the table I, line 125, columns 5 or 7, preferably of Table I B,
column 7, line 125. In one embodiment, it is preferred that the
nucleic acid molecule comprises as little as possible other
nucleotides not shown in any one of table I, line 125, columns 5 or
7, preferably of Table I B, column 7, line 125. In one embodiment,
the nucleic acid molecule comprises less than 500, 400, 300, 200,
100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a further
embodiment, the nucleic acid molecule comprises less than 30, 20 or
10 further nucleotides. In one embodiment, the nucleic acid
molecule use in the process of the invention is identical to a
sequences shown in table I, line 125, columns 5 or 7, preferably of
Table I B, column 7, line 125.
[5793] [0186.0.13.13] Also preferred is that one or more nucleic
acid molecule used in the process of the invention encodes a
polypeptide comprising a sequence shown in table II, line 125,
columns 5 or 7, preferably of Table II B, column 7, line 125. In
one embodiment, the nucleic acid molecule encodes less than 150,
130, 100, 80, 60, 50, 40 or 30 further amino acids. In a further
embodiment, the encoded polypeptide comprises less than 20, 15, 10,
9, 8, 7, 6 or 5 further amino acids. In one embodiment used in the
inventive process, the encoded polypeptide is identical to a
sequence shown in table II, line 125, columns 5 or 7, preferably of
Table II B, column 7, line 125.
[5794] [0187.0.13.13] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising a sequence shown in table II, line 125, columns 5 or 7,
preferably of Table II B, column 7, line 125 and comprises less
than 100 further nucleotides. In a further embodiment, said nucleic
acid molecule comprises less than 30 further nucleotides. In one
embodiment, the nucleic acid molecule used in the process is
identical to a coding sequence of a sequence shown in table I, line
125, columns 5 or 7, preferably of Table II B, column 7, line
125.
[5795] [0188.0.13.13] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide shown in table II,
line 125, columns 5 or 7 expressed under identical conditions.
[5796] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, columns 7, line 125.
[5797] [0189.0.13.13] Homologues of table I, line 125, columns 5 or
7 or of the derived sequences of table II, line 125, columns 5 or 7
also mean truncated sequences, cDNA, single-stranded DNA or RNA of
the coding and noncoding DNA sequence. Homologues of said sequences
are also understood as meaning derivatives, which comprise
noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[5798] [0190.0.0.13] to [0203.0.0.13]: see [0190.0.0.0] to
[0203.0.0.0]
[5799] [0204.0.13.13] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [5800] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide shown in table II, line 125, columns 5 or 7, preferably
of Table II B, column 7, line 125; or a fragment thereof conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof [5801] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule shown in table I, line 125, columns 5 or 7, preferably of
Table I B, column 7, line 125 or a fragment thereof conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [5802] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [5803]
d) nucleic acid molecule encoding a polypeptide whose sequence has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [5804] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [5805] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [5806] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [5807]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
the primers or primer pairs in table III, line 125, column 7 and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [5808] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [5809] j) nucleic acid molecule which encodes a
polypeptide comprising a consensus sequence shown in table IV, line
125, column 7 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [5810]
k) nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domaine of the polypeptide shown in table
II, line 125, columns 5 or 7, preferably of Table II B, column 7,
line 125 and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; and [5811] l)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (h) or of the nucleic
acid molecule shown in table I, line 125, columns 5 or 7,
preferably of Table I B, column 7, line 125 or a nucleic acid
molecule encoding, preferably at least the mature form of, a
polypeptide shown in table II, line 125, columns 5 or 7, preferably
of Table II B, column 7, line 125 and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over the sequence indicated in Table IA
or I B, columns 5 or 7, line 125, by one or more nucleotides. In
one embodiment, the nucleic acid molecule does not consist of the
sequence shown and indicated in Table I A or I B, columns 5 or 7,
line 125. In one embodiment, the nucleic acid molecule is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table I A or I B, columns 5 or 7, line 125. In another
embodiment, the nucleic acid molecule does not encode a polypeptide
of a sequence indicated in Table II A or II B, columns 5 or 7, line
125. In an other embodiment, the nucleic acid molecule of the
present invention is at least 30%, 40%, 50%, or 60% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table I A or I B, columns 5 or 7, line 125.
In a further embodiment the nucleic acid molecule does not encode a
polypeptide sequence as indicated in Table II A or II B, columns 5
or 7, line 125. Accordingly, in one embodiment, the nucleic acid
molecule of the differs at least in one or more residues from a
nucleic acid molecule indicated in Table I A or I B, columns 5 or
7, line 125. Accordingly, in one embodiment, the nucleic acid
molecule of the present invention encodes a polypeptide, which
differs at least in one or more amino acids from a polypeptide
indicated in Table II A or I B, columns 5 or 7, line 125. In
another embodiment, a nucleic acid molecule indicated in Table I A
or I B, columns 5 or 7, line 125 does not encode a protein of a
sequence indicated in Table II A or II B, columns 5 or 7, line 125.
Accordingly, in one embodiment, the protein encoded by a sequences
of a nucleic acid according to (a) to (l) does not consist of a
sequence as indicated in
[5812] Table II A or II B, columns 5 or 7, line 125. In a further
embodiment, the protein of the present invention is at least 30%,
40%, 50%, or 60% identical to a protein sequence indicated in Table
II A or II B, columns 5 or 7, line 125 and less than 100%,
preferably less than 99.999%, 99.99% or 99.9%, more preferably less
than 99%, 98%, 97%, 96% or 95% identical to a sequence as indicated
in Table I A or II B, columns 5 or 7, line 125.
[5813] [0205.0.0.13] to [0206.0.0.13]: see [0205.0.0.0] to
[0206.0.0.0]
[5814] [0207.0.13.13] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes are genes of the fatty acid metabolism, amino
acid metabolism, of glycolysis, of the tricarboxylic acid
metabolism or their combinations. As described herein, regulator
sequences or factors can have a positive effect on preferably the
gene expression of the genes introduced, thus increasing it. Thus,
an enhancement of the regulator elements may advantageously take
place at the transcriptional level by using strong transcription
signals such as promoters and/or enhancers. In addition, however,
an enhancement of translation is also possible, for example by
increasing mRNA stability or by inserting a translation enhancer
sequence.
[5815] [0208.0.0.13] to [0226.0.0.13]: see [0208.0.0.0] to
[00226.0.0.0]
[5816] [0227.0.13.13] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[5817] In addition to a sequence mentioned in table I, line 125,
columns 5 or 7 or its derivatives, it is advantageous additionally
to express and/or mutate further genes in the organisms. Especially
advantageously, additionally at least one further gene of the fatty
acid biosynthetic pathway such as for palmitate, palmitoleate,
oleate and/or linoleate or for erucic acid, arachinic acid,
pelagonic acid, brassylic acid and/or erucic acid amids is
expressed in the organisms such as plants or microorganisms. It is
also possible that the regulation of the natural genes has been
modified advantageously so that the gene and/or its gene product is
no longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the fatty acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine a sequences shown in table I, line 125,
columns 5 or 7 with genes which generally support or enhances to
growth or yield of the target organisms, for example genes which
lead to faster growth rate of microorganisms or genes which
produces stress-, pathogen, or herbicide resistant plants.
[5818] [0227.1.13.13] In addition to the sequence mentioned in
table I, line 125, columns 5 or 7 or its derivatives, it is
advantageous additionally to knock out and/or mutate further genes
in the organisms. Especially advantageously, additionally at least
one further gene of the fatty acid biosynthetic pathway for erucic
acid is reduced, deleted or in another way knocked out in the
organisms such as plants or microorganisms.
[5819] [0228.0.13.13] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the fatty acid
metabolism, in particular in monoenoic fatty acid synthesis.
[5820] [0229.0.13.13] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the saturated, poly unsaturated
fatty acid biosynthesis such as desaturases like
.DELTA.-4-desaturases, .DELTA.-5-desaturases,
.DELTA.-6-desaturases, .DELTA.-8-desaturases,
.DELTA.-9-desaturases, .DELTA.-12-desaturases,
.DELTA.-17-desaturases, .omega.-3-desaturases, elongases like
.DELTA.-5-elongases, .DELTA.-6-elongases, .DELTA.-9-elongases,
acyl-CoA-dehydrogenases, acyl-ACP-desaturases,
acyl-ACP-thioesterases, fatty acid acyl-transferases, acyl-CoA
lysophospholipid-acyltransferases, acyl-CoA carboxylases, fatty
acid synthases, fatty acid hydroxylases, acyl-CoA oxydases,
acetylenases, lipoxygenases, triacyl-lipases etc. as described in
WO 98/46765, WO 98/46763, WO 98/46764, WO 99/64616, WO 00/20603, WO
00/20602, WO 00/40705, US 20040172682, US 20020156254, U.S. Pat.
No. 6,677,145 US 20040053379 or US 20030101486. These genes lead to
an increased synthesis of the essential fatty acids.
[5821] [0230.0.0.13]: see [0230.0.0.0]
[5822] [0231.0.13.13] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a gadoleic acid degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[5823] [0232.0.0.13] to [0276.0.0.13]: see [0232.0.0.0] to
[0276.0.0.0]
[5824] [0277.0.13.13] The fatty acids produced can be isolated from
the organism by methods with which the skilled worker is familiar.
For example via extraction, salt precipitation and/or different
chromatography methods. The process according to the invention can
be conducted batchwise, semibatchwise or continuously. The
respective fine chemical produced in the process according to the
invention can be isolated as mentioned above from the organisms,
advantageously plants, in the form of their oils, fats, lipids
and/or free fatty acids. Fatty acids produced by this process can
be obtained by harvesting the organisms, either from the crop in
which they grow, or from the field. This can be done via pressing
or extraction of the plant parts, preferably the plant seeds.
Hexane is preferably used as solvent in the process, in which more
than 96% of the compounds produced in the process can be isolated.
Thereafter, the resulting products are processed further, i.e.
degummed, refined, bleached and/or deodorized.
[5825] [0278.0.0.13] to [0282.0.0.13]: see [0278.0.0.0] to
[0282.0.0.0]
[5826] [0283.0.13.13] Moreover, a native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an anti-YDR513W protein antibody or an
antibody against a polypeptide as shown in table II, line 125,
columns 5 or 7, which can be produced by standard techniques
utilizing the polypeptid of the present invention or charcterized
in the process of the present invention or fragment thereof, i.e.,
the polypeptide of this invention. Preferred are monoclonal
antibodies.
[5827] [0284.0.0.13] see [0284.0.0.0]
[5828] [0285.0.13.13] In one embodiment, the present invention
relates to a polypeptide having a sequence shown in table II, line
125, columns 5 or 7 or as coded by a nucleic acid molecule shown in
table I, line 125, columns 5 or 7 or functional homologues
thereof.
[5829] [0286.0.13.13] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
comprising or consisting of a consensus sequence shown in table IV,
line 125, column 7 and in one another embodiment, the present
invention relates to a polypeptide comprising or consisting of a
consensus sequence shown in table IV, line 125, column 7 whereby 20
or less, preferably 15 or 10, preferably 9, 8, 7, or 6, more
preferred 5 or 4, even more preferred 3, even more preferred 2,
even more preferred 1, most preferred 0 of the amino acids
positions indicated can be replaced by any amino acid.
[5830] [0287.0.0.13] to [0290.0.0.13]: see [0287.0.0.0] to
[0290.0.0.0]
[5831] [0291.0.13.13] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[5832] In one embodiment, said polypeptide of the invention
distinguishes over a sequence shown in Table II A or II B, line
125, columns 5 or 7 by one or more amino acids. In one embodiment,
polypeptide distinguishes form a sequence shown in Table II A or II
B, line 125, columns 5 or 7 by more than 5, 6, 7, 8 or 9 amino
acids, preferably by more than 10, 15, 20, 25 or 30 amino acids,
evenmore preferred are more than 40, 50, or 60 amino acids and,
preferably, the sequence of the polypeptide of the invention
distinguishes from a sequence shown in Table II A or II B, line
125, columns 5 or 7 by not more than 80% or 70% of the amino acids,
preferably not more than 60% or 50%, more preferred not more than
40% or 30%, even more preferred not more than 20% or 10%. In an
other embodiment, said polypeptide of the invention does not
consist of a sequence shown in Table II A or II B, line 125,
columns 5 or 7.
[5833] [0292.0.0.13]: see [0292.0.0.0]
[5834] [0293.0.13.13] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or used in the process of the invention and having
a sequence which distinguishes from the sequence as shown in Table
II A or II B, line 125, columns 5 or 7 by one or more amino acids.
In another embodiment, said polypeptide of the invention does not
consist of the sequence shown in Table II A or II B, line 125,
columns 5 or 7. In a further embodiment, said polypeptide of the
present invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by the nucleic acid molecules shown in Table I
A or I B, line 125, columns 5 or 7.
[5835] [0294.0.13.13] In one embodiment, the present invention
relates to a polypeptide having YDR513W protein activity, which
distinguishes over the sequence depicted in Table II A or II B,
line 125, columns 5 or 7 by one or more amino acids, preferably by
more than 5, 6, 7, 8 or 9 amino acids, preferably by more than 10,
15, 20, 25 or 30 amino acids, evenmore preferred are more than 40,
50, or 60 amino acids but even more preferred by less than 70% of
the amino acids, more preferred by less than 50%, even more
preferred my less than 30% or 25%, more preferred are 20% or 15%,
even more preferred are less than 10%.
[5836] [0295.0.0.13] to [0297.0.0.13]: see [0295.0.0.0] to
[0297.0.0.0]
[5837] [0297.1.13.13] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table II, columns 3, 5 or 7, lines 125, resp.
[5838] [0298.0.13.13] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
shown in table II, line 125, columns 5 or 7.
[5839] [0299.0.13.13] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, preferably at least
about 96%, 97%, 98%, 99% or more homologous to one of the amino
acid sequences as shown in table II, line 125, columns 5 or 7. The
preferred polypeptide of the present invention preferably possesses
at least one of the activities according to the invention and
described herein. A preferred polypeptide of the present invention
includes an amino acid sequence encoded by a nucleotide sequence
which hybridizes, preferably hybridizes under stringent conditions,
to a nucleotide sequence of table I, line 125, columns 5 or 7 or
which is homologous thereto, as defined above.
[5840] [0300.0.13.13] Accordingly the polypeptide of the present
invention can vary from the sequences shown in Table II, line 125,
columns 5 or 7 in amino acid sequence due to natural variation or
mutagenesis, as described in detail herein. Accordingly, the
polypeptide comprise an amino acid sequence which is at least about
35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about
75%, 80%, 85% or 90, and more preferably at least about 91%, 92%,
93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%,
99% or more homologous to an entire amino acid sequence shown in
Table II A or II B, line 125, columns 5 or 7.
[5841] [0301.0.0.13] see [0301.0.0.0]
[5842] [0302.0.13.13] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., the amino acid sequence shown in table II,
line 125, columns 5 or 7 or the amino acid sequence of a protein
homologous thereto, which include fewer amino acids than a full
length polypeptide of the present invention or used in the process
of the present invention or the full length protein which is
homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[5843] [0303.0.0.13] see [0303.0.0.0]
[5844] [0304.0.13.13] Manipulation of the nucleic acid molecule of
the invention may result in the production of protein indicated in
Table II, column 5, line 125 or being encoded by a nucleic acid
molecule indicated in Table I, column 5, line 125 or of its
homologs, e.g. as indicated in Table II, column 7, line 125, having
differences from the wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[5845] [0305.0.0.13] to [0308.0.0.13]: see [0305.0.0.0] to
[0308.0.0.0]
[5846] [0309.0.13.13] In one embodiment, an "YDR513W protein
(=polypeptide)" refers to a polypeptide having an amino acid
sequence corresponding to the polypeptide of the invention or used
in the process of the invention, whereas a "non-YDR513W
polypeptide" or "other polypeptide" refers to a polypeptide having
an amino acid sequence corresponding to a protein which is not
substantially homologous a polypeptide of the invention, preferably
which is not substantially homologous to a polypeptide having an
YDR513W protein activity, e.g., a protein which does not confer the
activity described herein and which is derived from the same or a
different organism.
[5847] [0310.0.0.13] to [0334.0.0.13]: see [0310.0.0.0] to
[0334.0.0.0]
[5848] [0335.0.13.13] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of the nucleic acid sequences of the table I, line 125, columns 5
or 7 and/or homologs thereof. As described inter alia in WO
99/32619, dsRNAi approaches are clearly superior to traditional
antisense approaches. The invention therefore furthermore relates
to double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived therefrom), bring about altered
metabolic activity by the reduction in the expression of thae
nucleic acid sequences of the table I, line 125, columns 5 or 7
and/or homologs thereof. In a double-stranded RNA molecule for
reducing the expression of an protein encoded by a nucleic acid
sequence of one of the table I, line 125, columns 5 or 7 and/or
homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[5849] [0336.0.0.13] to [0342.0.0.13]: see [0336.0.0.0] to
[0342.0.0.0]
[5850] [0343.0.13.13] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence of one of the sequences shown in table I, line 125,
columns 5 or 7 or its homolog is not necessarily required in order
to bring about effective reduction in the expression. The advantage
is, accordingly, that the method is tolerant with regard to
sequence deviations as may be present as a consequence of genetic
mutations, polymorphisms or evolutionary divergences. Thus, for
example, using the dsRNA, which has been generated starting from a
sequence of one of sequences shown in table I, line 125, columns 5
or 7 or homologs thereof of the one organism, may be used to
suppress the corresponding expression in another organism.
[5851] [0344.0.0.13] to [0361.0.0.13]: see [0344.0.0.0] to
[0361.0.0.0]
[5852] [0362.0.13.13] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. encoding a polypeptide having an
YDR513W protein activity or of a protein as indicated in Table II,
column 5, line 125 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, line 125 or of its homologs, e.g.
as indicated in Table II, column 7, line 125. Due to the above
mentioned activity the respective fine chemical content in a cell
or an organism is increased. For example, due to modulation or
manipulation, the cellular activity is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an YDR513W protein activity
e.g. for a protein as indicated in Table
[5853] II, column 5, line 125 or being encoded by a nucleic acid
molecule indicated in Table I, column 5, line 125 or of its
homologs, e.g. as indicated in Table II, column 7, line 125, means
herein that due to modulation or manipulation of the genome, the
activity of YDR513W or a YDR513W-like activity is increased in the
cell or organism or part thereof, e.g. of a protein as indicated in
Table II, column 5, line 125 or being encoded by a nucleic acid
molecule indicated in Table I, column 5, line 125 or of its
homologs, e.g. as indicated in Table II, column 7, line 125.
Examples are described above in context with the process of the
invention.
[5854] [0363.0.0.13] to [0373.0.0.13]: see [0363.0.0.0] to
[0373.0.0.0]
[5855] [0374.0.13.13] Transgenic plants comprising the fatty acids
synthesized in the process according to the invention can be
marketed directly without isolation of the compounds synthesized.
In the process according to the invention, plants are understood as
meaning all plant parts, plant organs such as leaf, stalk, root,
tubers or seeds or propagation material or harvested material or
the intact plant. In this context, the seed encompasses all parts
of the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue. The fatty acids produced in the
process according to the invention may, however, also be isolated
from the plant in the form of their free fatty acids or bound in or
to compounds. Fatty acids produced by this process can be harvested
by harvesting the organisms either from the culture in which they
grow or from the field. This can be done via expressing, grinding
and/or extraction, salt precipitation and/or ion-exchange
chromatography or other chromatographic methods of the plant parts,
preferably the plant seeds, plant fruits, plant tubers and the
like.
[5856] [0375.0.0.13] to [0376.0.0.13]: see [0375.0.0.0] to
[0376.0.0.0]
[5857] [0377.0.13.13] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced fatty acid and/or fatty acid composition or the produced
the respective fine chemical is isolated.
[5858] [0378.0.0.13]: see [0378.0.0.0]
[5859] [0379.0.13.13] In one embodiment, said fatty acid or
monoenoic fatty acid is the respective fine chemical.
[5860] [0380.0.13.13] The monoenoic fatty acids or the respective
fine chemical obtained in the process are suitable as starting
material for the synthesis of further products of value. For
example, they can be used in combination with each other or alone
for the production of pharmaceuticals, foodstuffs, animal feeds or
cosmetics. Accordingly, the present invention relates a method for
the production of a pharmaceuticals, food stuff, animal feeds,
nutrients or cosmetics comprising the steps of the process
according to the invention, including the isolation of the fatty
acid composition produced or the respective fine chemical produced
if desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the fatty acids produced in the
process or of the transgenic organisms in animal feeds, foodstuffs,
medicines, food supplements, cosmetics or pharmaceuticals.
[5861] [0381.0.0.13] to [0382.0.0.13]: see [0381.0.0.0] to
[0382.0.0.0]
[5862] [0383.0.13.13] For preparing fatty acid compound-containing
fine chemicals, in particular the respective fine chemical, it is
possible to use as fatty acid source organic compounds such as, for
example, oils, fats and/or lipids comprising fatty acids such as
fatty acids having a carbon back bone between C.sub.10- to
C.sub.16-carbon atoms and/or small organic acids such acetic acid,
propionic acid or butanoic acid as precursor compounds.
[5863] [0384.0.0.13]: see [0384.0.0.0]
[5864] [0385.0.13.13] The fermentation broths obtained in this way,
containing in particular gadoleic acid in mixtures with other
lipids, fats and/or oils, normally have a dry matter content of
from, 1% to 50%, preferably of 7.5 to 25% by weight. Sugar-limited
fermentation is additionally advantageous, at least at the end, but
especially over at least 30% of the fermentation time. This means
that the concentration of utilizable sugar in the fermentation
medium is kept at, or reduced to, 0 to 3 g/l during this time.
[5865] The fermentation broth is then processed further. Depending
on requirements, the biomass can be removed entirely or partly by
separation methods, such as, for example, centrifugation,
filtration, decantation or a combination of these methods, from the
fermentation broth or left completely in it. The fermentation broth
can then be thickened or concentrated by known methods, such as,
for example, with the aid of a rotary evaporator, thin-film
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. This concentrated fermentation broth can then be
worked up by freeze-drying, spray drying, spray granulation or by
other processes.
[5866] [0386.0.13.13] However, it is also possible to purify the
fatty acid produced further.
[5867] For this purpose, the product-containing composition is
subjected for example to a thin layder chromatography on silica gel
plates or to a chromatography such as a Florisil column (Bouhours
J. F., J. Chromatrogr. 1979, 169, 462), in which case the desired
product or the impurities are retained wholly or partly on the
chromatography resin. These chromatography steps can be repeated if
necessary, using the same or different chromatography resins. The
skilled worker is familiar with the choice of suitable
chromatography resins and their most effective use. An alternative
method to purify the fatty acids is for example crystallization in
the presence of urea. These methods can be combined with each
other.
[5868] [0387.0.0.13] to [0392.0.0.13]: see [0387.0.0.0] to
[0392.0.0.0]
[5869] [0393.0.13.13] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps:
(a) contacting e.g. hybridising, the nucleic acid molecules of a
sample, e.g. cells, tissues, plants or microorganisms or a nucleic
acid library, which can contain a candidate gene encoding a gene
product conferring an increase in the respective fine chemical
after expression, with the nucleic acid molecule of the present
invention; (b) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence shown in table I, line 125, columns 5 or 7,
preferably in Table I B, columns 5 or 7, line 125 and, optionally,
isolating the full length cDNA clone or complete genomic clone; (c)
introducing the candidate nucleic acid molecules in host cells,
preferably in a plant cell or a microorganism, appropriate for
producing the respective fine chemical; (d) expressing the
identified nucleic acid molecules in the host cells; (e) assaying
the the respective fine chemical level in the host cells; and (f)
identifying the nucleic acid molecule and its gene product which
expression confers an increase in the the respective fine chemical
level in the host cell after expression compared to the wild
type.
[5870] [0394.0.0.13] to [0399.0.0.13]: see [0394.0.0.0] to
[0399.0.0.0]
[5871] [0399.1.13.13] One can think to screen for increased fine
chemical production by for example resistance to drugs blocking
eicosenic, inparticular gadoleic acid synthesis and looking whether
this effect is dependent on a protein as indicated in Table II,
column 5, line 125 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, line 125 or of its homologs, e.g.
as indicated in Table II, column 7, line 125, eg by comparing the
phenotyp of nearly identical organisms with low and high activity
of a protein as indicated in Table II, column 5, line 125 or being
encoded by a nucleic acid molecule indicated in Table I, column 5,
line 125 or of its homologs, e.g. as indicated in Table II, column
7, line 125.
[5872] [0400.0.0.13] to [0416.0.0.13]: see [0400.0.0.0] to
[0416.0.0.0]
[5873] [0417.0.13.13] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the fatty acid production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect an organism such as a
microorganism or a plant against inhibitors, which block the fatty
acid, in particular the respective fine chemical, synthesis in said
organism. Examples of inhibitors or herbicides blocking the fatty
acid synthesis in organism such as microorganism or plants are for
example cerulenin, Thiolactomycin, Diazoborines or Triclosan, which
inhibit the fatty acids (beta-ketoacyl thioester synthetase
inhibitors) and sterol biosynthesis inhibitors,
aryloxyphenoxypropionates such as diclofop, fenoxaprop, haloxyfop,
fluazifop or quizalofop or cyclohexanediones such as clethodim or
sethoxydim
[(2-[1-{ethoxyimino}butyl]-542-{ethylthio}propyl]-3-hydroxy-2-cyclohexen--
1-one], which inhibit the plant acetyl-coenzyme A carboxylase or
thiocarbamates such as butylate, EPTC [=S-ethyl
dipropylcarbamothioat] or vernolate.
[5874] [0418.0.0.13] to [0423.0.0.13]: see [0418.0.0.0] to
[0423.0.0.0]
[5875] [0424.0.13.13] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other fatty acids, in
particular palmitic acid, palmitoleic acid, oleic acid, linoleic
acid, stearic acid and/or linolenic acid or erucic acid mixtures
thereof or mixtures of other fatty acids. Accordingly, the nucleic
acid of the invention, or the nucleic acid molecule identified with
the method of the present invention or the complement sequences
thereof, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the
antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the respective
fine chemical in a organism or part thereof, e.g. in a cell.
[5876] [0425.0.0.13] to [0435.0.0.13]: see [0425.0.0.0] to
[0435.0.0.0]
[5877] [0436.0.13.13] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of fatty acids can be
carried out by passing a plasmid DNA (or another vector DNA)
containing the desired nucleic acid sequence or nucleic acid
sequences through E. coli and other microorganisms (for example
Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are
not capable of maintaining the integrity of its genetic
information. Usual mutator strains have mutations in the genes for
the DNA repair system [for example mutHLS, mutD, mutT and the like;
for comparison, see Rupp, W. D. (1996) DNA repair mechanisms in
Escherichia coli and Salmonella, pp. 2277-2294, ASM: Washington].
The skilled worker knows these strains. The use of these strains is
illustrated for example in Greener, A. and Callahan, M. (1994)
Strategies 7; 32-34.
[5878] In-vitro mutation methods such as increasing the spontaneous
mutation rates by chemical or physical treatment are well known to
the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[5879] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[5880] Positive mutation events can be selected by screening the
organisms for the production of the desired fine chemical.
[0437.0.13.13] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces Cerevisiae and
Mortierella alpina
[5881] [0438.0.13.13] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology, VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[5882] [0439.0.13.13] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255-269) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[5883] [0440.0.13.13] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002,) teaches a homolog recombination system dependent
on rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this
system for homologous recombination are vectors derived from the
Ylp series. Plasmid vectors derived for example from the
2.mu.-Vector are known by the skilled worker and used for the
expression in yeasts. Other preferred vectors are for example
pART1, pCHY21 or pEVP11 as they have been described by McLeod et
al. (EMBO J. 1987, 6:729-736) and Hoffman et al. (Genes Dev. 5,
1991: 561-571.) or Russell et al. (J. Biol. Chem. 258, 1983:
143-149.). Other beneficial yeast vectors are plasmids of the REP,
REP-X, pYZ or RIP series.
[5884] [0441.0.0.13] to [0443.0.0.13]: see [0441.0.0.0] to
[0443.0.0.0]
[0444.0.13.13] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[5885] [0445.0.13.13] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[5886] [0446.0.0.13] to [0454.0.0.13]: see [0446.0.0.0] to
[0454.0.0.0]
[5887] [0455.0.13.13] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a fatty acid) can be determined by growing the
modified microorganisms or the modified plant under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the elevated production of
desired product (i.e. of the lipids or a fatty acid). These
analytical techniques are known to the skilled worker and comprise
spectroscopy, thin-layer chromatography, various types of staining
methods, enzymatic and microbiological methods and analytical
chromatography such as high-performance liquid chromatography (see,
for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2,
p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.,
(1987) "Applications of HPLC in Biochemistry" in: Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et
al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery
and purification", p. 469-714, VCH: Weinheim; Belter, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[5888] In addition to the abovementioned processes, plant lipids
are extracted from plant material as described by Cahoon et al.
(1999) Proc. Natl. Acad. Sci. USA 96 (22): 12935-12940 and Browse
et al. (1986) Analytic Biochemistry 152:141-145. The qualitative
and quantitative analysis of lipids or fatty acids is described by
Christie, William W., Advances in Lipid Methodology, Ayr/Scotland:
Oily Press (Oily Press Lipid Library; 2); Christie, William W., Gas
Chromatography and Lipids. A Practical Guide--Ayr, Scotland: Oily
Press, 1989, Repr. 1992, IX, 307 pp. (Oily Press Lipid Library; 1);
"Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952)--16
(1977) under the title: Progress in the Chemistry of Fats and Other
Lipids CODEN.
[5889] [0456.0.0.13]: see [0456.0.0.0]
[0457.0.13.13] Example 9
Purification of the Fatty Acid
[5890] [0458.0.13.13] One example is the analysis of fatty acids
(abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid
chromatography/mass spectrometry; TAG, triacylglycerol; TLC,
thin-layer chromatography).
[5891] The unambiguous detection for the presence of fatty acid
products can be obtained by analyzing recombinant organisms using
analytical standard methods: GC, GC-MS or TLC, as described on
several occasions by Christie and the references therein (1997, in:
Advances on Lipid Methodology, Fourth Edition: Christie, Oily
Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353).
[5892] The total fatty acids produced in the organism for example
in yeasts used in the inventive process can be analysed for example
according to the following procedure:
[5893] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods. After
disruption, the material must be centrifuged (1000.times.g, 10
min., 4.degree. C.) and washed once with 100 mM NaHCO.sub.3, pH 8.0
to remove residual medium and fatty acids. For preparation of the
fatty acid methyl esters (FAMES) the sediment is resuspended in
distilled water, heated for 10 minutes at 100.degree. C., cooled on
ice and recentrifuged, followed by extraction for one hour at
90.degree. C. in 0.5 M sulfuric acid in methanol with 2%
dimethoxypropane, which leads to hydrolyzed oil and lipid
compounds, which give transmethylated lipids.
[5894] The FAMES are then extracted twice with 2 ml petrolether,
washed once with 100 mM NaHCO.sub.3, pH 8.0 and once with distilled
water and dried with Na.sub.2SO.sub.4. The organic solvent can be
evaporated under a stream of Argon and the FAMES were dissolved in
50 .mu.l of petrolether. The samples can be separated on a ZEBRON
ZB-Wax capillary column (30 m, 0.32 mm, 0.25 .mu.m; Phenomenex) in
a Hewlett Packard 6850 gas chromatograph with a flame ionisation
detector. The oven temperature is programmed from 70.degree. C. (1
min. hold) to 200.degree. C. at a rate of 20.degree. C./min., then
to 250.degree. C. (5 min. hold) at a rate of 5.degree. C./min and
finally to 260.degree. C. at a rate of 5.degree. C./min. Nitrogen
is used as carrier gas (4.5 ml/min. at 70.degree. C.). The identity
of the resulting fatty acid methyl esters can be identified by
comparison with retention times of FAME standards, which are
available from commercial sources (i.e. Sigma).
[5895] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[5896] This is followed by heating at 100.degree. C. for 10 minutes
and, after cooling on ice, by resedimentation. The cell sediment is
hydrolyzed for one hour at 90.degree. C. with 1 M methanolic
sulfuric acid and 2% dimethoxypropane, and the lipids are
transmethylated. The resulting fatty acid methyl esters (FAMEs) are
extracted in petroleum ether. The extracted FAMEs are analyzed by
gas liquid chromatography using a capillary column (Chrompack, WCOT
Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and a temperature
gradient of from 170.degree. C. to 240.degree. C. in 20 minutes and
5 minutes at 240.degree. C. The identity of the fatty acid methyl
esters is confirmed by comparison with corresponding FAME standards
(Sigma). The identity and position of the double bond can be
analyzed further by suitable chemical derivatization of the FAME
mixtures, for example to give 4,4-dimethoxyoxazoline derivatives
(Christie, 1998) by means of GC-MS.
[5897] The methodology is described for example in Napier and
Michaelson, 2001, Lipids. 36(8):761-766; Sayanova et al., 2001,
Journal of Experimental Botany. 52(360):1581-1585, Sperling et al.,
2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al.,
1998, FEBS Letters. 439(3):215-218.
[5898] [0459.0.13.13] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously the fatty acids can be further purified with a
so-called RTHPLC. As eluent different an acetonitrile/water or
chloroform/acetonitrile mixtures are advantageously is used. For
example canola oil can be separated said HPLC method using an
RP-18-column (ET 250/3 Nucleosil 120-5 C.sub.18 Macherey and Nagel,
Duren, Germany). A chloroform/acetonitrile mixture (v/v 30:70) is
used as eluent. The flow rate is beneficial 0.8 ml/min. For the
analysis of the fatty acids an ELSD detector (evaporative
light-scattering detector) is used. MPLC, dry-flash chromatography
or thin layer chromatography are other beneficial chromatography
methods for the purification of fatty acids. If necessary, these
chromatography steps may be repeated, using identical or other
chromatography resins. The skilled worker is familiar with the
selection of suitable chromatography resin and the most effective
use for a particular molecule to be purified.
[5899] [0460.0.0.13]: see [0460.0.0.0]
[0461.0.13.13] Example 10
Cloning SEQ ID NO: 13791 for the Expression in Plants
[5900] [0462.0.0.13] see [0462.0.0.0]
[5901] [0463.0.13.13] SEQ ID NO: 13791 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[5902] [0464.0.0.13] to [0466.0.0.13]: see [0464.0.0.0] to
[0466.0.0.0]
[5903] [0467.0.13.13] The following primer sequences were selected
for the gene SEQ ID NO: 13791:
TABLE-US-00044 i) forward primer (SEQ ID NO: 13925) atggagacca
atttttcctt cgact ii) reverse primer (SEQ ID NO: 13926) ctattgaaat
accggcttca atattt
[5904] [0468.0.0.13] to [0479.0.0.13]: see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.13.13] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 13791
[5905] [0481.0.0.13] to [0513.0.0.13]: see [0481.0.0.0] to
[0513.0.0.0]
[5906] [0514.0.13.13] As an alternative, the fine chemicals can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55).
[5907] The results of the different plant analyses can be seen from
the table which follows:
TABLE-US-00045 TABLE 1 ORF Metabolite Method Min Max YDR513W
Gadoleic acid (C20:1) GC 1,46 1,53
[5908] [0515.0.0.13] Column 2 shows the metabolite analyzed.
Columns 4 and 5 shows the ratio of the analyzed metabolite between
the transgenic plants and the wild type; Increase of the
metabolites: Max: maximal x-fold (normalised to wild type)-Min:
minimal x-fold (normalised to wild type). Decrease of the
metabolites: Max: maximal x-fold (normalised to wild type) (minimal
decrease), Min: minimal x-fold (normalised to wild type) (maximal
decrease). Column 3 indicates the analytical method.
[5909] [0516.0.0.13] to [0552.0.0.13]: see [0516.0.0.0] to
[0552.0.0.0]
[5910] [0553.0.13.13] We claim: [5911] 1. A process for the
production of gadoleic acid, which comprises [5912] (a) increasing
or generating the activity of a protein as indicated in Table II,
columns 5 or 7, line 125 or a functional equivalent thereof in a
non-human organism, or in one or more parts thereof; and [5913] (b)
growing the organism under conditions which permit the production
of gadoleic acid in said organism. [5914] 2. A process for the
production of gadoleic acid, comprising the increasing or
generating in an organism or a part thereof the expression of at
least one nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [5915] a) nucleic acid
molecule encoding of a polypeptide as indicated in Table II,
columns 5 or 7, line 125 or a fragment thereof, which confers an
increase in the amount of gadoleic acid in an organism or a part
thereof; [5916] b) nucleic acid molecule comprising of a nucleic
acid molecule as indicated in Table I, columns 5 or 7, line 125;
[5917] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of gadoleic acid in an
organism or a part thereof; [5918] d) nucleic acid molecule which
encodes a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
gadoleic acid in an organism or a part thereof; [5919] e) nucleic
acid molecule which hybridizes with a nucleic acid molecule of (a)
to (c) under under stringent hybridisation conditions and
conferring an increase in the amount of gadoleic acid in an
organism or a part thereof; [5920] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table III,
columns 7, line 125 and conferring an increase in the amount of
gadoleic acid in an organism or a part thereof; [5921] g) nucleic
acid molecule encoding a polypeptide which is isolated with the aid
of monoclonal antibodies against a polypeptide encoded by one of
the nucleic acid molecules of (a) to (f) and conferring an increase
in the amount of gadoleic acid in an organism or a part thereof;
[5922] h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table IV, 7, line 125 and conferring an
increase in the amount of gadoleic acid in an organism or a part
thereof; and [5923] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of gadoleic acid in an organism or a part thereof. [5924] or
comprising a sequence which is complementary thereto. [5925] 3. The
process of claim 1 or 2, comprising recovering of the free or bound
gadoleic acid. [5926] 4. The process of any one of claims 1 to 3,
comprising the following steps: [5927] (a) selecting an organism or
a part thereof expressing a polypeptide encoded by the nucleic acid
molecule characterized in claim 2; [5928] (b) mutagenizing the
selected organism or the part thereof; [5929] (c) comparing the
activity or the expression level of said polypeptide in the
mutagenized organism or the part thereof with the activity or the
expression of said polypeptide of the selected organisms or the
part thereof;
[5930] (d) selecting the mutated organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
[5931] (e) optionally, growing and cultivating the organisms or the
parts thereof; and [5932] (f) recovering, and optionally isolating,
the free or bound gadoleic acid produced by the selected mutated
organisms or parts thereof. [5933] 5. The process of any one of
claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. [5934] 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [5935] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, line 125 or a
fragment thereof, which confers an increase in the amount of
gadoleic acid in an organism or a part thereof; [5936] b) nucleic
acid molecule comprising of a nucleic acid molecule as indicated in
Table I, columns 5 or 7, line 125; [5937] c) nucleic acid molecule
whose sequence can be deduced from a polypeptide sequence encoded
by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of gadoleic acid in an organism or a part thereof; [5938] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of gadoleic acid in an organism or a part
thereof; [5939] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of gadoleic
acid in an organism or a part thereof; [5940] f) nucleic acid
molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table III, column 7, line 125 and conferring an increase in the
amount of gadoleic acid in an organism or a part thereof; [5941] g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of gadoleic acid in an organism or a part
thereof; [5942] h) nucleic acid molecule encoding a polypeptide
comprising a consensus as indicated in Table IV, column 7, line 125
and conferring an increase in the amount of gadoleic acid in an
organism or a part thereof; and [5943] i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of gadoleic acid in an organism or a part thereof. [5944]
whereby the nucleic acid molecule distinguishes over the sequence
as indicated in Table I A, columns 5 or 7, line 125 by one or more
nucleotides. [5945] 7. A nucleic acid construct which confers the
expression of the nucleic acid molecule of claim 6, comprising one
or more regulatory elements. [5946] 8. A vector comprising the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7. [5947] 9. The vector as claimed in claim 8,
wherein the nucleic acid molecule is in operable linkage with
regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic, host. [5948] 10. A
host cell, which has been transformed stably or transiently with
the vector as claimed in claim 8 or 9 or the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 2 to 5. [5949] 11.
The host cell of claim 10, which is a transgenic host cell. [5950]
12. The host cell of claim 10 or 11, which is a plant cell, an
animal cell, a microorganism, or a yeast cell, a fungus cell, a
prokaryotic cell, an eukaryotic cell or an archaebacterium. [5951]
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. [5952] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table II A, columns 5 or 7, line 125 by one or more
amino acids 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. [5953] 16. A plant tissue,
propagation material, harvested material or a plant comprising the
host cell as claimed in claim 12 which is plant cell or an
Agrobacterium. [5954] 17. A method for screening for agonists and
antagonists of the activity of a polypeptide encoded by the nucleic
acid molecule of claim 6 conferring an increase in the amount of
gadoleic acid in an organism or a part thereof comprising: [5955]
(a) contacting cells, tissues, plants or microorganisms which
express the a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of gadoleic acid in an
organism or a part thereof with a candidate compound or a sample
comprising a plurality of compounds under conditions which permit
the expression the polypeptide; [5956] (b) assaying the gadoleic
acid level or the polypeptide expression level in the cell, tissue,
plant or microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [5957] (c)
identifying a agonist or antagonist by comparing the measured
gadoleic acid level or polypeptide expression level with a standard
gadoleic acid or polypeptide expression level measured in the
absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. [5958] 18. A process for
the identification of a compound conferring increased gadoleic acid
production in a plant or microorganism, comprising the steps:
[5959] (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of gadoleic acid in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
gadoleic acid in an organism or a part thereof; [5960] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [5961] 19. A method for the identification of a
gene product conferring an increase in gadoleic acid production in
a cell, comprising the following steps: [5962] (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in gadoleic
acid after expression with the nucleic acid molecule of claim 6;
[5963] (b) identifying the nucleic acid molecules, which hybridise
under relaxed stringent conditions with the nucleic acid molecule
of claim 6; [5964] (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing gadoleic acid;
[5965] (d) expressing the identified nucleic acid molecules in the
host cells; [5966] (e) assaying the gadoleic acid level in the host
cells; and [5967] (f) identifying nucleic acid molecule and its
gene product which expression confers an increase in the gadoleic
acid level in the host cell in the host cell after expression
compared to the wild type. [5968] 20. A method for the
identification of a gene product conferring an increase in gadoleic
acid production in a cell, comprising the following steps: [5969]
(a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the gadoleic acidamount or level
in an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; [5970]
(b) introducing the candidate nucleic acid molecules in host cells
appropriate for producing gadoleic acid; [5971] (c) expressing the
identified nucleic acid molecules in the host cells; [5972] (d)
assaying the gadoleic acidlevel in the host cells; and [5973] (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the gadoleic acid level in the
host cell after expression compared to the wild type. [5974] 21. A
method for the production of an agricultural composition comprising
the steps of the method of any one of claims 17 to 20 and
formulating the compound identified in any one of claims 17 to 20
in a form acceptable for an application in agriculture. [5975] 22.
A composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [5976] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
gadoleic acid after expression. [5977] 24. Use of the polypeptide
of claim 14 or the nucleic acid construct claim 7 or the gene
product identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of
gadoleic acid levels in an organism. [5978] 25. Cosmetical,
pharmaceutical, food or feed composition comprising the nucleic
acid molecule of claim 6, the polypeptide of claim 14, the nucleic
acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20. [5979] 26. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the protection of vegetable fats,
oils or waxes. [5980] 27. Use of the nucleic acid molecule of claim
6, the polypeptide of claim 14, the nucleic acid construct of claim
7, the vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the production of industrial oils,
fats or waxes. [5981] 28. Use of the nucleic acid molecule of claim
6, the polypeptide of claim 14, the nucleic acid construct of claim
7, the vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the production of detergents,
cleaning agents, cosmetics, dye additives, lubricating agents,
hydraulic oils, preservation agents, flavoring agents, plastic
softeners, formulation agents, flotation agents, wetting agents,
emulsifiers or lubricating agents. [5982] 29. The plant of claim
16, which has a low level of erucic acid. [5983] 30. The plant of
claim 16, which has a high level or erucic acid. [5984] 31. The
plant of any one of claim 16, 29 or 30, which has a low level of
gluconsinolate.
[5985] [0554.0.0.13] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[5986] [0000.0.0.14] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and corresponding embodiments as described herein
as follows.
[5987] [0001.0.0.14] to [0002.0.0.14]: see [0001.0.0.0] to
[0002.0.0.0]
[5988] [0002.1.14.14] L-alanine is used in various pharmaceutical
and veterinary applications. For example, it is included, together
with other amino acids, in preparations for infusion solutions or
preparations for parenteral administration as clinical preoperative
and postoperative foods, as well as an animal feed supplement.
Furthermore, alanine is used as a food additive on account of its
sweet taste. L-phenylalanine and L-aspartic acid have very
important markets as key components in the manufacture of the
sweetener aspartame. Aspartame (C.sub.14H.sub.18N.sub.2O.sub.5),
L-aspartyl-L-phenylalanine methyl ester, is a compound of three
components, which are methanol, aspartic acid and phenylalanine.
L-aspartic acid is further used as a flavoring agent.
[5989] The amino acid L-citrulline is a metabolite in the urea
cycle. Other amino acids in this cycle are L-arginine and
L-ornithine.L-citrulline is involved in liver detoxification of
ammonia, and has been shown to speed recover from fatigue. It has
also been utilized in the treatment of Ornithine Transcarbamylase
Deficiency and other Urea Cycle disorders. In cell metabolism,
L-arginine and L-citrulline might serve as endogenous N sources
(Ludwig et al., PLANT PHYSIOLOGY, Vol 101, Issue 2 429-434, 1993).
Glycine is a valuable compound of wide use as food additives for
processed foodstuffs and raw materials for agricultural chemicals
and medicines. Glycine is the simplest amino acid, and is used in
crop production as a chelating agent for micronutrients and has
been used as a nitrogen fertilizer, at least on an experimental
basis. As such, it is representative of amino acids used in crop
production. Practically all commercial glycine is produced by
synthetic processes such as the Strecker Synthesis, the reaction of
formaldehyde, ammonia, and hydrogen cyanide, and hydrolysis of the
resulting aminonitrile. Glycine is used as chelating/complexing
agent for cation nutrients, plant growth regulators, substrate for
microbiological products, fertilizer source of nitrogen. Serine is
a primary intermediate in the biosynthesis of a wide variety of
cellular metabolites including such economically important
compounds as choline, glycine, cysteine and tryptophan. In
addition, serine acts as a single carbon donor and is responsible
for 60% to 75% of the total need of the cell for Cl units through
the production of 5,10-methylenetetrahydrofolate from
tetrahydrofolate. These Cl units are used in a wide variety of
biosynthetic pathways including the synthesis of methionine,
inosine monophosphate, other purines and some pyrimidines (e.g.,
thymidine and hydroxymethyl cytidine).
[5990] The glycine-serine interconversion, catalysed by glycine
decarboxylase and serine hydroxymethyltransferase, is an important
reaction of primary metabolism in all organisms including plants,
by providing one-carbon units for many biosynthetic reactions. In
plants, in addition, it is an integral part of the photorespiratory
metabolic pathway and produces large amounts of photorespiratory
CO.sub.2 within mitochondria (Bauwe et al., Journal of Experimental
Botany, Vol. 54, No. 387, pp. 1523-1535, Jun. 1, 2003.)
[5991] The enzymatic conversion of phenylalanine to tyrosine is
known in eukaryotes. Human phenylalanine hydroxylase is
specifically expressed in the liver to convert L-phenylalanine to
L-tyrosine (Wang et al. J. Biol. Chem. 269 (12): 9137-46 (1994)).
Deficiency of the PAH enzyme causes classic phenylketonurea, a
common genetic disorder.
[5992] Tyrosine and homoserine and their derivatives are also used
in organic synthesis. For example, tyrosine is starting material in
the synthesis of chatecolamines or DOPA (dihydroxy-phenyl-alanine)
as well as a precursor of adrenaline, dopamine and norepinepherine.
A variety of beta-amino-gamma-keto acids can be prepared from
commercially available 1-homoserine. 5-Oxoproline, also named as
pyroglutamic acid PCA and slats like sodium-PCA, is used as
cosmetic ingredient, such as hair and skin conditioning agent. One
optical isomer of PCA (the L form) is a naturally occurring
component of mammalian tissue. 5-Oxoproline is further used as
templates in the synthesis of homochiral glutamate antagonists.
[5993] [0003.0.0.14] to [0008.0.0.14]: see [0003.0.0.0] to
[0008.0.0.0]
[5994] [0008.1.14.14]U.S. Pat. No. 5,498,532 disclose the
production of various L-amino acids like glutamic acid, glutamine,
lysine, threonine, isoleucine, valine, leucine, tryptophan,
phenylalanine, tyrosine, histidine, arginine, ornithine, citrulline
and proline by direct fermentation using, coryneform bacteria
belonging to the genus Corynbacterium or Brevibacterium, which are
inherently unable to assimilate lactose, but due to recombinant DNA
technology able to assimilate lactose, which represent the carbon
source.
[5995] An other method for producing amino acids such as homoserine
is disclosed in US 20010049126, which use a bacterium belonging to
the genus Escherichia which harbors a PTS, phosphotransferase
system, gene. The coproduction of glutamic acid and other amino
acids including lysine, aspartic acid, alanine by an auxotroph of
Bacillus methanolicus is described in U.S. Pat. No. 6,110,713.
According to the teaching of U.S. Pat. No. 5,677,156 L-aspartic
acid can be efficiently produced from maleic acid or fumaric acid
by adding the aspartase-containing microorganism, like
Brevibacterium flavum AB-41 strain (FERM BP-1498) and Eschirichia
coli ATCC 11303.
[5996] U.S. Pat. No. 5,354,672 discloses a method of producing
tyrosine, methionine, or phenylalanine by transiently incorporating
a DNA inversion gene into the host cell, Escherichia coli cells,
which induce hypersecretion of amino acids. Known is also the
production of citrulline in the small intestine as a product of
glutamine metabolism, or in the arginine biosynthetic pathway,
where ornithine carbamoyltransferases catalyse the production of
citrulline from carbamoyl-phosphate and ornithine. Benninghoff et
al. disclose the production of citrulline and ornithine by
interferon-gamma treated macrophages (International Immunology, Vol
3, 413-417, 1991).
[5997] There disclosed is a method for producing glycine in US
20030040085, which comprises subjecting an aqueous solution of
glycinonitrile to a hydrolysis reaction in a hydrolysis reaction
system under the action of a microbial enzyme, thereby converting
the glycinonitrile to glycine while by-producing ammonia.
[5998] US 20040157290 discloses a process for preparing a
serine-rich foreign protein comprising culturing a bacterium
containing the cysteine synthase (cysK) gene and a gene encoding
the foreign protein. US 20030079255 disclose the production of
Para-hydroxycinnamic acid by introducing genes encoding
phenylalanine ammonia-lyase from C. violaceum or R. glutinis
tyrosine into a host microorganism and as intermediates, tyrosine
and cinnamic acid are also produced.
[5999] Production of single cell protein and selected amino acids
by microbial fermentation is known, e.g., U.S. Pat. No. 4,652,527.
One amino acid which has been produced on an industrial scale is
lysine, see Tosaka et al., Trends in Biotechnology, 1: 70-74
(1983), Tosaka and Takinami, Progress in Industrial Microbiology,
Ch. 24, pp. 152-172 (Aida et al., 1986). Another example is
glutamic acid which has been produced using bacteria of the genera
Corynebacterium, Brevibacterium, Microbacterium, and Arothrobacter
by fermentation on molasses and starch hydrozylates. Aspartic acid
and alanine are produced by enzymatic means from fumaric acid and
ammonia. Bacillus species have been used in fermentation processes
to produce amino acids, Tosaka et al.; Tosaka and Takinami, as
named above.
[6000] [0009.0.14.14] As described above, the amino acids are
necessary for humans and many mammals, for example for livestock or
other applications in the health care area. L-aspartic acid is one
of the amino acids that is difficult to produce directly by
fermentation. Consequently today the enzyme catalyzed addition of
ammonia to fumaric acid is employed commercially in the production
of aspartic acid. As chelating agents, amino acids increase the
biological availability of different metals. Chelating agents in
general can enhance nutrient uptake, but may also increase the
uptake of toxic metals if those are also present. If cation
impurities are present in micronutrient sources (e.g. Cadmium),
chelation of those metals would make those contaminants more
readily assimilated by plants than in less available forms.
[6001] [0010.0.0.14] to [0011.0.0.14]: see [0010.0.0.0] to
[0011.0.0.0]
[6002] [0012.0.14.14] It is an object of the present invention to
develop an inexpensive process for the synthesis of 5-oxoproline,
alanine, aspartic acid, citrulline, glycine, homoserine,
phenylalanine, serine and/or tyrosine. Amino acids are (depending
on the organism) one of the most frequently limiting components of
food or feed.
[6003] [0013.0.0.14] see [0013.0.0.00]
[6004] [0014.0.14.14] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine. Accordingly, in the present invention, the
term "the fine chemical" as used herein relates to "5-oxoproline,
alanine, aspartic acid, citrulline, glycine, homoserine,
phenylalanine, serine and/or tyrosine". Further, the term "the fine
chemicals" as used herein also relates to fine chemicals comprising
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
homoserine, phenylalanine, serine and/or tyrosine.
[6005] [0015.0.14.14] In one embodiment, the term "the fine
chemical" or "the respective fine chemical" means 5-oxoproline,
alanine, aspartic acid, citrulline, glycine, homoserine,
phenylalanine, serine and/or tyrosine, preferably the amino acid of
the present invention in the L configuration, meaning
L-5-oxoproline, L-alanine, L-aspartic acid, L-citrulline,
L-glycine, L-homoserine, L-phenylalanine, L-serine and/or
L-tyrosine. Throughout the specification the term "the fine
chemical" means 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, homoserine, phenylalanine, serine and/or tyrosine,
preferably the amino acid of the present invention in the L
configuration, its salts, ester or amids in free form or bound to
proteins. In a preferred embodiment, the term "the fine chemical"
means L-5-oxoproline, L-alanine, L-aspartic acid, L-citrulline,
L-glycine, L-homoserine, L-phenylalanine, L-serine and/or
L-tyrosine in free form or its salts or bound to proteins.
[6006] [0016.0.14.14] Accordingly, the present invention relates to
a process for the production of the fine respective chemical
comprising [6007] (a) increasing or generating the activity of one
or more [6008] YDL127W, b1896, b0161, b0376, b0970, b1343 and/or
b3172 [6009] protein(s) or of a protein having the sequence of a
polypeptide encoded by a nucleic acid molecule indicated in Table
II, columns 5 or 7, lines 126 to 127 and/or 492 to 496; in a
non-human organism in one or more parts thereof and [6010] (b)
growing the organism under conditions which permit the production
of the fine chemical, meaning of 5-oxoproline or fine chemicals
comprising 5-oxoproline in said organism; [6011] or [6012] (a)
increasing or generating the activity of one or more
[6013] YIL150C, YFL050C, YER173W, YBL015W, b3008, b2095, b0236,
b0486, b1343, b1863, b2489, b2576, b3231 and/or b3767-protein(s) or
of a protein having the sequence of a polypeptide encoded by a
nucleic acid molecule indicated in Table II, columns 5 or 7, lines
128 to 133 and/or 497 to 504; in a non-human organism in one or
more parts thereof and [6014] (b) growing the organism under
conditions which permit the production of the fine chemical,
meaning of alanine or fine chemicals comprising alanine in said
organism; [6015] or [6016] (a) increasing or generating the
activity of one or more
[6017] YIL150C, YGR104C, YER173W, b1896, b0730, b0161, b0577,
b1343, b2023, b2507, b2576, b2753, b3116, b3169, b3172, b4129
and/or b4346-protein(s) or of a protein having the sequence of a
polypeptide encoded by a nucleic acid molecule indicated in Table
II, columns 5 or 7, lines 134 to 138 and/or 505 to 516; in a
non-human organism in one or more parts thereof and [6018] (b)
growing the organism under conditions which permit the production
of the fine chemical, meaning of aspartic acid or fine chemicals
comprising aspartic acid in said organism; [6019] or [6020] (a)
increasing or generating the activity of one or more
[6021] YOR245C, YLL013C, b0057, b0462, b1275, b1360, b2239, b2414,
b2426, b2489, b3160, b3241, b3926, b4214 and/or b4269--protein(s)
or of a protein having the sequence of a polypeptide encoded by a
nucleic acid molecule indicated in Table II, columns 5 or 7, lines
139 to 140 and/or 517 to 529;
in a non-human organism in one or more parts thereof and [6022] (b)
growing the organism under conditions which permit the production
of the fine chemical, meaning of citrulline or fine chemicals
comprising citrulline in said organism; [6023] or [6024] (a)
increasing or generating the activity of one or more
[6025] YOL123W, YFL050C, b0057, b0577, b2414, b2489, b2553 and/or
b2576--protein(s) or of a protein having the sequence of a
polypeptide encoded by a nucleic acid molecule indicated in Table
II, columns 5 or 7, lines 141 to 142 and/or 530 to 535; in a
non-human organism in one or more parts thereof and [6026] (b)
growing the organism under conditions which permit the production
of the fine chemical, meaning of glycine or fine chemicals
comprising glycine in said organism; or [6027] (a) increasing or
generating the activity of one or more
[6028] YEL046C and/or b3767--protein(s) or of a protein having the
sequence of a polypeptide encoded by a nucleic acid molecule
indicated in Table II, columns 5 or 7, lines 143 and/or 536;
in a non-human organism in one or more parts thereof and [6029] (b)
growing the organism under conditions which permit the production
of the fine chemical, meaning of homoserine or fine chemicals
comprising homoserine in said organism; or [6030] (a) increasing or
generating the activity of one or more
[6031] YPR138C, YJL072C, YHR130C, YGR101W, YER173W, YAL049C, b3462,
b3256, b1886, b1827, b1708, b1697, b0695, b0161, b1228, b2078,
b2414, b2576, b2796, b3919, b3938 and/or b3983--protein(s) or of a
protein having the sequence of a polypeptide encoded by a nucleic
acid molecule indicated in Table II, columns 5 or 7, lines 144 to
156 and/or 537 to 545;
in a non-human organism in one or more parts thereof and [6032] (b)
growing the organism under conditions which permit the production
of the fine chemical, meaning of phenylalanine or fine chemicals
comprising phenylalanine in said organism; or [6033] (a) increasing
or generating the activity of one or more
[6034] YOR261C, YLR082C, YLL009C, YKR057W, YIL150C, YER152C,
YEL045C, b3462, b1886, b1829, b0057, b0486, b2414, b2489, b2664,
b3064, b3116, b3160 and/or b3231-protein(s) or of a protein having
the sequence of a polypeptide encoded by a nucleic acid molecule
indicated in Table II, columns 5 or 7, lines 157 to 166 and/or 546
to 554;
in a non-human organism in one or more parts thereof and [6035] (b)
growing the organism under conditions which permit the production
of the fine chemical, meaning of serine or fine chemicals
comprising serine in said organism; or [6036] (a) increasing or
generating the activity of one or more
[6037] YOR350C, YIL150C, YHR130C, YFL050C, b1829, b1827, b0970,
b2491 and/or b3983--protein(s) or of a protein having the sequence
of a polypeptide encoded by a nucleic acid molecule indicated in
Table II, columns 5 or 7, lines 167 to 172 and/or 555 to 557;
in a non-human organism in one or more parts thereof and [6038] (b)
growing the organism under conditions which permit the production
of the fine chemical, meaning of tyrosine or fine chemicals
comprising tyrosine in said organism.
[6039] Accordingly, the present invention relates to a process for
the production of a fine chemical comprising [6040] (a) increasing
or generating the activity of one or more proteins having the
activity of a protein indicated in Table II, column 3, lines 126 to
172 and/or lines 492 to 557 or having the sequence of a polypeptide
encoded by a nucleic acid molecule indicated in Table I, column 5
or 7, lines 126 to 172 and/or lines 492 to 557, in a non-human
organism in one or more parts thereof and [6041] (b) growing the
organism under conditions which permit the production of the fine
chemical, in particular 5-oxoproline and/or alanine and/or aspartic
acid and/or citrulline and/or glycine and/or homoserine and/or
phenylalanine and/or serine and/or tyrosine.
[6042] [0016.1.14.14] Accordingly, the term "the fine chemical"
means in one embodiment "5-oxoproline" in relation to all sequences
listed in Table I to IV, lines 126 to 127 and/or 492 to 496 or
homologs thereof and
means in one embodiment "alanine" in relation to all sequences
listed in Tables I to IV, lines 128 to 133 and/or 497 to 504 or
homologs thereof and means in one embodiment "aspartic acid" in
relation to all sequences listed in Table I to
[6043] IV, lines 134 to 138 and/or 505 to 516, or homologs thereof
and
means in one embodiment "citrulline" in relation to all sequences
listed in Table I to IV, lines 139 to 140 and/or 517 to 529 or
homologs thereof and means in one embodiment "glycine" in relation
to all sequences listed in Table I to IV, lines 141 to 142 and/or
530 to 535 or homologs thereof and means in one embodiment
"homoserine" in relation to all sequences listed in Table I to IV,
lines 143 and/or 536 or homologs thereof and means in one
embodiment "phenylalanine" in relation to all sequences listed in
Table I to IV, lines 144 to 156 and/or 537 to 545 or homologs
thereof and means in one embodiment "serine" in relation to all
sequences listed in Table I to IV, lines 157 to 166 and/or 546 to
554 or homologs thereof and means in one embodiment "tyrosine" in
relation to all sequences listed in Table I to IV, lines 167 to 172
and/or 555 to 557. or homologs thereof.
[6044] Accordingly, in one embodiment the term "the fine chemical"
means "5-oxoproline" and "aspartic acid" in relation to all
sequences listed in Table I to IV, lines 127 and/or 137;
[6045] in one embodiment the term "the fine chemical" means
"alanine", "aspartic acid", "serine" and "tyrosine" in relation to
all sequences listed in Table I to IV, lines 128, 134, 161 and/or
168;
[6046] in one embodiment the term "the fine chemical" means
"alanine", "glycine" and "tyrosine" in relation to all sequences
listed in Table I to IV, lines 129, 142 and/or 170;
[6047] in one embodiment the term "the fine chemical" means
"phenylalanine" and "tyrosine" in relation to all sequences listed
in Table I to IV, lines 146 and/or 169;
[6048] in one embodiment the term "the fine chemical" means
"phenylalanine" and "tyrosine" in relation to all sequences listed
in Table I to IV, lines 153 and/or 172;
[6049] in one embodiment the term "the fine chemical" means
"serine" and "tyrosine" in relation to all sequences listed in
Table I to IV, lines 166 and/or 171;
[6050] in one embodiment the term "the fine chemical" means
"phenylalanine" and "serine" in relation to all sequences listed in
Table I to IV, lines 150 and/or 164;
[6051] in one embodiment the term "the fine chemical" means
"phenylalanine" and "serine" in relation to all sequences listed in
Table I to IV, lines 152 and/or 165;
[6052] in one embodiment the term "the fine chemical" means
"citrulline", "glycine" and "serine" in relation to all sequences
listed in Table I to IV, lines 517, 530 and/or 546;
[6053] in one embodiment the term "the fine chemical" means
"5-oxoproline", "aspartic acid" and "phenylalanine" in relation to
all sequences listed in Table I to IV, lines 492, 505 and/or
537;
[6054] in one embodiment the term "the fine chemical" means
"alanine" and "serine" in relation to all sequences listed in Table
I to IV, lines 498 and/or 547;
[6055] in one embodiment the term "the fine chemical" means
"glycine" and "aspartic acid" in relation to all sequences listed
in Table I to IV, lines 531 and/or 506;
[6056] in one embodiment the term "the fine chemical" means
"5-oxoproline" and "tyrosine" in relation to all sequences listed
in Table I to IV, lines 494 and/or 555;
[6057] in one embodiment the term "the fine chemical" means
"alanine", "5-oxoproline" and "aspartic acid" in relation to all
sequences listed in Table I to IV, lines 499, 495 and/or 507;
[6058] in one embodiment the term "the fine chemical" means
"citrulline", "glycine", "serine" and "phenylalanine" in relation
to all sequences listed in Table I to IV, lines 522, 532, 548
and/or 540;
[6059] in one embodiment the term "the fine chemical" means
"citrulline", "alanine", "glycine" and "serine" in relation to all
sequences listed in Table I to IV, lines 524, 501, 533 and/or
549;
[6060] in one embodiment the term "the fine chemical" means
"alanine", "glycine", "aspartic acid" and "phenylalanine" in
relation to all sequences listed in Table I to IV, lines 502, 535,
510 and/or 541;
[6061] in one embodiment the term "the fine chemical" means
"serine" and "aspartic acid" in relation to all sequences listed in
Table I to IV, lines 552 and/or 512;
[6062] in one embodiment the term "the fine chemical" means
"citrulline" and "serine" in relation to all sequences listed in
Table I to IV, lines 525 and/or 553;
[6063] in one embodiment the term "the fine chemical" means
"5-oxoproline" and "aspartic acid" in relation to all sequences
listed in Table I to IV, lines 496 and/or 514;
[6064] in one embodiment the term "the fine chemical" means
"alanine" and "serine" in relation to all sequences listed in Table
I to IV, lines 503 and/or 554;
[6065] in one embodiment the term "the fine chemical" means
"alanine" and "homoserine" in relation to all sequences listed in
Table I to IV, lines 504 and/or 536;
[6066] in one embodiment the term "the fine chemical" means
"phenylalanine" and "tyrosine" in relation to all sequences listed
in Table I to IV, lines 545 and/or 557;
[6067] in one embodiment the term "the fine chemical" means
"alanine", "aspartic acid" and "phenylalanine" in relation to all
sequences listed in Table I to IV, lines 130, 136 and/or 148;
[6068] Accordingly, the term "the fine chemical" can mean
"5-oxoproline", "alanine", "aspartic acid", "citrulline",
"glycine", "homoserine", "phenylalanine", "serine" and/or
"tyrosine", owing to circumstances and the context. In order to
illustrate that the meaning of the term "the fine chemical" means
"5-oxoproline", "alanine", "aspartic acid", "citrulline",
"glycine", "homoserine", "phenylalanine", "serine" and/or
"tyrosine" the term "the respective fine chemical" is also
used.
[6069] [0017.0.0.14] to [0018.0.0.14]: see [0017.0.0.0] to
[0018.0.0.0]
[6070] [0019.0.14.14] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the fine respective chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table II, column 3, lines 126 to 172 and/or lines 492
to 557 or encoded by nucleic acid molecule indicated in Table I,
columns 5 or 7, lines 126 to 172 and/or lines 492 to 557.
[6071] [0020.0.14.14] Surprisingly it was found, that the
transgenic expression of at least one of the Saccaromyces
cerevisiae protein(s) indicated in Table II, Column 3,
line 126 for 5-oxoproline and/or lines 128 to 131 for alanine
and/or lines 134 to 136 for aspartic acid and/or lines 139 to 140
for citrulline and/or lines 141 to 142 for glycine and/or line 143
for homoserine and/or lines 144 to 149 for phenylalanine and/or
lines 157 to 163 for serine and/or lines 167 to 170 for tyrosine in
Arabidopsis thaliana conferred an increase in the respective fine
chemical content of the transformed plants and/or at least one of
the Escherichia coli K12 proteins indicated in Table II, Column 3,
line 127 and/or 492 to 496 for 5-oxoproline and/or lines 132 to 133
and/or 497 to 504 for alanine and/or lines 137 to 138 and/or 505 to
516 for aspartic acid and/or lines 517 to 529 for citrulline and/or
lines 530 to 535 for glycine and/or line 536 for homoserine and/or
lines 150 to 156 and/or 537 to 545 for phenylalanine and/or lines
164 to 166 and/or 546 to 554 for serine and/or lines 171 to 172
and/or 555 to 557 for tyrosine in Arabidopsis thaliana conferred an
increase in the respective fine chemical content of the transformed
plants.
[6072] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 127 and 137 in Arabidopsis thaliana conferred an increase
in 5-oxoproline and/or aspartic acid (or the respective fine
chemical) content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of 5-oxoproline, in one embodiment, said protein or its
homologs are used for the production of aspartic acid; in one
embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of: 5-oxoproline and/or aspartic acid.
[6073] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein as indicated in Table II,
column 5, lines 128 and 134 and 161 and 168 in Arabidopsis thaliana
conferred an increase in alanine and/or aspartic acid and/or serine
and/or tyrosine (or the respective fine chemical) content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of alanine; in one embodiment,
said protein or its homologs are used for the production of
aspartic acid, in one embodiment, said protein or its homologs are
used for the production of serine, in one embodiment, said protein
or its homologs are used for the production of tyrosine, in one
embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of: alanine and/or aspartic acid and/or serine and/or
tyrosine.
[6074] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein as indicated in Table II,
column 5, lines 129 and 142 and 170 in Arabidopsis thaliana
conferred an increase in alanine and/or glycine and/or tyrosine (or
the respective fine chemical) content of the transformed plants.
Thus, in one embodiment, said protein or its homologs are used for
the production of alanine; in one embodiment, said protein or its
homologs are used for the production of glycine, in one embodiment,
said protein or its homologs are used for the production of
tyrosine, in one embodiment, said protein or its homologs are used
for the production of one or more fine chemical selected from the
group consisting of: alanine and/or glycine and/or tyrosine.
[6075] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein as indicated in Table II,
column 5, lines 146 and 169 in Arabidopsis thaliana conferred an
increase in phenylalanine and/or tyrosine (or the respective fine
chemical) content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of phenylalanine; in one embodiment, said protein or its
homologs are used for the production of tyrosine, in one
embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of: phenylalanine and/or tyrosine.
[6076] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 153 and 172 in Arabidopsis thaliana conferred an increase
in phenylalanine and/or tyrosine (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of
phenylalanine, in one embodiment, said protein or its homologs are
used for the production of tyrosine; in one embodiment, said
protein or its homologs are used for the production of one or more
fine chemical selected from the group consisting of: phenylalanine
and/or tyrosine.
[6077] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 166 and 171 in Arabidopsis thaliana conferred an increase
in serine and/or tyrosine (or the respective fine chemical) content
of the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of serine, in one
embodiment, said protein or its homologs are used for the
production of tyrosine; in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of: serine and/or tyrosine.
[6078] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 150 and 164 in Arabidopsis thaliana conferred an increase
in phenylalanine and/or serine (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of
phenylalanine, in one embodiment, said protein or its homologs are
used for the production of serine; in one embodiment, said protein
or its homologs are used for the production of one or more fine
chemical selected from the group consisting of: phenylalanine
and/or serine.
[6079] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 152 and 165 in Arabidopsis thaliana conferred an increase
in phenylalanine and/or serine (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of
phenylalanine, in one embodiment, said protein or its homologs are
used for the production of serine; in one embodiment, said protein
or its homologs are used for the production of one or more fine
chemical selected from the group consisting of: phenylalanine
and/or serine.
[6080] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 517 and 530 and 546 in Arabidopsis thaliana conferred an
increase in citrulline and/or glycine and/or serine (or the
respective fine chemical) content of the transformed plants. Thus,
in one embodiment, said protein or its homologs are used for the
production of citrulline; in one embodiment, said protein or its
homologs are used for the production of glycine, in one embodiment,
said protein or its homologs are used for the production of serine,
in one embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of: citrulline and/or glycine and/or serine.
[6081] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 492 and 505 and 537 in Arabidopsis thaliana conferred an
increase in 5-oxoproline and/or aspartic acid and/or phenylalanine
(or the respective fine chemical) content of the transformed
plants. Thus, in one embodiment, said protein or its homologs are
used for the production of 5-oxoproline; in one embodiment, said
protein or its homologs are used for the production of aspartic
acid, in one embodiment, said protein or its homologs are used for
the production of phenylalanine, in one embodiment, said protein or
its homologs are used for the production of one or more fine
chemical selected from the group consisting of: 5-oxoproline and/or
aspartic acid and/or phenylalanine.
[6082] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 498 and 547 in Arabidopsis thaliana conferred an increase
in alanine and/or serine (or the respective fine chemical) content
of the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of alanine; in one
embodiment, said protein or its homologs are used for the
production of serine, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of: alanine and/or serine.
[6083] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 531 and 506 in Arabidopsis thaliana conferred an increase
in glycine and/or aspartic acid (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of glycine; in
one embodiment, said protein or its homologs are used for the
production of aspartic acid, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of: glycine and/or aspartic
acid.
[6084] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 494 and 555 in Arabidopsis thaliana conferred an increase
in 5-oxoproline and/or tyrosine (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of
5-oxoproline; in one embodiment, said protein or its homologs are
used for the production of tyrosine, in one embodiment, said
protein or its homologs are used for the production of one or more
fine chemical selected from the group consisting of: 5-oxoproline
and/or tyrosine.
[6085] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 499 and 495 and 507 in Arabidopsis thaliana conferred an
increase in alanine and/or 5-oxoproline and/or aspartic acid (or
the respective fine chemical) content of the transformed plants.
Thus, in one embodiment, said protein or its homologs are used for
the production of alanine; in one embodiment, said protein or its
homologs are used for the production of 5-oxoproline, in one
embodiment, said protein or its homologs are used for the
production of aspartic acid, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of: alanine and/or 5-oxoproline
and/or aspartic acid.
[6086] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 522 and 532 and 548 and 540 in Arabidopsis thaliana
conferred an increase in citrulline and/or glycine and/or serine
and/or phenylalanine (or the respective fine chemical) content of
the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of citrulline; in one
embodiment, said protein or its homologs are used for the
production of glycine, in one embodiment, said protein or its
homologs are used for the production of serine, in one embodiment,
said protein or its homologs are used for the production of
phenylalanine, in one embodiment, said protein or its homologs are
used for the production of one or more fine chemical selected from
the group consisting of: citrulline and/or glycine and/or serine
and/or phenylalanine.
[6087] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 524 and 501 and 533 and 549 in Arabidopsis thaliana
conferred an increase in citrulline and/or alanine and/or glycine
and/or serine (or the respective fine chemical) content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of citrulline; in one
embodiment, said protein or its homologs are used for the
production of alanine, in one embodiment, said protein or its
homologs are used for the production of glycine, in one embodiment,
said protein or its homologs are used for the production of serine,
in one embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of: citrulline and/or alanine and/or glycine and/or
serine.
[6088] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 502 and 535 and 510 and 541 in Arabidopsis thaliana
conferred an increase in alanine and/or glycine and/or aspartic
acid and/or phenylalanine (or the respective fine chemical) content
of the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of alanine; in one
embodiment, said protein or its homologs are used for the
production of glycine, in one embodiment, said protein or its
homologs are used for the production of aspartic acid, in one
embodiment, said protein or its homologs are used for the
production of phenylalanine, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of: alanine and/or glycine
and/or aspartic acid and/or phenylalanine.
[6089] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 552 and 512 in Arabidopsis thaliana conferred an increase
in serine and/or aspartic acid (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of serine; in
one embodiment, said protein or its homologs are used for the
production of aspartic acid, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of: serine and/or aspartic
acid.
[6090] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 525 and 553 in Arabidopsis thaliana conferred an increase
in citrulline and/or serine (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of citrulline;
in one embodiment, said protein or its homologs are used for the
production of serine, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of: citrulline and/or
serine.
[6091] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 496 and 514 in Arabidopsis thaliana conferred an increase
in 5-oxoproline and/or aspartic acid (or the respective fine
chemical) content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of 5-oxoproline; in one embodiment, said protein or its
homologs are used for the production of aspartic acid, in one
embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of: 5-oxoproline and/or aspartic acid.
[6092] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 503 and 554 in Arabidopsis thaliana conferred an increase
in alanine and/or serine (or the respective fine chemical) content
of the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of alanine; in one
embodiment, said protein or its homologs are used for the
production of serine, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of: alanine and/or serine.
[6093] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 504 and 536 in Arabidopsis thaliana conferred an increase
in alanine and/or homoserine (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of alanine; in
one embodiment, said protein or its homologs are used for the
production of homoserine, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of: alanine and/or
homoserine.
[6094] Surprisingly it was found, that the transgenic expression of
the Escherichia coli K12 protein as indicated in Table II, column
5, lines 545 and 557 in Arabidopsis thaliana conferred an increase
in phenylalanine and/or tyrosine (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of
phenylalanine; in one embodiment, said protein or its homologs are
used for the production of tyrosine, in one embodiment, said
protein or its homologs are used for the production of one or more
fine chemical selected from the group consisting of: phenylalanine
and/or tyrosine.
[6095] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein as indicated in Table II,
column 5, lines 130 and 136 and 148 in Arabidopsis thaliana
conferred an increase in alanine and/or aspartic acid and/or
phenylalanine (or the respective fine chemical) content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of alanine;
[6096] in one embodiment, said protein or its homologs are used for
the production of aspartic acid, in one embodiment, said protein or
its homologs are used for the production of phenylalanine, in one
embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of: alanine and/or aspartic acid and/or
phenylalanine.
[6097] [0021.0.0.14] see [0021.0.0.0]
[6098] [0022.0.14.14] The sequence of YER152C from Saccharomyces
cerevisiae has been published in Dietrich, Nature 387 (6632 Suppl),
78-81, 1997, and Goffeau, Science 274 (5287), 546-547, 1996. It
seems to have an activity similar to tyrosine aminotransferase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity
similar to tyrosine aminotransferase from Saccaromyces cerevisiae,
preferably of the family of valine-pyruvate transaminase or its
homolog, e.g. as shown herein, for the production of the the
respective fine chemical, meaning of serine, in particular for
increasing the amount of serine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a similar to
tyrosine aminotransferase protein is increased or generated, e.g.
from Saccharomyces cerevisiae or a homolog thereof. The sequence of
YAL049C from Saccharomyces cerevisiae has been published in Bussey
et al., Proc. Natl. Acad. Sci. U.S.A. 92 (9), 3809-3813 (1995), and
Goffeau, Science 274 (5287), 546-547, 1996. It activity is not
characterized yet. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of a YAL049C protein from Saccaromyces cerevisiae, or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of phenylalanine, in particular for
increasing the amount of phenylalanine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a YAL049C protein
is increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof. The sequence of b3008 from Escherichia coli K12
has been published in Blattner et al., Science 277 (5331),
1453-1474, 1997, and its activity is being defined as a
cystathionine beta-lyase, PLP-dependent (beta-cystathionase),
preferably of the 0-succinylhomoserine (thiol)-lyase superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein of the
0-succinylhomoserine (thiol)-lyase superfamily, preferably such
protein is having a cystathionine beta-lyase, PLP-dependent
(beta-cystathionase) activity, e.g. as shown herein, for the
production of the respective fine chemical, meaning of alanine,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a cystathionine beta-lyase is increased
or generated, e.g. from E. coli or a homolog thereof. The sequence
of YOR261C from Saccharomyces cerevisiae has been published in
Dujon, et al., Nature 387 (6632 Suppl), 98-102 (1997), and Goffeau,
Science 274 (5287), 546-547, 1996 and its activity is being defined
as a proteasome regulatory particle subunit, preferably of the
mov-34 protein superfamily. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of mov-34 protein superfamily, preferably
such protein is having a proteasome regulatory particle subunit
activity or its homolog, e.g. as shown herein, for the production
of the the respective fine chemical, meaning of serine, in
particular for increasing the amount of serine, preferably of
serine in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a proteasome regulatory particle subunit
is increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof. The sequence of YOR245C from Saccharomyces
cerevisiae has been published in Dujon, et al., Nature 387 (6632
Suppl), 98-102 (1997), and Goffeau, Science 274 (5287), 546-547,
1996 and its activity is being defined as a Acyl-CoA:diacylglycerol
acyltransferase, preferably of the Caenorhabditis elegans
hypothetical protein KO7B1.4 superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of Caenorhabditis elegans
hypothetical protein KO7B1.4 superfamily, preferably such protein
is having a Acyl-CoA:diacylglycerol acyltransferase activity or its
homolog, e.g. as shown herein, for the production of the the
respective fine chemical, meaning of citrulline, in particular for
increasing the amount of citrulline in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a
Acyl-CoA:diacylglycerol acyltransferase is increased or generated,
e.g. from Saccharomyces cerevisiae or a homolog thereof. The
sequence of YOL123W from Saccharomyces cerevisiae has been
published in Dujon, et al., Nature 387 (6632 Suppl), 98-102 (1997),
and Goffeau, Science 274 (5287), 546-547, 1996 and its activity is
being defined as a cleavage and polyadenylation factor CF I
component involved in pre-mRNA 3'-end processing, preferably of the
heterogeneous nuclear ribonucleoprotein HRP1--yeast (Saccharomyces
cerevisiae) superfamily. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of heterogeneous nuclear ribonucleoprotein
HRP1--yeast (Saccharomyces cerevisiae) superfamily, preferably such
protein is having a cleavage and polyadenylation factor
[6099] CF I component involved in pre-mRNA 3'-end processing
activity or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, meaning of glycine, in particular
for increasing the amount of glycine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a cleavage and
polyadenylation factor CF I component involved in pre-mRNA 3'-end
processing is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof. The sequence of YLR082C from
Saccharomyces cerevisiae has been published in Johnston, Nature 387
(6632 Suppl), 87-90, (1997), and Goffeau, Science 274 (5287),
546-547, 1996, and Goffeau, Science 274 (5287), 546-547, 1996 and
its activity is being defined as a suppressor of Rad53 null
lethality. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a suppressor of Rad53 null lethality activity or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of serine, in particular for increasing the
amount of serine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a suppressor of Rad53 null
lethality is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof. The sequence of b0695 from
Escherichia coli K12 has been published in Blattner et al., Science
277(5331), 1453-1474, 1997, and its activity is being defined as
sensory histidine kinase in two-component regulatory system,
preferably of the sensor histidine kinase homology superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
sensor histidine kinase homology superfamily, preferably such
protein is having a sensory histidine kinase in two-component
regulatory system activity from E. coli or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of phenylalanine, in particular for increasing the amount
of phenylalanine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a sensory histidine kinase in
two-component regulatory system is increased or generated, e.g.
from E. coli or a homolog thereof.
[6100] The sequence of b0730 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as transcriptional regulator of
succinylCoA synthetase operon and/or fatty acyl response regulator,
preferably of the transcription regulator GntR superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
transcription regulator GntR superfamily, preferably such protein
is having a transcriptional regulator of succinylCoA synthetase
operon and/or fatty acyl response regulator activity from E. coli
or its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of aspartic acid, in particular
for increasing the amount of aspartic acid in free or bound form in
an organism or a part thereof, as mentioned. In one embodiment, in
the process of the present invention the activity of a
transcriptional regulator of succinylCoA synthetase operon and/or
fatty acyl response regulator is increased or generated, e.g. from
E. coli or a homolog thereof. The sequence of b1697 from
Escherichia coli K12 has been published in Blattner et al., Science
277(5331), 1453-1474, 1997, and its activity is being defined as an
electron transfer flavoprotein subunit with ETFP adenine
nucleotide-binding like domain, preferably of the electron transfer
flavoprotein beta chain superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of electron transfer
flavoprotein beta chain superfamily, preferably such protein is
having a electron transfer flavoprotein subunit with ETFP adenine
nucleotide-binding like domain activity from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of phenylalanine, in particular for
increasing the amount of phenylalanine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of an electron
transfer flavoprotein subunit with ETFP adenine nucleotide-binding
like domain is increased or generated, e.g. from E. coli or a
homolog thereof. The sequence of b1708 from Escherichia coli K12
has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as a
lipoprotein, preferably of the conserved hypothetical protein
H11314 superfamily. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of conserved hypothetical protein H11314 superfamily,
preferably such protein is having a lipoprotein activity from E.
coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of phenylalanine, in
particular for increasing the amount of phenylalanine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a lipoprotein is increased or generated, e.g. from E. coli or a
homolog thereof. The sequence of b1827 from Escherichia coli K12
has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as a
transcriptional repressor with DNA-binding Winged helix domain
(IcIR family), preferably of the acetate operon repressor
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of acetate operon repressor superfamily, preferably such
protein is having a transcriptional repressor with DNA-binding
Winged helix domain (IcIR family) activity from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of phenylalanine and/or tyrosine, in
particular for increasing the amount of phenylalanine and/or
tyrosine, preferably of phenylalanine and/or tyrosine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a transcriptional repressor protein with a DNA-binding Winged helix
domain (IcIR family) is increased or generated, e.g. from E. coli
or a homolog thereof. The sequence of b1829 from Escherichia coli
K12 has been published in Blattner, Science 277(5331), 1453-1474,
1997, and its activity is being defined as a heat shock protein
with protease activity, preferably of the heat-shock protein htpX
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the heat-shock protein htpX superfamily, preferably
such protein is having a heat shock protein activity with protease
activity from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of serine
and/or tyrosine, in particular for increasing the amount of serine
and/or tyrosine, preferably of serine and/or tyrosine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a heat shock protein with protease is increased or generated, e.g.
from E. coli or a homolog thereof. The sequence of b1886 from
Escherichia coli K12 has been published in Blattner, Science
277(5331), 1453-1474, 1997, and its activity is being defined as a
methyl-accepting chemotaxis protein II and/or aspartate sensor
receptor, preferably of the methyl-accepting chemotaxis protein
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the methyl-accepting chemotaxis protein superfamily,
preferably such protein is having a methyl-accepting chemotaxis
protein II and/or aspartate sensor receptor activity from E. coli
or its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of serine and/or phenylalanine,
in particular for increasing the amount of serine and/or
phenylalanine, preferably of serine and/or phenylalanine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a methyl-accepting chemotaxis protein II and/or aspartate sensor
receptor is increased or generated, e.g. from E. coli or a homolog
thereof. The sequence of b1896 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a trehalose-6-phosphate synthase,
preferably of the Schizosaccharomyces pombe
alpha,alpha-trehalose-phosphate synthase (UdP-forming) superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Schizosaccharomyces pombe alpha,alpha-trehalose-phosphate
synthase (UdP-forming) superfamily, preferably such protein is
having a trehalose-6-phosphate synthase activity from E. coli or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of 5-oxoproline and/or aspartic
acid, in particular for increasing the amount of 5-oxoproline
and/or aspartic acid, preferably of 5-oxoproline and/or aspartic
acid in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a trehalose-6-phosphate synthase is
increased or generated, e.g. from E. coli or a homolog thereof. The
sequence of b2095 from Escherichia coli K12 has been published in
Blattner, Science 277(5331), 1453-1474, 1997, and its activity is
being defined as a tagatose-6-phosphate kinase, preferably of the
Escherichia probable tagatose 6-phosphate kinase gatZ superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Escherichia probable tagatose 6-phosphate kinase gatZ
superfamily, preferably such protein is having a
tagatose-6-phosphate kinase activity from E. coli or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, meaning of phenylalanine, in particular for increasing
the amount of alanine, preferably of alanine in free or bound form
in an organism or a part thereof, as mentioned. In one embodiment,
in the process of the present invention the activity of a
tagatose-6-phosphate kinase is increased or generated, e.g. from E.
coli or a homolog thereof. The sequence of b3256 from Escherichia
coli K12 has been published in Blattner,
[6101] Science 277(5331), 1453-1474, 1997, and its activity is
being defined as a acetyl CoA carboxylase and/or biotin carboxylase
subunit, preferably of the biotin carboxylase superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the biotin carboxylase superfamily, preferably such protein is
having a acetyl CoA carboxylase and/or biotin carboxylase subunit
activity from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of
phenylalanine, in particular for increasing the amount of
phenylalanine, preferably of phenylalanine in free or bound form in
an organism or a part thereof, as mentioned. In one embodiment, in
the process of the present invention the activity of a acetyl CoA
carboxylase and/or biotin carboxylase subunit is increased or
generated, e.g. from E. coli or a homolog thereof. The sequence of
b3462 from Escherichia coli K12 has been published in Blattner,
Science 277(5331), 1453-1474, 1997, and its activity is being
defined as a integral membrane cell division protein, preferably of
the cell division protein ftsX superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the cell division protein
ftsX superfamily, preferably such protein is having a integral
membrane cell division protein activity from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of serine and/or phenylalanine, in
particular for increasing the amount of serine and/or
phenylalanine, preferably of serine and/or phenylalanine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a integral membrane cell division protein is increased or
generated, e.g. from E. coli or a homolog thereof. The sequence of
YBL015W from Saccharomyces cerevisiae has been published in
Goffeau, Science 274 (5287), 546-547, 1996, and in Feldmann, EMBO
J., 13, 5795-5809, 1994 and its activity is being defined as a
mannose-containing glycoprotein and/or as an acetyl-CoA hydrolase,
preferably of the acetyl-CoA hydrolase superfamily. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of the acetyl-CoA hydrolase
superfamily, preferably such protein is having a mannose-containing
glycoprotein and/or as an acetyl-CoA hydrolase activity or its
homolog, e.g. as shown herein, for the production of the the
respective fine chemical, meaning of alanine, in particular for
increasing the amount of alanine, preferably of alanine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a a mannose-containing glycoprotein and/or as an acetyl-CoA
hydrolase is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof. The sequence of YDL127W from
Saccharomyces cerevisiae has been published in Jacq et al., Nature
387 (6632 Suppl), 75-78, 1997 and Goffeau, Science 274 (5287),
546-547, 1996, and its activity is being defined as a G1/S-specific
cyclin PCL2 (Cyclin HCS26 homolog). Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of the G1/S-specific cyclin PCL2 (Cyclin
HCS26 homolog) or its homolog, e.g. as shown herein, for the
production of the the respective fine chemical, meaning of
5-oxoproline, in particular for increasing the amount of
5-oxoproline, preferably of 5-oxoproline in free or bound form in
an organism or a part thereof, as mentioned. In one embodiment, in
the process of the present invention the activity of a
G1/S-specific cyclin PCL2 (Cyclin HCS26 homolog) is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog thereof.
The sequence of YEL045C from Saccharomyces cerevisiae has been
published in Dietrich, Nature 387 (6632 Suppl), 78-81, 1997. It
activity is not characterized yet. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of a YEL045C protein from Saccaromyces
cerevisiae, or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of serine, in
particular for increasing the amount of serine, preferably of
serine in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a YEL045C protein is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog thereof.
The sequence of YLL009C from Saccharomyces cerevisiae has been
published in Johnston et al., Nature 387 (6632 Suppl), 87-90, 1997
and Goffeau, Science 274 (5287), 546-547, 1996, and its activity is
being defined as a cytochrome c oxidase copper chaperone.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the cytochrome c oxidase copper chaperone or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of serine, in particular for increasing the amount of
serine preferably of serine in free or bound form in an organism or
a part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a cytochrome c oxidase copper
chaperone is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof. The sequence of YER173W from
Saccharomyces cerevisiae has been published in Dietrich et al.,
Nature 387 (6632 Suppl), 78-81, 1997, and Goffeau et al., Science
274 (5287), 546-547, 1996, and its activity is being defined as a
checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of Checkpoint protein, involved in the
activation of the DNA damage and meiotic pachytene checkpoints or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of alanine and/or aspartic acid,
of alanine and or phenylalanine, of aspartic acid and/or
phenylalanine, of alanine and/or aspartic acid and/or
phenylalanine, in particular for increasing the amount of one or
two or all amino acid(s) selected from the group consisting of
alanine and/or aspartic acid and/or phenylalanine, preferably of
one or two or all amino acid(s) selected from the group consisting
of alanine and/or aspartic acid and/or phenylalanine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a Checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints is increased or generated, e.g.
from Saccharomyces cerevisiae or a homolog thereof. The sequence of
YFL050C from Saccharomyces cerevisiae has been published in
Murakami et al., Nat. Genet. 10 (3), 261-268, 1995, and Goffeau et
al., Science 274 (5287), 546-547, 1996, and its activity is being
defined as a di- and/or trivalent inorganic cation transporter,
preferably of the magnesium and cobalt transport protein
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the magnesium and cobalt transport protein superfamily,
preferably such protein is having a di- and/or trivalent inorganic
cation transporter activity or its homolog, e.g. as shown herein,
for the production of the the respective fine chemical, meaning of
alanine and/or glycine, of alanine and or tyrosine, of glycine
and/or tyrosine, of alanine and/or glycine and/or tyrosine, in
particular for increasing the amount of one or two or all amino
acid(s) selected from the group consisting of alanine and/or
glycine and/or tyrosine, preferably of one or two or all amino
acid(s) selected from the group consisting of alanine and/or
glycine and/or tyrosine in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a di- and/or trivalent
inorganic cation transporter is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof. The sequence of
YGR101W from Saccharomyces cerevisiae has been published in
Tettelin et al., Nature 387 (6632 Suppl), 81-84 (1997) and Goffeau
et al., Science 274 (5287), 546-547, 1996, and its activity is
being defined as a rhomboid protease. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of rhomboid protease or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of phenylalanine, in particular for
increasing the amount of phenylalanine, preferably of phenylalanine
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a rhomboid protease is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog thereof.
The sequence of YGR104C from Saccharomyces cerevisiae has been
published in Thompson et al., Cell 73:1361-1375(1993) and its
activity is being defined as a RNA polymerase II suppressor protein
SRB5 from yeast; and/or as an suppressor of RNA polymerase B SRB5,
preferably of the RNA polymerase II suppressor protein SRB5
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the RNA polymerase II suppressor protein SRB5
superfamily, preferably such protein is having a RNA polymerase II
suppressor protein SRB5 from yeast; and/or a suppressor of RNA
polymerase B SRB5 activity or its homolog, e.g. as shown herein,
for the production of the the respective fine chemical, meaning of
aspartic acid, in particular for increasing the amount of aspartic
acid, preferably of aspartic acid in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a RNA polymerase
II suppressor protein SRB5 from yeast and/or as an suppressor of
RNA polymerase B SRB5 is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof. The sequence of
YHR130C from Saccharomyces cerevisiae has been published in
Johnston et al., Science 265:2077-2082(1994). Its activity is not
characterized yet. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of a YHR130C protein from Saccaromyces cerevisiae, or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of phenylalanine and/or tyrosine, in
particular for increasing the amount of phenylalanine and/or
tyrosine, preferably of phenylalanine and/or tyrosine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a YHR130C protein is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof. The sequence of
YIL150C from Saccharomyces cerevisiae has been published in
Churcher et al., Nature 387 (6632 Suppl), 84-87 (1997) and Goffeau
et al., Science 274 (5287), 546-547, 1996, and its activity is
being defined as a chromatin binding protein, required for S-phase
(DNA synthesis) initiation or completion. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of chromatin binding protein,
required for S-phase (DNA synthesis) initiation or completion or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of alanine and aspartic acid,
alanine and serine, alanine and tyrosine, aspartic acid and serine,
aspartic acid and tyrosine, serine and tyrosine, alanine and
aspartic acid and serine, alanine and aspartic acid and tyrosine,
alanine and serine and tyrosine, aspartic acid and serine and
tyrosine and/or alanine and aspartic acid and serine and tyrosine,
in particular for increasing the amount of alanine and aspartic
acid, alanine and serine, alanine and tyrosine, aspartic acid and
serine, aspartic acid and tyrosine, serine and tyrosine, alanine
and aspartic acid and serine, alanine and aspartic acid and
tyrosine, alanine and serine and tyrosine, aspartic acid and serine
and tyrosine and/or alanine and aspartic acid and serine and
tyrosine, preferably of alanine and aspartic acid, alanine and
serine, alanine and tyrosine, aspartic acid and serine, aspartic
acid and tyrosine, serine and tyrosine, alanine and aspartic acid
and serine, alanine and aspartic acid and tyrosine, alanine and
serine and tyrosine, aspartic acid and serine and tyrosine and/or
alanine and aspartic acid and serine and tyrosine in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a chromatin binding protein, required for S-phase (DNA synthesis)
initiation or completion is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof. The sequence of
YJL072C from Saccharomyces cerevisiae has been published in
Goffeau, Science 274 (5287), 546-547 (1996) and Galibert, EMBO J.
15 (9), 2031-2049 (1996) and its activity is being defined as a
subunit of the GINS complex required for chromosomal DNA
replication, preferably of the Saccharomyces cerevisiae probable
membrane protein YJL072c superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the Saccharomyces cerevisiae
probable membrane protein YJL072c superfamily, preferably such
protein is having a subunit of the GINS complex required for
chromosomal DNA replication activity or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of phenylalanine, in particular for increasing the amount of
phenylalanine, preferably of phenylalanine in free or bound form in
an organism or a part thereof, as mentioned. In one embodiment, in
the process of the present invention the activity of a subunit of
the GINS complex required for chromosomal DNA replication is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof. The sequence of YKR057W from Saccharomyces
cerevisiae has been published in Goffeau, Science 274 (5287),
546-547 (1996) and Dujon, Nature 369 (6479), 371-378 (1994) and its
activity is being defined as a ribosomal protein, similar to S21
ribosomal proteins, involved in ribosome biogenesis and
translation, preferably of the rat ribosomal protein S21
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the rat ribosomal protein S21 superfamily, preferably
such protein is having a ribosomal protein, similar to S21
ribosomal proteins, involved in ribosome biogenesis and translation
activity or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, meaning of serine, in particular
for increasing the amount of serine, preferably of serine in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a ribosomal protein, similar to S21 ribosomal proteins,
involved in ribosome biogenesis and translation is increased or
generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof. The sequence of
YLL013C from Saccharomyces cerevisiae has been published in
Johnston, Nature 387 (6632 Suppl), 87-90 (1997) and Goffeau et al.,
Science 274 (5287), 546-547, 1996, and its activity is being
defined as a member of the PUF protein family, which is named for
the founding members, pumilio and Fbf. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of member of the PUF protein
family, which is named for the founding members, pumilio and Fbf or
its homolog, e.g. as shown herein, for the production of the the
respective fine chemical, meaning of citrulline, in particular for
increasing the amount of citrulline, preferably of citrulline in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of a member of the PUF protein family, which is named for
the founding members, pumilio and Fbf is increased or generated,
e.g. from Saccharomyces cerevisiae or a homolog thereof. The
sequence of YOR350C from Saccharomyces cerevisiae has been
published in Goffeau, Science 274 (5287), 546-547 (1996) and Dujon,
Nature 369 (6479), 371-378 (1994) and its activity is not been
characterized yet. It seems to be member of the Saccharomyces
cerevisiae MNE1 protein superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the Saccharomyces cerevisiae
MNE1 protein superfamily, preferably such protein is having a
YOR350C activity or its homolog, e.g. as shown herein, for the
production of the the respective fine chemical, meaning of
tyrosine, in particular for increasing the amount of tyrosine,
preferably of tyrosine in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a YOR350C protein is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof. The sequence of YPR138C from Saccharomyces
cerevisiae has been published in Goffeau, Science 274 (5287),
546-547 (1996) and Bussey, Nature 387 (6632 Suppl), 103-105 (1997)
and its activity is being defined as a subunit of the NH4+
transporter, preferably of the ammonium transport protein and/or
ammonium transporter nrgA superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the ammonium transport
protein and/or ammonium transporter nrgA superfamily, preferably
such protein is having a sNH4+ transporter activity or its homolog,
e.g. as shown herein, for the production of the the respective fine
chemical, meaning of phenylalanine, in particular for increasing
the amount of phenylalanine, preferably of phenylalanine in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a NH4+ transporter is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof. The sequence of
YEL046C from Saccharomyces cerevisiae has been published in
Goffeau, Science 274 (5287), 546-547 (1996) and Dietrich, Nature
387 (6632 Suppl), 78-81 (1997) and its activity is being defined as
a low specificity L-threonine aldolase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the low specificity
L-threonine aldolase or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of homoserine,
in particular for increasing the amount of homoserine, preferably
of homoserine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a low specificity L-threonine
aldolase is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof.
[6102] The sequence of b0057 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has not been characterized yet. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of the b0057 protein from E.
coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of citrulline, glycine and/or
serine, in particular for increasing the amount of citrulline, in
particular for increasing the amount of glycine, in particular for
increasing the amount of serine, in particular for increasing the
amount of citrulline and glycine, in particular for increasing the
amount of citrulline and serine, in particular for increasing the
amount of glycine and serine, in particular for increasing the
amount of citrulline and glycine and serine, preferably of
citrulline, glycine and/or serine in free or bound form in an
organism or a part thereof, as mentioned.
[6103] The sequence of b0161 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a periplasmic serine protease
(heat shock protein), preferably of the Helicobacter serine
proteinase superfamily. Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of the periplasmic serine protease (heat shock protein)
from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of
5-oxoproline, aspartic acid and/or phenylalanine, in particular for
increasing the amount of 5-oxoproline, in particular for increasing
the amount of aspartic acid, in particular for increasing the
amount of phenylalanine, in particular for increasing the amount of
5-oxoproline and aspartic acid, in particular for increasing the
amount of 5-oxoproline and phenylalanine, in particular for
increasing the amount of aspartic acid and phenylalanine, in
particular for increasing the amount of 5-oxoproline and aspartic
acid and phenylalanine, preferably of 5-oxoproline, aspartic acid
and/or phenylalanine in free or bound form in an organism or a part
thereof, as mentioned.
[6104] The sequence of b0236 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a Peptide chain release factor
homolog, preferably of the translation releasing factor
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a Peptide chain release factor homolog from E. coli or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of alanine, in particular for
increasing the amount of alanine, preferably alanine in free or
bound form in an organism or a part thereof, as mentioned.
[6105] The sequence of 0376 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a beta-lactamase/D-ala
carboxypeptidase, penicillin-binding protein, preferably of the
Escherichia coli beta-lactamase superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of a beta-lactamase/D-ala
carboxypeptidase, penicillin-binding protein from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of 5-oxoproline, in particular for
increasing the amount of 5-oxoproline, preferably 5-oxoproline in
free or bound form in an organism or a part thereof, as
mentioned.
[6106] The sequence of b0462 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a acridine efflux pump
protein, preferably of the acriflavin resistance protein
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a acridine efflux pump protein from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of citrulline, in particular for increasing
the amount of citrulline, preferably citrulline in free or bound
form in an organism or a part thereof, as mentioned.
[6107] The sequence of b0486 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a putative amino-acid/amine
transport protein (APC family), preferably of the probable membrane
protein ybaT superfamily. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of the putative amino-acid/amine transport
protein (APC family) from E. coli or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of alanine and/or serine, in particular for increasing the amount
of alanine, in particular for increasing the amount of serine, in
particular for increasing the amount of alanine and serine,
preferably of alanine and/or serine in free or bound form in an
organism or a part thereof, as mentioned.
[6108] The sequence of b0577 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a putative transport protein.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the putative transport protein from E. coli or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of glycine and/or aspartic acid, in particular for
increasing the amount of glycine, in particular for increasing the
amount of aspartic acid, in particular for increasing the amount of
glycine and aspartic acid, preferably of glycine and/or aspartic
acid in free or bound form in an organism or a part thereof, as
mentioned.
[6109] The sequence of b0970 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a probable glutamate receptor,
preferably of the Escherichia coli ybhL protein superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the probable glutamate receptor from E. coli or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, meaning of 5-oxoproline and/or tyrosine, in particular
for increasing the amount of 5-oxoproline, in particular for
increasing the amount of tyrosine, in particular for increasing the
amount of 5-oxoproline and tyrosine, preferably of 5-oxoproline
and/or tyrosine in free or bound form in an organism or a part
thereof, as mentioned.
[6110] The sequence of b1228 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity has not been characterized yet. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of the b1228 protein from E.
coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of phenylalanine, in
particular for increasing the amount of phenylalanine, preferably
phenylalanine in free or bound form in an organism or a part
thereof, as mentioned.
[6111] The sequence of b1275 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a transcriptional regulator of
biosynthesis of L-cysteine and regulator of sulfur assimilation
(LysR familiy), preferably of the regulatory protein lysR
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a transcriptional regulator of biosynthesis of
L-cysteine and regulator of sulfur assimilation (LysR familiy) from
E. coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of citrulline, in particular
for increasing the amount of citrulline, preferably citrulline in
free or bound form in an organism or a part thereof, as
mentioned.
[6112] The sequence of b1343 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a ATP-dependent RNA helicase,
stimulated by 23S rRNA. Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of the ATP-dependent RNA helicase, stimulated by 23S
rRNA from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of alanine,
5-oxoproline and/or aspartic acid, in particular for increasing the
amount of alanine, in particular for increasing the amount of
5-oxoproline, in particular for increasing the amount of aspartic
acid, in particular for increasing the amount of alanine and
5-oxoproline, in particular for increasing the amount of alanine
and aspartic acid, in particular for increasing the amount of
5-oxoproline and aspartic acid, in particular for increasing the
amount of alanine and 5-oxoproline and aspartic acid, preferably of
alanine, 5-oxoproline and/or aspartic acid in free or bound form in
an organism or a part thereof, as mentioned.
[6113] The sequence of b1360 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a putative DNA replication
protein, preferably of the DNA replication protein dnaC
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a putative DNA replication protein from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of citrulline, in particular for increasing
the amount of citrulline, preferably citrulline in free or bound
form in an organism or a part thereof, as mentioned.
[6114] The sequence of b1863 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a Crossover junction
endodeoxyribonuclease, preferably of the DNA repair protein ruvC
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a Crossover junction endodeoxyribonuclease from E. coli
or its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of alanine, in particular for
increasing the amount of alanine, preferably alanine in free or
bound form in an organism or a part thereof, as mentioned.
[6115] The sequence of b2023 from Escherichia coli K12 has been
published in Blattner et al.,
[6116] Science 277 (5331), 1453-1474, 1997, and its activity is
being defined as a glutamine amidotransferase subunit of imidazole
glycerol phosphate synthase heterodimer, preferably of the
amidotransferase hisH, trpG homology superfamily. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of a glutamine
amidotransferase subunit of imidazole glycerol phosphate synthase
heterodimer from E. coli or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, meaning of aspartic
acid, in particular for increasing the amount of aspartic acid,
preferably aspartic acid in free or bound form in an organism or a
part thereof, as mentioned.
[6117] The sequence of b2078 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a sensory histidine kinase in
two-component regulatory system, preferably of the sensor histidine
kinase homology, envZ protein superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of a sensory histidine kinase in
two-component regulatory system from E. coli or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, meaning of phenylalanine, in particular for increasing
the amount of phenylalanine, preferably phenylalanine in free or
bound form in an organism or a part thereof, as mentioned.
[6118] The sequence of b2239 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a glycerophosphodiester
phosphodiesterase. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of a glycerophosphodiester phosphodiesterase from E. coli
or its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of citrulline, in particular for
increasing the amount of citrulline, preferably citrulline in free
or bound form in an organism or a part thereof, as mentioned.
[6119] The sequence of b2414 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a subunit of cysteine synthase
A and O-acetylserine sulfhydrolase A, PLP-dependent enzyme,
preferably of the threonine dehydratase superfamily. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product with an activity of the subunit of
cysteine synthase A and O-acetylserine sulfhydrolase A,
PLP-dependent enzyme from E. coli or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of citrulline, glycine, serine and/or phenylalanine, in particular
for increasing the amount of citrulline, in particular for
increasing the amount of glycine, in particular for increasing the
amount of serine, in particular for increasing the amount of
phenylalanine, in particular for increasing the amount of
citrulline and glycine, in particular for increasing the amount of
citrulline and serine, in particular for increasing the amount of
citrulline and phenylalanine, in particular for increasing the
amount of glycine and serine, in particular for increasing the
amount of glycine and phenylalanine, in particular for increasing
the amount of serine and phenylalanine, in particular for
increasing the amount of citrulline and glycine and serine, in
particular for increasing the amount of citrulline and glycine and
phenylalanine, in particular for increasing the amount of
citrulline and serine and phenylalanine, in particular for
increasing the amount of citrulline and glycine and phenylalanine,
in particular for increasing the amount of citrulline and glycine
and serine and phen, preferably of citrulline, glycine, serine
and/or phenylalanine in free or bound form in an organism or a part
thereof, as mentioned.
[6120] The sequence of b2426 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a putative oxidoreductase with
NAD(P)-binding domain, preferably of the ribitol dehydrogenase,
short-chain alcohol dehydrogenase homology superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
putative oxidoreductase with NAD(P)-binding domain from E. coli or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of citrulline, in particular for
increasing the amount of citrulline, preferably citrulline in free
or bound form in an organism or a part thereof, as mentioned.
[6121] The sequence of b2489 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a hydrogenase Fe--S subunit,
preferably of the psbG protein superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the hydrogenase Fe--S subunit
from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of citrulline,
alanine, glycine and/or serine, in particular for increasing the
amount of citrulline, in particular for increasing the amount of
alanine, in particular for increasing the amount of glycine, in
particular for increasing the amount of serine, in particular for
increasing the amount of citrulline and alanine, in particular for
increasing the amount of citrulline and glycine, in particular for
increasing the amount of citrulline and serine, in particular for
increasing the amount of alanine and glycine, in particular for
increasing the amount of alanine and serine, in particular for
increasing the amount of glycine and serine, in particular for
increasing the amount of citrulline and alanine and glycine, in
particular for increasing the amount of citrulline and alanine and
serine, in particular for increasing the amount of citrulline and
glycine and serine, in particular for increasing the amount of
alanine and glycine and serine, in particular for increasing the
amount of citrulline and alanine and glycine and serine, preferably
of citrulline, alanine, glycine and/or serine in free or bound form
in an organism or a part thereof, as mentioned.
[6122] The sequence of b2491 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a transcriptional activator
for expression of hydrogenase 4 genes, interacts with sigma 54 (EBP
family). Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
transcriptional activator for expression of hydrogenase 4 genes,
interacts with sigma 54 (EBP family) from E. coli or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, meaning of tyrosine, in particular for increasing the
amount of tyrosine, preferably tyrosine in free or bound form in an
organism or a part thereof, as mentioned.
[6123] The sequence of b2507 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a GMP synthetase (glutamine
aminotransferase), preferably of the GMP synthase
(glutamine-hydrolyzing), trpG homology superfamily. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of a GMP synthetase
(glutamine aminotransferase) from E. coli or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of aspartic acid, in particular for increasing the amount
of aspartic acid, preferably aspartic acid in free or bound form in
an organism or a part thereof, as mentioned.
[6124] The sequence of b2553 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a regulatory protein P-II for
glutamine synthetase, preferably of the regulatory protein P-II
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a regulatory protein P-II for glutamine synthetase from
E. coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of glycine, in particular for
increasing the amount of glycine, preferably glycine in free or
bound form in an organism or a part thereof, as mentioned.
[6125] The sequence of b2576 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a ATP-dependent RNA helicase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the ATP-dependent RNA helicase from E. coli or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of alanine, glycine, aspartic acid and/or phenylalanine, in
particular for increasing the amount of alanine, in particular for
increasing the amount of glycine, in particular for increasing the
amount of aspartic acid, in particular for increasing the amount of
phenylalanine, in particular for increasing the amount of alanine
and glycine, in particular for increasing the amount of alanine and
aspartic acid, in particular for increasing the amount of alanine
and phenylalanine, in particular for increasing the amount of
glycine and aspartic acid, in particular for increasing the amount
of glycine and phenylalanine, in particular for increasing the
amount of aspartic acid and phenylalanine, in particular for
increasing the amount of alanine and glycine and aspartic acid, in
particular for increasing the amount of alanine and glycine and
phenylalanine, in particular for increasing the amount of alanine
and aspartic acid and phenylalanine, in particular for increasing
the amount of glycine and aspartic acid and phenylalanine, in
particular for increasing the amount of alanine and glycine and
aspartic acid and phenylalanine, preferably of alanine, glycine,
aspartic acid and/or phenylalanine in free or bound form in an
organism or a part thereof, as mentioned.
[6126] The sequence of b2664 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a putative transcriptional
repressor with DNA-binding Winged helix domain (GntR familiy),
preferably of the probable transcription regulator gabP
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a putative transcriptional repressor with DNA-binding
Winged helix domain (GntR familiy) from E. coli or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, meaning of serine, in particular for increasing the
amount of serine, preferably serine in free or bound form in an
organism or a part thereof, as mentioned.
[6127] The sequence of b2753 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a aminopeptidase in alkaline
phosphatase isozyme conversion, preferably of the alkaline
phosphatase isozyme conversion protein superfamily. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of a aminopeptidase in
alkaline phosphatase isozyme conversion from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of aspartic acid, in particular for
increasing the amount of aspartic acid, preferably aspartic acid in
free or bound form in an organism or a part thereof, as
mentioned.
[6128] The sequence of b2796 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a putative serine transport
protein (HAAAP family), preferably of the threonine-serine permease
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a putative serine transport protein (HAAAP family) from
E. coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of phenylalanine, in
particular for increasing the amount of phenylalanine, preferably
phenylalanine in free or bound form in an organism or a part
thereof, as mentioned.
[6129] The sequence of b3064 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a putative 0-sialoglycoprotein
endopeptidase, with actin-like ATPase domain, preferably of the
0-sialoglycoprotein endopeptidase superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of a putative
O-sialoglycoprotein endopeptidase, with actin-like ATPase domain
from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of serine, in
particular for increasing the amount of serine, preferably serine
in free or bound form in an organism or a part thereof, as
mentioned.
[6130] The sequence of b3116 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a L-threonine/L-serine
permease, anaerobically inducible (HAAAP family), preferably of the
threonine-serine permease superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the L-threonine/L-serine
permease, anaerobically inducible (HAAAP family) from E. coli or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of serine and/or aspartic acid,
in particular for increasing the amount of serine, in particular
for increasing the amount of aspartic acid, in particular for
increasing the amount of serine and aspartic acid, preferably of
serine and/or aspartic acid in free or bound form in an organism or
a part thereof, as mentioned.
[6131] The sequence of b3160 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a putative monooxygenase with
luciferase-like ATPase activity, preferably of the ynbW protein
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the putative monooxygenase with luciferase-like ATPase
activity from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of citrulline
and/or serine, in particular for increasing the amount of
citrulline, in particular for increasing the amount of serine, in
particular for increasing the amount of citrulline and serine,
preferably of citrulline and/or serine in free or bound form in an
organism or a part thereof, as mentioned.
[6132] The sequence of b3169 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a transcription
termination-antitermination factor, preferably of the Escherichia
coli transcription factor nusA superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of a transcription
termination-antitermination factor from E. coli or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, meaning of aspartic acid, in particular for increasing
the amount of aspartic acid, preferably aspartic acid in free or
bound form in an organism or a part thereof, as mentioned.
[6133] The sequence of b3172 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a argininosuccinate
synthetase, preferably of the argininosuccinate synthase
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the argininosuccinate synthetase from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of 5-oxoproline and/or aspartic acid, in
particular for increasing the amount of 5-oxoproline, in particular
for increasing the amount of aspartic acid, in particular for
increasing the amount of 5-oxoproline and aspartic acid, preferably
of 5-oxoproline and/or aspartic acid in free or bound form in an
organism or a part thereof, as mentioned.
[6134] The sequence of b3231 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a 50S ribosomal subunit
protein L13, preferably of the Escherichia coli ribosomal protein
L13 superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the 50S ribosomal subunit protein L13 from E. coli or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of alanine and/or serine, in
particular for increasing the amount of alanine, in particular for
increasing the amount of serine, in particular for increasing the
amount of alanine and serine, preferably of alanine and/or serine
in free or bound form in an organism or a part thereof, as
mentioned.
[6135] The sequence of b3241 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a putative membrane located
multidrug resistance protein. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of a putative membrane located multidrug
resistance protein from E. coli or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of citrulline, in particular for increasing the amount of
citrulline, preferably citrulline in free or bound form in an
organism or a part thereof, as mentioned.
[6136] The sequence of b3767 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a Acetolactate synthase
isozyme II large subunit. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of the Acetolactate synthase isozyme II
large subunit from E. coli or its homolog, e.g. as shown herein,
for the production of the respective fine chemical, meaning of
alanine and/or homoserine, in particular for increasing the amount
of alanine, in particular for increasing the amount of homoserine,
in particular for increasing the amount of alanine and homoserine,
preferably of alanine and/or homoserine in free or bound form in an
organism or a part thereof, as mentioned.
[6137] The sequence of b3919 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a triosephosphate isomerase,
preferably of the triose-phosphate isomerase superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
triosephosphate isomerase from E. coli or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of phenylalanine, in particular for increasing the amount
of phenylalanine, preferably phenylalanine in free or bound form in
an organism or a part thereof, as mentioned.
[6138] The sequence of b3926 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a glycerol kinase, preferably
of the xylulokinase superfamily. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of a glycerol kinase from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of citrulline, in particular for increasing
the amount of citrulline, preferably citrulline in free or bound
form in an organism or a part thereof, as mentioned.
[6139] The sequence of b3938 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a transcriptional repressor
for methionine biosynthesis, preferably of the metJ protein
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a transcriptional repressor for methionine biosynthesis
from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of
phenylalanine, in particular for increasing the amount of
phenylalanine, preferably phenylalanine in free or bound form in an
organism or a part thereof, as mentioned.
[6140] The sequence of b3983 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a 50S ribosomal subunit
protein L12, preferably of the Escherichia coli ribosomal protein
L11 superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the 50S ribosomal subunit protein L12 from E. coli or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of phenylalanine and/or tyrosine,
in particular for increasing the amount of phenylalanine, in
particular for increasing the amount of tyrosine, in particular for
increasing the amount of phenylalanine and tyrosine, preferably of
phenylalanine and/or tyrosine in free or bound form in an organism
or a part thereof, as mentioned.
[6141] The sequence of b4129 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a lysine tRNA synthetase,
inducible, preferably of the lysine-tRNA ligase superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
lysine tRNA synthetase, inducible from E. coli or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, meaning of aspartic acid, in particular for increasing
the amount of aspartic acid, preferably aspartic acid in free or
bound form in an organism or a part thereof, as mentioned.
[6142] The sequence of b4214 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a ammonium transport system
structural protein, preferably of the Aquifex aeolicus cysQ protein
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a ammonium transport system structural protein from E.
coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of citrulline, in particular
for increasing the amount of citrulline, preferably citrulline in
free or bound form in an organism or a part thereof, as
mentioned.
[6143] The sequence of b4269 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a putative alcohol
dehydrogenase with NAD(P)-binding and GroES domains. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product with an activity of a putative alcohol
dehydrogenase with NAD(P)-binding and GroES domains from E. coli or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of citrulline, in particular for
increasing the amount of citrulline, preferably citrulline in free
or bound form in an organism or a part thereof, as mentioned.
[6144] The sequence of b4346 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a component of
5-methylcytosine-specific restriction enzyme McrBC, preferably of
the 5-methylcytosine-specific restriction enzyme B superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
component of 5-methylcytosine-specific restriction enzyme McrBC
from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of aspartic
acid, in particular for increasing the amount of aspartic acid,
preferably aspartic acid in free or bound form in an organism or a
part thereof, as mentioned.
[6145] [0023.0.14.14] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content. In one
embodiment, the homolog of the any one of the polypeptides
indicated in Table II, column 3, line 126 is a homolog having the
same or a similar activity. In particular an increase of activity
confers an increase in the content of the respective fine chemical
in the organisms, preferably of 5-oxoproline. In one embodiment,
the homolog is a homolog with a sequence as indicated in Table I or
II, column 7, line 126. In one embodiment, the homolog of one of
the polypeptides indicated in Table II, column 3, line 126, is
derived from an eukaryotic. In one embodiment, the homolog is
derived from Fungi. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 126, is derived from
Ascomyceta. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 126, is derived from
Saccharomycotina. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 126, is derived from
Saccharomycetes. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 126, is a homolog being
derived from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 126, is a homolog
having the same or a similar activity being derived from
Saccharomycetaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 126, is a homolog having the
same or a similar activity being derived from Saccharomycetes.
[6146] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 128, 129, 130
or 131 resp., is a homolog having the same or a similar activity.
In particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of alanine. In one embodiment, the homolog is a homolog
with a sequence as indicated in Table I or II, column 7, lines 128,
129, 130 or 131 resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table II, column 3, lines 128, 129, 130
or 131 resp., is derived from an eukaryotic. In one embodiment, the
homolog is derived from Fungi. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 128, 129, 130 or
131 resp., is derived from Ascomyceta. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines
128, 129, 130 or 131 resp., is derived from Saccharomycotina. In
one embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 128, 129, 130 or 131 resp., is derived from
Saccharomycetes. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 128, 129, 130 or 131 resp.,
is a homolog being derived from Saccharomycetales. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 128, 129, 130 or 131 resp., is a homolog having the
same or a similar activity being derived from Saccharomycetaceae.
In one embodiment, the homolog of a polypeptide indicated in Table
II, column 3, lines 128, 129, 130 or 131 resp., is a homolog having
the same or a similar activity being derived from
Saccharomycetes.
[6147] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 134, 135 or 136
resp., is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of aspartic acid. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
lines 134, 135 or 136 resp. In one embodiment, the homolog of one
of the polypeptides indicated in Table II, column 3, lines 134, 135
or 136 resp., is derived from an eukaryotic. In one embodiment, the
homolog is derived from Fungi. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 134, 135 or 136
resp., is derived from Ascomyceta. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 134, 135 or
136 resp., is derived from Saccharomycotina. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines
134, 135 or 136 resp., is derived from Saccharomycetes. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 134, 135 or 136 resp., is a homolog being derived
from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 134, 135 or 136
resp., is a homolog having the same or a similar activity being
derived from Saccharomycetaceae. In one embodiment, the homolog of
a polypeptide indicated in Table II, column 3, lines 134, 135 or
136 resp., is a homolog having the same or a similar activity being
derived from Saccharomycetes.
[6148] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 139 or 140
resp., is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of citrulline. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
lines 139 or 140 resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table II, column 3, lines 139 or 140
resp., is derived from an eukaryotic. In one embodiment, the
homolog is derived from Fungi. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 139 or 140
resp., is derived from Ascomyceta. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 139 or 140
resp., is derived from Saccharomycotina. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 139
or 140 resp., is derived from Saccharomycetes. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
139 or 140 resp., is a homolog being derived from
Saccharomycetales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 139 or 140 resp., is a
homolog having the same or a similar activity being derived from
Saccharomycetaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 139 or 140 resp., is a
homolog having the same or a similar activity being derived from
Saccharomycetes.
[6149] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 141 or 142
resp., is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of glycine. In one embodiment, the homolog is a homolog
with a sequence as indicated in Table I or II, column 7, lines 141
or 142 resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table II, column 3, lines 141 or 142
resp., is derived from an eukaryotic. In one embodiment, the
homolog is derived from Fungi. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 141 or 142
resp., is derived from Ascomyceta. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 141 or 142
resp., is derived from Saccharomycotina. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 141
or 142 resp., is derived from Saccharomycetes. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
141 or 142 resp., is a homolog being derived from
Saccharomycetales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 141 or 142 resp., is a
homolog having the same or a similar activity being derived from
Saccharomycetaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 141 or 142 resp., is a
homolog having the same or a similar activity being derived from
Saccharomycetes.
[6150] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 143, is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms, preferably of
homoserine. In one embodiment, the homolog is a homolog with a
sequence as indicated in Table I or II, column 7, line 143. In one
embodiment, the homolog of one of the polypeptides indicated in
Table II, column 3, line 143, is derived from an eukaryotic. In one
embodiment, the homolog is derived from Fungi. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, line
143, is derived from Ascomyceta. In one embodiment, the homolog of
a polypeptide indicated in Table II, column 3, line 143, is derived
from Saccharomycotina. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 143, is derived
from Saccharomycetes. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 143, is a homolog
being derived from Saccharomycetales. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 143,
is a homolog having the same or a similar activity being derived
from Saccharomycetaceae. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, line 143, is a homolog
having the same or a similar activity being derived from
Saccharomycetes.
[6151] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 144, 145, 146,
147, 148 or 149 resp., is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of the respective fine chemical in the organisms,
preferably of phenylalanine. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
lines 144, 145, 146, 147, 148 or 149 resp. In one embodiment, the
homolog of one of the polypeptides indicated in Table II, column 3,
lines 144, 145, 146, 147, 148 or 149 resp., is derived from an
eukaryotic. In one embodiment, the homolog is derived from Fungi.
In one embodiment, the homolog of a polypeptide indicated in Table
II, column 3, lines 144, 145, 146, 147, 148 or 149 resp., is
derived from Ascomyceta. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 144, 145, 146,
147, 148 or 149 resp., is derived from Saccharomycotina. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 144, 145, 146, 147, 148 or 149 resp., is derived
from Saccharomycetes. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 144, 145, 146,
147, 148 or 149 resp., is a homolog being derived from
Saccharomycetales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 144, 145, 146, 147, 148 or
149 resp., is a homolog having the same or a similar activity being
derived from Saccharomycetaceae. In one embodiment, the homolog of
a polypeptide indicated in Table II, column 3, lines 144, 145, 146,
147, 148 or 149 resp., is a homolog having the same or a similar
activity being derived from Saccharomycetes.
[6152] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 157, 158, 159,
160, 161, 162 or 163 resp., is a homolog having the same or a
similar activity. In particular an increase of activity confers an
increase in the content of the respective fine chemical in the
organisms, preferably of serine. In one embodiment, the homolog is
a homolog with a sequence as indicated in Table I or II, column 7,
lines 157, 158, 159, 160, 161, 162 or 163 resp. In one embodiment,
the homolog of one of the polypeptides indicated in Table II,
column 3, lines 157, 158, 159, 160, 161, 162 or 163 resp., is
derived from an eukaryotic. In one embodiment, the homolog is
derived from Fungi. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 157, 158, 159, 160, 161, 162
or 163 resp., is derived from Ascomyceta. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines
157, 158, 159, 160, 161, 162 or 163 resp., is derived from
Saccharomycotina. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 157, 158, 159, 160, 161, 162
or 163 resp., is derived from Saccharomycetes. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
157, 158, 159, 160, 161, 162 or 163 resp., is a homolog being
derived from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 157, 158, 159,
160, 161, 162 or 163 resp., is a homolog having the same or a
similar activity being derived from Saccharomycetaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 157, 158, 159, 160, 161, 162 or 163 resp., is a
homolog having the same or a similar activity being derived from
Saccharomycetes.
[6153] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 167, 168, 169
or 170 resp., is a homolog having the same or a similar activity.
In particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of tyrosine. In one embodiment, the homolog is a homolog
with a sequence as indicated in Table I or II, column 7, lines 167,
168, 169 or 170 resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table II, column 3, lines 167, 168, 169
or 170 resp., is derived from an eukaryotic. In one embodiment, the
homolog is derived from Fungi. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 167, 168, 169 or
170 resp., is derived from Ascomyceta. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines
167, 168, 169 or 170 resp., is derived from Saccharomycotina. In
one embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 167, 168, 169 or 170 resp., is derived from
Saccharomycetes. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 167, 168, 169 or 170 resp.,
is a homolog being derived from Saccharomycetales. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 167, 168, 169 or 170 resp., is a homolog having the
same or a similar activity being derived from Saccharomycetaceae.
In one embodiment, the homolog of a polypeptide indicated in Table
II, column 3, lines 167, 168, 169 or 170 resp., is a homolog having
the same or a similar activity being derived from
Saccharomycetes.
[6154] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 127 and/or 492
to 496 resp. is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms preferably
of 5-oxoproline. In one embodiment, the homolog is a homolog with a
sequence as indicated in Table I or II, column 7, lines 127 and/or
492 to 496 resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table II, column 3, line lines 127 and/or
492 to 496 resp. is derived from an bacteria. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
127 and/or 492 to 496 resp. is derived from Proteobacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 127 and/or 492 to 496 resp. is a homolog having the
same or a similar activity being derived from Gammaproteobacteria.
In one embodiment, the homolog of a polypeptide indicated in Table
II, column 3, lines 127 and/or 492 to 496 resp. is derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line lines 127 and/or 492 to 496
resp. is a homolog being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line lines 127 and/or 492 to 496 resp. is a homolog
having the same or a similar activity and being derived from
Escherichia.
[6155] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 132 to 133
and/or 497 to 504 resp. is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of the respective fine chemical in the organisms,
preferably of alanine. In one embodiment, the homolog is a homolog
with a sequence as indicated in Table I or II, column 7, lines 132
to 133 and/or 497 to 504 resp. In one embodiment, the homolog of
one of the polypeptides indicated in Table II, column 3, lines 132
to 133 and/or 497 to 504 resp. is derived from an bacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 132 to 133 and/or 497 to 504 resp. is derived from
Proteobacteria. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 132 to 133 and/or 497 to 504
resp. is a homolog having the same or a similar activity being
derived from Gammaproteobacteria. In one embodiment, the homolog of
a polypeptide indicated in Table II, column 3, lines 132 to 133
and/or 497 to 504 resp. is derived from Enterobacteriales. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 132 to 133 and/or 497 to 504 resp. is a homolog
being derived from Enterobacteriaceae. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 132
to 133 and/or 497 to 504 resp. is a homolog having the same or a
similar activity and being derived from Escherichia.
[6156] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 137 to 138
and/or 505 to 516 resp. is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of the respective fine chemical in the organisms,
preferably of aspartic acid. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
lines 137 to 138 and/or 505 to 516 resp. In one embodiment, the
homolog of one of the polypeptides indicated in Table II, column 3,
lines 137 to 138 and/or 505 to 516 resp. is derived from an
bacteria. In one embodiment, the homolog of a polypeptide indicated
in Table II, column 3, lines 137 to 138 and/or 505 to 516 resp. is
derived from Proteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 137 to 138
and/or 505 to 516 resp. is a homolog having the same or a similar
activity being derived from Gammaproteobacteria. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
137 to 138 and/or 505 to 516 resp. is derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 137 to 138 and/or 505 to 516
resp. is a homolog being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 137 to 138 and/or 505 to 516 resp. is a homolog
having the same or a similar activity and being derived from
Escherichia.
[6157] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 517 to 529
resp. is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of citrulline. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
lines 517 to 529 resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table II, column 3, lines 517 to 529
resp. is derived from an bacteria. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 517 to 529
resp. is derived from Proteobacteria. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 517
to 529 resp. is a homolog having the same or a similar activity
being derived from Gammaproteobacteria. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 517
to 529 resp. is derived from Enterobacteriales. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
517 to 529 resp. is a homolog being derived from
Enterobacteriaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 517 to 529 resp. is a
homolog having the same or a similar activity and being derived
from Escherichia.
[6158] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 530 to 535
resp. is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of glycine. In one embodiment, the homolog is a homolog
with a sequence as indicated in Table I or II, column 7, lines 530
to 535 resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table II, column 3, lines 530 to 535
resp. is derived from an bacteria. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 530 to 535
resp. is derived from Proteobacteria. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 530
to 535 resp. is a homolog having the same or a similar activity
being derived from Gammaproteobacteria. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 530
to 535 resp. is derived from Enterobacteriales. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
530 to 535 resp. is a homolog being derived from
Enterobacteriaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 530 to 535 resp. is a
homolog having the same or a similar activity and being derived
from Escherichia.
[6159] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 536 resp. is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms, preferably of
homoserine. In one embodiment, the homolog is a homolog with a
sequence as indicated in Table I or II, column 7, line 536 resp. In
one embodiment, the homolog of one of the polypeptides indicated in
Table II, column 3, line 536 resp. is derived from an bacteria. In
one embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 536 resp. is derived from Proteobacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 536 resp. is a homolog having the same or a similar
activity being derived from Gammaproteobacteria. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, line
536 resp. is derived from Enterobacteriales. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 536
resp. is a homolog being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 536 resp. is a homolog having the same or a similar
activity and being derived from Escherichia.
[6160] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 150 to 156
and/or 537 to 545 resp. is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of the respective fine chemical in the organisms,
preferably of phenylalanine. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
lines 150 to 156 and/or 537 to 545 resp. In one embodiment, the
homolog of one of the polypeptides indicated in Table II, column 3,
lines 150 to 156 and/or 537 to 545 resp. is derived from an
bacteria. In one embodiment, the homolog of a polypeptide indicated
in Table II, column 3, lines 150 to 156 and/or 537 to 545 resp. is
derived from Proteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 150 to 156
and/or 537 to 545 resp. is a homolog having the same or a similar
activity being derived from Gammaproteobacteria. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
150 to 156 and/or 537 to 545 resp. is derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 150 to 156 and/or 537 to 545
resp. is a homolog being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 150 to 156 and/or 537 to 545 resp. is a homolog
having the same or a similar activity and being derived from
Escherichia.
[6161] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 164 to 166
and/or 546 to 554 resp. is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of the respective fine chemical in the organisms,
preferably of serine. In one embodiment, the homolog is a homolog
with a sequence as indicated in Table I or II, column 7, lines 164
to 166 and/or 546 to 554 resp. In one embodiment, the homolog of
one of the polypeptides indicated in Table II, column 3, lines 164
to 166 and/or 546 to 554 resp. is derived from an bacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 164 to 166 and/or 546 to 554 resp. is derived from
Proteobacteria. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 164 to 166 and/or 546 to 554
resp., is a homolog having the same or a similar activity being
derived from Gammaproteobacteria. In one embodiment, the homolog of
a polypeptide indicated in Table II, column 3, lines 164 to 166
and/or 546 to 554 resp. is derived from Enterobacteriales. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 164 to 166 and/or 546 to 554 resp. is a homolog
being derived from Enterobacteriaceae. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 164
to 166 and/or 546 to 554 resp. is a homolog having the same or a
similar activity and being derived from Escherichia.
[6162] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 171 to 172
and/or 555 to 557 resp. is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of the respective fine chemical in the organisms,
preferably of tyrosine. In one embodiment, the homolog is a homolog
with a sequence as indicated in Table I or II, column 7, lines 171
to 172 and/or 555 to 557 resp. In one embodiment, the homolog of
one of the polypeptides indicated in Table II, column 3, lines 171
to 172 and/or 555 to 557 resp. is derived from an bacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 171 to 172 and/or 555 to 557 resp. is derived from
Proteobacteria. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 171 to 172 and/or 555 to 557
resp., is a homolog having the same or a similar activity being
derived from Gammaproteobacteria. In one embodiment, the homolog of
a polypeptide indicated in Table II, column 3, lines 171 to 172
and/or 555 to 557 resp. is derived from Enterobacteriales. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 171 to 172 and/or 555 to 557 resp. is a homolog
being derived from Enterobacteriaceae. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, lines 171
to 172 and/or 555 to 557 resp. is a homolog having the same or a
similar activity and being derived from Escherichia.
[6163] [0023.1.14.14] Homologs of the polypeptide indicated in
Table II, column 3, lines 126 to 172 and/or lines 492 to 557,
resp., may be the polypeptides encoded by the nucleic acid
molecules indicated in Table I, column 7, lines 126 to 172 and/or
lines 492 to 557, resp., or may be the polypeptides indicated in
Table II, column 7, lines 126 to 172 and/or lines 492 to 557, resp.
Homologs of the polypeptides polypeptide indicated in Table II,
column 3, lines 126 to 172 and/or lines 492 to 557, resp., may be
the polypeptides encoded by the nucleic acid molecules indicated in
Table I, column 7, lines 126 to 172 and/or lines 492 to 557, resp.,
or may be the polypeptides indicated in Table II, column 7, lines
126 to 172 and/or lines 492 to 557, resp.
[6164] Homologs of the polypeptides indicated in Table II, column
3, lines 126 to 127 and/or 492 to 496 resp. may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 126 to 127 and/or 492 to 496 respectively or may be the
polypeptides indicated in Table II, column 7, lines 126 to 127
and/or 492 to 496 resp., having a 5-oxoproline content- and/or
amount-increasing activity.
[6165] Homologs of the polypeptides indicated in Table II, column
3, lines 128 to 133 and/or 497 to 504 resp. may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 128 to 133 and/or 497 to 504 respectively or may be the
polypeptides indicated in Table II, column 7, lines 128 to 133
and/or 497 to 504 resp., having a alanine content- and/or
amount-increasing activity.
[6166] Homologs of the polypeptides indicated in Table II, column
3, lines 134 to 138 and/or 505 to 516 resp. may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 134 to 138 and/or 505 to 516 respectively or may be the
polypeptides indicated in Table II, column 7, lines 134 to 138
and/or 505 to 516 resp., having a aspartic acid content- and/or
amount-increasing activity.
[6167] Homologs of the polypeptides indicated in Table II, column
3, lines 139 to 140 and/or 517 to 529 resp. may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 139 to 140 and/or 517 to 529 respectively or may be the
polypeptides indicated in Table II, column 7, lines 139 to 140
and/or 517 to 529 resp., having a citrulline content- and/or
amount-increasing activity.
[6168] Homologs of the polypeptides indicated in Table II, column
3, lines 141 to 142 and/or 530 to 535 resp. may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 141 to 142 and/or 530 to 535 respectively or may be the
polypeptides indicated in Table II, column 7, lines 141 to 142
and/or 530 to 535 resp., having a glycine content- and/or
amount-increasing activity.
[6169] Homologs of the polypeptides indicated in Table II, column
3, lines 143 and/or 536 resp. may be the polypeptides encoded by
the nucleic acid molecules indicated in Table I, column 7, lines
143 and/or 536 resp. or may be the polypeptides indicated in Table
II, column 7, lines 143 and/or 536 resp., having a homoserine
content- and/or amount-increasing activity.
[6170] Homologs of the polypeptides indicated in Table II, column
3, lines 144 to 156 and/or 537 to 545 resp. may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 144 to 156 and/or 537 to 545 respectively or may be the
polypeptides indicated in Table II, column 7, lines 144 to 156
and/or 537 to 545 resp., having a phenylalanine content- and/or
amount-increasing activity.
[6171] Homologs of the polypeptides indicated in Table II, column
3, lines 157 to 166 and/or 546 to 554 resp. may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 157 to 166 and/or 546 to 554 respectively or may be the
polypeptides indicated in Table II, column 7, lines 157 to 166
and/or 546 to 554 resp. having a serine content- and/or
amount-increasing activity.
[6172] Homologs of the polypeptides indicated in Table II, column
3, lines 167 to 172 and/or 555 to 557 resp. may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 167 to 172 and/or 555 to 557 resp. respectively or may be
the polypeptides indicated in Table II, column 7, lines 167 to 172
and/or 555 to 557 resp., having a tyrosine content- and/or
amount-increasing activity.
[6173] [0024.0.0.14] see [0024.0.0.0]
[6174] [0025.0.14.14] In accordance with the invention, a protein
or polypeptide has the "activity of an protein of the invention",
e.g. the activity of a protein indicated in Table II, column 3,
lines 126 to 127 and/or 492 to 496 for 5-oxoproline and/or lines
128 to 133 and/or 497 to 504 for alanine and/or lines 134 to 138
and/or 505 to 516 for aspartic acid and/or lines 139 to 140 and/or
517 to 529 for citrulline and/or lines 141 to 142 and/or 530 to 535
for glycine and/or lines 143 and/or 536 for homoserine and/or lines
144 to 156 and/or 537 to 545 for phenylalanine and/or lines 157 to
166 and/or 546 to 554 for serine and/or lines 167 to 172 and/or 555
to 557 for tyrosine, resp.,
[6175] if its de novo activity, or its increased expression
directly or indirectly leads to an increased amino acid level, in
particular to a increased 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., in the organism or a part thereof, preferably in a
cell of said organism. In a preferred embodiment, the protein or
polypeptide has the above-mentioned additional activities of a
protein indicated in Table II, column 3, lines 126 to 127 and/or
492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines
134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to
529 and/or lines 141 to 142 and/or 530 to 535 and/or lines 143
and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or lines
157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to
557, resp.
[6176] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of any one of the proteins indicated in Table II, column
3, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., or which has at least
10% of the original enzymatic activity, preferably 20%,
particularly preferably 30%, most particularly preferably 40% in
comparison to any one of the proteins indicated in Table II, column
3, line 126 and/or 128 to 131 and/or 134 to 136 and/or lines 139 to
140 and/or lines 141 to 142 and/or line 143 and/or lines 144 to 149
and/or lines 157 to 163 and/or lines 167 to 170 resp. of
Saccharomyces cerevisiae and/or any one of the proteins indicated
in Table II, column 3, lines 127 and/or 492 to 496 and/or lines 132
to 133 and/or 497 to 504 and/or lines 137 to 138 and/or 505 to 516
and/or lines 517 to 529 and/or lines 530 to 535 and/or line 536
and/or lines 150 to 156 and/or 537 to 545 and/or lines 164 to 166
and/or 546 to 554 and/or lines 171 to 172 and/or 555 to 557 resp.
of E. coli K12.
[6177] [0025.1.0.14] to [0033.0.0.14]: see [0025.1.0.0] to
[0033.0.0.0]
[6178] [0034.0.14.14] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of an protein having the activity of a
protein as indicated in Table II, column 3, lines 126 to 127 and/or
492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines
134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to
529 and/or lines 141 to 142 and/or 530 to 535 and/or lines 143
and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or lines
157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to
557, resp., or being encoded by a nucleic acid molecule indicated
in Table I, column 5, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., or its
homologs, e.g. as indicated in Table I, column 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., its biochemical or genetical causes and
therefore shows the increased amount of the fine chemical.
[6179] [0035.0.0.14] to [0044.0.0.14]: see [0035.0.0.0] to
[0044.0.0.0]
[6180] [0045.0.14.14] In one embodiment, the activity of the
Saccharomyces cerevisiae protein YAL049C or its homologs, e.g. an
activity of a YAL049C protein, e.g. as indicated in Table I,
columns 5 or 7, line 149, is increased conferring an increase of
the respective fine chemical, preferably of the phenylalanine
between 30% and 74% or more.
[6181] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YBL015W or its homologs, e.g. an activity of a
mannose-containing glycoprotein and/or as an acetyl-CoA hydrolase,
preferably of the acetyl-CoA hydrolase superfamily, e.g. as
indicated in Table I, columns 5 or 7, line 131, is increased
conferring an increase of the respective fine chemical, preferably
of the alanine between 29% and 204% or more.
[6182] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YDL127W or its homologs, e.g. an activity of a
G1/S-specific cyclin PCL2 (Cyclin HCS26 homolog), e.g. as indicated
in Table I, columns 5 or 7, line 126, is increased conferring an
increase of the respective fine chemical, preferably of the
5-oxoproline between 40% and 127% or more.
[6183] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YEL045C or its homologs, e.g. an activity of a
YEL045C protein, e.g. as indicated in Table I, columns 5 or 7, line
163, is increased conferring an increase of the respective fine
chemical, preferably of the serine between 23% and 80% or more.
[6184] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YEL046C or its homologs, e.g. an activity of a
low specificity L-threonine aldolase, e.g. as indicated in Table I,
columns 5 or 7, line 143, is increased conferring an increase of
the respective fine chemical, preferably of the homoserine between
44% and 177% or more.
[6185] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER152C or its homologs, e.g. an activity
similar to tyrosine aminotransferase, e.g. as indicated in Table I,
columns 5 or 7, line 162, is increased conferring an increase of
the respective fine chemical, preferably of the serine between 23%
and 75% or more.
[6186] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER173W or its homologs, e.g. an activity of a
checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints, e.g. as indicated in Table I,
columns 5 or 7, line 130, is increased conferring an increase of
the respective fine chemical, preferably of the alanine between 21%
and 132% or more. In one embodiment, the activity of the
Saccharomyces cerevisiae protein YER173W or its homologs, e.g. an
activity of a checkpoint protein, involved in the activation of the
DNA damage and meiotic pachytene checkpoints, e.g. as indicated in
Table I, columns 5 or 7, line 136, is increased conferring an
increase of the respective fine chemical, preferably of the
aspartic acid between 64% and 93% or more. In one embodiment, the
activity of the Saccharomyces cerevisiae protein YER173W or its
homologs, e.g. an activity of a checkpoint protein, involved in the
activation of the DNA damage and meiotic pachytene checkpoints,
e.g. as indicated in Table I, columns 5 or 7, lines 130 and/or 136,
is increased conferring an increase of the respective fine
chemical, preferably of alanine and aspartic acid between 21% and
132%, preferably between 64% and 132% or more. In one embodiment,
the activity of the Saccharomyces cerevisiae protein YER173W or its
homologs, e.g. an activity of a checkpoint protein, involved in the
activation of the DNA damage and meiotic pachytene checkpoints,
e.g. as indicated in Table I, columns 5 or 7, line 148, is
increased conferring an increase of the respective fine chemical,
preferably of the phenylalanine between 44% and 99% or more. In one
embodiment, the activity of the Saccharomyces cerevisiae protein
YER173W or its homologs, e.g. an activity of a checkpoint protein,
involved in the activation of the DNA damage and meiotic pachytene
checkpoints, e.g. as indicated in Table I, columns 5 or 7, lines
130 and/or 148, is increased conferring an increase of the
respective fine chemical, preferably of alanine and phenylalanine
between 21% and 132%, preferably between 44% and 132% or more. In
one embodiment, the activity of the Saccharomyces cerevisiae
protein YER173W or its homologs, e.g. an activity of a checkpoint
protein, involved in the activation of the DNA damage and meiotic
pachytene checkpoints, e.g. as indicated in Table I, columns 5 or
7, lines 136 and/or 148, is increased conferring an increase of the
respective fine chemical, preferably of aspartic acid and
phenylalanine between 44% and 99%, preferably between 64% and 99%
or more. In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER173W or its homologs, e.g. an activity of a
checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints, e.g. as indicated in Table I,
columns 5 or 7, lines 130 and/or 136 and/or 148, is increased
conferring an increase of the respective fine chemical, preferably
of alanine and aspartic acid and phenylalanine between 21% and
132%, preferably 44% and 132%, more preferably between 64% and 132%
or more.
[6187] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YFL050C or its homologs, e.g. an activity of a
di- and/or trivalent inorganic cation transporter, preferably of
the magnesium and cobalt transport protein superfamily, e.g. as
indicated in Table I, columns 5 or 7, line 129, is increased
conferring an increase of the respective fine chemical, preferably
of the alanine between 19% and 104% or more. In one embodiment, the
activity of the Saccharomyces cerevisiae protein YFL050C or its
homologs, e.g. an activity of a di- and/or trivalent inorganic
cation transporter, preferably of the magnesium and cobalt
transport protein superfamily, e.g. as indicated in Table I,
columns 5 or 7, line 142, is increased conferring an increase of
the respective fine chemical, preferably of the glycine between 36%
and 74% or more. In one embodiment, the activity of the
Saccharomyces cerevisiae protein YFL050C or its homologs, e.g. an
activity of a di- and/or trivalent inorganic cation transporter,
preferably of the magnesium and cobalt transport protein
superfamily, e.g. as indicated in Table I, columns 5 or 7, lines
129 and/or 142, is increased conferring an increase of the
respective fine chemical, preferably of alanine and glycine between
19% and 104%, preferably between 36% and 104% or more. In one
embodiment, the activity of the Saccharomyces cerevisiae protein
YFL050C or its homologs, e.g. an activity of a di- and/or trivalent
inorganic cation transporter, preferably of the magnesium and
cobalt transport protein superfamily, e.g. as indicated in Table I,
columns 5 or 7, line 170, is increased conferring an increase of
the respective fine chemical, preferably of the tyrosine between
42% and 56% or more. In one embodiment, the activity of the
Saccharomyces cerevisiae protein YFL050C or its homologs, e.g. an
activity of a di- and/or trivalent inorganic cation transporter,
preferably of the magnesium and cobalt transport protein
superfamily, e.g. as indicated in Table I, columns 5 or 7, lines
129 and/or 170, is increased conferring an increase of the
respective fine chemical, preferably of alanine and tyrosine
between 19% and 104%, preferably between 42% and 104% or more. In
one embodiment, the activity of the Saccharomyces cerevisiae
protein YFL050C or its homologs, e.g. an activity of a di- and/or
trivalent inorganic cation transporter, preferably of the magnesium
and cobalt transport protein superfamily, involved in the
activation of the DNA damage and meiotic pachytene checkpoints,
e.g. as indicated in Table I, columns 5 or 7, lines 142 and/or 170,
is increased conferring an increase of the respective fine
chemical, preferably of glycine and tyrosine between 36% and 74%,
preferably between 42% and 74% or more. In one embodiment, the
activity of the Saccharomyces cerevisiae protein YFL050C or its
homologs, e.g. an activity of a di- and/or trivalent inorganic
cation transporter, preferably of the magnesium and cobalt
transport protein superfamily, e.g. as indicated in Table I,
columns 5 or 7, lines 129 and/or 142 and/or 170, is increased
conferring an increase of the respective fine chemical, preferably
of alanine and glycine and tyrosine between 19% and 104%,
preferably 36% and 104%, more preferably between 42% and 104% or
more.
[6188] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YGR101W or its homologs, e.g. an activity of a
rhomboid protease, e.g. as indicated in Table I, columns 5 or 7,
line 147, is increased conferring an increase of the respective
fine chemical, preferably of the phenylalanine between 40% and 92%
or more.
[6189] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YGR104C or its homologs, e.g. an activity of a
rhomboid protease, e.g. as indicated in Table I, columns 5 or 7,
line 135, is increased conferring an increase of the respective
fine chemical, preferably of the aspartic acid between 108% and
126% or more.
[6190] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YHR130C or its homologs, e.g. an activity of a
YHR130C protein, e.g. as indicated in Table I, columns 5 or 7, line
146, is increased conferring an increase of the respective fine
chemical, preferably of the phenylalanine between 77% and 77%,
preferably of 77% or more.
[6191] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YHR130C or its homologs, e.g. an activity of a
YHR130C protein, e.g. as indicated in Table I, columns 5 or 7, line
169, is increased conferring an increase of the respective fine
chemical, preferably of the tyrosine between 51% and 126% or more.
In one embodiment, the activity of the Saccharomyces cerevisiae
protein YHR130C or its homologs, e.g. an activity of a YHR130C
protein, e.g. as indicated in Table I, columns 5 or 7, lines 146
and/or 169, is increased conferring an increase of the respective
fine chemical, preferably of phenylalanine and tyrosine between 51%
and 126%, preferably between 77% and 126%, more preferably of 126%
or more.
[6192] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YIL150C or its homologs, e.g. an activity of a
chromatin binding protein, required for S-phase (DNA synthesis)
initiation or completion, e.g. as indicated in Table I, columns 5
or 7, line 128, is increased conferring an increase of the
respective fine chemical, preferably of the alanine between 47% and
328% or more. In one embodiment, the activity of the Saccharomyces
cerevisiae protein YIL150C or its homologs, e.g. an activity of a
chromatin binding protein, required for S-phase (DNA synthesis)
initiation or completion, e.g. as indicated in Table I, columns 5
or 7, line 134, is increased conferring an increase of the
respective fine chemical, preferably of the aspartic acid between
308% and 308%, preferably 308% or more. In one embodiment, the
activity of the Saccharomyces cerevisiae protein YIL150C or its
homologs, e.g. an activity of a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion, e.g. as
indicated in Table I, columns 5 or 7, lines 128 and/or 134, is
increased conferring an increase of the respective fine chemical,
preferably of alanine and aspartic acid between 47% and 328%,
preferably between 308% and 328% or more. In one embodiment, the
activity of the Saccharomyces cerevisiae protein YIL150C or its
homologs, e.g. an activity of a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion, e.g. as
indicated in Table I, columns 5 or 7, line 161, is increased
conferring an increase of the respective fine chemical, preferably
of the serine between 30% and 322% or more. In one embodiment, the
activity of the Saccharomyces cerevisiae protein YIL150C or its
homologs, e.g. an activity of a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion, e.g. as
indicated in Table I, columns 5 or 7, lines 128 and/or 161, is
increased conferring an increase of the respective fine chemical,
preferably of alanine and serine between 30% and 328%, preferably
between 47% and 328% or more. In one embodiment, the activity of
the Saccharomyces cerevisiae protein YIL150C or its homologs, e.g.
an activity of a chromatin binding protein, required for S-phase
(DNA synthesis) initiation or completion, involved in the
activation of the DNA damage and meiotic pachytene checkpoints,
e.g. as indicated in Table I, columns 5 or 7, lines 134 and/or 161,
is increased conferring an increase of the respective fine
chemical, preferably of aspartic acid and serine between 30% and
322%, preferably between 308% and 328% or more. In one embodiment,
the activity of the Saccharomyces cerevisiae protein YIL150C or its
homologs, e.g. an activity of a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion, e.g. as
indicated in Table I, columns 5 or 7, lines 128 and/or 134 and/or
161, is increased conferring an increase of the respective fine
chemical, preferably of alanine and aspartic acid and serine
between 30% and 328%, preferably 47% and 328%, more preferably
between 308% and 328% or more. In one embodiment, the activity of
the Saccharomyces cerevisiae protein YIL150C or its homologs, e.g.
an activity of a chromatin binding protein, required for S-phase
(DNA synthesis) initiation or completion, e.g. as indicated in
Table I, columns 5 or 7, line 168, is increased conferring an
increase of the respective fine chemical, preferably of the
tyrosine between 698% and 698%, preferably 698% or more. In one
embodiment, the activity of the Saccharomyces cerevisiae protein
YIL150C or its homologs, e.g. an activity of a chromatin binding
protein, required for S-phase (DNA synthesis) initiation or
completion, e.g. as indicated in Table I, columns 5 or 7, lines 128
and/or 168, is increased conferring an increase of the respective
fine chemical, preferably of alanine and tyrosine between 47% and
698%, preferably between 698% and 698% or more. In one embodiment,
the activity of the Saccharomyces cerevisiae protein YIL150C or its
homologs, e.g. an activity of a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion, involved in
the activation of the DNA damage and meiotic pachytene checkpoints,
e.g. as indicated in Table I, columns 5 or 7, lines 134 and/or 168,
is increased conferring an increase of the respective fine
chemical, preferably of aspartic acid and tyrosine between 308% and
698%, preferably between 698% and 698% or more. In one embodiment,
the activity of the Saccharomyces cerevisiae protein YIL150C or its
homologs, e.g. an activity of a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion, involved in
the activation of the DNA damage and meiotic pachytene checkpoints,
e.g. as indicated in Table I, columns 5 or 7, lines 161 and/or 168,
is increased conferring an increase of the respective fine
chemical, preferably of serine and tyrosine between 30% and 698%,
preferably between 698% and 698% or more. In one embodiment, the
activity of the Saccharomyces cerevisiae protein YIL150C or its
homologs, e.g. an activity of a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion, e.g. as
indicated in Table I, columns 5 or 7, lines 128 and/or 134 and/or
161, is increased conferring an increase of the respective fine
chemical, preferably of alanine and aspartic acid and tyrosine
between 47% and 698%, preferably 308% and 698%, more preferably
between 698% and 698% or more. In one embodiment, the activity of
the Saccharomyces cerevisiae protein YIL150C or its homologs, e.g.
an activity of a chromatin binding protein, required for S-phase
(DNA synthesis) initiation or completion, e.g. as indicated in
Table I, columns 5 or 7, lines 134 and/or 161 and/or 168, is
increased conferring an increase of the respective fine chemical,
preferably of aspartic acid serine and tyrosine between 30% and
698%, preferably 308% and 698%, more preferably between 698% and
698% or more. In one embodiment, the activity of the Saccharomyces
cerevisiae protein YIL150C or its homologs, e.g. an activity of a
chromatin binding protein, required for S-phase (DNA synthesis)
initiation or completion, e.g. as indicated in Table I, columns 5
or 7, lines 128 and/or 161 and/or 168, is increased conferring an
increase of the respective fine chemical, preferably of alanine and
serine and tyrosine between 30% and 698%, preferably 47% and 698%,
more preferably between 698% and 698% or more. In one embodiment,
the activity of the Saccharomyces cerevisiae protein YIL150C or its
homologs, e.g. an activity of a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion, e.g. as
indicated in Table I, columns 5 or 7, lines 128 and/or 134 and/or
161 and/or 168, is increased conferring an increase of the
respective fine chemical, preferably of alanine and aspartic acid
and serine and tyrosine between 30% and 698%, preferably 47% and
698%, more preferably between 308% and 698%, or more.
[6193] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YJL072C or its homologs, e.g. an activity of a
subunit of the GINS complex required for chromosomal DNA
replication, preferably of the Saccharomyces cerevisiae probable
membrane protein YJL072c superfamily, e.g. as indicated in Table I,
columns 5 or 7, line 145, is increased conferring an increase of
the respective fine chemical, preferably of the phenylalanine
between 25% and 204% or more.
[6194] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YKR057W or its homologs, e.g. an activity of a
ribosomal protein, similar to S21 ribosomal proteins, involved in
ribosome biogenesis and translation, preferably of the rat
ribosomal protein S21 superfamily, e.g. as indicated in Table I,
columns 5 or 7, line 160, is increased conferring an increase of
the respective fine chemical, preferably of the serine between 24%
and 170% or more.
[6195] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YLL009C or its homologs, e.g. an activity of a
cytochrome c oxidase copper chaperone, e.g. as indicated in Table
I, columns 5 or 7, line 159, is increased conferring an increase of
the respective fine chemical, preferably of the serine between 27%
and 84% or more.
[6196] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YLL013C or its homologs, e.g. an activity as a
PUF protein family, which is named for the founding members,
pumilio and Fbf, e.g. as indicated in Table I, columns 5 or 7, line
140, is increased conferring an increase of the respective fine
chemical, preferably of the citrulline between 30% and 44% or
more.
[6197] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YLR082C or its homologs, e.g. an activity as a
suppressor of Rad53 null lethality, e.g. as indicated in Table I,
columns 5 or 7, line 158, is increased conferring an increase of
the respective fine chemical, preferably of the serine between 22%
and 61% or more.
[6198] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOL123W or its homologs, e.g. an activity of a
cleavage and polyadenylation factor CF I component involved in
pre-mRNA 3'-end processing, preferably of the heterogeneous nuclear
ribonucleoprotein HRP1--yeast (Saccharomyces cerevisiae)
superfamily, e.g. as indicated in Table I, columns 5 or 7, line 141
is increased conferring an increase of the respective fine
chemical, preferably of glycine between 35% and 105% or more.
[6199] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR245C or its homologs, e.g. an activity of a
Acyl-CoA:diacylglycerol acyltransferase, preferably of the
Caenorhabditis elegans hypothetical protein KO7B1.4 superfamily,
e.g. as indicated in Table I, columns 5 or 7, line 139 is increased
conferring an increase of the respective fine chemical, preferably
of citrulline between 47% and 69% or more.
[6200] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR261C or its homologs, e.g. an activity of a
proteasome regulatory particle subunit, preferably of the mov-34
protein superfamily, e.g. as indicated in Table I, columns 5 or 7,
line 157 is increased conferring an increase of the respective fine
chemical, preferably of serine between 28% and 40% or more.
[6201] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR350C or its homologs, e.g. an activity which
is not been characterized yet, but seems to be member of the
Saccharomyces cerevisiae MNE1 protein superfamily, e.g. as
indicated in Table I, columns 5 or 7, line 167 is increased
conferring an increase of the respective fine chemical, preferably
of tyrosine between 67% and 177% or more.
[6202] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YPR138C or its homologs, e.g. an activity of a
subunit of the NH4+ transporter, preferably of the ammonium
transport protein and/or ammonium transporter nrgA superfamily,
e.g. as indicated in Table I, columns 5 or 7, line 144 is increased
conferring an increase of the respective fine chemical, preferably
of phenylalanine between 49% and 93% or more.
[6203] In one embodiment, the activity of the Escherichia coli K12
protein b0695 or its homologs, e.g. an activity of a sensory
histidine kinase in two-component regulatory system, preferably of
the sensor histidine kinase homology superfamily, e.g. as indicated
in Table I, columns 5 or 7, line 156, is increased conferring an
increase of the respective fine chemical, preferably of the
phenylalanine between 36% and 74% or more.
[6204] In one embodiment, the activity of the Escherichia coli K12
protein b0730 or its homologs, e.g. an activity of a
transcriptional regulator of succinylCoA synthetase operon and/or
fatty acyl response regulator, preferably of the transcription
regulator GntR superfamily, e.g. as indicated in Table I, columns 5
or 7, line 138, is increased conferring an increase of the
respective fine chemical, preferably of the aspartic acid between
47% and 268% or more.
[6205] In one embodiment, the activity of the Escherichia coli K12
protein b1708 or its homologs, e.g. an activity of a lipoprotein,
preferably of the conserved hypothetical protein H11314
superfamily, e.g. as indicated in Table I, columns 5 or 7, line
154, is increased conferring an increase of the respective fine
chemical, preferably of the phenylalanine between 48% and 237% or
more.
[6206] In one embodiment, the activity of the Escherichia coli K12
protein b1827 or its homologs, e.g. an activity of a
transcriptional repressor with DNA-binding Winged helix domain
(IcIR family), preferably of the acetate operon repressor
superfamily, e.g. as indicated in Table I, columns 5 or 7, line
153, is increased conferring an increase of the respective fine
chemical, preferably of the phenylalanine between 40% and 293% or
more.
[6207] In one embodiment, the activity of the Escherichia coli K12
protein b1827 or its homologs, e.g. an activity of a
transcriptional repressor with DNA-binding Winged helix domain
(IcIR family), preferably of the acetate operon repressor
superfamily, e.g. as indicated in Table I, columns 5 or 7, line
172, is increased conferring an increase of the respective fine
chemical, preferably of the tyrosine between 40% and 228% or
more.
[6208] In one embodiment, the activity of the Escherichia coli K12
protein b1827 or its homologs, e.g. an activity of a
transcriptional repressor with DNA-binding Winged helix domain
(IcIR family), preferably of the acetate operon repressor
superfamily, e.g. as indicated in Table I, columns 5 or 7, lines
153 and/or 172, is increased conferring an increase of the
respective fine chemical, preferably of the phenylalanine and
tyrosine between 40% and 293%, preferably between 40% and 293% or
more.
[6209] In one embodiment, the activity of the Escherichia coli K12
protein b1829 or its homologs, e.g. an activity of a heat shock
protein with protease activity, preferably of the heat-shock
protein htpX superfamily, e.g. as indicated in Table I, columns 5
or 7, line 166, is increased conferring an increase of the
respective fine chemical, preferably of the serine between 24% and
166% or more. In one embodiment, the activity of the Escherichia
coli K12 protein b1829 or its homologs, e.g. an activity of a heat
shock protein with protease activity, preferably of the heat-shock
protein htpX superfamily, e.g. as indicated in Table I, columns 5
or 7, line 171, is increased conferring an increase of the
respective fine chemical, preferably of the tyrosine between 56%
and 741% or more.
[6210] In one embodiment, the activity of the Escherichia coli K12
protein b1829 or its homologs, e.g. an activity of a heat shock
protein with protease activity, preferably of the heat-shock
protein htpX superfamily, e.g. as indicated in Table I, columns 5
or 7, lines 166 and/or 171, is increased conferring an increase of
the respective fine chemical, preferably of the serine and tyrosine
between 24% and 741%, preferably between 56% and 741% or more.
[6211] In one embodiment, the activity of the Escherichia coli K12
protein b2095 or its homologs, e.g. an activity of a
tagatose-6-phosphate kinase, preferably of the Escherichia probable
tagatose 6-phosphate kinase gatZ superfamily, e.g. as indicated in
Table I, columns 5 or 7, line 133, is increased conferring an
increase of the respective fine chemical, preferably of the alanine
between 22% and 33% or more.
[6212] In one embodiment, the activity of the Escherichia coli K12
protein b3008 or its homologs, e.g. an activity of a cystathionine
beta-lyase, PLP-dependent (beta-cystathionase), preferably of the
0-succinylhomoserine (thiol)-lyase superfamily, e.g. as indicated
in Table I, columns 5 or 7, line 132, is increased conferring an
increase of the respective fine chemical, preferably of the alanine
between 27% and 71% or more.
[6213] In one embodiment, the activity of the Escherichia coli K12
protein b3256 or its homologs, e.g. an activity of an acetyl CoA
carboxylase and/or biotin carboxylase subunit, preferably of the
biotin carboxylase superfamily, e.g. as indicated in Table I,
columns 5 or 7, line 151, is increased conferring an increase of
the respective fine chemical, preferably of the phenylalanine
between 44% and 50% or more.
[6214] In one embodiment, the activity of the Escherichia coli K12
protein b1697 or its homologs, e.g. an activity of an electron
transfer flavoprotein subunit with ETFP adenine nucleotide-binding
like domain, preferably of the electron transfer flavoprotein beta
chain superfamily, e.g. as indicated in Table I, columns 5 or 7,
line 155, is increased conferring an increase of the respective
fine chemical, preferably of the phenylalanine between 30% and 188%
or more.
[6215] In one embodiment, the activity of the Escherichia coli K12
protein b1886 or its homologs, e.g. an activity of a
methyl-accepting chemotaxis protein II and/or aspartate sensor
receptor, preferably of the methyl-accepting chemotaxis protein
superfamily e.g. as indicated in Table I, columns 5 or 7, line 152,
is increased conferring an increase of the respective fine
chemical, preferably of the phenylalanine between 33% and 196% or
more.
[6216] In one embodiment, the activity of the Escherichia coli K12
protein b1886 or its homologs, e.g. an activity of a
methyl-accepting chemotaxis protein II and/or aspartate sensor
receptor, preferably of the methyl-accepting chemotaxis protein
superfamily, e.g. as indicated in Table I, columns 5 or 7, line
165, is increased conferring an increase of the respective fine
chemical, preferably of the serine between 25% and 111% or
more.
[6217] In one embodiment, the activity of the Escherichia coli K12
protein b1886 or its homologs, e.g. an activity of a
methyl-accepting chemotaxis protein II and/or aspartate sensor
receptor, preferably of the methyl-accepting chemotaxis protein
superfamily, e.g. as indicated in Table I, columns 5 or 7, lines
152 and/or 165, is increased conferring an increase of the
respective fine chemical, preferably of the phenylalanine and
serine between 25% and 196%, preferably between 33% and 196% or
more. In one embodiment, the activity of the Escherichia coli K12
protein b1896 or its homologs, e.g. an activity of a
trehalose-6-phosphate synthase, preferably of the
[6218] Schizosaccharomyces pombe alpha,alpha-trehalose-phosphate
synthase (UdP-forming) superfamily, e.g. as indicated in Table I,
columns 5 or 7, line 127, is increased conferring an increase of
the respective fine chemical, preferably of the 5-oxoproline
between 34% and 150% or more.
[6219] In one embodiment, the activity of the Escherichia coli K12
protein b1896 or its homologs, e.g. an activity of a
trehalose-6-phosphate synthase, preferably of the
Schizosaccharomyces pombe alpha,alpha-trehalose-phosphate synthase
(UdP-forming) superfamily, e.g. as indicated in Table I, columns 5
or 7, line 137, is increased conferring an increase of the
respective fine chemical, preferably of the aspartic acid between
90% and 255% or more. In one embodiment, the activity of the
Escherichia coli K12 protein b1896 or its homologs, e.g. an
activity of a trehalose-6-phosphate synthase, preferably of the
Schizosaccharomyces pombe alpha,alpha-trehalose-phosphate synthase
(UdP-forming) superfamily, e.g. as indicated in Table I, columns 5
or 7, lines 127 and/or 137, is increased conferring an increase of
the respective fine chemical, preferably of the 5-oxoproline and
aspartic acid between 34% and 255%, preferably between 90% and 255%
or more.
[6220] In one embodiment, the activity of the Escherichia coli K12
protein b3462 or its homologs, e.g. an activity of integral
membrane cell division protein, preferably of the cell division
protein ftsX superfamily, e.g. as indicated in Table I, columns 5
or 7, line 150, is increased conferring an increase of the
respective fine chemical, preferably of the phenylalanine between
26% and 140% or more.
[6221] In one embodiment, the activity of the Escherichia coli K12
protein b3462 or its homologs, e.g. an activity of integral
membrane cell division protein, preferably of the cell division
protein ftsX superfamily, e.g. as indicated in Table I, columns 5
or 7, line 164, is increased conferring an increase of the
respective fine chemical, preferably of the serine between 26% and
64% or more. In one embodiment, the activity of the Escherichia
coli K12 protein b3462 or its homologs, e.g. an activity of
integral membrane cell division protein, preferably of the cell
division protein ftsX superfamily, e.g. as indicated in Table I,
columns 5 or 7, lines 127 and/or 137, is increased conferring an
increase of the respective fine chemical, preferably of the
phenylalanine and serine between 26% and 140%, preferably between
26% and 140% or more.
[6222] In case the activity of the Escherichia coli K12 protein
b0057 or its homologs e.g. a protein with an activity as defined in
[0022.0.14.14],e.g. as indicated in Table II, columns 5 or 7, line
517 and 530 and 546, is increased, preferably, in one embodiment
the increase of the fine chemical, preferably of citrulline between
33% and 119%, preferably of glycine between 43% and 67%, preferably
of serine between 32% and 61%, preferably of citrulline and serine
between 32% and 119%, preferably of citrulline and glycine between
33% and 119%, preferably of glycine and serine between 32% and 67%,
preferably of citrulline and glycine and serine between 32% and
119% or more is conferred.
[6223] In case the activity of the Escherichia coli K12 protein
b0161 or its homologs e.g. a periplasmic serine protease e.g. as
indicated in Table II, columns 5 or 7, lines 492 and 505 and 537,
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of 5-oxoproline between 45% and 123% or
more, preferably of aspartic acid between 57% and 116% or more,
preferably of phenylalanine between 37% and 668% or more,
preferably of 5-oxoproline and aspartic acid between 45% and 123%
or more, preferably of 5-oxoproline and phenylalanine between 37%
and 668% or more, preferably of aspartic acid and phenylalanine
between 37% and 668% or more, preferably of 5-oxoproline and
aspartic acid and phenylalanine between 37% and 668% or more is
conferred.
[6224] In case the activity of the Escherichia coli K12 protein
b0236 or its homologs, as indicated in Table II, columns 5 or 7,
line 497, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of alanine between 22%
and 41% or more is conferred.
[6225] In case the activity of the Escherichia coli K12 protein
b0376 or its homologs e.g. a beta-lactamase/D-ala carboxypeptidase,
penicilling binding protein e.g. as indicated in Table II, columns
5 or 7, line 493, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of 5-oxoproline between
41% and 100% or more is conferred.
[6226] In case the activity of the Escherichia coli K12 protein
b0462 or its homologs, as indicated in Table II, columns 5 or 7,
line 518, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 36%
and 44% or more is conferred.
[6227] In case the activity of the Escherichia coli K12 protein
b0486 or its homologs e.g. a amino-acid/amine transport protein
(APC family) e.g. as indicated in Table II, columns 5 or 7, line
498 and 547, is increased, preferably, in one embodiment the
increase of the fine respective chemical, preferably of alanine
between 44% and 52%, preferably of serine between 27% and 49%,
preferably of alanine and serine between 27% and 52% or more, is
conferred.
[6228] In case the activity of the Escherichia coli K12 protein
b0577 or its homologs e.g. a putative transport protein e.g. as
indicated in Table II, columns 5 or 7, line 506 and531, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of glycine between 36% and 50%, preferably of
aspartic acid between 56% and 65%, preferably of glycine and
aspartic acid between 36% and 65%, or more is conferred.
[6229] In case the activity of the Escherichia coli K12 protein
b0970 or its homologs e.g. a glutamate receptor e.g. as indicated
in Table II, columns 5 or 7, line 494 and 555, is increased,
preferably, in one embodiment the increase of the fine respective
chemical, preferably of 5-oxoproline between 37% and 203%,
preferably of tyrosine between 35% and 498%, preferably of
5-oxoproline and tyrosine between 35% and 498%, or more, is
conferred.
[6230] In case the activity of the Escherichia coli K12 protein
b1228 or its homologs, as indicated in Table II, columns 5 or 7,
line 538, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of phenylalanine between
35% and 44% or more is conferred.
[6231] In case the activity of the Escherichia coli K12 protein
b1275 or its homologs, as indicated in Table II, columns 5 or 7,
line 519, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 32%
and 50% or more is conferred.
[6232] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1343 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 495 and 499 and 507, is increased, e.g.
the activity of a protein involved in rRNA processing and/or
translation is increased, preferred the activity of a ATP-dependent
RNA helicase, stimulated by 23S rRNA or its homolog is increased.
Preferably, an increase of the respective fine chemical, preferably
of 5-oxoproline between 37% and 87%, preferably of alanine between
27% and 44%, preferably of aspartic acid between 76% and 136%,
preferably of alanine and 5-oxoproline between 27% and 87%,
preferably of alanine and aspartic acid between 27% and 136%,
preferably of 5-oxoproline and aspartic acid between 37% and 136%,
preferably of alanine and 5-oxoproline and aspartic acid between
27% and 136%, or more is conferred.
[6233] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1360 or a protein with the activity defined as
putative DNA replication protein or its homologs, e.g.
transcriptional regulator or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 520, is increased, preferably, in
one embodiment an increase of the fine chemical, preferably of
citrulline between 34% and 72% or more is conferred.
[6234] In case the activity of the Escherichia coli K12 protein
b1863 or its homologs, as indicated in Table II, columns 5 or 7,
line 500, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of alanine between 22%
and 45%, or more is conferred.
[6235] In case the activity of the Escherichia coli K12 protein
b2023 or its homologs, as indicated in Table II, columns 5 or 7,
line 508, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of aspartic acid between
56% and 81%, or more is conferred.
[6236] In case the activity of the Escherichia coli K12 protein
b2078 or its homologs, as indicated in Table II, columns 5 or 7,
line 539, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of phenylalanine between
26% and 89%, or more is conferred.
[6237] In case the activity of the Escherichia coli K12 protein
b2239 or its homologs, as indicated in Table II, columns 5 or 7,
line 521, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 37%
and 73%, or more is conferred.
[6238] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2414 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 522 and 532 and 548 and 540, is increased,
e.g. the activity of a protein of the threonine
dehydratase-superfamily is increased preferably the activity of a
protein involved in amino acid biosynthesis, biosynthesis of the
cysteine-aromatic group, degradation of amino acids of the
cysteine-aromatic group, nitrogen and sulfur
utilizationbiosynthesis of the aspartate family, degradation of
amino acids of the aspartate group, biosynthesis of sulfuric acid
and L-cysteine derivatives, biosynthesis of secondary products
derived from primary amino acids, biosynthesis of secondary
products derived from glycine, L-serine and L-alanine, pyridoxal
phosphate binding is increased, preferred the activity of a subunit
of cysteine synthase A and O-acetylserine sulfhydrolase A,
PLP-dependent enzyme or its homolog is increased. Preferably, an
increase of the respective fine chemical, preferably of citrulline
between 31% and 40%, preferably of glycine between 38% and 62%,
preferably of serine between 27% and 53%, preferably of
phenylalanine between 27% and 197%, preferably of citrulline and
glycine between 31% and 62%, preferably of citrulline and serine
between 27% and 53%, preferably of citrulline and phenylalanine
between 27% and 197%, preferably of glycine and serine between 27%
and 62%, preferably of glycine and phenylalanine between 27% and
197%, preferably of serine and phenylalanine between 27% and 197%,
preferably of citrulline and glycine and serine between 27% and
62%, preferably of citrulline and glycine and phenylalanine between
27% and 197%, preferably of citrulline and serine and phenylalanine
between 27% and 197%, preferably of glycine and serine and
phenylalanine between 27% and 197%, preferably of citrulline and
glycine and serine and phenylalanine between 27% and 197%, or more
is conferred. In one embodiment, in case the activity of the
Escherichia coli K12 protein b2426 or its homologs, e.g. as
indicated in Table II, columns 5 or 7, line 523, is increased, e.g.
the activity of a oxidoreductase with NAD(P)-binding domain is
increased. Preferably, an increase of the respective fine chemical,
preferably of citrulline between 32% and 41% or more is
conferred.
[6239] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2489 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 524, 533, 549 and 501, is increased, e.g.
the activity of a hydrogenase Fe--S subunit is increased.
Preferably, an increase of the respective fine chemical, preferably
of citrulline between 33% and 60%, preferably of glycine between
33% and 78%, preferably of serine between 23% and 47%, preferably
of alanine between 21% and 27%, preferably of citrulline and
glycine between 33% and 78%, preferably of citrulline and serine
between 23% and 60%, preferably of citrulline and alanine between
21% and 60%, preferably of glycine and serine between 23% and 78%,
preferably of glycine and alanine between 21% and 78%, preferably
of serine and alanine between 21% and 47%, preferably of citrulline
and glycine and serine between 23% and 78%, preferably of
citrulline and glycine and alanine between 21% and 78%, preferably
of citrulline and serine and alanine between 21% and 60%,
preferably of glycine and serine and alanine between 21% and 78%,
preferably of citrulline and glycine and serine and alanine between
21% and 78%, or more is conferred.
[6240] In case the activity of the Escherichia coli K12 protein
b2491 or its homologs, as indicated in Table II, columns 5 or 7,
line 556, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of tyrosine between 40%
and 89%, or more is conferred.
[6241] In case the activity of the Escherichia coli K12 protein
b2507 or its homologs, as indicated in Table II, columns 5 or 7,
line 509, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of aspartic acid between
49% and 120%, or more is conferred.
[6242] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2553 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 534, is increased, e.g. the activity of a
regulatory protein P-II for glutamine synthetase is increased.
Preferably, an increase of the respective fine chemical, preferably
of glycine between 36% and 83% or more is conferred.
[6243] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2576 or its homologs, e.g. as indicated in Table
III, columns 5 or 7, line 502 and 535 and 510 and 541, is
increased, e.g. the activity of a protein with an activity as
defined in [0022.0.14.14] is increased, an increase of the
respective fine chemical, preferably of alanine between 24% and
49%, preferably of glycine between 40% and 73%, preferably of
aspartic acid between 52% and 103%, preferably of phenylalanine
between 23% and 41%, preferably of alanine and glycine between 24%
and 73%, preferably of alanine and aspartic acid between 24% and
103%, preferably of alanine and phenylalanine between 23% and 49%,
preferably of glycine and aspartic acid between 40% and 103%,
preferably of glycine and phenylalanine between 23% and 73%,
preferably of aspartic acid and phenylalanine between 23% and 103%,
preferably of alanine and glycine and aspartic acid between 24% and
103%, preferably of alanine and glycine and phenylalanine between
23% and 73%, preferably of alanine and aspartic acid and
phenylalanine between 23% and 103%, preferably of glycine and
aspartic acid and phenylalanine between 23% and 103%, preferably of
alanine and glycine and aspartic acid and phenylalanine between 23%
and 103%, or more is conferred.
[6244] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2664 or a protein with the activity defined as
putative transcriptional repressor with DNA-binding Winged helix
domain (GntR familiy) or its homologs, e.g. transcriptional
regulator, e.g. as indicated in Table III, columns 5 or 7, line
550, is increased, preferably, in one embodiment an increase of the
fine chemical serine between 25% and 231% or more is conferred.
[6245] In case the activity of the Escherichia coli K12 protein
b2753 or its homologs, as indicated in Table II, columns 5 or 7,
line 511, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of aspartic acid between
56% and 154%, or more is conferred.
[6246] In case the activity of the Escherichia coli K12 protein
b2796 or its homologs, as indicated in Table II, columns 5 or 7,
line 542, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of phenylalanine between
33% and 105%, or more is conferred.
[6247] In case the activity of the Escherichia coli K12 protein
b3064 or its homologs, as indicated in Table II, columns 5 or 7,
line 551, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of serine between 39% and
84%, or more is conferred.
[6248] In case the activity of the Escherichia coli K12 protein
b3116 or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 512 and 552, is increased, e.g. the activity of a
L-threonine/L-serine permease, anaerobically inducible (HAAAP
family) is increased, preferably, an increase of the respective
fine chemical, preferably of serine between 35% and 70%, preferably
of aspartic acid between 50% and 130%, preferably of serine and
aspartic acid between 35% and 130%, or more is conferred.
[6249] In case the activity of the Escherichia coli K12 protein
b3160 or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 525 and 553, is increased, e.g. the activity of a
monooxygenase with luciferase-like ATPase activity is increased,
preferably, an increase of the respective fine chemical, preferably
of citrulline between 34% and 48%, preferably of serine between 31%
and 60%, preferably of citrulline and serine between 31% and 60% or
more is conferred.
[6250] In case the activity of the Escherichia coli K12 protein
b3169 or its homologs e.g. a transcription
termination-antitermination factor e.g. as indicated in Table II,
columns 5 or 7, line 513, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
aspartic acid between 66% and 168% or more is conferred.
[6251] In case the activity of the Escherichia coli K12 protein
b3172 or its homologs, as indicated in Table II, columns 5 or 7,
line 496 and 514, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of aspartic acid between
52% and 234%, preferably of 5-oxoproline between 44% and 94%,
preferably of aspartic acid and 5-oxoproline between 44% and 234%,
or more is conferred.
[6252] In case the activity of the Escherichia coli K12 protein
b3231 or its homologs e.g. a 50S ribosomal subunit protein L13 e.g.
as indicated in Table II, columns 5 or 7, line 503 and 554, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of alanine between 21% and 35%, preferably of
serine between 23% and 49%, preferably of alanine and serine
between 21% and 49% or more is conferred.
[6253] In case the activity of the Escherichia coli K12 protein
b3241 or its homologs, as indicated in Table II, columns 5 or 7,
line 526, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 38%
and 70%, or more is conferred.
[6254] In case the activity of the Escherichia coli K12 protein
b3767 or its homologs, as indicated in Table II, columns 5 or 7,
line 504 and 536, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of alanine between 24%
and 70%, preferably of homoserine between 24% and 71%, preferably
of alanine and homoserine between 24% and 71%, or more is
conferred.
[6255] In case the activity of the Escherichia coli K12 protein
b3919 or its homologs e.g. an triosephosphate isomerase e.g. as
indicated in Table II, columns 5 or 7, line 543, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of phenylalanine between 30% and 188% or more is
conferred.
[6256] In case the activity of the Escherichia coli K12 protein
b3926 or its homologs, as indicated in Table II, columns 5 or 7,
line 527, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 70%
and 94%, or more is conferred.
[6257] In case the activity of the Escherichia coli K12 protein
b3938 or its homologs, as indicated in Table II, columns 5 or 7,
line 544, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of phenylalanine between
34% and 40%, or more is conferred.
[6258] In case the activity of the Escherichia coli K12 protein
b3983 or its homologs, e.g. the activity of a Escherichia coli
ribosomal protein L11 superfamily, preferably a protein with with a
t50S ribosomal subunit protein L12 activity, e.g. as indicated in
Table II, columns 5 or 7, line 545 and 557, is increased conferring
an increase of the respective fine chemical, preferably
phenylalanine between 37% and 266%, preferably tyrosine between 44%
and 357%, preferably phenylalanine and tyrosine between 37% and
357% or more is conferred.
[6259] In case the activity of the Escherichia coli K12 protein
b4129 or its homologs e.g. a lysine tRNA synthetase e.g. as
indicated in Table II, columns 5 or 7, line 515, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of aspartic acid between 53% and 141% or more is
conferred.
[6260] In case the activity of the Escherichia coli K12 protein
b4214 or its homologs, as indicated in Table II, columns 5 or 7,
line 528, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 32%
and 102%, or more is conferred.
[6261] In case the activity of the Escherichia coli K12 protein
b4269 or its homologs, as indicated in Table II, columns 5 or 7,
line 529, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 31%
and 63%, or more is conferred.
[6262] In case the activity of the Escherichia coli K12 protein
b4346 or its homologs e.g. a component of 5-methylcytosine-specific
restriction enzyme McrBC e.g. as indicated in
[6263] Table II, columns 5 or 7, line 516, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of aspartic acid between 63% and 94% or more is
conferred.
[6264] [0046.0.14.14] In one embodiment, the activity of the
Saccaromyces cerevisiae protein YAL049C or its homologs, e.g. an
activity of a YAL049C protein, e.g. as indicated in Table I,
columns 5 or 7, line 149, confers an increase of the respective
fine chemical and of further amino acids or their precursors. In
one embodiment, the activity of the Saccaromyces cerevisiae protein
YBL015W or its homologs, e.g. an activity of a mannose-containing
glycoprotein and/or as an acetyl-CoA hydrolase, preferably of the
hacetyl-CoA hydrolase superfamily, e.g. as indicated in Table I,
columns 5 or 7, line 131, confers an increase of the respective
fine chemical and of further amino acid activity having-compounds
or their precursors. In one embodiment, the activity of the
Saccaromyces cerevisiae protein YDL127W or its homologs, e.g. an
activity of a G1/S-specific cyclin PCL2 (Cyclin HCS26 homolog),
e.g. as indicated in Table I, columns 5 or 7, line 126, confers an
increase of the respective fine chemical and of further amino acid
activity having-compounds or their precursors.
[6265] In one embodiment, the activity of the Saccaromyces
cerevisiae protein YEL045C or its homologs, e.g. an activity of a
YEL045C protein, e.g. as indicated in Table I, columns 5 or 7, line
163, confers an increase of the respective fine chemical and of
further amino acid activity having-compounds or their
precursors.
[6266] In one embodiment, the activity of the Saccaromyces
cerevisiae protein YEL046C or its homologs, e.g. an activity of a
low specificity L-threonine aldolase, e.g. as indicated in Table I,
columns 5 or 7, line 143, confers an increase of the respective
fine chemical and of further amino acid activity having-compounds
or their precursors. In one embodiment, the activity of the
Saccaromyces cerevisiae protein YER152C or its homologs, e.g. an
activity similar to tyrosine aminotransferase, e.g. as indicated in
Table I, columns 5 or 7, line 162, confers an increase of the
respective fine chemical and of further amino acid activity
having-compounds or their precursors. In one embodiment, the
activity of the Saccaromyces cerevisiae protein YER173W or its
homologs, e.g. an activity of a checkpoint protein, involved in the
activation of the DNA damage and meiotic pachytene checkpoints,
e.g. as indicated in Table I, columns 5 or 7, lines 130 and/or 136
and/or 148, confers an increase of the respective fine chemical and
of further amino acid activity having-compounds or their
precursors. In one embodiment, the activity of the Saccaromyces
cerevisiae protein YFL050C or its homologs, e.g. an activity of a
di- and/or trivalent inorganic cation transporter, preferably of
the magnesium and cobalt transport protein superfamily, e.g. as
indicated in Table I, columns 5 or 7, lines 129 and/or 142 and/or
170, confers an increase of the respective fine chemical and of
further amino acid activity having-compounds or their
precursors.
[6267] In one embodiment, the activity of the Saccaromyces
cerevisiae protein YGR101W or its homologs, e.g. an activity of a
rhomboid protease, e.g. as indicated in Table I, columns 5 or 7,
line 147, confers an increase of the respective fine chemical and
of further amino acid activity having-compounds or their
precursors. In one embodiment, the activity of the Saccaromyces
cerevisiae protein YGR104C or its homologs, e.g. an activity of a
rhomboid protease, e.g. as indicated in Table I, columns 5 or 7,
line 135, confers an increase of the respective fine chemical and
of further amino acid activity having-compounds or their
precursors. In one embodiment, the activity of the Saccaromyces
cerevisiae protein YHR130C or its homologs, e.g. an activity of a
YHR130C protein, e.g. as indicated in Table I, columns 5 or 7,
lines 146 and/or 169, confers an increase of the respective fine
chemical and of further amino acid activity having-compounds or
their precursors. In one embodiment, the activity of the
Saccaromyces cerevisiae protein YIL150C or its homologs, e.g. an
activity of a chromatin binding protein, required for S-phase (DNA
synthesis) initiation or completion, e.g. as indicated in Table I,
columns 5 or 7, lines 128 and/or 134 and/or 161 and/or 168, confers
an increase of the respective fine chemical and of further amino
acid activity having-compounds or their precursors. In one
embodiment, the activity of the Saccaromyces cerevisiae protein
YJL072C or its homologs, e.g. an activity of a subunit of the GINS
complex required for chromosomal DNA replication, preferably of the
Saccharomyces cerevisiae probable membrane protein YJL072c
superfamily, e.g. as indicated in Table I, columns 5 or 7, line
145, confers an increase of the respective fine chemical and of
further amino acid activity having-compounds or their
precursors.
[6268] In one embodiment, the activity of the Saccaromyces
cerevisiae protein YKR057W or its homologs, e.g. an activity of a
ribosomal protein, similar to S21 ribosomal proteins, involved in
ribosome biogenesis and translation, preferably of the rat
ribosomal protein S21 superfamily, e.g. as indicated in Table I,
columns 5 or 7, line 160, confers an increase of the respective
fine chemical and of further amino acid activity having-compounds
or their precursors. In one embodiment, the activity of the
Saccaromyces cerevisiae protein YLL009C or its homologs, e.g. an
activity of a cytochrome c oxidase copper chaperone, e.g. as
indicated in Table I, columns 5 or 7, line 159, confers an increase
of the respective fine chemical and of further amino acid activity
having-compounds or their precursors. In one embodiment, the
activity of the Saccaromyces cerevisiae protein YLL013C or its
homologs, e.g. an activity as a PUF protein family, which is named
for the founding members, pumilio and Fbf, e.g. as indicated in
Table I, columns 5 or 7, line 140, confers an increase of the
respective fine chemical and of further amino acid activity
having-compounds or their precursors. In one embodiment, the
activity of the Saccaromyces cerevisiae protein YLR082C or its
homologs, e.g. an activity as a suppressor of Rad53 null lethality,
e.g. as indicated in Table I, columns 5 or 7, line 158, confers an
increase of the respective fine chemical and of further amino acid
activity having-compounds or their precursors. In one embodiment,
the activity of the Saccaromyces cerevisiae protein YOL123W or its
homologs, e.g. an activity of a cleavage and polyadenylation factor
CF I component involved in pre-mRNA 3'-end processing, preferably
of the heterogeneous nuclear ribonucleoprotein HRP1--yeast
(Saccharomyces cerevisiae) superfamily, e.g. as indicated in Table
I, columns 5 or 7, line 141, confers an increase of the respective
fine chemical and of further amino acid activity having-compounds
or their precursors. In one embodiment, the activity of the
Saccaromyces cerevisiae protein YOR245C or its homologs, e.g. an
activity of a Acyl-CoA:diacylglycerol acyltransferase, preferably
of the Caenorhabditis elegans hypothetical protein KO7B1.4
superfamily, e.g. as indicated in Table I, columns 5 or 7, line
139, confers an increase of the respective fine chemical and of
further amino acid activity having-compounds or their precursors.
In one embodiment, the activity of the Saccaromyces cerevisiae
protein YOR261C or its homologs, e.g. an activity of a proteasome
regulatory particle subunit, preferably of the mov-34 protein
superfamily, e.g. as indicated in Table I, columns 5 or 7, line
157, confers an increase of the respective fine chemical and of
further amino acid activity having-compounds or their precursors.
In one embodiment, the activity of the Saccaromyces cerevisiae
protein YOR350C or its homologs, e.g. an activity which is not been
characterized yet, but seems to be member of the Saccharomyces
cerevisiae MNE1 protein superfamily, e.g. as indicated in Table I,
columns 5 or 7, line 167, confers an increase of the respective
fine chemical and of further amino acid activity having-compounds
or their precursors. In one embodiment, the activity of the
Saccaromyces cerevisiae protein YPR138C or its homologs, e.g. an
activity of a subunit of the NH4+ transporter, preferably of the
ammonium transport protein and/or ammonium transporter nrgA
superfamily, e.g. as indicated in Table I, columns 5 or 7, line
144, confers an increase of the respective fine chemical and of
further amino acid activity having-compounds or their
precursors.
[6269] In one embodiment, the activity of the Escherichia coli K12
protein b0695 or its homologs, e.g. an activity of a sensory
histidine kinase in two-component regulatory system, preferably of
the sensor histidine kinase homology superfamily, e.g. as indicated
in Table I, columns 5 or 7, line 156, confers an increase of the
respective fine chemical and of further amino acid activity-having
compounds or their precursors. In one embodiment, the activity of
the Escherichia coli K12 protein b0730 or its homologs, e.g. an
activity of a transcriptional regulator of succinylCoA synthetase
operon and/or fatty acyl response regulator, preferably of the
transcription regulator GntR superfamily, e.g. as indicated in
Table I, columns 5 or 7, line 138, confers an increase of the
respective fine chemical and of further amino acid activity-having
compounds or their precursors. In one embodiment, the activity of
the Escherichia coli K12 protein b1708 or its homologs, e.g. an
activity of a lipoprotein, preferably of the conserved hypothetical
protein H11314 superfamily, e.g. as indicated in Table I, columns 5
or 7, line 154, confers an increase of the respective fine chemical
and of further amino acid activity-having compounds or their
precursors. In one embodiment, the activity of the Escherichia coli
K12 protein b1827 or its homologs, e.g. an activity of a
transcriptional repressor with DNA-binding Winged helix domain
(IcIR family), preferably of the acetate operon repressor
superfamily, e.g. as indicated in Table I, columns 5 or 7, lines
153 and/or 172, confers an increase of the respective fine chemical
and of further amino acid activity-having compounds or their
precursors.
[6270] In one embodiment, the activity of the Escherichia coli K12
protein b1829 or its homologs, e.g. an activity of a heat shock
protein with protease activity, preferably of the heat-shock
protein htpX superfamily, e.g. as indicated in Table I, columns 5
or 7, lines 166 and/or 171, confers an increase of the respective
fine chemical and of further amino acid activity-having compounds
or their precursors. In one embodiment, the activity of the
Escherichia coli K12 protein b2095 or its homologs, e.g. an
activity of a tagatose-6-phosphate kinase, preferably of the
Escherichia probable tagatose 6-phosphate kinase gatZ superfamily,
e.g. as indicated in Table I, columns 5 or 7, line 133, confers an
increase of the respective fine chemical and of further amino acid
activity-having compounds or their precursors. In one embodiment,
the activity of the Escherichia coli K12 protein b3008 or its
homologs, e.g. an activity of a cystathionine beta-lyase,
PLP-dependent (beta-cystathionase), preferably of the
0-succinylhomoserine (thiol)-lyase superfamily, e.g. as indicated
in Table I, columns 5 or 7, line 132, confers an increase of the
respective fine chemical and of further amino acid activity-having
compounds or their precursors. In one embodiment, the activity of
the Escherichia coli K12 protein b3256 or its homologs, e.g. an
activity of an acetyl CoA carboxylase and/or biotin carboxylase
subunit, preferably of the biotin carboxylase superfamily, e.g. as
indicated in Table I, columns 5 or 7, line 151, confers an increase
of the respective fine chemical and of further amino acid
activity-having compounds or their precursors. In one embodiment,
the activity of the Escherichia coli K12 protein b1697 or its
homologs, e.g. an activity of an electron transfer flavoprotein
subunit with ETFP adenine nucleotide-binding like domain,
preferably of the electron transfer flavoprotein beta chain
superfamily, e.g. as indicated in Table I, columns 5 or 7, line
155, confers an increase of the respective fine chemical and of
further amino acid activity-having compounds or their precursors.
In one embodiment, the activity of the Escherichia coli K12 protein
b1886 or its homologs, e.g. an activity of a methyl-accepting
chemotaxis protein II and/or aspartate sensor receptor, preferably
of the methyl-accepting chemotaxis protein superfamily, e.g. as
indicated in Table I, columns 5 or 7, lines 152 and/or 165, confers
an increase of the respective fine chemical and of further amino
acid activity-having compounds or their precursors. In one
embodiment, the activity of the Escherichia coli K12 protein b1896
or its homologs, e.g. an activity of a trehalose-6-phosphate
synthase, preferably of the Schizosaccharomyces pombe
alpha,alpha-trehalose-phosphate synthase (UdP-forming) superfamily,
e.g. as indicated in Table I, columns 5 or 7, lines 127 and/or 137,
confers an increase of the respective fine chemical and of further
amino acid activity-having compounds or their precursors. In one
embodiment, the activity of the Escherichia coli K12 protein b3462
or its homologs, e.g. an activity of integral membrane cell
division protein, preferably of the cell division protein ftsX
superfamily, e.g. as indicated in Table I, columns 5 or 7, lines
150 and/or 164, confers an increase of the respective fine chemical
and of further amino acid activity-having compounds or their
precursors.
[6271] In case the activity of the Escherichia coli K12 protein
b0057 or its homologs e.g. a protein with an activity as defined in
[0022.0.14.14],e.g. as indicated in Table II, columns 5 or 7, line
517 and 530 and 546, is increased, preferably, in one embodiment
the increase of the fine chemical, preferably of citrulline between
33% and 119%, preferably of glycine between 43% and 67%, preferably
of serine between 32% and 61%, preferably of citrulline and serine
between 32% and 119%, preferably of citrulline and glycine between
33% and 119%, preferably of glycine and serine between 32% and 67%,
preferably of citrulline and glycine and serine between 32% and
119% and of further amino acids or their precursors or more is
conferred.
[6272] In case the activity of the Escherichia coli K12 protein
b0161 or its homologs e.g. a periplasmic serine protease e.g. as
indicated in Table II, columns 5 or 7, lines 492 and 505 and 537,
is increased, preferably, in one embodiment the increase of the
fine chemical, preferably of 5-oxoproline between 45% and 123% or
more, preferably of aspartic acid between 57% and 116% or more,
preferably of phenylalanine between 37% and 668% or more,
preferably of 5-oxoproline and aspartic acid between 45% and 123%
or more, preferably of 5-oxoproline and phenylalanine between 37%
and 668% or more, preferably of aspartic acid and phenylalanine
between 37% and 668% or more, preferably of 5-oxoproline and
aspartic acid and phenylalanine between 37% and 668% and of further
amino acids or their precursors or more is conferred.
[6273] In case the activity of the Escherichia coli K12 protein
b0236 or its homologs, as indicated in Table II, columns 5 or 7,
line 497, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of alanine between 22%
and 41% and of further amino acids or their precursors or more is
conferred.
[6274] In case the activity of the Escherichia coli K12 protein
b0376 or its homologs e.g. a beta-lactamase/D-ala carboxypeptidase,
penicilling binding protein e.g. as indicated in Table II, columns
5 or 7, line 493, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of 5-oxoproline between
41% and 100% and of further amino acids or their precursors or more
is conferred.
[6275] In case the activity of the Escherichia coli K12 protein
b0462 or its homologs, as indicated in Table II, columns 5 or 7,
line 518, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 36%
and 44% and of further amino acids or their precursors or more is
conferred.
[6276] In case the activity of the Escherichia coli K12 protein
b0486 or its homologs e.g. a amino-acid/amine transport protein
(APC family) e.g. as indicated in Table II, columns 5 or 7, line
498 and 547, is increased, preferably, in one embodiment the
increase of the fine respective chemical, preferably of alanine
between 44% and 52%, preferably of serine between 27% and 49%,
preferably of alanine and serine between 27% and 52% and of further
amino acids or their precursors or more, is conferred.
[6277] In case the activity of the Escherichia coli K12 protein
b0577 or its homologs e.g. a putative transport protein e.g. as
indicated in Table II, columns 5 or 7, line 506 and531, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of glycine between 36% and 50%, preferably of
aspartic acid between 56% and 65%, preferably of glycine and
aspartic acid between 36% and 65%, and of further amino acids or
their precursors or more is conferred.
[6278] In case the activity of the Escherichia coli K12 protein
b0970 or its homologs e.g. a glutamate receptor e.g. as indicated
in Table II, columns 5 or 7, line 494 and 555, is increased,
preferably, in one embodiment the increase of the fine respective
chemical, preferably of 5-oxoproline between 37% and 203%,
preferably of tyrosine between 35% and 498%, preferably of
5-oxoproline and tyrosine between 35% and 498%, and of further
amino acids or their precursors or more, is conferred.
[6279] In case the activity of the Escherichia coli K12 protein
b1228 or its homologs, as indicated in Table II, columns 5 or 7,
line 538, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of phenylalanine between
35% and 44% and of further amino acids or their precursors or more
is conferred.
[6280] In case the activity of the Escherichia coli K12 protein
b1275 or its homologs, as indicated in Table II, columns 5 or 7,
line 519, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 32%
and 50% and of further amino acids or their precursors or more is
conferred.
[6281] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1343 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 495 and 499 and 507, is increased, e.g.
the activity of a protein involved in rRNA processing and/or
translation is increased, preferred the activity of a ATP-dependent
RNA helicase, stimulated by 23S rRNA or its homolog is increased.
Preferably, an increase of the respective fine chemical, preferably
of 5-oxoproline between 37% and 87%, preferably of alanine between
27% and 44%, preferably of aspartic acid between 76% and 136%,
preferably of alanine and 5-oxoproline between 27% and 87%,
preferably of alanine and aspartic acid between 27% and 136%,
preferably of 5-oxoproline and aspartic acid between 37% and 136%,
preferably of alanine and 5-oxoproline and aspartic acid between
27% and 136%, and of further amino acids or their precursors or
more is conferred.
[6282] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1360 or a protein with the activity defined as
putative DNA replication protein or its homologs, e.g.
transcriptional regulator or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 520, is increased, preferably, in
one embodiment an increase of the fine chemical, preferably of
citrulline between 34% and 72% and of further amino acids or their
precursors or more is conferred.
[6283] In case the activity of the Escherichia coli K12 protein
b1863 or its homologs, as indicated in Table II, columns 5 or 7,
line 500, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of alanine between 22%
and 45%, and of further amino acids or their precursors or more is
conferred.
[6284] In case the activity of the Escherichia coli K12 protein
b2023 or its homologs, as indicated in Table II, columns 5 or 7,
line 508, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of aspartic acid between
56% and 81%, or more is conferred.
[6285] In case the activity of the Escherichia coli K12 protein
b2078 or its homologs, as indicated in Table II, columns 5 or 7,
line 539, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of phenylalanine between
26% and 89%, or more is conferred.
[6286] In case the activity of the Escherichia coli K12 protein
b2239 or its homologs, as indicated in Table II, columns 5 or 7,
line 521, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 37%
and 73%, and of further amino acids or their precursors or more is
conferred.
[6287] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2414 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 522 and 532 and 548 and 540, is increased,
e.g. the activity of a protein of the threonine
dehydratase-superfamily is increased preferably the activity of a
protein involved in amino acid biosynthesis, biosynthesis of the
cysteine-aromatic group, degradation of amino acids of the
cysteine-aromatic group, nitrogen and sulfur
utilizationbiosynthesis of the aspartate family, degradation of
amino acids of the aspartate group, biosynthesis of sulfuric acid
and L-cysteine derivatives, biosynthesis of secondary products
derived from primary amino acids, biosynthesis of secondary
products derived from glycine, L-serine and L-alanine, pyridoxal
phosphate binding is increased, preferred the activity of a subunit
of cysteine synthase A and O-acetylserine sulfhydrolase A,
PLP-dependent enzyme or its homolog is increased. Preferably, an
increase of the respective fine chemical, preferably of citrulline
between 31% and 40%, preferably of glycine between 38% and 62%,
preferably of serine between 27% and 53%, preferably of
phenylalanine between 27% and 197%, preferably of citrulline and
glycine between 31% and 62%, preferably of citrulline and serine
between 27% and 53%, preferably of citrulline and phenylalanine
between 27% and 197%, preferably of glycine and serine between 27%
and 62%, preferably of glycine and phenylalanine between 27% and
197%, preferably of serine and phenylalanine between 27% and 197%,
preferably of citrulline and glycine and serine between 27% and
62%, preferably of citrulline and glycine and phenylalanine between
27% and 197%, preferably of citrulline and serine and phenylalanine
between 27% and 197%, preferably of glycine and serine and
phenylalanine between 27% and 197%, preferably of citrulline and
glycine and serine and phenylalanine between 27% and 197%, or more
is conferred.
[6288] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2426 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 523, is increased, e.g. the activity of a
oxidoreductase with NAD(P)-binding domain is increased. Preferably,
an increase of the respective fine chemical, preferably of
citrulline between 32% and 41% and of further amino acids or their
precursors or more is conferred.
[6289] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2489 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 524, 533, 549 and 501, is increased, e.g.
the activity of a hydrogenase Fe--S subunit is increased.
Preferably, an increase of the respective fine chemical, preferably
of citrulline between 33% and 60%, preferably of glycine between
33% and 78%, preferably of serine between 23% and 47%, preferably
of alanine between 21% and 27%, preferably of citrulline and
glycine between 33% and 78%, preferably of citrulline and serine
between 23% and 60%, preferably of citrulline and alanine between
21% and 60%, preferably of glycine and serine between 23% and 78%,
preferably of glycine and alanine between 21% and 78%, preferably
of serine and alanine between 21% and 47%, preferably of citrulline
and glycine and serine between 23% and 78%, preferably of
citrulline and glycine and alanine between 21% and 78%, preferably
of citrulline and serine and alanine between 21% and 60%,
preferably of glycine and serine and alanine between 21% and 78%,
preferably of citrulline and glycine and serine and alanine between
21% and 78%, and of further amino acids or their precursors or more
is conferred.
[6290] In case the activity of the Escherichia coli K12 protein
b2491 or its homologs, as indicated in Table II, columns 5 or 7,
line 556, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of tyrosine between 40%
and 89%, or more is conferred.
[6291] In case the activity of the Escherichia coli K12 protein
b2507 or its homologs, as indicated in Table II, columns 5 or 7,
line 509, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of aspartic acid between
49% and 120%, or more is conferred.
[6292] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2553 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 534, is increased, e.g. the activity of a
regulatory protein P-II for glutamine synthetase is increased.
Preferably, an increase of the respective fine chemical, preferably
of glycine between 36% and 83% and of further amino acids or their
precursors or more is conferred.
[6293] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2576 or its homologs, e.g. as indicated in Table
III, columns 5 or 7, line 502 and 535 and 510 and 541, is
increased, e.g. the activity of a protein with an activity as
defined in [0022.0.14.14] is increased, an increase of the
respective fine chemical, preferably of alanine between 24% and
49%, preferably of glycine between 40% and 73%, preferably of
aspartic acid between 52% and 103%, preferably of phenylalanine
between 23% and 41%, preferably of alanine and glycine between 24%
and 73%, preferably of alanine and aspartic acid between 24% and
103%, preferably of alanine and phenylalanine between 23% and 49%,
preferably of glycine and aspartic acid between 40% and 103%,
preferably of glycine and phenylalanine between 23% and 73%,
preferably of aspartic acid and phenylalanine between 23% and 103%,
preferably of alanine and glycine and aspartic acid between 24% and
103%, preferably of alanine and glycine and phenylalanine between
23% and 73%, preferably of alanine and aspartic acid and
phenylalanine between 23% and 103%, preferably of glycine and
aspartic acid and phenylalanine between 23% and 103%, preferably of
alanine and glycine and aspartic acid and phenylalanine between 23%
and 103%, or more is conferred.
[6294] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2664 or a protein with the activity defined as
putative transcriptional repressor with DNA-binding Winged helix
domain (GntR familiy) or its homologs, e.g. transcriptional
regulator, e.g. as indicated in Table III, columns 5 or 7, line
550, is increased, preferably, in one embodiment an increase of the
fine chemical serine between 25% and 231% and of further amino
acids or their precursors or more is conferred.
[6295] In case the activity of the Escherichia coli K12 protein
b2753 or its homologs, as indicated in Table II, columns 5 or 7,
line 511, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of aspartic acid between
56% and 154%, or more is conferred.
[6296] In case the activity of the Escherichia coli K12 protein
b2796 or its homologs, as indicated in Table II, columns 5 or 7,
line 542, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of phenylalanine between
33% and 105%, or more is conferred.
[6297] In case the activity of the Escherichia coli K12 protein
b3064 or its homologs, as indicated in Table II, columns 5 or 7,
line 551, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of serine between 39% and
84%, and of further amino acids or their precursors or more is
conferred.
[6298] In case the activity of the Escherichia coli K12 protein
b3116 or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 512 and 552, is increased, e.g. the activity of a
L-threonine/L-serine permease, anaerobically inducible (HAAAP
family) is increased, preferably, an increase of the respective
fine chemical, preferably of serine between 35% and 70%, preferably
of aspartic acid between 50% and 130%, preferably of serine and
aspartic acid between 35% and 130%, and of further amino acids or
their precursors or more is conferred.
[6299] In case the activity of the Escherichia coli K12 protein
b3160 or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 525 and 553, is increased, e.g. the activity of a
monooxygenase with luciferase-like ATPase activity is increased,
preferably, an increase of the respective fine chemical, preferably
of citrulline between 34% and 48%, preferably of serine between 31%
and 60%, preferably of citrulline and serine between 31% and 60%
and of further amino acids or their precursors or more is
conferred.
[6300] In case the activity of the Escherichia coli K12 protein
b3169 or its homologs e.g. a transcription
termination-antitermination factor e.g. as indicated in Table II,
columns 5 or 7, line 513, is increased, preferably, in one
embodiment the increase of the fine chemical, preferably of
aspartic acid between 66% and 168% or more is conferred.
[6301] In case the activity of the Escherichia coli K12 protein
b3172 or its homologs, as indicated in Table II, columns 5 or 7,
line 496 and 514, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of aspartic acid between
52% and 234%, preferably of 5-oxoproline between 44% and 94%,
preferably of aspartic acid and 5-oxoproline between 44% and 234%,
and of further amino acids or their precursors or more is
conferred.
[6302] In case the activity of the Escherichia coli K12 protein
b3231 or its homologs e.g. a 50S ribosomal subunit protein L13 e.g.
as indicated in Table II, columns 5 or 7, line 503 and 554, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of alanine between 21% and 35%, preferably of
serine between 23% and 49%, preferably of alanine and serine
between 21% and 49% and of further amino acids or their precursors
or more is conferred.
[6303] In case the activity of the Escherichia coli K12 protein
b3241 or its homologs, as indicated in Table II, columns 5 or 7,
line 526, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 38%
and 70%, and of further amino acids or their precursors or more is
conferred.
[6304] In case the activity of the Escherichia coli K12 protein
b3767 or its homologs, as indicated in Table II, columns 5 or 7,
line 504 and 536, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of alanine between 24%
and 70%, preferably of homoserine between 24% and 71%, preferably
of alanine and homoserine between 24% and 71%, and of further amino
acids or their precursors or more is conferred.
[6305] In case the activity of the Escherichia coli K12 protein
b3919 or its homologs e.g. an triosephosphate isomerase e.g. as
indicated in Table II, columns 5 or 7, line 543, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of phenylalanine between 30% and 188% and of further
amino acids or their precursors or more is conferred.
[6306] In case the activity of the Escherichia coli K12 protein
b3926 or its homologs, as indicated in Table II, columns 5 or 7,
line 527, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 70%
and 94%, and of further amino acids or their precursors or more is
conferred.
[6307] In case the activity of the Escherichia coli K12 protein
b3938 or its homologs, as indicated in Table II, columns 5 or 7,
line 544, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of phenylalanine between
34% and 40%, and of further amino acids or their precursors or more
is conferred.
[6308] In case the activity of the Escherichia coli K12 protein
b3983 or its homologs, e.g. the activity of a Escherichia coli
ribosomal protein L11 superfamily, preferably a protein with with a
t50S ribosomal subunit protein L12 activity, e.g. as indicated in
Table II, columns 5 or 7, line 545 and 557, is increased conferring
an increase of the respective fine chemical, preferably
phenylalanine between 37% and 266%, preferably tyrosine between 44%
and 357%, preferably phenylalanine and tyrosine between 37% and
357% and of further amino acids or their precursors or more is
conferred.
[6309] In case the activity of the Escherichia coli K12 protein
b4129 or its homologs e.g. a lysine tRNA synthetase e.g. as
indicated in Table II, columns 5 or 7, line 515, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of aspartic acid between 53% and 141% and of further
amino acids or their precursors or more is conferred.
[6310] In case the activity of the Escherichia coli K12 protein
b4214 or its homologs, as indicated in Table II, columns 5 or 7,
line 528, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 32%
and 102%, and of further amino acids or their precursors or more is
conferred.
[6311] In case the activity of the Escherichia coli K12 protein
b4269 or its homologs, as indicated in Table II, columns 5 or 7,
line 529, e.g. protein with an activity as defined in
[0022.0.14.14], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of citrulline between 31%
and 63%, and of further amino acids or their precursors or more is
conferred.
[6312] In case the activity of the Escherichia coli K12 protein
b4346 or its homologs e.g. a component of 5-methylcytosine-specific
restriction enzyme McrBC e.g. as indicated in
[6313] Table II, columns 5 or 7, line 516, is increased,
preferably, in one embodiment the increase of the fine chemical,
preferably of aspartic acid between 63% and 94% and of further
amino acids or their precursors or more is conferred.
[6314] [0047.0.0.14] to [0048.0.0.14]: see [0047.0.0.0] to
[0048.0.0.0]
[6315] [0049.0.14.14]
[6316] A protein having an activity conferring an increase in the
amount or level of 5-oxoproline chemical preferably has the
structure of the polypeptide described herein, in particular of a
polypeptides comprising a consensus sequence as indicated in Table
IV, columns 7, lines 126 to 127 and/or 492 to 496 of a polypeptide
as indicated in Table II, columns 5 or 7, lines 126 to 127 and/or
492 to 496 the functional homologues thereof as described herein,
or is encoded by the nucleic acid molecule characterized herein or
the nucleic acid molecule according to the invention, for example
by a nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 or its herein described
functional homologues and has the herein mentioned activity.
[6317] A protein having an activity conferring an increase in the
amount or level of alanine chemical preferably has the structure of
the polypeptide described herein, in particular of a polypeptides
comprising a consensus sequence as indicated in Table IV, columns
7, lines 128 to 133 and/or 497 to 504 of a polypeptide as indicated
in Table II, columns 5 or 7, lines 128 to 133 and/or 497 to 504 the
functional homologues thereof as described herein, or is encoded by
the nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 128 to 133
and/or 497 to 504 or its herein described functional homologues and
has the herein mentioned activity.
[6318] A protein having an activity conferring an increase in the
amount or level of aspartic acid chemical preferably has the
structure of the polypeptide described herein, in particular of a
polypeptides comprising a consensus sequence as indicated in Table
IV, columns 7, lines 134 to 138 and/or 505 to 516 of a polypeptide
as indicated in Table II, columns 5 or 7, lines 134 to 138 and/or
505 to 516 the functional homologues thereof as described herein,
or is encoded by the nucleic acid molecule characterized herein or
the nucleic acid molecule according to the invention, for example
by a nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 134 to 138 and/or 505 to 516 or its herein described
functional homologues and has the herein mentioned activity.
[6319] A protein having an activity conferring an increase in the
amount or level of citrulline chemical preferably has the structure
of the polypeptide described herein, in particular of a
polypeptides comprising a consensus sequence as indicated in Table
IV, columns 7, lines 139 to 140 and/or 517 to 529 of a polypeptide
as indicated in Table II, columns 5 or 7, lines 139 to 140 and/or
517 to 529 the functional homologues thereof as described herein,
or is encoded by the nucleic acid molecule characterized herein or
the nucleic acid molecule according to the invention, for example
by a nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 139 to 140 and/or 517 to 529 or its herein described
functional homologues and has the herein mentioned activity.
[6320] A protein having an activity conferring an increase in the
amount or level of glycine chemical preferably has the structure of
the polypeptide described herein, in particular of a polypeptides
comprising a consensus sequence as indicated in Table IV, columns
7, lines 141 to 142 and/or 530 to 535 of a polypeptide as indicated
in Table II, columns 5 or 7, lines 141 to 142 and/or 530 to 535 the
functional homologues thereof as described herein, or is encoded by
the nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 141 to 142
and/or 530 to 535 or its herein described functional homologues and
has the herein mentioned activity.
[6321] A protein having an activity conferring an increase in the
amount or level of homoserine chemical preferably has the structure
of the polypeptide described herein, in particular of a
polypeptides comprising a consensus sequence as indicated in Table
IV, columns 7, lines 143 and/or 536 of a polypeptide as indicated
in Table II, columns 5 or 7, lines 143 and/or 536 the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 143 and/or
536 or its herein described functional homologues and has the
herein mentioned activity.
[6322] A protein having an activity conferring an increase in the
amount or level of phenylalanine chemical preferably has the
structure of the polypeptide described herein, in particular of a
polypeptides comprising a consensus sequence as indicated in Table
IV, columns 7, lines 144 to 156 and/or 537 to 545 of a polypeptide
as indicated in Table II, columns 5 or 7, lines 144 to 156 and/or
537 to 545 the functional homologues thereof as described herein,
or is encoded by the nucleic acid molecule characterized herein or
the nucleic acid molecule according to the invention, for example
by a nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 144 to 156 and/or 537 to 545 or its herein described
functional homologues and has the herein mentioned activity.
[6323] A protein having an activity conferring an increase in the
amount or level of serine chemical preferably has the structure of
the polypeptide described herein, in particular of a polypeptides
comprising a consensus sequence as indicated in Table IV, columns
7, lines 157 to 166 and/or 546 to 554 of a polypeptide as indicated
in Table II, columns 5 or 7, lines 157 to 166 and/or 546 to 554 the
functional homologues thereof as described herein, or is encoded by
the nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 157 to 166
and/or 546 to 554 or its herein described functional homologues and
has the herein mentioned activity.
[6324] A protein having an activity conferring an increase in the
amount or level of tyrosine chemical preferably has the structure
of the polypeptide described herein, in particular of a
polypeptides comprising a consensus sequence as indicated in Table
IV, columns 7, lines 167 to 172 and/or 555 to 557 of a polypeptide
as indicated in Table II, columns 5 or 7, lines 167 to 172 and/or
555 to 557 the functional homologues thereof as described herein,
or is encoded by the nucleic acid molecule characterized herein or
the nucleic acid molecule according to the invention, for example
by a nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 167 to 172 and/or 555 to 557 or its herein described
functional homologues and has the herein mentioned activity.
[6325] [0050.0.14.14] For the purposes of the present invention
"the respective fine chemical" also encompass the corresponding
salts, such as, for example, the potassium, amonium or sodium salts
of the respective fine chemical or the respective amino acid
hydrochloride or sulfateof 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine.
[6326] [0051.0.0.14] to [0052.0.0.14]: see [0051.0.0.0] to
[0052.0.0.0]
[6327] [0053.0.14.14] In one embodiment, the process of the present
invention comprises one or more of the following steps:
a) stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 126
to 127 and/or 492 to 496 for 5-oxoproline and/or lines 128 to 133
and/or 497 to 504 for alanine and/or lines 134 to 138 and/or 505 to
516 for aspartic acid and/or lines 139 to 140 and/or 517 to 529 for
citrulline and/or lines 141 to 142 and/or 530 to 535 for glycine
and/or lines 143 and/or 536 for homoserine and/or lines 144 to 156
and/or 537 to 545 for phenylalanine and/or lines 157 to 166 and/or
546 to 554 for serine and/or lines 167 to 172 and/or 555 to 557 for
tyrosine resp., or its homologs activity, e.g. as indicated in
Table II, columns 5 or 7, lines 126 to 127 and/or 492 to 496 for
5-oxoproline and/or lines 128 to 133 and/or 497 to 504 for alanine
and/or lines 134 to 138 and/or 505 to 516 for aspartic acid and/or
lines 139 to 140 and/or 517 to 529 for citrulline and/or lines 141
to 142 and/or 530 to 535 for glycine and/or lines 143 and/or 536
for homoserine and/or lines 144 to 156 and/or 537 to 545 for
phenylalanine and/or lines 157 to 166 and/or 546 to 554 for serine
and/or lines 167 to 172 and/or 555 to 557 for tyrosine resp.,
having herein-mentioned the respective fine chemical-increasing
activity; b) stabilizing a mRNA conferring the increased expression
of a protein encoded by the nucleic acid molecule of the invention,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., or of a mRNA encoding the
polypeptide of the present invention having herein-mentioned the
respective fine chemical increasing activity; c) increasing the
specific activity of a protein conferring the increased expression
of a protein encoded by the nucleic acid molecule of the invention
or of the polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., or decreasing the inhibitory
regulation of the polypeptide of the invention; d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 126 to 127 and/or
492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines
134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to
529 and/or lines 141 to 142 and/or 530 to 535 and/or lines 143
and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or lines
157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to
557, resp., or its homologs activity, e.g. as indicated in Table
II, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp.; e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., by adding one or more exogenous
inducing factors to the organisms or parts thereof; f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned the respective fine chemical increasing
activity, e.g. of a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
or its homologs activity, e.g. as indicated in Table II, columns 5
or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp.; g) increasing the copy
number of a gene conferring the increased expression of a nucleic
acid molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned the respective fine chemical increasing
activity, e.g. of a polypeptide havingan an activity of a protein
as indicated in Table II, column 3, lines 126 to 127 and/or 492 to
496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to
138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp.; h) Increasing the
expression of the endogenous gene encoding the polypeptide of the
invention, e.g. a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
or its homologs activity, e.g. as indicated in Table II, columns 5
or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., by adding positive
expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; i)
Modulating growth conditions of an organism in such a manner, that
the expression or activity of the gene encoding the protein of the
invention or the protein itself is enhanced for example
microorganisms or plants can be grown under a higher temperature
regime leading to an enhanced expression of heat shock proteins,
e.g. the heat shock protein of the invention, which can lead an
enhanced the fine chemical production; and/or j) selecting of
organisms with especially high activity of the proteins of the
invention from natural or from mutagenized resources and breeding
them into the target organisms, eg the elite crops.
[6328] [0054.0.14.14] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein according to Table II, column 3, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp.
[6329] [0055.0.0.14] to [0071.0.0.14]: see [0055.0.0.0] to
[0071.0.0.0]
[6330] [0072.0.14.14] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, homoserine, phenylalanine, serine and/or tyrosine further
amino acids or the respective precursors.
[6331] [0073.0.14.14] Accordingly, in one embodiment, the process
according to the invention relates to a process which comprises:
[6332] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [6333] b) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table II, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., or of a polypeptide being encoded by the nucleic acid
molecule of the present invention and described below, i.e.
conferring an increase of the respective fine chemical in the
organism, preferably in a microorganism, a non-human animal, a
plant or animal cell, a plant or animal tissue or a plant, [6334]
c) growing the organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a
plant, under conditions which permit the production of the
respective fine chemical in the organism, preferably the
microorganism, the plant cell, the plant tissue or the plant; and
[6335] d) if desired, recovering, optionally isolating, the free
and/or bound the respective fine chemical and, optionally further
free and/or bound amino acids synthetized by the organism, the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue or the plant.
[6336] [0074.0.0.14] to [0084.0.0.14]: see [0075.0.0.0] to
[0084.0.0.0]
[6337] [0085.0.14.14] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [6338] a) a nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., or a derivative thereof, or [6339] b) a genetic regulatory
element, for example a promoter, which is functionally linked to
the nucleic acid sequence as indicated in Table I, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., or a derivative thereof, or [6340]
c) (a) and (b) is/are not present in its/their natural genetic
environment or has/have been modified by means of genetic
manipulation methods, it being possible for the modification to be,
by way of example, a substitution, addition, deletion, inversion or
insertion of one or more nucleotide. "Natural genetic environment"
means the natural chromosomal locus in the organism of origin or
the presence in a genomic library. In the case of a genomic
library, the natural, genetic environment of the nucleic acid
sequence is preferably at least partially still preserved. The
environment flanks the nucleic acid sequence at least on one side
and has a sequence length of at least 50 bp, preferably at least
500 bp, particularly preferably at least 1000 bp, very particularly
preferably at least 5000 bp.
[6341] [0086.0.0.14] to [0088.1.0.14]: see [0086.0.0.0] to
[0088.1.0.0]
[6342] [0089.0.0.14] to [0097.0.0.14]: see [0089.0.0.0] to
[0097.0.0.0]
[6343] [0098.0.14.14] In a preferred embodiment, the respective
fine chemical (5-oxoproline, alanine, aspartic acid, citrulline,
glycine, homoserine, phenylalanine, serine and/or tyrosine) are
produced in accordance with the invention and, if desired, are
isolated. The production of further amino acids and of amino acid
mixtures or mixtures with other compounds by the process according
to the invention is advantageous.
[6344] [0099.0.0.14] to [0102.0.0.14]: see [0099.0.0.0] to
[0102.0.0.0]
[6345] [0103.0.14.14] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [6346] a) nucleic acid molecule encoding, preferably
at least the mature form, of a polypeptide having a sequence as
indicated in Table II, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [6347] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table I, columns 5 or 7, lines 126 to 127 and/or
492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines
134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to
529 and/or lines 141 to 142 and/or 530 to 535 and/or lines 143
and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or lines
157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to
557, resp.; [6348] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [6349] d) nucleic
acid molecule encoding a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [6350] e) nucleic acid molecule which hybridizes with
a nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[6351] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[6352] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [6353] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers pairs having a sequence as indicated in
Table III, columns 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [6354] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from an
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [6355] j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence having a sequences as
indicated in Table IV, columns 7, lines 126 to 127 and/or 492 to
496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to
138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [6356] k) nucleic
acid molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a
domaine of a polypeptide indicated in Table II, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; and [6357] l) nucleic acid molecule which is obtainable by
screening a suitable library under stringent conditions with a
probe comprising one of the sequences of the nucleic acid molecule
of (a) to (k), preferably to (a) to (c), or with a fragment of at
least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500
nt of the nucleic acid molecule characterized in (a) to (k),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
or which comprises a sequence which is complementary thereto.
[6358] [00103.1.0.14.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., by one or more nucleotides. In one embodiment,
the nucleic acid molecule used in the process of the invention does
not consist of the sequence shown in indicated in Table I A,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp. In one embodiment,
the nucleic acid molecule used in the process of the invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table I A, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp. In another embodiment, the nucleic acid molecule
does not encode a polypeptide of a sequence indicated in Table II
A, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp.
[6359] [00103.2.0.14.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., by one or more nucleotides. In one embodiment,
the nucleic acid molecule used in the process of the invention does
not consist of the sequence shown in indicated in Table I B,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp. In one embodiment,
the nucleic acid molecule used in the process of the invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table I B, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp. In another embodiment, the nucleic acid molecule
does not encode a polypeptide of a sequence indicated in Table II
B, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp.
[6360] [0104.0.14.14] In one embodiment, the nucleic acid molecule
of the invention distinguishes over the sequence indicated in Table
I, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., by one or more
nucleotides. In one embodiment, the nucleic acid molecule of the
invention does not consist of the sequence shown in indicated in
Table I, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp. In one
embodiment, the nucleic acid molecule of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table I, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp. In another embodiment, the nucleic acid molecule
does not encode a polypeptide of a sequence indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp.
[6361] [0105.0.0.14] to [0107.0.0.14]: see [0105.0.0.0] to
[0107.0.0.0]
[6362] [0108.0.14.14] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., nucleic acid molecules which are derived from a amino acid
sequences as indicated in Table II, columns 5 or 7, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., or from polypeptides comprising the
consensus sequence as indicated in Table IV, columns 7, lines 126
to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., or their derivatives or homologues
encoding polypeptides with the enzymatic or biological activity of
a polypeptide as indicated in Table I, column 3, 5 or 7, lines 126
to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., or e.g. conferring a increase of the
respective fine chemical after increasing its expression or
activity are advantageously increased in the process according to
the invention.
[6363] [0109.0.0.14]: see [0109.0.0.0]
[6364] [0110.0.14.14] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
used in the process of the invention, e.g. of a protein as
indicated in II, column 5, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 126 to 127 and/or 492 to 496 and/or lines 128 to
133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., or of its
homologs, e.g. as indicated in Table II, column 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., can be determined from generally accessible
databases.
[6365] [0111.0.0.14]: see [0111.0.0.0]
[6366] [0112.0.14.14] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table I, column 3, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., or having the sequence of a polypeptide
as indicated in Table II, columns 5 and 7, lines 126 to 127 and/or
492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines
134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to
529 and/or lines 141 to 142 and/or 530 to 535 and/or lines 143
and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or lines
157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to
557, resp., and conferring a 5-oxoproline and/or alanine and/or
aspartic acid and/or citrulline and/or glycine and/or homoserine
and/or phenylalanine and/or serine and/or tyrosine increase.
[6367] [0113.0.0.14] to [0120.0.0.14]: see [0113.0.0.0] to
[0120.0.0.0]
[6368] [0121.0.14.14] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., or the functional
homologues thereof as described herein, preferably conferring
above-mentioned activity, i.e. conferring a increase of the
respective fine chemical after increasing its activity.
[6369] [0122.0.0.14] to [0127.0.0.14]: see [0122.0.0.0] to
[0127.0.0.0]
[6370] [0128.0.14.14] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, column 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table I, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., or the sequences derived from
sequences as indicated in Table II, columns 5 or 7, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp.
[6371] [0129.0.14.14] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequences indicated in Table IV,
column 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to
133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., are derived from
said alignments.
[6372] [0130.0.14.14] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of amino
acids, e.g. of 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, homoserine, phenylalanine, serine and/or tyrosine after
increasing its expression or activity of the protein comprising
said fragment.
[6373] [0131.0.0.14] to [0138.0.0.14]: see [0131.0.0.0] to
[0138.0.0.0]
[6374] [0139.0.14.14] Polypeptides having above-mentioned activity,
i.e. conferring an increase of the respective fine chemical level,
derived from other organisms, can be encoded by other DNA sequences
which hybridize to a sequences indicated in Table I, columns 5 or
7, preferably Table I B, lines 126 to 127 and/or 492 to 496 for
5-oxoproline and/or lines 128 to 133 and/or 497 to 504 for alanine
and/or lines 134 to 138 and/or 505 to 516 for aspartic acid and/or
lines 139 to 140 and/or 517 to 529 for citrulline and/or lines 141
to 142 and/or 530 to 535 for glycine and/or lines 143 and/or 536
for homoserine and/or lines 144 to 156 and/or 537 to 545 for
phenylalanine and/or lines 157 to 166 and/or 546 to 554 for serine
and/or lines 167 to 172 and/or 555 to 557 for tyrosine resp., under
relaxed hybridization conditions and which code on expression for
peptides having the respective fine chemical, in particular, of
5-oxoproline and/or alanine and/or aspartic acid and/or citrulline
and/or glycine and/or homoserine and/or phenylalanine and/or serine
and/or tyrosine resp., increasing activity.
[6375] [0140.0.0.14] to [0146.0.0.14]: see [0140.0.0.0] to
[0146.0.0.0]
[6376] [0147.0.14.14] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., preferably of
Table I B, columns 5 or 7, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
is one which is sufficiently complementary to one of said
nucleotide sequences s such that it can hybridize to one of said
nucleotide sequences thereby forming a stable duplex. Preferably,
the hybridisation is performed under stringent hybridization
conditions. However, a complement of one of the herein disclosed
sequences is preferably a sequence complement thereto according to
the base pairing of nucleic acid molecules well known to the
skilled person. For example, the bases A and G undergo base pairing
with the bases T and U or C, resp. and visa versa. Modifications of
the bases can influence the base-pairing partner.
[6377] [0148.0.14.14] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7,
preferably Table I B, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
preferably Table I B or a portion thereof and preferably has above
mentioned activity, in particular, of arginine and/or glutamate
and/or proline and/or glutamine increasing activity after
increasing the activity or an activity of a product of a gene
encoding said sequences or their homologs.
[6378] [0149.0.14.14] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., preferably of Table I B, columns 5
or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., or a portion thereof and
encodes a protein having above-mentioned activity and as indicated
in indicated in Table II.
[6379] [00149.1.14.14] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., preferably of
Table I B, columns 5 or 7, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
has further one or more of the activities annotated or known for
the a protein as indicated in Table II, column 3, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp.
[6380] [0150.0.14.14] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table I, columns 5 or 7, preferably
Table I B, lines 126 to 127 and/or 492 to 496 and/or lines 128 to
133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., for example a
fragment which can be used as a probe or primer or a fragment
encoding a biologically active portion of the polypeptide of the
present invention or of a polypeptide used in the process of the
present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of arginine and/or glutamate and/or proline
and/or glutamine, resp., if its activity is increased. The
nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table I, columns
5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., an anti-sense sequence
of one of the sequences, e.g., as indicated in Table I, columns 5
or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., or naturally occurring
mutants thereof. Primers based on a nucleotide of invention can be
used in PCR reactions to clone homologues of the polypeptide of the
invention or of the polypeptide used in the process of the
invention, e.g. as the primers described in the examples of the
present invention, e.g. as shown in the examples. A PCR with the
primer pairs indicated in Table III, column 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., will result in a fragment of a polynucleotide
sequence as indicated in Table I, columns 5 or 7 lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., or its gene product.
[6381] [0151.0.0.14] see [0151.0.0.0]
[6382] [0152.0.14.14] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., such that the protein or portion thereof
maintains the ability to participate in the respective fine
chemical production, in particular an activity increasing the level
of 5-oxoproline and/or alanine and/or aspartic acid and/or
citrulline and/or glycine and/or homoserine and/or phenylalanine
and/or serine and/or tyrosine as mentioned above or as described in
the examples in plants or microorganisms is comprised.
[6383] [0153.0.14.14] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table I, columns 5 or 7, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., such that the protein or portion thereof
is able to participate in the increase of the respective fine
chemical production. In one embodiment, a protein or portion
thereof as indicated in Table II, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., has for example an activity of a polypeptide
indicated in Table II, column 3, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp.
[6384] [0154.0.14.14] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., and has above-mentioned activity, e.g.
conferring preferably the increase of the respective fine
chemical.
[6385] [0155.0.0.14] to [0156.0.0.14]: see [0155.0.0.0] to
[0156.0.0.0]
[6386] [0157.0.14.14] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., (and portions thereof) due to degeneracy of the genetic code
and thus encode a polypeptide of the present invention, in
particular a polypeptide having above mentioned activity, e.g.
conferring an increase in the respective fine chemical in a
organism, e.g. as that polypeptides comprising the consensus
sequences as indicated in Table IV, column 7 lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., or of the polypeptide as indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., or their
functional homologues. Advantageously, the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, a consensus sequences as indicated in Table IV,
column 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to
133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., or of the
polypeptide as indicated in Table II, columns 5 or 7, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., or the functional homologues. In a still
further embodiment, the nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
encodes a full length protein which is substantially homologous to
an amino acid sequence comprising a consensus sequence as indicated
in Table IV, column 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., or of
a polypeptide as indicated in Table II, columns 5 or 7, lines 126
to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., or the functional homologues thereof.
However, in a preferred embodiment, the nucleic acid molecule of
the present invention does not consist of a sequence as indicated
in Table I A, columns 5 or 7 lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
preferably as indicated in Table I A, columns 5 or 7, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp. Preferably the nucleic acid molecule of
the invention is a functional homologue or identical to a nucleic
acid molecule indicated in Table I B, columns 5 or 7, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp.
[6387] [0158.0.0.14] to [0160.0.0.14]: see [0158.0.0.0] to
[0160.0.0.0]
[6388] [0161.0.14.14] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp. The nucleic acid molecule is preferably at least
20, 30, 50, 100, 250 or more nucleotides in length.
[6389] [0162.0.0.14] see [0162.0.0.0]
[6390] [0163.0.14.14] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 126 to 127 and/or
492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines
134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to
529 and/or lines 141 to 142 and/or 530 to 535 and/or lines 143
and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or lines
157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to
557, resp., corresponds to a naturally-occurring nucleic acid
molecule of the invention. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein). Preferably, the nucleic acid molecule encodes a natural
protein having above-mentioned activity, e.g. conferring the
respective fine chemical increase after increasing the expression
or activity thereof or the activity of an protein of the invention
or used in the process of the invention.
[6391] [0164.0.0.14] see [0164.0.0.0]
[6392] [0165.0.14.14] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp.
[6393] [0166.0.0.14] to [0167.0.0.14]: see [0166.0.0.0] to
[0167.0.0.0]
[6394] [0168.0.14.14] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., preferably of Table II B, column
7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino add sequence as indicated in Table II, columns 5 or 7, lines
126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to
504 and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to
140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535
and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to
545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to
172 and/or 555 to 557, resp., preferably of Table II B, column 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., and is capable of participation in
the increase of production of the respective fine chemical after
increasing its activity, e.g. its expression. Preferably, the
protein encoded by the nucleic acid molecule is at least about 60%
identical to a sequence as indicated in Table II, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., preferably of Table II B, column
7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., more preferably at least
about 70% identical to one of the sequences as indicated in Table
II, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., preferably of
Table II B, column 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., even
more preferably at least about 80%, 90%, or 95% homologous to a
sequence as indicated in Table II, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., preferably of Table II B, column 7, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., and most preferably at least about 96%,
97%, 98%, or 99% identical to the sequence as indicated in Table
II, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., preferably of
Table II B, column 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.
[6395] [0169.0.0.14] to [0172.0.0.14]: see [0169.0.0.0] to
[0172.0.0.0]
[6396] [0173.0.14.14] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 21744 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 21744 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[6397] [0174.0.0.14] see [0174.0.0.0]
[6398] [0175.0.14.14] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 21745 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 21745 by the above program algorithm with the
above parameter set, has a 80% homology.
[6399] [0176.0.14.14] Functional equivalents derived from one of
the polypeptides as indicated in Table II, columns 5 or 7, lines
126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to
504 and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to
140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535
and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to
545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to
172 and/or 555 to 557, resp., according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., according to the
invention and are distinguished by essentially the same properties
as a polypeptide as indicated in Table II, columns 5 or 7, lines
126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to
504 and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to
140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535
and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to
545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to
172 and/or 555 to 557, resp.
[6400] [0177.0.14.14] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., preferably of Table I B, column 7, lines
126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to
504 and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to
140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535
and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to
545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to
172 and/or 555 to 557, resp., according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of a polypeptide as indicated in Table II,
columns 5 or 7 lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., according to the
invention and encode polypeptides having essentially the same
properties as a polypeptide as indicated in Table II, columns 5 or
7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., preferably of Table I B,
column 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to
133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp.
[6401] [0178.0.0.14]: see [0178.0.0.0]
[6402] [0179.0.14.14] A nucleic acid molecule encoding an
homologous to a protein sequence as indicated in Table II, columns
5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., preferably of Table II
B, column 7 lines 126 to 127 and/or 492 to 496 and/or lines 128 to
133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., can be created by
introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of the nucleic acid molecule
of the present invention, in particular as indicated in Table I,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., such that one or
more amino acid substitutions, additions or deletions are
introduced into the encoded protein. Mutations can be introduced
into the encoding sequences of a sequences as indicated in Table I,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[6403] [0180.0.0.14] to [0183.0.0.14]: see [0180.0.0.0] to
[0183.0.0.0]
[6404] [0184.0.14.14] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table I, preferably Table I
B, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., or of the nucleic
acid sequences derived from a sequences as indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., comprise also
allelic variants with at least approximately 30%, 35%, 40% or 45%
homology, by preference at least approximately 50%, 60% or 70%,
more preferably at least approximately 90%, 91%, 92%, 93%, 94% or
95% and even more preferably at least approximately 96%, 97%, 98%,
99% or more homology with one of the nucleotide sequences shown or
the abovementioned derived nucleic acid sequences or their
homologues, derivatives or analogues or parts of these. Allelic
variants encompass in particular functional variants which can be
obtained by deletion, insertion or substitution of nucleotides from
the sequences shown, preferably from a sequence as indicated in
Table I, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., or
from the derived nucleic acid sequences, the intention being,
however, that the enzyme activity or the biological activity of the
resulting proteins synthesized is advantageously retained or
increased.
[6405] [0185.0.14.14] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table I, preferably Table I B, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp. In one embodiment, it is preferred that the
nucleic acid molecule comprises as little as possible other
nucleotides not shown in any one of sequences as indicated in Table
I, preferably table I B, columns 5 or 7, lines 126 to 127 and/or
492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines
134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to
529 and/or lines 141 to 142 and/or 530 to 535 and/or lines 143
and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or lines
157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to
557, resp. In one embodiment, the nucleic acid molecule comprises
less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or 40 further
nucleotides. In a further embodiment, the nucleic acid molecule
comprises less than 30, 20 or 10 further nucleotides. In one
embodiment, a nucleic acid molecule used in the process of the
invention is identical to a sequences as indicated in Table I,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp.,
[6406] [0186.0.14.14] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II,
preferably table II B, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp. In one embodiment, the nucleic acid molecule encodes less
than 150, 130, 100, 80, 60, 50, 40 or 30 further amino acids. In a
further embodiment, the encoded polypeptide comprises less than 20,
15, 10, 9, 8, 7, 6 or 5 further amino acids. In one embodiment, the
encoded polypeptide used in the process of the invention is
identical to the sequences as indicated in Table II, preferably
table II B, columns 5 or 7, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp.
[6407] [0187.0.14.14] In one embodiment, a nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence as indicated in Table II, preferably Table II B, columns 5
or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp.
[6408] [0188.0.14.14] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., and is expressed
under identical conditions.
[6409] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in Ti
B, columns 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp.
[6410] [0189.0.14.14 Homologues of a sequences as indicated in
Table I, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., or of
a derived sequences as indicated in Table II, columns 5 or 7 lines
126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to
504 and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to
140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535
and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to
545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to
172 and/or 555 to 557, resp., also mean truncated sequences, cDNA,
single-stranded DNA or RNA of the coding and noncoding DNA
sequence. Homologues of said sequences are also understood as
meaning derivatives, which comprise noncoding regions such as, for
example, UTRs, terminators, enhancers or promoter variants. The
promoters upstream of the nucleotide sequences stated can be
modified by one or more nucleotide substitution(s), insertion(s)
and/or deletion(s) without, however, interfering with the
functionality or activity either of the promoters, the open reading
frame (=ORF) or with the 3'-regulatory region such as terminators
or other 3' regulatory regions, which are far away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[6411] [0190.0.0.14] to [0203.0.0.14]: see [0190.0.0.0] to
[0203.0.0.0]
[6412] [0204.0.14.14] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule which comprises a nucleic acid
molecule selected from the group consisting of: [6413] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, preferably Table II B,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., or a fragment
thereof conferring an increase in the amount of the respective fine
chemical, in particular, of 5-oxoproline (lines 126 to 127 and/or
492 to 496) and/or alanine (lines 128 to 133 and/or 497 to 504)
and/or aspartic acid (lines 134 to 138 and/or 505 to 516) and/or
citrulline (lines 139 to 140 and/or 517 to 529) and/or glycine
(lines 141 to 142 and/or 530 to 535) and/or homoserine (lines 143
and/or 536) and/or phenylalanine (lines 144 to 156 and/or 537 to
545) and/or serine (lines 157 to 166 and/or 546 to 554) and/or
tyrosine (lines 167 to 172 and/or 555 to 557), resp., in an
organism or a part thereof [6414] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., or a fragment thereof conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [6415] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [6416] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[6417] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [6418] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [6419] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [6420] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table III, column 7, lines 126 to 127 and/or 492 to
496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to
138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., and conferring an increase in the amount of the respective
fine chemical, in particular, of 5-oxoproline (lines 126 to 127
and/or 492 to 496) and/or alanine (lines 128 to 133 and/or 497 to
504) and/or aspartic acid (lines 134 to 138 and/or 505 to 516)
and/or citrulline (lines 139 to 140 and/or 517 to 529) and/or
glycine (lines 141 to 142 and/or 530 to 535) and/or homoserine
(lines 143 and/or 536) and/or phenylalanine (lines 144 to 156
and/or 537 to 545) and/or serine (lines 157 to 166 and/or 546 to
554) and/or tyrosine (lines 167 to 172 and/or 555 to 557), resp.,
in an organism or a part thereof; [6421] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from a expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[6422] j) nucleic acid molecule which encodes a polypeptide
comprising a consensus sequence as indicated in Table IV, columns 5
or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., and conferring an
increase in the amount of the respective fine chemical, in
particular, of 5-oxoproline (lines 126 to 127 and/or 492 to 496)
and/or alanine (lines 128 to 133 and/or 497 to 504) and/or aspartic
acid (lines 134 to 138 and/or 505 to 516) and/or citrulline (lines
139 to 140 and/or 517 to 529) and/or glycine (lines 141 to 142
and/or 530 to 535) and/or homoserine (lines 143 and/or 536) and/or
phenylalanine (lines 144 to 156 and/or 537 to 545) and/or serine
(lines 157 to 166 and/or 546 to 554) and/or tyrosine (lines 167 to
172 and/or 555 to 557), resp., in an organism or a part thereof;
[6423] k) nucleic acid molecule encoding the amino acid sequence of
a polypeptide encoding a domaine of a polypeptide as indicated in
Table II, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., and
conferring an increase in the amount of the respective fine
chemical, in particular, of 5-oxoproline (lines 126 to 127 and/or
492 to 496) and/or alanine (lines 128 to 133 and/or 497 to 504)
and/or aspartic acid (lines 134 to 138 and/or 505 to 516) and/or
citrulline (lines 139 to 140 and/or 517 to 529) and/or glycine
(lines 141 to 142 and/or 530 to 535) and/or homoserine (lines 143
and/or 536) and/or phenylalanine (lines 144 to 156 and/or 537 to
545) and/or serine (lines 157 to 166 and/or 546 to 554) and/or
tyrosine (lines 167 to 172 and/or 555 to 557), resp., in an
organism or a part thereof; and [6424] l) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (h) or of a nucleic acid molecule as indicated in Table
I, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., or a nucleic acid
molecule encoding, preferably at least the mature form of, a
polypeptide as indicated in Table II, columns 5 or 7, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[6425] or which encompasses a sequence which is complementary
thereto; [6426] whereby, preferably, the nucleic acid molecule
according to (a) to (l) distinguishes over the sequence indicated
in Table IA, columns 5 or 7, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
by one or more nucleotides. In one embodiment, the nucleic acid
molecule does not consist of the sequence shown and indicated in
Table I A or I B, columns 5 or 7, lines 126 to 127 and/or 492 to
496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to
138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp. [6427] In one embodiment, the nucleic acid molecule is less
than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table I A or I B, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp. [6428] In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table II A or II B, columns 5 or 7, lines 126 to 127 and/or 492 to
496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to
138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp. [6429] In an other embodiment, the nucleic acid molecule of
the present invention is at least 30%, 40%, 50%, or 60% identical
and less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table I A or I B, columns 5 or 7, lines 126
to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp. [6430] In a further embodiment the nucleic
acid molecule does not encode a polypeptide sequence as indicated
in Table II A or II B, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp. [6431] Accordingly, in one embodiment, the nucleic acid
molecule of the differs at least in one or more residues from a
nucleic acid molecule indicated in Table I A or I B, columns 5 or
7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp. [6432] Accordingly, in
one embodiment, the nucleic acid molecule of the present invention
encodes a polypeptide, which differs at least in one or more amino
acids from a polypeptide indicated in Table II A or I B, columns 5
or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp. [6433] In another
embodiment, a nucleic acid molecule indicated in Table I A or I B,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., does not encode a
protein of a sequence indicated in Table II A or II B, columns 5 or
7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp. [6434] Accordingly, in
one embodiment, the protein encoded by a sequences of a nucleic
acid according to (a) to (l) does not consist of a sequence as
indicated in Table II A or II B, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp. [6435] In a further embodiment, the protein of
the present invention is at least 30%, 40%, 50%, or 60% identical
to a protein sequence indicated in Table II A or II B, columns 5 or
7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., and less than 100%,
preferably less than 99.999%, 99.99% or 99.9%, more preferably less
than 99%, 985, 97%, 96% or 95% identical to a sequence as indicated
in Table I A or II B, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp.
[6436] [0205.0.0.14] to [0226.0.0.14]: see [0205.0.0.0] to
[0226.0.0.0]
[6437] [0227.0.14.14] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[6438] In addition to a sequence indicated in Table I, columns 5 or
7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., or its derivatives, it
is advantageous additionally to express and/or mutate further genes
in the organisms. Especially advantageously, additionally at least
one further gene of the amino acid biosynthetic pathway such as for
amino acid precursors is expressed in the organisms such as plants
or microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the amino acids desired since, for example, feedback
regulations no longer exist to the same extent or not at all. In
addition it might be advantageously to combine a sequence as
indicated in Table I, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., with genes which generally support or enhances to growth or
yield of the target organisms, for example genes which lead to
faster growth rate of microorganisms or genes which produces
stress-, pathogen, or herbicide resistant plants.
[6439] [0228.0.0.14] to [0230.0.0.14]: see [0228.0.0.0] to
[0230.0.0.0]
[6440] [00231.0.14.14] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a protein degrading 5-oxoproline
and/or alanine and/or aspartic acid and/or citrulline and/or
glycine and/or homoserine and/or phenylalanine and/or serine and/or
tyrosine resp., is attenuated, in particular by reducing the rate
of expression of the corresponding gene.
[6441] [0232.0.0.14] to [0282.0.0.14]: see [0232.0.0.0] to
[0282.0.0.0]
[6442] [0283.0.14.14] Moreover, a native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table II,
column 3, lines 126 to 127 and/or 492 to 496 and/or lines 128 to
133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., or an antibody
against a polypeptide as indicated in Table II, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., which can be produced by standard
techniques utilizing polypeptides comprising or consisting of above
mentioned sequences, e.g. the polypeptid of the present invention
or fragment thereof. Preferred are monoclonal antibodies.
[6443] [0284.0.0.14] see [0284.0.0.0]
[6444] [0285.0.14.14] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., or as coded by a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., or functional homologues
thereof.
[6445] [0286.0.14.14] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp. In another
embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table IV, column 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., whereby 20 or
less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred
5 or 4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table IV, column 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp.
[6446] [0287.0.0.14] to [0290.0.0.14]: see [0287.0.0.0] to
[0290.0.0.0]
[6447] [0291.0.14.14] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. In one embodiment, said polypeptide of the
invention distinguishes over a sequence as indicated in Table II A
or II B, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., by one
or more amino acids. In one embodiment, polypeptide distinguishes
form a sequence as indicated in Table II A or II B, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., by more than 1, 2, 3, 4, 5, 6, 7,
8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or 30
amino acids, evenmore preferred are more than 40, 50, or 60 amino
acids and, preferably, the sequence of the polypeptide of the
invention distinguishes from a sequence as indicated in Table II A
or II B, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., by not
more than 80% or 70% of the amino acids, preferably not more than
60% or 50%, more preferred not more than 40% or 30%, even more
preferred not more than 20% or 10%. In an other embodiment, said
polypeptide of the invention does not consist of a sequence as
indicated in Table II A or II B, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp.
[6448] [0292.0.0.14]: see [0292.0.0.0]
[6449] [0293.0.14.14] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, the polypeptide of
the invention has a sequence which distinguishes from a sequence as
indicated in Table II A or II B, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., by one or more amino acids. In an other
embodiment, said polypeptide of the invention does not consist of
the sequence as indicated in Table II A or II B, columns 5 or 7,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp.
[6450] In a further embodiment, said polypeptide of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by a nucleic acid molecules as indicated in
Table I A or IB, columns 5 or 7, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp.
[6451] [0294.0.14.14] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
which distinguishes over a sequence as indicated in Table II A or
Table II B, columns 5 or 7, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
by one or more amino acids, preferably by more than 5, 6, 7, 8 or 9
amino acids, preferably by more than 10, 15, 20, 25 or 30 amino
acids, evenmore preferred are more than 40, 50, or 60 amino acids
but even more preferred by less than 70% of the amino acids, more
preferred by less than 50%, even more preferred my less than 30% or
25%, more preferred are 20% or 15%, even more preferred are less
than 10%.
[6452] [0295.0.0.14] to [0297.0.0.14]: see [0295.0.0.0] to
[0297.0.0.0]
[6453] [0297.1.14.14] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table II, columns 3, 5 or 7, lines 126 to 127 and/or
492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines
134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to
529 and/or lines 141 to 142 and/or 530 to 535 and/or lines 143
and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or lines
157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to
557, resp.
[6454] [0298.0.14.14] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table II, columns 5 or 7, lines 126 to 127 and/or
492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines
134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to
529 and/or lines 141 to 142 and/or 530 to 535 and/or lines 143
and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or lines
157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to
557, resp., such that the protein or portion thereof maintains the
ability to confer the activity of the present invention. The
portion of the protein is preferably a biologically active portion
as described herein. Preferably, the polypeptide used in the
process of the invention has an amino acid sequence identical to a
sequence as indicated in Table II, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp.
[6455] [0299.0.14.14] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to
504 and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to
140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535
and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to
545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to
172 and/or 555 to 557, resp.
[6456] The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table I, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., or
which is homologous thereto, as defined above.
[6457] [0300.0.14.14] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., in amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of a sequence as indicated in Table
II A or II B, columns 5 or 7, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp.
[6458] [0301.0.0.14] see [0301.0.0.0]
[6459] [0302.0.14.14] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table II, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., or the
amino acid sequence of a protein homologous thereto, which include
fewer amino acids than a full length polypeptide of the present
invention or used in the process of the present invention or the
full length protein which is homologous to an polypeptide of the
present invention or used in the process of the present invention
depicted herein, and exhibit at least one activity of polypeptide
of the present invention or used in the process of the present
invention.
[6460] [0303.0.0.14] see [0303.0.0.0]
[6461] [0304.0.14.14] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., but having
differences in the sequence from said wild-type protein. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity in relation to the wild type protein.
[6462] [0305.0.0.14] to [0308.0.0.14]: see [0305.0.0.0] to
[0308.0.0.0]
[6463] [0309.0.14.14] In one embodiment, an reference to a "protein
(=polypeptide)" of the invention or as indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., refers to a
polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas a "non-polypeptide of the invention" or "other
polypeptide" not being indicated in Table II, columns 5 or 7, lines
126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to
504 and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to
140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535
and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to
545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to
172 and/or 555 to 557, resp., refers to a polypeptide having an
amino acid sequence corresponding to a protein which is not
substantially homologous a polypeptide of the invention, preferably
which is not substantially homologous to a as indicated in Table
II, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines
128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to
516 and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to
142 and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144
to 156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., e.g., a protein
which does not confer the activity described herein or annotated or
known for as indicated in Table II, column 3, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., and which is derived from the same or a
different organism. In one embodiment, a "non-polypeptide of the
invention" or "other polypeptide" not being indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., does not confer
an increase of the respective fine chemical in an organism or part
thereof.
[6464] [0310.0.0.14] to [0334.0.0.14]: see [0310.0.0.0] to
[0334.0.0.0]
[6465] [0335.0.14.14] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table I, columns 5 or
7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., and/or homologs thereof.
As described inter alia in WO 99/32619, dsRNAi approaches are
clearly superior to traditional antisense approaches. The invention
therefore furthermore relates to double-stranded RNA molecules
(dsRNA molecules) which, when introduced into an organism,
advantageously into a plant (or a cell, tissue, organ or seed
derived therefrom), bring about altered metabolic activity by the
reduction in the expression of a nucleic acid sequences as
indicated in Table I, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., and/or homologs thereof. In a double-stranded RNA molecule
for reducing the expression of an protein encoded by a nucleic acid
sequence of one of the sequences as indicated in Table I, columns 5
or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133
and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or
lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or
530 to 535 and/or lines 143 and/or 536 and/or lines 144 to 156
and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554 and/or
lines 167 to 172 and/or 555 to 557, resp., and/or homologs thereof,
one of the two RNA strands is essentially identical to at least
part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[6466] [0336.0.0.14] to [0342.0.0.14]: see [0336.0.0.0] to
[0342.0.0.0]
[6467] [0343.0.14.14] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., or its homolog is not necessarily required in
order to bring about effective reduction in the expression. The
advantage is, accordingly, that the method is tolerant with regard
to sequence deviations as may be present as a consequence of
genetic mutations, polymorphisms or evolutionary divergences. Thus,
for example, using the dsRNA, which has been generated starting
from a sequence as indicated in Table I, columns 5 or 7, lines 126
to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., or homologs thereof of the one organism,
may be used to suppress the corresponding expression in another
organism.
[6468] [0344.0.0.14] to [0361.0.0.14]: see [0344.0.0.0] to
[0361.0.0.0]
[6469] [0362.0.14.14] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., e.g.
encoding a polypeptide having protein activity, as indicated in
Table II, columns 3, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.
[6470] Due to the above mentioned activity the respective fine
chemical content in a cell or an organism is increased. For
example, due to modulation or manipulation, the cellular activity
of the polypeptide of the invention or nucleic acid molecule of the
invention is increased, e.g. due to an increased expression or
specific activity of the subject matters of the invention in a cell
or an organism or a part thereof. Transgenic for a polypeptide
having an activity of a polypeptide as indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., means herein that
due to modulation or manipulation of the genome, an activity as
annotated for a polypeptide as indicated in Table II, column 3,
lines 126 to 127 and/or 492 to 496 and/or lines 128 to 133 and/or
497 to 504 and/or lines 134 to 138 and/or 505 to 516 and/or lines
139 to 140 and/or 517 to 529 and/or lines 141 to 142 and/or 530 to
535 and/or lines 143 and/or 536 and/or lines 144 to 156 and/or 537
to 545 and/or lines 157 to 166 and/or 546 to 554 and/or lines 167
to 172 and/or 555 to 557, resp., e.g. having a sequence as
indicated in Table II, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., is increased in a cell or an organism or a part thereof.
Examples are described above in context with the process of the
invention
[6471] [0363.0.0.14] to [0384.0.0.14]: see [0363.0.0.0] to
[0384.0.0.0]
[6472] [0385.0.14.14] The fermentation broths obtained in this way,
containing in particular 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine, normally have a dry matter content of from 7.5 to 25% by
weight. Sugar-limited fermentation is additionally advantageous, at
least at the end, but especially over at least 30% of the
fermentation time. This means that the concentration of utilizable
sugar in the fermentation medium is kept at, or reduced to, 0 to 3
g/l during this time. The fermentation broth is then processed
further. Depending on requirements, the biomass can be removed
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation or a combination of these
methods, from the fermentation broth or left completely in it. The
fermentation broth can then be thickened or concentrated by known
methods, such as, for example, with the aid of a rotary evaporator,
thin-film evaporator, falling film evaporator, by reverse osmosis
or by nanofiltration. This concentrated fermentation broth can then
be worked up by freeze-drying, spray drying, spray granulation or
by other processes.
[6473] An other method for purification the amino acids of the
invention, in particular 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine is a process by means of electrodialysis as described in
U.S. Pat. No. 6,551,803.
[6474] [0386.0.0.14] to [0392.0.0.14]: see [0386.0.0.0] to
[0392.0.0.0]
[6475] [0393.0.4.4] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [6476] q) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [6477] r) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table I, preferably Table I B, columns 5 or 7, lines 126 to 127
and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504 and/or
lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140 and/or
517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or lines
143 and/or 536 and/or lines 144 to 156 and/or 537 to 545 and/or
lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172 and/or
555 to 557, resp., and, optionally, isolating the full length cDNA
clone or complete genomic clone; [6478] s) introducing the
candidate nucleic acid molecules in host cells, preferably in a
plant cell or a microorganism, appropriate for producing the fine
chemical; [6479] t) expressing the identified nucleic acid
molecules in the host cells; [6480] u) assaying the the fine
chemical level in the host cells; and [6481] v) identifying the
nucleic acid molecule and its gene product which expression confers
an increase in the the fine chemical level in the host cell after
expression compared to the wild type.
[6482] [0394.0.0.14] to [0399.0.0.14]: see [0394.0.0.0] to
[0399.0.0.0]
[6483] [0399.1.14.14] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the fine
chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., or a homolog
thereof, e.g. comparing the phenotyp of nearly identical organisms
with low and high activity of a protein as indicated in Table II,
columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or lines 128
to 133 and/or 497 to 504 and/or lines 134 to 138 and/or 505 to 516
and/or lines 139 to 140 and/or 517 to 529 and/or lines 141 to 142
and/or 530 to 535 and/or lines 143 and/or 536 and/or lines 144 to
156 and/or 537 to 545 and/or lines 157 to 166 and/or 546 to 554
and/or lines 167 to 172 and/or 555 to 557, resp., after incubation
with the drug.
[6484] [0400.0.0.14] to [0423.0.0.14]: see [0400.0.0.0] to
[0423.0.0.0]
[6485] [0424.0.14.14] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the fine chemical or of the fine
chemical and one or more other amino acids, in particular
Threonine, Glutamin, Glutamic acid, Valine, Aspargine,
[6486] Methionine, Cysteine, Leucine, Proline, Tryptophan, Valine,
Isoleucine and, Arginine. Accordingly, the nucleic acid of the
invention, or the nucleic acid molecule identified with the method
of the present invention or the complement sequences thereof, the
polypeptide of the invention, the nucleic acid construct of the
invention, the organisms, the host cell, the microorganisms, the
plant, plant tissue, plant cell, or the part thereof of the
invention, the vector of the invention, the antagonist identified
with the method of the invention, the antibody of the present
invention, the antisense molecule of the present invention, can be
used for the reduction of the respective fine chemical in a
organism or part thereof, e.g. in a cell.
[6487] [0425.0.0.14] to [0460.0.0.14]: see [0425.0.0.0] to
[0460.0.0.0]
[0461.0.14.14] Example 10
Cloning SEQ ID NO: 18718 for the Expression in Plants
[6488] [0462.0.0.14] see [0462.0.0.0]
[6489] [0463.0.14.14] SEQ ID NO: 18718 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[6490] [0464.0.0.14] to [0466.0.0.14]: see [0464.0.0.0] to
[0466.0.0.0]
[6491] [0467.0.14.14] The following primer sequences were selected
for the gene SEQ ID NO:
[6492] 18718:
TABLE-US-00046 i) forward primer (SEQ ID NO: 18874) atgaaataca
aggaaatcaa tttcttc ii) reverse primer (SEQ ID NO: 18875) ctacatagtt
atgttattgg tgatcg
[6493] [0468.0.0.14] to [0479.0.0.14]: see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.14.14] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 18718
[6494] [0481.0.0.14] to [0513.0.0.14]: see [0481.0.0.0] to
[0513.0.0.0]
[6495] [0514.0.14.14] As an alternative, the amino acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55).
[6496] The results of the different plant analyses can be seen from
the table 1 which follows:
TABLE-US-00047 ORF Metabolite Method Min Max YAL049C Phenylalanine
GC + LC 1.30 1.74 YBL015W Alanine GC 1.29 3.04 YDL127W 5-Oxoproline
GC 1.40 2.27 YEL045C Serine GC 1.23 1.80 YEL046C Homoserine GC 1.44
2.77 YER152C Serine GC 1.23 1.75 YER173W Alanine GC 1.21 2.32
YER173W Aspartic Acid GC 1.64 1.93 YER173W Phenylalanine GC 1.44
1.99 YFL050C Alanine GC 1.19 2.04 YFL050C Glycine GC 1.36 1.74
YFL050C Tyrosine GC 1.42 1.56 YGR101W Phenylalanine GC 1.40 1.92
YGR104C Aspartic Acid GC 2.08 2.26 YHR130C Phenylalanine LC 1.77
1.77 YHR130C Tyrosine GC + LC 1.51 2.26 YIL150C Alanine GC 1.47
4.28 YIL150C Serine GC 1.30 4.22 YIL150C Aspartic Acid GC 4.08 4.08
YIL150C Tyrosine GC 7.98 7.98 YJL072C Phenylalanine GC 1.25 3.04
YKR057W Serine GC 1.24 2.70 YLL009C Serine GC 1.27 1.84 YLL013C
Citrulline LC 1.30 1.44 YLR082C Serine GC 1.22 1.61 YOL123W Glycine
GC 1.35 2.05 YOR245C Citrulline LC 1.47 1.69 YOR261C Serine GC 1.28
1.40 YOR350C Tyrosine GC 1.67 2.77 YPR138C Phenylalanine GC + LC
1.49 1.93 b0695 Phenylalanine GC + LC 1.36 1.74 b0730 Aspartic Acid
GC 1.47 3.68 b1708 Phenylalanine GC 1.48 3.37 b1827 Phenylalanine
GC 1.40 3.93 b1827 Tyrosine GC 1.40 3.28 b1829 Serine GC 1.24 2.66
b1829 Tyrosine GC + LC 1.56 8.41 b2095 Alanine GC 1.22 1.33 b3008
Alanine GC 1.27 1.71 b3256 Phenylalanine GC 1.44 1.50 b1697
Phenylalanine GC 1.30 2.88 b1886 Phenylalanine LC 1.33 2.96 b1886
Serine GC 1.25 2.11 b1896 5-Oxoproline GC 1.34 2.50 b1896 Aspartic
acid GC 1.90 3.55 b3462 Serine GC 1.26 1.64 b3462 Phenylalanine GC
+ LC 1.26 2.40 b0057 Citrulline LC 1.33 2.19 b0057 Glycine GC 1.43
1.67 b0057 Serine GC 1.32 1.61 b0161 5-Oxoproline GC 1.45 2.23
b0161 Aspartic acid GC 1.57 2.16 b0161 Phenylalanine LC + GC 1.37
7.68 b0236 Alanine GC 1.22 1.41 b0376 5-Oxoproline GC 1.41 2.00
b0462 Citrulline LC 1.36 1.44 b0486 Alanine GC 1.44 1.52 b0486
Serine GC 1.27 1.49 b0577 Glycine GC 1.36 1.50 b0577 Aspartic acid
GC 1.56 1.65 b0970 5-Oxoproline GC 1.37 3.03 b0970 Tyrosine GC 1.35
5.98 b1228 Phenylalanine GC 1.35 1.44 b1275 Citrulline LC 1.32 1.50
b1343 Alanine GC 1.27 1.44 b1343 5-Oxoproline GC 1.37 1.87 b1343
Aspartic acid GC 1.76 2.36 b1360 Citrulline LC 1.34 1.72 b1863
Alanine GC 1.22 1.45 b2023 Aspartic acid GC 1.56 1.81 b2078
Phenylalanine LC + GC 1.26 1.89 b2239 Citrulline LC 1.37 1.73 b2414
Citrulline LC 1.31 1.40 b2414 Glycine GC 1.38 1.62 b2414 Serine GC
1.27 1.53 b2414 Phenylalanine GC 1.27 2.97 b2426 Citrulline LC 1.32
1.41 b2489 Citrulline LC 1.33 1.60 b2489 Alanine GC 1.21 1.27 b2489
Glycine GC 1.33 1.78 b2489 Serine GC 1.23 1.47 b2491 Tyrosine GC
1.40 1.89 b2507 Aspartic acid GC 1.49 2.20 b2553 Glycine GC 1.36
1.83 b2576 Alanine GC 1.24 1.49 b2576 Glycine GC 1.40 1.73 b2576
Aspartic acid GC 1.52 2.03 b2576 Phenylalanine GC 1.23 1.41 b2664
Serine GC 1.25 3.31 b2753 Aspartic acid GC 1.56 2.54 b2796
Phenylalanine LC 1.33 2.05 b3064 Serine GC 1.39 1.84 b3116 Serine
GC 1.35 1.70 b3116 Aspartic acid GC 1.50 2.30 b3160 Citrulline LC
1.34 1.48 b3160 Serine GC 1.31 1.60 b3169 Aspartic acid GC 1.66
2.68 b3172 5-Oxoproline GC 1.44 1.94 b3172 Aspartic acid GC 1.52
3.34 b3231 Alanine GC 1.21 1.35 b3231 Serine GC 1.23 1.49 b3241
Citrulline LC 1.38 1.70 b3767 Alanine GC 1.24 1.70 b3767 Homoserine
GC 1.24 1.71 b3919 Phenylalanine GC 1.30 2.88 b3926 Citrulline LC
1.70 1.94 b3938 Phenylalanine LC + GC 1.34 1.40 b3983 Phenylalanine
LC + GC 1.37 3.66 b3983 Tyrosine LC + GC 1.44 4.57 b4129 Aspartic
acid GC 1.53 2.41 b4214 Citrulline LC 1.32 2.02 b4269 Citrulline LC
1.31 1.63 b4346 Aspartic acid GC 1.63 1.94
[6497] [0515.0.14.14] Column 2 shows the amino acid analyzed.
Columns 4 and 5 shows the ratio of the analyzed amino acid between
the transgenic plants and the wild type; Increase of the
metabolites: Max: maximal x-fold (normalised to wild type)-Min:
minimal x-fold (normalised to wild type). Decrease of the
metabolites: Max: maximal x-fold (normalised to wild type) (minimal
decrease), Min: minimal x-fold (normalised to wild type) (maximal
decrease). Column 6 indicates the analytical method.
[6498] [0516.0.0.14] to [0552.0.0.14]: see [0516.0.0.0] to
[0552.0.0.0]
[0552.1.14.14]: Example 15
Metabolite Profiling Info from Zea mays
[6499] Zea mays plants were engineered, grown and analyzed as
described in Example 14c. The results of the different Zea mays
plants analysed can be seen from Table 2 which follows:
TABLE-US-00048 TABLE 2 ORF_NAME Metabolite Min Max YAL049C
Phenylalanine 1.78 1.86 YIL150C Alanine 1.63 3.17 YIL150C Serine
1.25 2.02 YKR057W Serine 2.05 12.64 YIL150C Aspartic acid 2.11 3.56
YIL150C Tyrosine 1.19 2.09 YEL046C Homoserine 2.04 2.45 YLR082C
Serine 1.63 2.84 b0970 5-Oxoproline 1.17 3.04 b0970 Tyrosine 1.53
5.05 b1829 Serine 1.53 2.12 b1896 5-Oxoproline 1.58 2.48 b1896
Aspartic acid 2.24 4.06 b2553 Glycine 2.70 23.32 b2664 Serine 1.70
3.47 b3116 Serine 1.45 2.45 b3116 Aspartic acid 1.91 2.20 b3172
5-Oxoproline 1.43 2.94 b3172 Aspartic acid 1.39 4.99
[6500] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in phenylalanine and/or
alanine and/or serine and/or aspartic acid and/or tyrosine and/or
5-oxoproline and/or homoserine and/or glycine in genetically
modified corn plants expressing the Saccharomyces cerevisiae
nucleic acid sequence
[6501] YAL049C, YIL150C, YKR057W, YEL046C or YLR082C or E. coli
nucleic acid sequence b0970, b1829, b1896, b2553, b2664, b3116 or
b3172 resp.
[6502] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YAL049C or its homologs, its activity has not
been characterized yet, is increased in corn plants, preferably, an
increase of the fine chemical phenylalanine between 78% and 86% is
conferred.
[6503] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein
[6504] YIL150C or its homologs, e.g. "a chromatin binding protein,
required for S-phase (DNA synthesis) initiation or completion", is
increased in corn plants, preferably, an increase of the fine
chemical alanine between 63% and 217% is conferred and/or an
increase of the fine chemical serine between 25% and 102% is
conferred and/or an increase of the fine chemical aspartic acid
between 111% and 256% is conferred and/or an increase of the fine
chemical tyrosine between 19% and 109% is conferred.
[6505] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YKR057W or a ribosomal protein, similar to S21
ribosomal proteins, involved in ribosome biogenesis and translation
or its homologs, is increased in corn plants, preferably, an
increase of the fine chemical serine between 105% and 1164% is
conferred.
[6506] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YEL046C or its homologs, e.g. "an activity of a
low specificity L-threonine aldolase", is increased in corn plants,
preferably, an increase of the fine chemical homoserine between
104% and 145% is conferred.
[6507] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YLR082C or its homologs, e.g. "an activity as a
suppressor of Rad53 null lethality", is increased in corn plants,
preferably, an increase of the fine chemical serine between 63% and
184% is conferred.
[6508] In one embodiment, in case the activity of the E. coli
protein b0970 or its homologs, e.g. "the activity of a glutamate
receptor", is increased in corn plants, preferably, an increase of
the fine chemical 5-oxoproline between 17% and 204% is conferred
and/or an increase of the fine chemical tyrosine between 53% and
405% is conferred.
[6509] In one embodiment, in case the activity of the E. coli
protein b1829 or its homologs, e.g. "an activity of a heat shock
protein with protease activity, preferably of the heat-shock
protein htpX superfamily", is increased in corn plants, preferably,
an increase of the fine chemical serine between 53% and 112% is
conferred.
[6510] In one embodiment, in case the activity of the E. coli
protein b1896 or its homologs, e.g. "an activity of a
trehalose-6-phosphate synthase, preferably of the
[6511] Schizosaccharomyces pombe alpha,alpha-trehalose-phosphate
synthase (UdP-forming) superfamily", is increased in corn plants,
preferably, an increase of the fine chemical 5-oxoproline between
58% and 148% is conferred and/or an increase of the fine chemical
aspartic acid between 124% and 306% is conferred.
[6512] In one embodiment, in case the activity of the E. coli
protein b2553 or its homologs, e.g. "the activity of a regulatory
protein P-II for glutamine synthetase", is increased in corn
plants, preferably, an increase of the fine chemical glycine
between 170% and 2232% is conferred.
[6513] In one embodiment, in case the activity of the E. coli
protein b2664 or its homologs, e.g. "a protein with the activity
defined as putative transcriptional repressor with DNA-binding
Winged helix domain (GntR familiy) or its homologs, e.g.
transcriptional regulator", is increased in corn plants,
preferably, an increase of the fine chemical serine between 70% and
247% is conferred.
[6514] In one embodiment, in case the activity of the E. coli
protein b3116 or its homologs, e.g. "the activity of a
L-threonine/L-serine permease, anaerobically inducible (HAAAP
family)", is increased in corn plants, preferably, an increase of
the fine chemical serine between 45% and 145% is conferred and/or
an increase of the fine chemical aspartic acid between 91% and 120%
is conferred.
[6515] In one embodiment, in case the activity of the E. coli
protein b3172 or its homologs, e.g. "the activity of an
argininosuccinate synthetase", is increased in corn plants,
preferably, an increase of the fine chemical 5-oxoproline between
43% and 194% is conferred and/or an increase of the fine chemical
aspartic acid between 39% and 399% is conferred.
[6516] [0552.2.0.14]: see [0552.2.0.0]
[6517] [0553.0.14.14] [6518] 1. A process for the production of
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
homoserine, phenylalanine, serine and/or tyrosine resp., which
comprises (a) increasing or generating the activity of a protein as
indicated in Table II, columns 5 or 7, lines 126 to 127 and/or 492
to 496 and/or lines 128 to 133 and/or 497 to 504 and/or lines 134
to 138 and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529
and/or lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or
536 and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to
166 and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557,
resp., or a functional equivalent thereof in a non-human organism,
or in one or more parts thereof; and (b) growing the organism under
conditions which permit the production of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine resp. in said organism. [6519] 2. A process
for the production of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., comprising the increasing or generating in an
organism or a part thereof the expression of at least one nucleic
acid molecule comprising a nucleic acid molecule selected from the
group consisting of: a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 126 to
127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., or a fragment thereof, which confers an
increase in the amount of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof; b) nucleic acid
molecule comprising of a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine resp., in an organism or a part thereof; d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof; e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof; f) nucleic acid
molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table III, column 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., and
conferring an increase in the amount of the 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine resp., in an organism or a part thereof; g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof; h) nucleic acid
molecule encoding a polypeptide comprising a consensus sequence as
indicated in Table IV, column 7, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
and conferring an increase in the amount of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine resp., in an organism or a part thereof; and
i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of 5-oxoproline,
alanine, aspartic acid, citrulline, glycine, homoserine,
phenylalanine, serine and/or tyrosine resp., in an organism or a
part thereof. or comprising a sequence which is complementary
thereto. [6520] 3. The process of claim 1 or 2, comprising
recovering of the free or bound 5-oxoproline, alanine, aspartic
acid, citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp. [6521] 4. The process of any one of claims 1 to 3,
comprising the following steps: (a) selecting an organism or a part
thereof expressing a polypeptide encoded by the nucleic acid
molecule characterized in claim 2; (b) mutagenizing the selected
organism or the part thereof; (c) comparing the activity or the
expression level of said polypeptide in the mutagenized organism or
the part thereof with the activity or the expression of said
polypeptide of the selected organisms or the part thereof; (d)
selecting the mutated organisms or parts thereof, which comprise an
increased activity or expression level of said polypeptide compared
to the selected organism or the part thereof; (e) optionally,
growing and cultivating the organisms or the parts thereof; and (f)
recovering, and optionally isolating, the free or bound
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
homoserine, phenylalanine, serine and/or tyrosine resp., produced
by the selected mutated organisms or parts thereof. [6522] 5. The
process of any one of claims 1 to 4, wherein the activity of said
protein or the expression of said nucleic acid molecule is
increased or generated transiently or stably. [6523] 6. An isolated
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: a) nucleic acid molecule encoding of
a polypeptide as indicated in Table II, columns 5 or 7, lines 126
to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., or a fragment thereof, which confers an
increase in the amount of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof; b) nucleic acid
molecule comprising of a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine resp., in an organism or a part thereof; d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof; e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof; f) nucleic acid
molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table III, column 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., and
conferring an increase in the amount of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine resp., in an organism or a part thereof; g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof; h) nucleic acid
molecule encoding a polypeptide comprising a consensus sequence as
indicated in Table IV, column 7, lines 126 to 127 and/or 492 to 496
and/or lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138
and/or 505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or
lines 141 to 142 and/or 530 to 535 and/or lines 143 and/or 536
and/or lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166
and/or 546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp.,
and conferring an increase in the amount of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine resp., in an organism or a part thereof; and
i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of 5-oxoproline,
alanine, aspartic acid, citrulline, glycine, homoserine,
phenylalanine, serine and/or tyrosine resp., in an organism or a
part thereof. whereby the nucleic acid molecule distinguishes over
the sequence as indicated in Table I A, columns 5 or 7, lines 126
to 127 and/or 492 to 496 and/or lines 128 to 133 and/or 497 to 504
and/or lines 134 to 138 and/or 505 to 516 and/or lines 139 to 140
and/or 517 to 529 and/or lines 141 to 142 and/or 530 to 535 and/or
lines 143 and/or 536 and/or lines 144 to 156 and/or 537 to 545
and/or lines 157 to 166 and/or 546 to 554 and/or lines 167 to 172
and/or 555 to 557, resp., by one or more nucleotides. [6524] 7. A
nucleic acid construct which confers the expression of the nucleic
acid molecule of claim 6, comprising one or more regulatory
elements. [6525] 8. A vector comprising the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7.
[6526] 9. The vector as claimed in claim 8, wherein the nucleic
acid molecule is in operable linkage with regulatory sequences for
the expression in a prokaryotic or eukaryotic, or in a prokaryotic
and eukaryotic, host. [6527] 10. A host cell, which has been
transformed stably or transiently with the vector as claimed in
claim 8 or 9 or the nucleic acid molecule as claimed in claim 6 or
the nucleic acid construct of claim 7 or produced as described in
claim any one of claims 2 to 5. [6528] 11. The host cell of claim
10, which is a transgenic host cell. [6529] 12. The host cell of
claim 10 or 11, which is a plant cell, an animal cell, a
microorganism, or a yeast cell, a fungus cell, a prokaryotic cell,
an eukaryotic cell or an archaebacterium. [6530] 13. A process for
producing a polypeptide, wherein the polypeptide is expressed in a
host cell as claimed in any one of claims 10 to 12. [6531] 14. A
polypeptide produced by the process as claimed in claim 13 or
encoded by the nucleic acid molecule as claimed in claim 6 whereby
the polypeptide distinguishes over a sequence as indicated in Table
II A, columns 5 or 7, lines 126 to 127 and/or 492 to 496 and/or
lines 128 to 133 and/or 497 to 504 and/or lines 134 to 138 and/or
505 to 516 and/or lines 139 to 140 and/or 517 to 529 and/or lines
141 to 142 and/or 530 to 535 and/or lines 143 and/or 536 and/or
lines 144 to 156 and/or 537 to 545 and/or lines 157 to 166 and/or
546 to 554 and/or lines 167 to 172 and/or 555 to 557, resp., by one
or more amino acids [6532] 15. An antibody, which binds
specifically to the polypeptide as claimed in claim 14. [6533] 16.
A plant tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [6534] 17. A method for screening for agonists
and antagonists of the activity of a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, homoserine, phenylalanine, serine and/or tyrosine resp.,
in an organism or a part thereof comprising:
(a) contacting cells, tissues, plants or microorganisms which
express the a polypeptide encoded by the nucleic acid molecule of
claim 5 conferring an increase in the amount of 5-oxoproline,
alanine, aspartic acid, citrulline, glycine, homoserine,
phenylalanine, serine and/or tyrosine resp., in an organism or a
part thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; (b) assaying the 5-oxoproline, alanine, aspartic
acid, citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., level or the polypeptide expression level in the
cell, tissue, plant or microorganism or the media the cell, tissue,
plant or microorganisms is cultured or maintained in; and (c)
identifying a agonist or antagonist by comparing the measured
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
homoserine, phenylalanine, serine and/or tyrosine resp., level or
polypeptide expression level with a standard 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine resp., or polypeptide expression level
measured in the absence of said candidate compound or a sample
comprising said plurality of compounds, whereby an increased level
over the standard indicates that the compound or the sample
comprising said plurality of compounds is an agonist and a
decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[6535] 18. A process for the identification of a compound
conferring increased 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., production in a plant or microorganism, comprising
the steps: (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, homoserine, phenylalanine, serine and/or tyrosine resp.,
in an organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with dais readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine resp., in an organism or a part thereof; (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [6536] 19. A method for the identification of a
gene product conferring an increase in 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine resp., production in a cell, comprising the
following steps: (a) contacting the nucleic acid molecules of a
sample, which can contain a candidate gene encoding a gene product
conferring an increase in 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., after expression with the nucleic acid molecule of
claim 6; (b) identifying the nucleic acid molecules, which
hybridise under relaxed stringent conditions with the nucleic acid
molecule of claim 6; (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing 5-oxoproline,
alanine, aspartic acid, citrulline, glycine, homoserine,
phenylalanine, serine and/or tyrosine resp.; (d) expressing the
identified nucleic acid molecules in the host cells; (e) assaying
the 5-oxoproline, alanine, aspartic acid, citrulline, glycine,
homoserine, phenylalanine, serine and/or tyrosine resp., level in
the host cells; and (f) identifying nucleic acid molecule and its
gene product which expression confers an increase in the
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
homoserine, phenylalanine, serine and/or tyrosine resp., level in
the host cell in the host cell after expression compared to the
wild type. [6537] 20. A method for the identification of a gene
product conferring an increase in 5-oxoproline, alanine, aspartic
acid, citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., production in a cell, comprising the following
steps: (a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, homoserine, phenylalanine,
serine and/or tyrosine resp., amount or level in an organism or a
part thereof after expression, and which are at least 20% homolog
to the nucleic acid molecule of claim 6; (b) introducing the
candidate nucleic acid molecules in host cells appropriate for
producing 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, homoserine, phenylalanine, serine and/or tyrosine resp.,;
(c) expressing the identified nucleic acid molecules in the host
cells; (d) assaying the 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosineresp., level in the host cells; and (e) identifying nucleic
acid molecule and its gene product which expression confers an
increase in the 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, homoserine, phenylalanine, serine and/or tyrosine resp.,
level in the host cell after expression compared to the wild type.
[6538] 21. A method for the production of an agricultural
composition comprising the steps of the method of any one of claims
17 to 20 and formulating the compound identified in any one of
claims 17 to 20 in a form acceptable for an application in
agriculture. [6539] 22. A composition comprising the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of any one of claim 8 or 9, an
antagonist or agonist identified according to claim 17, the
compound of claim 18, the gene product of claim 19 or 20, the
antibody of claim 15, and optionally an agricultural acceptable
carrier. [6540] 23. Use of the nucleic acid molecule as claimed in
claim 6 for the identification of a nucleic acid molecule
conferring an increase of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, homoserine, phenylalanine, serine and/or
tyrosine resp., after expression. [6541] 24. Use of the polypeptide
of claim 14 or the nucleic acid construct claim 7 or the gene
product identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
homoserine, phenylalanine, serine and/or tyrosine resp., levels in
an organism. [6542] 25. Food or feed composition comprising the
nucleic acid molecule of claim 6, the polypeptide of claim 14, the
nucleic acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20. [6543] 26. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the protection of a plant against
a 5-oxoproline and/or alanine and/or aspartic acid and/or
citrulline and/or glycine and/or homoserine and/or phenylalanine
and/or serine and/or tyrosine synthesis inhibiting herbicide.
[6544] [0554.0.0.14] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[6545] [0000.0.0.15] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[6546] [0001.0.0.15] see [0001.0.0.0]
[6547] [0002.0.15.15] Oils and fats, which chemically are glycerol
esters of fatty acids (triacylglycerols (TAGs)), play a major role
in nutrition but more and more in nonfood applications such as
lubricants, hydraulic oil, biofuel, or oleochemicals for coatings,
plasticizer, soaps, and detergents (W. Lohs and W. Friedt, in
Designer Oil Crops, D. J. Murphy, Ed. (VCH, Weinheirn, Gerrnany,
1993)). The ideal oil for industrial application would consist of a
particular type of fatty acid that could be supplied constantly at
a competitively low price as compared with raw materials based on
mineral oil products. Furthermore, such a fatty acid may have a
reactive group in addition to the carboxyl function to provide an
additional target for chemical modifications (Topfer et al.,
Science, Vol. 268, 681-686, 1995).
[6548] [0003.0.15.15] Fatty acids and triglycerides have numerous
applications in the food and feed industry, in cosmetics and in the
drug sector. Depending on whether they are free saturated or
unsaturated fatty acids or triglycerides with an increased content
of saturated or unsaturated fatty acids, they are suitable for the
most varied applications; thus, for example, polyunsaturated fatty
acids (=PUFAs) are added to infant formula to increase its
nutritional value. The various fatty acids and triglycerides are
mainly obtained from microorganisms such as fungi or from
oil-producing plants including phytoplankton and algae, such as
soybean, oilseed rape, sunflower and others, where they are usually
obtained in the form of their triacylglycerides.
[6549] [0004.0.15.15] Principally microorganisms such as
Mortierella or oil producing plants such as soybean, rapeseed or
sunflower or algae such as Crytocodinium or Phaeodactylum are a
common source for oils containing fatty acids, where they are
usually obtained in the form of their triacyl glycerides.
Alternatively, they are obtained advantageously from animals, such
as fish. The free fatty acids are prepared advantageously by
hydrolysis with a strong base such as potassium or sodium
hydroxide.
[6550] [0005.0.15.15] Further sources of fatty acids are membrane
lipids of organisms. Preferably lipids are phopholipids and/or
glycolipids, more preferably glycerophospholipids, galactolipids
and/or sphingolipids.
[6551] [0006.0.15.15.] Margaric acid was first mentioned in the
early 1800s. 1813 M. E. Chevreul discovered that fats are composed
of fatty acids and named one of these "margaric acid" because it
glistened with lustrous pearly drops that reminded him of the Greek
word for pearl, margaron or margarites. In the middle of the 1800s
W. H. Heintz showed that "margaric acid" discovered by Chevreul was
an indefinite mixture of palmitic and stearic acids.
[6552] Today, the term "margaric acid" is the trivial name for
heptadecanoic acid (17:0), which is naturally occurring in minor
amounts.
[6553] The fatty acid with odd number of carbon atoms is present in
trace amounts in plants, in triglycerides from Brazil-nut oil,
Dracocephalum moldavica oil, Poppy-seed, Palm, Almond, Sunflower or
Soyabean. Margaric acid can be isolated from tallow (1%), specially
from subcutaneous adipose tissue in subcutaneous fat from
lambs.
[6554] Margaric acid can be ingredient of satiety agents or
fungicide composition. It is further used as ingredient in
cosmetics, pharmaceuticals and in feed and food, like baking
adjuvants as disclosed in US 20030143312 or accordind to US
20040097392 as component in surfactant systems.
[6555] The heptadecanoic acid is mainly used as an internal
standard in quantification of fatty acids. It can be further useful
in treatment of neurological diseases which may be caused by yeast,
fungi or prions based on yeast or fungal etiology (U.S. Pat. No.
6,652,866) or in antikeratolytic-wound healing compositions (U.S.
Pat. No. 5,641,814). Heptadecanoic acid was produced up to now in
higher amount primarily by organic synthesis.
[6556] [0007.0.15.15.] 2-Hydroxy fatty acids are synthesised in
animal and plant tissues, and are often major constituents of the
sphingolipids. Sphingolipids with 2-hydroxy fatty acid are found in
most organisms including plants, yeast, worms, vertebrate animals,
and some bacterial species.
[6557] In plants more than 95% of the fatty acid component of the
ceramides and sphingolipids is alpha-hydroxylated. The acyl groups
of ceramides tend to consist of long-chain (C16 up to C26 but
occasionally longer) odd- and even-numbered saturated or monoenoic
fatty acids and related 2-D-hydroxy fatty acids, both in plant and
animal tissues.
[6558] Typical plant sphingolipids are made up by the long-chain
sphingosine backbone which is glycosylated and amide-linked to an
usually hydroxylated (very-)-long-chain fatty acid, called
cerebroside. Cerebrosides are essential constituents of the plasma
membrane involved in various physiological functions including
signaling, exocytosis, anchoring of proteins, and vesicular protein
transport (Matthes et al., Z. Naturforsch. 57C, 843-852, 2002).
[6559] In mammals, 2-hydroxysphingolipids are present abundantly in
brain because the major myelin lipids galactosylceramides and
sulfatides contain 2-hydroxy fatty acids. In mammals, 2-hydroxy
fatty acid-containing sphingolipids are uniquely abundant in
nervous and epidermal tissues. In mammalian central and peripheral
nervous systems, galactosylceramides and sulfatides (3-sulfate
ester of galactosylceramide) are major lipid components of myelin.
These glycosphingolipids contain a high proportion (about 50%) of
2-hydroxy fatty acid and are critical components of myelin (4,
5).
[6560] In the yeast Saccharomyces cerevisiae most sphingolipids
contain 2-hydroxy fatty acid. COS7 cells expressing human FA2H
contained 3-20-fold higher levels of 2-hydroxyceramides (C16, C18,
C24, and C24:1) and 2-hydroxy fatty acids compared with control
cells (Alderson et al., J. Biol. Chem. Vol. 279, No. 47,
48562-48568, 2004).
[6561] The 2-hydroxylation occurs during de novo ceramide synthesis
and is catalyzed by fatty acid 2-hydroxylase (also known as fatty
acid alpha-hydroxylase). No free hydroxy fatty acid or hydroxy
fatty acid CoA has ever been reported; the hydroxylated product
always appeared as a component of ceramide or cerebroside (Hoshi et
al., J. Biol. Chem. 248, 4123-4130, 1973). The alpha-hydroxylation
involves the direct hydroxylation of a sphingolipid-bound fatty
acid. (Kayal et al., J. Biol. Chem. Vol. 259, No. 6, 3548-3553,
1984).
[6562] Hydroxylated fatty adds initiate inflammation in the soft
tissues and regulate the immune response.
[6563] The 2-hydroxyl group in sphingolipids has a profound effect
in the lipid organization within membranes because of its
hydrogenbonding capability.
[6564] Alpha-hydroxy-palmitic acid (hC 16:0) is mainly a building
block of plant sphingolipids, for example soy glucosylceramide
(GIcCer), which consists predominantly of a 4,8-sphingadiene
backbone and alpha-hydroxy-palmitic acid. Soy GIcCer suppress
tumorigenesis and gene expression in mice (Symolon et al., J. Nutr.
2004 May; 134(5):1157-61).
[6565] A monoglucosecerebroside (pinelloside) with strong
antimicrobial properties (against Gram-positive and -negative
bacteria and against fungi) was described in the tuber of
[6566] PineIla ternata (Araceae), one component of decoctions used
in traditional Chinese medicine (Chen et al., Phytochemistry 2003,
64, 903). Its structure was shown to include a glucose moiety and
the unusual 4,11-sphingadienine linked to a 2-hydroxy-palmitic
acid.
[6567] Another hydioxylated fatty acid being a building block of
cerrebrosides is the 2-hydroxy-nervonic acid (2-OH--C24:1).
2-hydroxy-15-tetracosenoic acid (hydroxynervonic acid) is
constituent of the ceramide part of cerebrosides
(glycosphingolipides found mainly in nervous tissue and in little
amount in plants). The occurrence of 2-hydroxy nervonic acid is
characteristic for the leaf cerebrosides of some chilling-resistant
cereals (Sperling et al., BBA 1632, 1-15, 2003).
[6568] The hydroxylated fatty add may be used in a method for
producing fats or oils according to US 20030054509 or in
lanolin-free cosmetic composition according to US 20040166130.
[6569] [0008.0.15.15] Whether oils with unsaturated or with
saturated fatty acids are preferred depends on the intended
purpose; thus, for example, lipids with unsaturated fatty acids,
specifically polyunsaturated fatty acids, are preferred in human
nutrition since they have a positive effect on the cholesterol
level in the blood and thus on the possibility of heart disease.
They are used in a variety of dietetic foodstuffs or medicaments.
In addition PUFAs are commonly used in food, feed and in the
cosmetic industry. Poly unsaturated .omega.-3- and/or
.omega.-6-fatty acids are an important part of animal feed and
human food. Because of the common composition of human food
polyunsaturated .omega.-3-fatty acids, which are an essential
component of fish oil, should be added to the food to increase the
nutritional value of the food; thus, for example, polyunsaturated
fatty acids such as DHA or EPA are added as mentioned above to
infant formula to increase its nutritional value. The true
essential fatty acids linoleic and linolenic fatty acid have a lot
of positive effects in the human body such as a positive effect on
healthy heart, arteries and skin. They bring for example relieve
from eczema, diabetic neuropathy or PMS and cyclical breast
pain.
[6570] [0009.0.15.15.] Further poly unsaturated .omega.-3- and/or
.omega.-6-fatty acids important part of animal feed and human food
are delta 7,10 hexadecadienic acid (16:2(n-6)) and delta 7, 10, 13
hexadecatrienic acid (16:3(n-3)). Hexadecadienic acid is a minor
component of some seed and fish oils, and of plant leaves but the
precursor of hexadecatrienic acid 16:3(n-3), which is a common
constituent of leaf lipids. This acid is known to occur in
photosynthetic leaves, such as for example Arabidopsis thaliana,
rape leaves, fern lipid, ginko leaves, potato leaves, tomato leaves
and spinach. It may also occur in the leaves of Brassicaceae
plants, such as horse radish, cabbage, turnip, Chinese mustard,
cauliflower and watercress.
[6571] In higher plants, the galactolipids contain a high
proportion of polyunsaturated fatty acids, up to 95% of which can
be linolenic acid (18:3(n-3)). In this instance, the most abundant
molecular species of mono- and digalactosyldiacylglycerols must
have 18:3 at both sn-I and sn-2 positions of the glycerol backbone.
Plants such as the pea, which have 18:3 as almost the only fatty
acid in the monogalactosyldiacylglycerols, have been termed "18:3
plants". Other species, and Arabidopsis thaliana is an example,
contain appreciable amounts of hexadecatrienoic acid (16:3(n-3)) in
the monogalactosyldiacylglycerols, and they are termed "16:3
plants".
[6572] As mentioned, polyunsaturated fatty acid are further used in
the cosmetic industry. The application US 20030039672 discloses a
cosmetic method for treating aged, sensitive, dry, flaky, wrinkled
and/or photodamaged skin through topical application of a
composition which comprises an unsaturated C16 fatty acid having at
least three double bonds, which may be preferably hexadecatrienoic
acid.
[6573] [0010.0.15.15.] On account of their positive properties
there has been no shortage of attempts in the past to make
available genes which participate in the synthesis of fatty acids
or triglycerides for the production of oils in various organisms
having a modified content of unsaturated fatty acids.
[6574] [0011.0.15.15] Methods of recombinant DNA technology have
also been used for some years to improve the oil content in
microorganisms or plants by amplifying individual fatty acid
biosynthesis genes and investigating the effect on fatty acid
production. For example in WO 91/13972 a .DELTA.-9-desaturase is
described, which is involved in the synthesis of polyunsaturated
fatty acids. In WO 93/11245 a .DELTA.-15-desaturase and in WO
94/11516 a .DELTA.-12-desaturase is claimed. Other desaturases are
described, for example, in EP-A-0 550 162, WO 94/18337, WO
97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250, Stukey et al.,
J. Biol. Chem., 265, 1990: 20144-20149, Wada et al., Nature 347,
1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. To date,
however, the various desaturases have been only inadequately
characterized biochemically since the enzymes in the form of
membrane-bound proteins are isolable and characterizable only with
very great difficulty (McKeon et al., Methods in Enzymol. 71, 1981:
12141-12147, Wang et al., Plant Physiol. Biochem., 26, 1988:
777-792). Generally, membrane-bound desaturases are characterized
by introduction into a suitable organism, which is then
investigated for enzyme activity by means of analysis of starting
materials and products. With regard to the effectiveness of the
expression of desaturases and their effect on the formation of
polyunsaturated fatty acids it may be noted that through expression
of a desaturases and elongases as described to date only low
contents of poly-unsaturated fatty acids/lipids have been achieved.
Therefore, an alternative and more effective pathway with higher
product yield is desirable.
[6575] [0012.0.15.15] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes which
participate in the biosynthesis of fatty acids and make it possible
to produce certain fatty acids specifically on an industrial scale
without unwanted byproducts forming. In the selection of genes for
biosynthesis two characteristics above all are particularly
important. On the one hand, there is as ever a need for improved
processes for obtaining the highest possible contents of
polyunsaturated fatty acids on the other hand as less as possible
byproducts should be produced in the production process.
[6576] [0013.0.0.15] see [0013.0.0.0]
[6577] [0014.0.15.15] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is heptadecanoic acid (C17:0,
margaric acid) and/or 2-hydroxy palmitic acid (2-OH--C16:0,
alfa-hydroxy palmitic acid, C16:0 OH) and/or
2-hydroxy-tetracosenoic-acid (2-hydroxy-15-tetracosenoic acid,
hydroxynervonic acid, alfa-hydroxy-tetracosenoic-acid, C24:1 (n-9)
OH, 2-hydroxy-cis 9-tetracosenoic-acid, delta 9
hydroxy-tetracosenoic-acid) and/or hexadecadienoic acid, preferably
delta 7,10 hexadecadienoic acid (C16:2 (n-6), cis 7-cis
10-hexadecadienoic acid) and/or hexadecatrienoic acid, preferably
delta 7,10,13 hexadecatrienoic acid (C16:3 (n-3), cis 7-cis 10-cis
13-hexadecatrienoic acid, hiragonic acid) or tryglycerides, lipids,
oils or fats containing heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid. Accordingly, in the present invention, the
term "the fine chemical" as used herein relates to "heptadecanoic
acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or
tryglycerides, lipids, oils and/or fats containing heptadecanoic
acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid". Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising heptadecanoic acid and/or 2-hydroxy palmitic
acid and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic
acid, preferably delta 7,10 hexadecadienoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid and/or triglycerides, lipids, oils and/or fats containing
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid.
[6578] [0015.0.15.15] In one embodiment, the term "the fine
chemical" means heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or
tryglycerides, lipids, oils and/or fats containing heptadecanoic
acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid. Throughout
the specification the term "the fine chemical" means heptadecanoic
acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or
tryglycerides, lipids, oils and/or fats containing heptadecanoic
acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic, heptadecanoic acid
and/or 2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid
and/or hexadecadienoic acid, preferably delta 7,10 hexadecadienoic
acid and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and its salts, ester, thioester or
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid in free form
or bound to other compounds such as triglycerides, glycolipids,
phospholipids etc. In a preferred embodiment, the term "the fine
chemical" means heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid, in free form
or its salts or bound to a glycerol backbone or to
glycerol-3-phosphate backbone or to a sphingosine-phosphate
backbone or to sphingosine-mono- or oligosaccharide backbone or to
a glycerol-3-mono- or disaccharide backbone. Triglycerides, lipids,
oils, fats or lipid mixture thereof shall mean any triglyceride,
lipid, oil and/or fat containing any bound or free heptadecanoic
acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid for example
sphingolipids, phosphoglycerides, lipids, glycerophospholipids,
galactolipids, glycolipids such as glycosphingolipids,
phospholipids such as phosphatidylethanolamine,
phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol or diphosphatidylglycerol, or as
monoacylglyceride, diacylglyceride or triacylglyceride or other
fatty acid esters such as acetyl-Coenzym A thioester, which contain
further saturated or unsaturated fatty acids in the fatty acid
molecule.
[6579] [0016.0.15.15] Accordingly, the present invention relates to
a process comprising [6580] (a) increasing or generating the
activity of of one or more [6581] YDR513W and/or YER156C and/or
YLR255C and/or b1829 protein(s) and/or [6582] YER173W protein
and/or [6583] YFR042W protein and/or [6584] YOR317W protein and/or
[6585] YGL205W and/or YIL150C and/or YKL132C and/or YOR344C and/or
b3430 and/or b0161 and/or b0758 and/or b0057 and/or b1097 and/or
b2078 and/or b3231 protein(s) or of a protein having the sequence
of a polypeptide indicated in Table II A or II B, column 3, lines
174 to 185, 558 to 563 or having the sequence of a polypeptide
encoded by a nucleic acid molecule indicated in Table IA or IB,
column 5 or 7, lines 174 to 185, 558 to 563, [6586] in a non-human
organism in one or more parts thereof and [6587] (b) growing the
organism under conditions which permit the production of the fine
chemical, thus, heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid or fine
chemicals comprising heptadecanoic acid and/or 2-hydroxy palmitic
acid and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic
acid, preferably delta 7,10 hexadecadienoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid and/or 24:1 fatty acid, preferably C24:1 delta 15 fatty acid,
in said organism.
[6588] Accordingly, the present invention relates to a process for
the production of the respective fine chemical comprising [6589]
(a) increasing or generating the activity of one or more protein(s)
having the activity of a protein indicated in Table IIA or IIB,
Column 3, lines 174 to 185, 558 to 563 or having the sequence of a
polypeptide encoded by a nucleic acid molecule indicated in Table
IA or IB, columns 5 or 7, lines 174 to 185, 558 to 563 in a
non-human organism in one or more parts thereof and [6590] (b)
growing the organism under conditions which permit the production
of the fine chemical, thus, fatty acid, in particular heptadecanoic
acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid.
[6591] [0016.1.15.15] Accordingly, the term "the fine chemical"
means in one embodiment "heptadecanoic acid" in relation to all
sequences listed in Table I to IV, lines 174 to 177 or homologs
thereof and
[6592] means in one embodiment "2-hydroxy palmitic acid" in
relation to all sequences listed in Tables I to IV, line 178, 558
and 559 or homologs thereof and
[6593] means in one embodiment "2-Hydroxy-tetracosenoic acid" in
relation to all sequences listed in Table IA or IB, line 179,
and
[6594] means in one embodiment "hexadecadienoic acid, preferably
delta 7,10 hexadecadienoic acid" in relation to all sequences
listed in Table I to IV, line 180 and means in one embodiment
"hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid" in relation to all sequences listed in Table I to IV, lines
181 to 185, and means in one embodiment "tetracosenoic acid" in
relation to all sequences listed in Table I to IV, lines 560 to 563
or homologs thereof.
[6595] Accordingly, the term "the fine chemical" can mean
"heptadecanoic acid" ("C17:0", "margaric acid") and/or "2-hydroxy
palmitic acid" ("2-OH--C16:0", "alfa-hydroxy palmitic acid", "C16:0
OH") and/or "2-hydroxy-tetracosenoic-acid"
("2-hydroxy-15-tetracosenoic acid", "hydroxynervonic acid",
"alfa-hydroxy-tetracosenoic-acid", "C24:1 (n-9) OH", "2-hydroxy-cis
9-tetracosenoic-acid", "delta 9 hydroxy-tetracosenoic-acid") and/or
"hexadecadienoic acid", preferably "delta 7,10 hexadecadienoic
acid" ("C16:2 (n-6)", "cis 7-cis 10-hexadecadienoic acid") and/or
"hexadecatrienoic acid", preferably "delta 7,10,13 hexadecatrienoic
acid" ("C16:3 (n-3)", "cis 7-cis 10-cis 13-hexadecatrienoic acid",
"hiragonic acid"), owing to circumstances and the context. In order
to illustrate that the meaning of the term "the fine chemical"
means "heptadecanoic acid" ("C17:0", "margaric acid") and/or
"2-hydroxy palmitic acid" ("2-OH--C16:0", "alfa-hydroxy palmitic
acid", "C16:0 OH") and/or "2-hydroxy-tetracosenoic-acid"
("2-hydroxy-15-tetracosenoic acid", "hydroxynervonic acid",
"alfa-hydroxy-tetracosenoic-acid", "C24:1 (n-9) OH", "2-hydroxy-cis
9-tetracosenoic-acid", "delta 9 hydroxy-tetracosenoic-acid") and/or
"hexadecadienoic acid", preferably "delta 7,10 hexadecadienoic
acid" ("C16:2 (n-6)", "cis 7-cis 10-hexadecadienoic acid") and/or
"hexadecatrienoic acid", preferably "delta 7,10,13 hexadecatrienoic
acid" ("C16:3 (n-3)", "cis 7-cis 10-cis 13-hexadecatrienoic acid",
"hiragonic acid") and/or 24:1 fatty acid, preferably C24:1 delta 15
fatty acid the term "the respective fine chemical" is also
used.
[6596] [0017.0.0.15] see [0017.0.0.0]
[6597] [0018.0.0.15] see [0018.0.0.0]
[6598] [0019.0.15.15] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the fine respective chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table IIA or IIB, column 3, lines 174 to 185, 558 to
563 or encoded by nucleic acid molecule indicated in Table IA or
IB, columns 5 or 7, lines 174 to 185, 558 to 563.
[6599] [0020.0.15.15] Surprisingly it was found, that the
transgenic expression of the Saccharomyces cerevisiae protein(s)
YDR513W and/or YER156C and/or YLR255C as indicated in Table II,
Column 3 or 5, lines 174 to 176 respectively and/or the Escherichia
coli K12 protein(s) b1829 as indicated in Table I, Column 3 or 5,
line 177 in Arabidopsis thaliana conferred an increase in the
heptadecanoic acid content of the transformed plants. Thus, in one
embodiment, said protein(s) or its homologs as indicated in Table
II, Column 7, line 174 to 177 are used for the production of
heptadecanoic acid.
[6600] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein YER173W as indicated in Table
II, Column 3 or 5, line 178 in Arabidopsis thaliana conferred an
increase in the 2-hydroxy palmitic acid content of the transformed
plants. Thus, in one embodiment, said protein or its homologs as
indicated in Table II, Column 7, line 178 are used for the
production of 2-hydroxy palmitic acid.
[6601] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein YFR042W as indicated in Table
II, Column 3 or 5, line 179 in Arabidopsis thaliana conferred an
increase in the 2-hydroxy-tetracosenoic-acid content of the
transformed plants. Thus, in one embodiment, said protein(s) or its
homologs as indicated in Table II, Column 7, line 179 are used for
the production of 2-hydroxy-tetracosenoic-acid.
[6602] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein YOR317W as indicated in Table
II, Column 3 or 5, line 180 in Arabidopsis thaliana conferred an
increase in the hexadecadienoic acid, preferably delta 7,10
hexadecadienoic acid content of the transformed plants. Thus, in
one embodiment, said protein(s) or its homologs as indicated in
Table II, Column 7, line 180 are used for the production of
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic
acid.
[6603] Surprisingly it was found, that the transgenic expression of
the Saccharomyces cerevisiae protein(s) YGL205W and/or YIL150C
and/or YKL132C and/or YOR344C as indicated in Table II, Column 3 or
5, line 181 to 184 and/or the Escherichia coli K12 protein b3430 as
indicated in Table II, Column 3 or 5, line 185 respectively in
Arabidopsis thaliana conferred an increase in the hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic content of the
transformed plants. Thus, in one embodiment, said protein(s) or its
homologs as indicated in Table II, Column 7, line 181 to 185 are
used for the production of hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic. Surprisingly it was found, that the
transgenic expression of the Escherichia coli protein b 0161 and/or
b0758 as indicated in Table IIA or IIB, Column 3 or 5, line 558,
559 in Arabidopsis thaliana conferred an increase in the
2-hydroxypalmitic acid content of the transformed plants. Thus, in
one embodiment, said protein(s) or its homologs as indicated in
Table II, column 7, line 558, 559 are used for the production of
2-hydroxypalmitic acid.
[6604] Surprisingly it was found, that the transgenic expression of
the Escherichia coli protein b0057, b1097, b2078 or b3231 as
indicated in Table IIA or IIB, Column 3 or 5, line 560 to 563 in
Arabidopsis thaliana conferred an increase in the C24:1 fatty acid
content of the transformed plants. Thus, in one embodiment, said
protein(s) or its homologs as indicated in Table II, column 7, line
560 to 563 are used for the production of C24:1 fatty acid.
[6605] [0021.0.15.15] see [0021.0.0.0]
[6606] [0022.0.15.15] The sequence of YDR513W from Saccharomyces
cerevisiae has been published in Jacq et al., Nature 387 (6632
Suppl), 75-78 (1997) and in Goffeau et al., Science 274 (5287),
546-547, 1996 and its cellular activity has been characterized as a
glutathione reductase, preferably of the glutaredoxin superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the glutaredoxin superfamily, preferably a protein with a
glutathione reductase activity or its homolog, e.g. as shown
herein, from Saccharomyces cerevisiae or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
heptadecanoic acid and/or tryglycerides, lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, oils
and/or fats containing heptadecanoic acid, in particular for
increasing the amount of heptadecanoic acid and/or tryglycerides,
lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing margaric acid,
preferably heptadecanoic acid in free or bound form in an organism
or a part thereof, as mentioned. In one further embodiment the
YDR513W protein expression is increased together with the increase
of another gene of the lipid biosynthesis pathway, preferably with
a gene encoding a protein being involved in the production of fatty
acids or encoding a fatty acid transporter protein or a compound,
which functions as a sink for the respective fatty acid. In one
embodiment, in the process of the present invention the activity of
a protein of the glutaredoxin superfamily, preferably of a
glutathione reductase is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof. The sequence of
YER156C from Saccharomyces cerevisiae has been published in
Dietrich, et al. (Nature 387 (6632 Suppl), 78-81 (1997)), and its
activity has not been characterized yet. It seems to have a
activity similar to Arabidopsis thaliana hypothetical protein
F2K15.180. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein with the activity
of a YER156C from Saccharomyces cerevisiae or of a Arabidopsis
thaliana hypothetical protein F2K15.180 or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of heptadecanoic acid and/or tryglycerides, lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing heptadecanoic acid
and/or conjugates, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one further embodiment the
YER156C protein expression is increased together with the increase
of another gene of the lipid biosynthesis pathway, preferably with
a gene encoding a protein being involved in the production of fatty
acids or encoding a fatty acid transporter protein or a compound,
which functions as a sink for the respective fatty acid. In one
embodiment, in the process of the present invention said activity,
e.g. of a YER156C protein is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof. The sequence of
YLR255C from Saccharomyces cerevisiae has been submitted by R;
Johnson to the EMBL Data Library, (February 1995), and its activity
has not been characterized yet, but having preferably an activity
of Saccharomyces hypothetical protein YLR255c superfamily
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
protein of the Saccharomyces hypothetical protein YLR255c
superfamily, preferably of a a gene product with an activity of a
YLR255C protein from Saccharomyces cerevisiae or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, meaning of heptadecanoic acid and/or tryglycerides,
lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing heptadecanoic acid in
free or bound form in an organism or a part thereof, as mentioned.
In one further embodiment the YLR255C protein expression is
increased together with the increase of another gene of the lipid
biosynthesis pathway, preferably with a gene encoding a protein
being involved in the production of fatty acids or encoding a fatty
acid transporter protein or a compound, which functions as a sink
for the resprective fatty acid. In one embodiment, in the process
of the present invention the activity of a Saccharomyces
hypothetical protein YLR255c superfamily protein, preferably
YLR255C protein, is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof.
[6607] The sequence of b1829 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a heat shock protein with protease
activity, preferably of the heat-shock protein htpX superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the heat-shock protein htpX superfamily, preferably such protein is
having a heat shock protein activity with protease activity from E.
coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of heptadecanoic acid and/or
tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
margaric acid, in particular for increasing the amount of margaric
acid and/or tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
heptadecanoic acid, preferably heptadecanoic acid in free or bound
form in an organism or a part thereof, as mentioned. In one further
embodiment the b1829 protein expression is increased together with
the increase of another gene of the lipid biosynthesis pathway,
preferably with a gene encoding a protein being involved in the
production of fatty acids or encoding a fatty acid transporter
protein or a compound, which functions as a sink for the respective
fatty acid. In one embodiment, in the process of the present
invention the activity of a heat shock protein with protease
activity is increased or generated, e.g. from E. coli or a homolog
thereof.
[6608] The sequence of YER173W from Saccharomyces cerevisiae has
been published in Dietrich et al., Nature 387 (6632 Suppl), 78-81,
1997, and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is being defined as a checkpoint protein, involved in
the activation of the DNA damage and meiotic pachytene checkpoints.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
Checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of 2-hydroxy-palmitic acid and/or tryglycerides, lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, oils
and/or fats containing 2-hydroxy-palmitic acid, in particular for
increasing the amount of 2-hydroxy-palmitic acid and/or
tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
2-hydroxy-palmitic acid in free or bound form in an organism or a
part thereof, as mentioned. In one further embodiment the YER173w
protein expression is increased together with the increase of
another gene of the lipid biosynthesis pathway, preferably with a
gene encoding a protein being involved in the production of
glucosylceramide, preferably of monoglucosecerebroside. In one
embodiment, in the process of the present invention the activity of
a Checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints is increased or generated, e.g.
from Saccharomyces cerevisiae or a homolog thereof.
[6609] The sequence of YFR042W from Saccharomyces cerevisiae has
been published in Murakami et al., Nat. Genet. 10 (3), 261-268
(1995) and Goffeau et al., Science 274 (5287), 546-547, 1996, and
has a putative cellular function of a "protein, which is required
for cell viability", preferably of the Saccharomyces cerevisiae
probable membrane protein YFR042w superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a a gene product with an activity of the Saccharomyces
cerevisiae probable membrane protein YFR042w superfamily,
preferably a gene product with a YFR042W protein activity from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of tetracosenoic acid
(nervonic acid, C24:1 delta 15), preferably
2-hydroxy-tetracosenoic-acid and/or tryglycerides, lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing tetracosenoic acid
(nervonic acid, C24:1 delta 15), preferably 2-hydroxy-tetracosenoic
acid, in particular for increasing the amount of tetracosenoic acid
(nervonic acid), preferably 2-hydroxy-tetracosenoic acid and/or
tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
tetracosenoic acid (nervonic acid), preferably
2-hydroxy-tetracosenoic acid, preferably tetracosenoic acid
(nervonic acid), preferably 2-hydroxy-tetracosenoic acid in free or
bound form in an organism or a part thereof, as mentioned. In one
further embodiment the YFR042W protein expression is increased
together with the increase of another gene of the lipid
biosynthesis pathway, preferably with a gene encoding a protein
being involved in the production of lipids, preferably of
cerebroside. In one embodiment, in the process of the present
invention the activity of a YFR042W protein is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[6610] The sequence of YOR317W from Saccharomyces cerevisiae has
been published in Dujon et al., Nature 387 (6632 Suppl), 98-102
(1997) and in Goffeau et al., Science 274 (5287), 546-547, 1996 and
its cellular activity has characterized as a long chain fatty
acyl:CoA synthetase, preferably of the long-chain-fatty-acid-CoA
ligase superfamily. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of the long-chain-fatty-acid-CoA ligase superfamily,
preferably a protein with a long chain fatty acyl:CoA synthetase
activity or its homolog, e.g. as shown herein, from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the fine chemical, meaning of hexadecadienoic acid (C16:2,
preferably C16:2 delta 7,10) and/or tryglycerides, lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing hexadecadienoic acid, in
particular for increasing the amount of hexydecadienoic acid and/or
tryglycerides, lipids, oils and/or fats containing hexadecadienoic
acid, preferably hexydecadienoic acid in free or bound form in an
organism or a part thereof, as mentioned. In one further embodiment
the YOR317W protein expression is increased together with the
increase of another gene of the lipid biosynthesis pathway,
preferably with a gene encoding a protein being involved in the
production of galactolipids, preferably of
monogalactosyldiacylglycerol. In one embodiment, in the process of
the present invention the activity of a protein of the
long-chain-fatty-acid-CoA ligase superfamily, preferably of a long
chain fatty acyl:CoA synthetase is increased or generated, e.g.
from Saccharomyces cerevisiae or a homolog thereof.
[6611] The sequence of YGL205W from Saccharomyces cerevisiae has
been published in Tettelin et al., Nature 387 (6632 Suppl), 81-84
(1997) and in Goffeau et al., Science 274 (5287), 546-547, 1996 and
its cellular activity has characterized as fatty-acyl coenzyme A
oxidase, preferably of the acyl-CoA oxidase superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the acyl-CoA oxidase superfamily, preferably a protein with a
fatty-acyl coenzyme A oxidase activity or its homolog, e.g. as
shown herein, from Saccharomyces cerevisiae or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
hexydecatrienoic acid (C16:3, preferably C16:2 delta 7,10,13)
and/or tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
hexydecatrienoic acid, in particular for increasing the amount of
hexydecatrienoic acid and/or tryglycerides, lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, oils
and/or fats containing hexydecatrienoic acid, preferably
hexydecatrienoic acid in free or bound form in an organism or a
part thereof, as mentioned. In one further embodiment the YGL205W
protein expression is increased together with the increase of
another gene of the lipid biosynthesis pathway, preferably with a
gene encoding a protein being involved in the production of
galactolipids, preferably of monogalactosyldiacylglycerol. In one
embodiment, in the process of the present invention the activity of
a protein of the acyl-CoA oxidase superfamily, preferably of a
fatty-acyl coenzyme A oxidase is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[6612] The sequence of YIL150C from Saccharomyces cerevisiae has
been published in Churcher et al., Nature 387 (6632 Suppl), 84-87
(1997) and in Goffeau et al., Science 274 (5287), 546-547, 1996 and
seems to have a putative cellular activity of a its "protein
required for S-phase (DNA synthesis) initiation or completion
and/or chromatin binding protein".
[6613] Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with the activity of
a YIL150C protein, from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or tryglycerides, lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, oils
and/or fats containing hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid, in particular for increasing the
amount of hexadecatrienoic acid and/or tryglycerides, lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing hexadecatrienoic acid,
preferably hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid in free or bound form in an organism or a
part thereof, as mentioned. In one further embodiment the YIL150C
protein expression is increased together with the increase of
another gene of the lipid biosynthesis pathway, preferably with a
gene encoding a protein being involved in the production of
galactolipids, preferably of monogalactosyldiacylglycerol. In one
embodiment, in the process of the present invention the activity of
a YIL150C protein is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[6614] The sequence of YKL132C from Saccharomyces cerevisiae has
been published in Dujon et al., Nature 369 (6479), 371-378 (1994)
and in Goffeau et al., Science 274 (5287), 546-547, 1996 and its
cellular activity has characterized as folyl-polyglutamate
synthase, preferably of the folylpolyglutamate synthase
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the folylpolyglutamate synthase superfamily, preferably
a protein with a folyl-polyglutamate synthase activity or its
homolog, e.g. as shown herein, from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of hexydecatrienoic acid preferably delta 7,10,13
hexadecatrienoic and/or tryglycerides, lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, oils
and/or fats containing hexydecatrienoic acid, in particular for
increasing the amount of hexydecatrienoic acid preferably delta
7,10,13 hexadecatrienoic and/or tryglycerides, lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, oils
and/or fats containing hexydecatrienoic acid, preferably
hexydecatrienoic acid in free or bound form in an organism or a
part thereof, as mentioned. In one further embodiment the YKL132C
protein expression is increased together with the increase of
another gene of the lipid biosynthesis pathway, preferably with a
gene encoding a protein being involved in the production of
galactolipids, preferably of monogalactosyldiacylglycerol. In one
embodiment, in the process of the present invention the activity of
a protein of the folylpolyglutamate synthase superfamily,
preferably of a folyl-polyglutamate synthase is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[6615] The sequence of YOR344C from Saccharomyces cerevisiae has
been published in Dujon et al., Nature 369 (6479), 371-378 (1994)
and in Goffeau et al., Science 274 (5287), 546-547, 1996 and its
cellular activity has characterized as a serine-rich protein,
putatively involved in glycolytic gene expression. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of a serine-rich protein,
putatively involved in glycolytic gene expression activity or its
homolog, e.g. as shown herein, from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of hexydecatrienoic acid preferably delta 7,10,13
hexadecatrienoic acid and/or tryglycerides, lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, oils
and/or fats containing hexydecatrienoic acid preferably delta
7,10,13 hexadecatrienoic acid, in particular for increasing the
amount of hexydecatrienoic acid preferably delta 7,10,13
hexadecatrienoic acid and/or tryglycerides, lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, oils
and/or fats containing hexydecatrienoic acid, preferably preferably
delta 7,10,13 hexadecatrienoic acid in free or bound form in an
organism or a part thereof, as mentioned. In one further embodiment
the YOR344C protein expression is increased together with the
increase of another gene of the lipid biosynthesis pathway,
preferably with a gene encoding a protein being involved in the
production of galactolipids, preferably of
monogalactosyldiacylglycerol. In one embodiment, in the process of
the present invention the activity of a serine-rich protein,
putatively involved in glycolytic gene expression is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[6616] The sequence of b3430 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a glucose-1-phosphate
adenylyltransferase, preferably of the a glucose-1-phosphate
adenylyltransferase superfamily. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of the a glucose-1-phosphate
adenylyltransferase superfamily, preferably such protein is having
a a glucose-1-phosphate adenylyltransferase activity from E. coli
or its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of hexydecatrienoic acid (C16:3,
preferably C16:3 delta 7,10, 13) and/or tryglycerides, lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing hexydecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid, in particular for
increasing the amount of hexydecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid and/or tryglycerides, lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing hexydecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid in free or bound
form in an organism or a part thereof, as mentioned. In one further
embodiment the b3430 protein expression is increased together with
the increase of another gene of the lipid biosynthesis pathway,
preferably with a gene encoding a protein being involved in the
production of galactolipids, preferably of
monogalactosyldiacylglycerol In one embodiment, in the process of
the present invention the activity of a a glucose-1-phosphate
adenylyltransferase is increased or generated, e.g. from E. coli or
a homolog thereof.
[6617] The sequence of b0057 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity has not been characterized yet. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a b0057 protein from E. coli or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of tetracosenoic acid (nervonic acid, C24:1 delta 15), preferably
2-hydroxy-tetracosenoic-acid and/or tryglycerides, lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing tetracosenoic acid
(nervonic acid, C24:1 delta 15), preferably 2-hydroxy-tetracosenoic
acid, in particular for increasing the amount of tetracosenoic acid
(nervonic acid), preferably 2-hydroxy-tetracosenoic acid and/or
tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
tetracosenoic acid (nervonic acid), preferably
2-hydroxy-tetracosenoic acid, preferably tetracosenoic acid
(nervonic acid), preferably 2-hydroxy-tetracosenoic acid in free or
bound form in an organism or a part thereof, as mentioned. In one
further embodiment the b0057 protein expression is increased
together with the increase of another gene of the lipid
biosynthesis pathway, preferably with a gene encoding a protein
being involved in the production of lipids, preferably of
cerebroside. In one embodiment, in the process of the present
invention the activity of a b0057 protein is increased or
generated, e.g. from Echerischia Coli or a homolog thereof.
[6618] The sequence of b0161 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a protein having periplasmic serine
protease (heat shock protein) activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of a b0161 protein from E. coli
or its homolog, e.g. as shown herein, for the production of the
respective fine chemical, meaning of 2-hydroxy-palmitic acid and/or
tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
2-hydroxy-palmitic acid, in particular for increasing the amount of
2-hydroxy-palmitic acid and/or tryglycerides, lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, oils
and/or fats containing 2-hydroxy-palmitic acid in free or bound
form in an organism or a part thereof, as mentioned. In one further
embodiment the b0161 protein expression is increased together with
the increase of another gene of the lipid biosynthesis pathway,
preferably with a gene encoding a protein being involved in the
production of glucosylceramide, preferably of
monoglucosecerebroside. In one embodiment, in the process of the
present invention the activity of a protein having periplasmic
serine protease (heat shock protein) activity is increased or
generated, e.g. from Escherichia Coli.
[6619] The sequence of b0758 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a protein having galactose-1-phosphate
uridylyltransferase activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of a b0758 protein from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of 2-hydroxy-palmitic acid and/or
tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
2-hydroxy-palmitic acid, in particular for increasing the amount of
2-hydroxy-palmitic acid and/or tryglycerides, lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, oils
and/or fats containing 2-hydroxy-palmitic acid in free or bound
form in an organism or a part thereof, as mentioned. In one further
embodiment the b0758 protein expression is increased together with
the increase of another gene of the lipid biosynthesis pathway,
preferably with a gene encoding a protein being involved in the
production of glucosylceramide, preferably of
monoglucosecerebroside. In one embodiment, in the process of the
present invention the activity of a protein having
galactose-1-phosphate uridylyltransferase activity is increased or
generated, e.g. from Escherichia Coli.
[6620] The sequence of b1097 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a protein having thymidylate kinase
activity. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a b1097 protein from E. coli or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of tetracosenoic acid (nervonic acid, C24:1 delta 15),
preferably 2-hydroxy-tetracosenoic-acid and/or tryglycerides,
lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing tetracosenoic acid
(nervonic acid, C24:1 delta 15), preferably 2-hydroxy-tetracosenoic
acid, in particular for increasing the amount of tetracosenoic acid
(nervonic acid), preferably 2-hydroxy-tetracosenoic acid and/or
tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
tetracosenoic acid (nervonic acid), preferably
2-hydroxy-tetracosenoic acid, preferably tetracosenoic acid
(nervonic acid), preferably 2-hydroxy-tetracosenoic acid in free or
bound form in an organism or a part thereof, as mentioned. In one
further embodiment the b1097 protein expression is increased
together with the increase of another gene of the lipid
biosynthesis pathway, preferably with a gene encoding a protein
being involved in the production of lipids, preferably of
cerebroside. In one embodiment, in the process of the present
invention the activity of a b0057 protein is increased or
generated, e.g. from Echerischia Coli or a homolog thereof.
[6621] The sequence of b2078 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a protein having histidine kinase
activity. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a b2078 protein from E. coli or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of tetracosenoic acid (nervonic acid, C24:1 delta 15),
preferably 2-hydroxy-tetracosenoic-acid and/or tryglycerides,
lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing tetracosenoic acid
(nervonic acid, C24:1 delta 15), preferably 2-hydroxy-tetracosenoic
acid, in particular for increasing the amount of tetracosenoic acid
(nervonic acid), preferably 2-hydroxy-tetracosenoic acid and/or
tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
tetracosenoic acid (nervonic acid), preferably
2-hydroxy-tetracosenoic acid, preferably tetracosenoic acid
(nervonic acid), preferably 2-hydroxy-tetracosenoic acid in free or
bound form in an organism or a part thereof, as mentioned. In one
further embodiment the b2078 protein expression is increased
together with the increase of another gene of the lipid
biosynthesis pathway, preferably with a gene encoding a protein
being involved in the production of lipids, preferably of
cerebroside. In one embodiment, in the process of the present
invention the activity of a b2078 protein is increased or
generated, e.g. from Echerischia Coli or a homolog thereof.
[6622] The sequence of b3231 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a protein having 50S ribosomal subunit
protein L13 activity. Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of a b3231 protein from E. coli or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of tetracosenoic acid (nervonic acid, C24:1 delta 15),
preferably 2-hydroxy-tetracosenoic-acid and/or tryglycerides,
lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing tetracosenoic acid
(nervonic acid, C24:1 delta 15), preferably 2-hydroxy-tetracosenoic
acid, in particular for increasing the amount of tetracosenoic acid
(nervonic acid), preferably 2-hydroxy-tetracosenoic acid and/or
tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
tetracosenoic acid (nervonic acid), preferably
2-hydroxy-tetracosenoic acid, preferably tetracosenoic acid
(nervonic acid), preferably 2-hydroxy-tetracosenoic acid in free or
bound form in an organism or a part thereof, as mentioned. In one
further embodiment the b3231 protein expression is increased
together with the increase of another gene of the lipid
biosynthesis pathway, preferably with a gene encoding a protein
being involved in the production of lipids, preferably of
cerebroside. In one embodiment, in the process of the present
invention the activity of a b3231 protein is increased or
generated, e.g. from Echerischia Coli or a homolog thereof.
[6623] [0023.0.15.15] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[6624] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, lines 174,
175 or 176 resp. is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of the respective fine chemical in the organisms,
preferably of heptadecanoic acid. In one embodiment, the homolog is
a homolog with a sequence as indicated in Table I or II, column 7,
lines 174, 175 and/or 176. In one embodiment, the homolog of one of
the polypeptides indicated in Table IIA or IIB, column 3, lines
174, 175 or 176 resp., is derived from an eukaryotic. In one
embodiment, the homolog is derived from Fungi. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, lines
174, 175 or 176 resp., is derived from Ascomyceta. In one
embodiment, the homolog of a polypeptide indicated in Table IIA or
IIB, column 3, lines 174, 175 or 176 resp., is derived from
Saccharomycotina. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, lines 174, 175 or 176
resp., is derived from Saccharomycetes. In one embodiment, the
homolog of a polypeptide indicated in Table IIA or IIB, column 3,
lines 174, 175 or 176 resp., is a homolog being derived from
Saccharomycetales. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, lines 174, 175 or 176
resp., is a homolog having the same or a similar activity being
derived from Saccharomycetaceae. In one embodiment, the homolog of
a polypeptide indicated in Table IIA or IIB, column 3, lines 174,
175 or 176 resp., is a homolog having the same or a similar
activity being derived from Saccharomycetes.
[6625] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 178, 558
or 559 is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of 2-hydroxy palmitic acid. In one embodiment, the
homolog is a homolog with a sequence as indicated in Table I or II,
column 7, line 178, 558 or 559. In one embodiment, the homolog of
one of the polypeptides indicated in Table IIA or IIB, column 3,
line 178, 558 or 559 is derived from an eukaryotic. In one
embodiment, the homolog is derived from Fungi. In one embodiment,
the homolog of a polypeptide indicated in Table IIA or IIB, column
3, line 178, 558 or 559 is derived from Ascomyceta. In one
embodiment, the homolog of a polypeptide indicated in Table IIA or
IIB, column 3, line 178, 558 or 559 is derived from
Saccharomycotina. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 178, 558 or 559 is
derived from Saccharomycetes. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, line 178, 558
or 559 is a homolog being derived from Saccharomycetales. In one
embodiment, the homolog of a polypeptide indicated in Table IIA or
IIB, column 3, line 178, 558 or 559 is a homolog having the same or
a similar activity being derived from Saccharomycetaceae. In one
embodiment, the homolog of a polypeptide indicated in Table IIA or
IIB, column 3, line 178, 558 or 559 is a homolog having the same or
a similar activity being derived from Saccharomycetes.
[6626] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 558 or
559 is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms,
preferably of 2-hydroxy palmitic acid. In one embodiment, the
homolog is a homolog with a sequence as indicated in Table I or II,
column 7, line 558 or 559. In one embodiment, the homolog of one of
the polypeptides indicated in Table IIA or IIB, column 3, line 558
or 559 is derived from a bacteria. In one embodiment, the homolog
is derived from Proteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, line 558 or
559 is derived from Gammaproteobacteria. In one embodiment, the
homolog of a polypeptide indicated in Table IIA or IIB, column 3,
line 558 or 559 is derived from Enterobacteriales. In one
embodiment, the homolog of a polypeptide indicated in Table IIA or
IIB, column 3, line 558 or 559 is derived from Enterobacteriaceae.
In one embodiment, the homolog of a polypeptide indicated in Table
IIA or IIB, column 3, line 558 or 559 is a homolog being derived
from Escherichia.
[6627] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 179 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms, preferably of
2-hydroxy-tetracosenoic-acid. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
line 179. In one embodiment, the homolog of one of the polypeptides
indicated in Table IIA or IIB, column 3, line 179 is derived from
an eukaryotic. In one embodiment, the homolog is derived from
Fungi. In one embodiment, the homolog of a polypeptide indicated in
Table IIA or IIB, column 3, line 179 is derived from Ascomyceta. In
one embodiment, the homolog of a polypeptide indicated in Table IIA
or IIB, column 3, line 179 is derived from Saccharomycotina. In one
embodiment, the homolog of a polypeptide indicated in Table IIA or
IIB, column 3, line 179 is derived from Saccharomycetes. In one
embodiment, the homolog of a polypeptide indicated in Table IIA or
IIB, column 3, line 179 is a homolog being derived from
Saccharomycetales. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 179 is a homolog
having the same or a similar activity being derived from
Saccharomycetaceae. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 179 is a homolog
having the same or a similar activity being derived from
Saccharomycetes.
[6628] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 180 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms, preferably of
hexadecadienoic acid, preferably delta 7, 10 hexadecadienoic acid.
In one embodiment, the homolog is a homolog with a sequence as
indicated in Table I or II, column 7, line 180. In one embodiment,
the homolog of one of the polypeptides indicated in Table IIA or
IIB, column 3, line 180 is derived from an eukaryotic. In one
embodiment, the homolog is derived from Fungi. In one embodiment,
the homolog of a polypeptide indicated in Table IIA or IIB, column
3, line 180 is derived from Ascomyceta. In one embodiment, the
homolog of a polypeptide indicated in Table IIA or IIB, column 3,
line 180 is derived from Saccharomycotina. In one embodiment, the
homolog of a polypeptide indicated in Table IIA or IIB, column 3,
line 180 is derived from Saccharomycetes. In one embodiment, the
homolog of a polypeptide indicated in Table IIA or IIB, column 3,
line 180 is a homolog being derived from Saccharomycetales. In one
embodiment, the homolog of a polypeptide indicated in Table IIA or
IIB, column 3, line 180 is a homolog having the same or a similar
activity being derived from Saccharomycetaceae. In one embodiment,
the homolog of a polypeptide indicated in Table IIA or IIB, column
3, line 180 is a homolog having the same or a similar activity
being derived from Saccharomycetes.
[6629] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, lines 181,
182, 183 or 184 resp., is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of the respective fine chemical in the organisms,
preferably of hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid. In one embodiment, the homolog is a homolog
with a sequence as indicated in Table I or II, column 7, lines 181,
182, 183 or 184 resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table IIA or IIB, column 3, lines 181,
182, 183 or 184 resp., is derived from an eukaryotic. In one
embodiment, the homolog is derived from Fungi. In one embodiment,
the homolog of a polypeptide indicated in Table IIA or IIB, column
3, lines 181, 182, 183 or 184 resp., is derived from Ascomyceta. In
one embodiment, the homolog of a polypeptide indicated in Table IIA
or IIB, column 3, lines 181, 182, 183 or 184 resp., is derived from
Saccharomycotina. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, lines 181, 182, 183 or 184
resp., is derived from Saccharomycetes. In one embodiment, the
homolog of a polypeptide indicated in Table IIA or IIB, column 3,
lines 181, 182, 183 or 184 resp., is a homolog being derived from
Saccharomycetales. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, lines 181, 182, 183 or 184
resp., is a homolog having the same or a similar activity being
derived from
[6630] Saccharomycetaceae. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, lines 181,
182, 183 or 184 resp., is a homolog having the same or a similar
activity being derived from Saccharomycetes.
[6631] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 177 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms preferably of
heptadecanoic acid. In one embodiment, the homolog is a homolog
with a sequence as indicated in Table I or II, column 7, line 177,
resp. In one embodiment, the homolog of one of the polypeptides
indicated in Table IIA or IIB, column 3, line 177 is derived from
an bacteria. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 177 is derived from
Proteobacteria. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 177 is a homolog
having the same or a similar activity being derived from
Gammaproteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, line 177 is
derived from Enterobacteriales. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, line 177 is a
homolog being derived from Enterobacteriaceae. In one embodiment,
the homolog of a polypeptide indicated in Table IIA or IIB, column
3, line 177 is a homolog having the same or a similar activity and
being derived from Escherichia.
[6632] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 185 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms, preferably of
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid. In one embodiment, the homolog is a homolog with a sequence
as indicated in Table I or II, column 7, line 185. In one
embodiment, the homolog of one of the polypeptides indicated in
Table IIA or IIB, column 3, line 185 resp. is derived from a
bacteria. In one embodiment, the homolog of a polypeptide indicated
in Table IIA or IIB, column 3, line 185 resp. is derived from
Proteobacteria. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 185 is a homolog
having the same or a similar activity being derived from
Gammaproteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, line 185 is
derived from Enterobacteriales. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, line 185 is a
homolog being derived from Enterobacteriaceae. In one embodiment,
the homolog of a polypeptide indicated in Table IIA or IIB, column
3, lines 185 is a homolog having the same or a similar activity and
being derived from Escherichia.
[6633] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 560 to 563 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms, preferably of C24:1
fatty acid, preferably C24:1 delta 15 fatty acid. In one
embodiment, the homolog is a homolog with a sequence as indicated
in Table I or II, column 7, line 560 to 563. In one embodiment, the
homolog of one of the polypeptides indicated in Table IIA or IIB,
column 3, line 560 to 563 resp. is derived from an bacteria. In one
embodiment, the homolog of a polypeptide indicated in Table IIA or
IIB, column 3, line 560 to 563 resp. is derived from
Proteobacteria. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 560 to 563 is a
homolog having the same or a similar activity being derived from
Gammaproteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, line 560 to
563 is derived from Enterobacteriales. In one embodiment, the
homolog of a polypeptide indicated in Table IIA or IIB, column 3,
line 560 to 563 is a homolog being derived from Enterobacteriaceae.
In one embodiment, the homolog of a polypeptide indicated in Table
IIA or IIB, column 3, lines 560 to 563 is a homolog having the same
or a similar activity and being derived from Escherichia.
[6634] [0023.1.15.15] Homologs of the polypeptide indicated in
Table IIA or IIB, column 3, lines 174 to 185, 558 to 563 may be the
polypeptides encoded by the nucleic acid molecules indicated in
Table IA or IB, column 7, lines 174 to 185, 558 to 563, resp., or
may be the polypeptides indicated in Table IIA or IIB, column 7,
lines 174 to 185, 558 to 563, resp. Homologs of the polypeptides
polypeptide indicated in Table IIA or IIB, column 3, lines 174 to
185, 558 to 563 may be the polypeptides encoded by the nucleic acid
molecules indicated in Table IA or IB, column 7, lines 174 to 185,
558 to 563 or may be the polypeptides indicated in Table IIA or
IIB, column 7, lines 174 to 185, 558 to 563.
[6635] Homologs of the polypeptides indicated in Table IIA of IIB,
column 3, lines 174 to 177 may be the polypeptides encoded by the
nucleic acid molecules indicated in Table IA or IB, column 7, lines
174 to 177, respectively or may be the polypeptides indicated in
Table IIA or IIB, column 7, lines 174 to 177, having a
heptadecanoic acid content- and/or amount-increasing activity.
[6636] Homologs of the polypeptides indicated in Table IIA or IIB,
column 3, line 179 may be the polypeptides encoded by the nucleic
acid molecules indicated in Table IA or IB, column 7, line 179,
respectively or may be the polypeptides indicated in Table IIA or
IIB, column 7, line 179, having a 2-hydroxy-tetracosenoic-acid
content- and/or amount-increasing activity.
[6637] Homologs of the polypeptides indicated in Table IIA or IIB,
column 3, line 180 may be the polypeptides encoded by the nucleic
acid molecules indicated in Table IA or IB, column 7, line 180,
respectively or may be the polypeptides indicated in Table IIA or
IIB, column 7, line 180, having a hexadecadienoic acid, preferably
delta 7,10 hexadecadienoic acid content- and/or amount-increasing
activity.
[6638] Homologs of the polypeptides indicated in Table IIA or IIB,
column 3, lines 181 to 185 may be the polypeptides encoded by the
nucleic acid molecules indicated in Table IA or IB, column 7, lines
181 to 185, respectively or may be the polypeptides indicated in
Table IIA or IIB, column 7, lines 181 to 185, having a
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid content- and/or amount-increasing activity.
[6639] Homologs of the polypeptides indicated in Table IIA or IIB,
column 3, lines 558, 559 may be the polypeptides encoded by the
nucleic acid molecules indicated in Table IA or IB, column 7, lines
558, 559, respectively or may be the polypeptides indicated in
Table IIA or IIB, column 7, lines 558, 559 having
2-hydroxy-palmitic acid content- and/or amount-increasing
activity.
[6640] Homologs of the polypeptides indicated in Table IIA or IIB,
column 3, lines 560 to 563 may be the polypeptides encoded by the
nucleic acid molecules indicated in Table IA or IB, column 7, lines
560 to 563, respectively or may be the polypeptides indicated
in
[6641] Table IIA or IIB, column 7, lines 560 to 563, having a C24:1
fatty acid, preferably C24:1 delta 15 fatty acid content- and/or
amount-increasing activity.
[6642] [0024.0.0.15] see [0024.0.0.0]
[6643] [0025.0.15.15] In accordance with the invention, a protein
or polypeptide has the "activity of an protein of the invention",
e.g. the activity of a protein indicated in Table IIA or IIB,
column 3, lines 174 to 177 and/or line 178 and/or line 179 and/or
line 180 and/or lines 181 to 185 and/or 558, 559 and/or 560 to 563
resp., if its de novo activity, or its increased expression
directly or indirectly leads to an increased fatty acid level, in
particular to a increased heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid and/or 2-hydroxy-palmitic acid and/or C24:1
fatty acid, preferably delta 7,10 hexadecadienoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid resp., in the organism or a part thereof, preferably in a cell
of said organism. In a preferred embodiment, the protein or
polypeptide has the above-mentioned additional activities of a
protein indicated in Table IIA or IIB, column 3, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or 558, 559 and/or 560 to 563 resp. Throughout the
specification the activity or preferably the biological activity of
such a protein or polypeptide or an nucleic acid molecule or
sequence encoding such protein or polypeptide is identical or
similar if it still has the biological or enzymatic activity of any
one of the proteins indicated in Table IIA or IIB, column 3, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 resp., or which has at least 10% of the original
enzymatic activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to any one of the
proteins indicated in Table IIA or IIB, column 3, lines 174 to 176,
or 178, or 179, or 180 or 181 to 184 of Saccharomyces cerevisiae
and/or any one of the proteins indicated in Table IIA or IIB,
column 3, lines 177 or 185 or 558, 559 or 560 to 563 of E. coli
K12.
[6644] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table IA
or IB, column 4 and is expressed in an organism, which is
evolutionary distant to the origin organism. For example origin and
expressing organism are derived from different families, orders,
classes or phylums whereas origin and the organism indicated in
Table IA or IB, column 4 are derived from the same families,
orders, classes or phylums.
[6645] [0026.0.0.15] to [0033.0.0.15] see [0026.0.0.0] to
[0033.0.0.0]
[6646] [0034.0.15.15] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of an protein having the activity of a
protein as indicated in Table IIA or IIB, column 3, lines 174 to
177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp., or
being encoded by a nucleic acid molecule indicated in Table IA or
IB, column 5, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., or its homologs, e.g. as indicated
in Table I, column 7, lines 174 to 177 and/or line 178 and/or line
179 and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560
to 563 resp., its biochemical or genetical causes and therefore
shows the increased amount of the fine chemical.
[6647] [0035.0.0.15] to [0036.0.0.15] see [0035.0.0.0] to
[0036.0.0.0]
[6648] [0037.0.15.15] A series of mechanisms exists via which a
modification of the a protein, e.g. the polypeptide of the
invention can directly or indirectly affect the yield, production
and/or production efficiency of the fatty acid.
[6649] [0038.0.0.15] to [0044.0.0.15] see [0038.0.0.0] to
[0044.0.0.0]
[6650] [0045.0.15.15] In one embodiment, the activity of the
Saccharomyces cerevisiae protein YDR513W or its homologs, e.g. an
activity of a glutathione reductase, preferably of the glutaredoxin
superfamily, e.g. as indicated in Table II, columns 5 or 7, line
174, is increased conferring an increase of the respective fine
chemical, preferably of the heptadecanoic acid between 24% and 97%
or more.
[6651] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER156C or its homologs, e.g. an activity of a
YER156C protein, e.g. as indicated in Table II, columns 5 or 7,
line 175, is increased conferring an increase of the respective
fine chemical, preferably of the heptadecanoic acid between 20% and
49% or more.
[6652] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER173W or its homologs, e.g. an activity of a
checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints, e.g. as indicated in Table II,
columns 5 or 7, line 178, is increased conferring an increase of
the respective fine chemical, preferably of the 2-hydroxy palmitic
acid between 26% and 394% or more.
[6653] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YFR042W or its homologs, e.g. an activity of a
protein, which is required for cell viability, preferably of the
Saccharomyces cerevisiae probable membrane protein YFR042w
superfamily, e.g. as indicated in Table II, columns 5 or 7, line
179, is increased conferring an increase of the respective fine
chemical, preferably of the 2-hydroxy-tetracosenoic-acid between
28% and 56% or more.
[6654] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YGL205W or its homologs, e.g. an activity of a
fatty-acyl coenzyme A oxidase, preferably of the acyl-CoA oxidase
superfamily, e.g. as indicated in Table II, columns 5 or 7, line
181, is increased conferring an increase of the respective fine
chemical, preferably of the hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid between 14% and 16% or more.
[6655] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YIL150C or its homologs, e.g. an activity of a
protein required for S-phase (DNA synthesis) initiation or
completion and/or chromatin binding protein, e.g. as indicated in
Table II, columns 5 or 7, line 182, is increased conferring an
increase of the respective fine chemical, preferably of the
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid between 150% and 205%, preferably of 224% or more.
[6656] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YKL132C or its homologs, e.g. an activity of a
folyl-polyglutamate synthase, preferably of the folylpolyglutamate
synthase superfamily, e.g. as indicated in Table II, columns 5 or
7, line 183, is increased conferring an increase of the respective
fine chemical, preferably of the hexadecatrienoic acid, preferably
delta 7,10,13 hexadecatrienoic acid between 13% and 56% or
more.
[6657] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YLR255C or its homologs, e.g. an activity of of
Saccharomyces hypothetical protein YLR255c superfamily, e.g. as
indicated in Table II, columns 5 or 7, line 176, is increased
conferring an increase of the respective fine chemical, preferably
of heptadecanoic acid between 20% and 27% or more.
[6658] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR317W or its homologs, e.g. an activity of a
long chain fatty acyl:CoA synthetase, preferably of the
long-chain-fatty-acid-CoA ligase superfamily, e.g. as indicated in
Table II, columns 5 or 7, line 180, is increased conferring an
increase of the respective fine chemical, preferably of the
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
between 19% and 69% or more.
[6659] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR344C or its homologs, e.g. an activity of a
serine-rich protein, putatively involved in glycolytic gene
expression, e.g. as indicated in Table II, columns 5 or 7, line
184, is increased conferring an increase of the respective fine
chemical, preferably of the hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid between 12% and 20% or more.
[6660] In one embodiment, the activity of the Escherichia coli K12
protein b1829 or its homologs, e.g. an activity of a heat shock
protein with protease activity, preferably of the heat-shock
protein htpX superfamily, e.g. as indicated in Table II, columns 5
or 7, line 177, is increased conferring an increase of the
respective fine chemical, preferably of the heptadecanoic acid
between 20% and 133% or more.
[6661] In one embodiment, the activity of the Escherichia coli K12
protein b3430 or its homologs, e.g. an activity of a
transcriptional regulator of glucose-1-phosphate
adenylyltransferase, preferably of the a glucose-1-phosphate
adenylyltransferase superfamily, e.g. as indicated in Table II,
columns 5 or 7, line 185, is increased conferring an increase of
the respective fine chemical, preferably of the hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid between 12%
and 20% or more.
[6662] In one embodiment, the activity of the Escherichia coli K12
protein b0057 or its homologs, e.g. as indicated in Table II,
columns 5 or 7, line 560, is increased conferring an increase of
the respective fine chemical, preferably of the C24:1 fatty acid
between 22% and 37% or more.
[6663] In one embodiment, the activity of the Escherichia coli K12
protein b0161 or its homologs, e.g. an activity of a periplasmic
serine protease (heat shock protein), e.g. as indicated in Table
II, columns 5 or 7, line 558, is increased conferring an increase
of the respective fine chemical, preferably of the
2-hydroxy-palmitic acid between 21% and 48% or more.
[6664] In one embodiment, the activity of the Escherichia coli K12
protein b0758 or its homologs, e.g. an activity of a
galactose-1-phosphate uridylyltransferase protein, e.g. as
indicated in Table II, columns 5 or 7, line 559, is increased
conferring an increase of the respective fine chemical, preferably
of the 2-hydroxy-palmitic acid between 19% and 38% or more.
[6665] In one embodiment, the activity of the Escherichia coli K12
protein b1097 or its homologs, e.g. an activity of a thymidylate
kinase protein, e.g. as indicated in Table II, columns 5 or 7, line
561, is increased conferring an increase of the respective fine
chemical, preferably of the C24:1 fatty acid between 23% and 41% or
more.
[6666] In one embodiment, the activity of the Escherichia coli K12
protein b2078 or its homologs, e.g. an activity of a sensory
histidine kinase in two-component regulatory system protein, e.g.
as indicated in Table II, columns 5 or 7, line 562, is increased
conferring an increase of the respective fine chemical, preferably
of the the C24:1 fatty acid between 23% and 41% or more.
[6667] In one embodiment, the activity of the Escherichia coli K12
protein b3231 or its homologs, e.g. an activity of a 50S ribosomal
subunit protein L13 protein, e.g. as indicated in Table II, columns
5 or 7, line 563, is increased conferring an increase of the
respective fine chemical, preferably of the the C24:1 fatty acid
between 23% and 48% or more.
[6668] [0046.0.15.15] In one embodiment, the activity of the
Saccharomyces cerevisiae protein YDR513W or its homologs, e.g. an
activity of a glutathione reductase, preferably of the glutaredoxin
superfamily, e.g. as indicated in Table II, columns 5 or 7, line
174, confers an increase of the respective fine chemical and of
further fatty acid activity having-compounds or lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, or their
precursors.
[6669] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER156C or its homologs, e.g. an activity of a
YER156C protein, e.g. as indicated in Table II, columns 5 or 7,
line 175, confers an increase of the respective fine chemical and
of further fatty acid activity having-compounds or lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, or their precursors.
[6670] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER173W or its homologs, e.g. an activity of a
checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints, e.g. as indicated in Table II,
columns 5 or 7, line 178, confers an increase of the respective
fine chemical and of further fatty acid activity having-compounds
or lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, or their precursors.
[6671] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YFR042W or its homologs, e.g. an activity of a
protein, which is required for cell viability, preferably of the
Saccharomyces cerevisiae probable membrane protein YFR042w
superfamily, e.g. as indicated in Table II, columns 5 or 7, line
179, confers an increase of the respective fine chemical and of
further fatty acid activity having-compounds or lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, or their
precursors.
[6672] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YGL205W or its homologs, e.g. an activity of a
fatty-acyl coenzyme A oxidase, preferably of the acyl-CoA oxidase
superfamily, e.g. as indicated in Table II, columns 5 or 7, line
181, confers an increase of the respective fine chemical and of
further fatty acid activity having-compounds or lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, or their
precursors.
[6673] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YIL150C or its homologs, e.g. an activity of a
protein required for S-phase (DNA synthesis) initiation or
completion and/or chromatin binding protein, e.g. as indicated in
Table II, columns 5 or 7, line 182, confers an increase of the
respective fine chemical and of further fatty acid activity
having-compounds or lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, or their precursors.
[6674] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YKL132C or its homologs, e.g. an activity of a
folyl-polyglutamate synthase, preferably of the folylpolyglutamate
synthase superfamily, e.g. as indicated in Table II, columns 5 or
7, line 183, confers an increase of the respective fine chemical
and of further fatty acid activity having-compounds or lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, or their precursors.
[6675] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YLR255C or its homologs, e.g. an activity of the
Saccharomyces hypothetical protein YLR255c superfamily, e.g. as
indicated in Table II, columns 5 or 7, line 176, confers an
increase of the respective fine chemical and of further fatty acid
activity having-compounds or lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, or their
precursors.
[6676] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR317W or its homologs, e.g. an activity of a
long chain fatty acyl:CoA synthetase, preferably of the
long-chain-fatty-acid-CoA ligase superfamily, e.g. as indicated in
Table II, columns 5 or 7, line 180, confers an increase of the
respective fine chemical and of further fatty acid activity
having-compounds or lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, or their precursors.
[6677] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR344C or its homologs, e.g. an activity of a
serine-rich protein, putatively involved in glycolytic gene
expression, e.g. as indicated in Table II, columns 5 or 7, line
184, confers an increase of the respective fine chemical and of
further fatty acid activity having-compounds or lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, or their
precursors.
[6678] In one embodiment, the activity of the Escherichia coli K12
protein b1829 or its homologs, e.g. an activity of a heat shock
protein with protease activity, preferably of the heat-shock
protein htpX superfamily, e.g. as indicated in Table II, columns 5
or 7, line 177, confers an increase of the respective fine chemical
and of further fatty acid activity having-compounds or lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, or their precursors.
[6679] In one embodiment, the activity of the Escherichia coli K12
protein b3430 or its homologs, e.g. an activity of a
transcriptional regulator of glucose-1-phosphate
adenylyltransferase, preferably of the a glucose-1-phosphate
adenylyltransferase superfamily, e.g. as indicated in Table II,
columns 5 or 7, line 185, confers an increase of the respective
fine chemical and of further fatty acid activity having-compounds
or lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, compounds or their precursors.
[6680] In one embodiment, the activity of the Escherichia coli K12
protein b0057 or its homologs, e.g. as indicated in Table II,
columns 5 or 7, line 560, confers an increase of the respective
fine chemical and of further fatty acid activity having-compounds
or lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, compounds or their precursors.
[6681] In one embodiment, the activity of the Escherichia coli K12
protein b0161 or its homologs, e.g. a periplasmic serine protease
(heat shock protein) activity, e.g. as indicated in Table II,
columns 5 or 7, line 558, confers an increase of the respective
fine chemical and of further fatty acid activity having-compounds
or lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, compounds or their precursors.
[6682] In one embodiment, the activity of the Escherichia coli K12
protein b0758 or its homologs, e.g. a galactose-1-phosphate
uridylyltransferase activity, e.g. as indicated in Table II,
columns 5 or 7, line 559, confers an increase of the respective
fine chemical and of further fatty acid activity having-compounds
or lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, compounds or their precursors.
[6683] In one embodiment, the activity of the Escherichia coli K12
protein b1097 or its homologs, e.g. a thymidylate kinase activity,
e.g. as indicated in Table II, columns 5 or 7, line 561, confers an
increase of the respective fine chemical and of further fatty acid
activity having-compounds or lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, compounds
or their precursors.
[6684] In one embodiment, the activity of the Escherichia coli K12
protein b2078 or its homologs, e.g. a sensory histidine kinase in
two-component regulatory system activity, e.g. as indicated in
Table II, columns 5 or 7, line 562, confers an increase of the
respective fine chemical and of further fatty acid activity
having-compounds or lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, compounds or their
precursors.
[6685] In one embodiment, the activity of the Escherichia coli K12
protein b3231 or its homologs, e.g. a 50S ribosomal subunit protein
L13 activity, e.g. as indicated in Table II, columns 5 or 7, line
563, confers an increase of the respective fine chemical and of
further fatty acid activity having-compounds or lipids, preferably
glycerophospholipids, sphingolipids and/or galactolipids, compounds
or their precursors.
[6686] [0047.0.0.15] and [0048.0.0.15] see [0047.0.0.0] and
[0048.0.0.0]
[6687] [0049.0.15.15] A protein having an activity conferring an
increase in the amount or level of the heptadecanoic acid
preferably has the structure of the polypeptide described herein.
In a particular embodiment, the polypeptides used in the process of
the present invention or the polypeptide of the present invention
comprises the sequence of a consensus sequence as shown in SEQ ID
NO: 15686, 15687 and/or 15688 or 19697, 15698, 15699, 15700, 15701
and/or 15702 or 15703, 15704 and/or 15705 and/or the sequence of a
consensus sequence as indicated in Table IV, columns 5 or 7, lines
174 to 177 and/or the sequence of a polypeptide as indicated in
Table II, columns 5 or 7, lines 174 to 177, or of a functional
homologue thereof as described herein, or of a polypeptide encoded
by the nucleic acid molecule characterized herein or the nucleic
acid molecule according to the invention, for example by a nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines 174 to
177 or its herein described functional homologues and has the
herein mentioned activity conferring an increase in the
heptadecanoic acid level.
[6688] A protein having an activity conferring an increase in the
amount or level of the 2-hydroxy-tetracosenoic-acid preferably has
the structure of the polypeptide described herein. In a particular
embodiment, the polypeptides used in the process of the present
invention or the polypeptide of the present invention comprises the
sequence of a consensus sequence as shown in SEQ ID NO: 15691
and/or 15692 and/or the sequence of a consensus sequence as
indicated in Table IV, columns 5 or 7, line 179 and/or the sequence
of a polypeptide as indicated in Table II, columns 5 or 7, line 179
or of a functional homologue thereof as described herein, or of a
polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, line 179 or its herein described functional homologues and
has the herein mentioned activity conferring an increase in the
2-hydroxy-tetracosenoic-acid level.
[6689] A protein having an activity conferring an increase in the
amount or level of the hexadecadienoic acid, preferably delta 7,10
hexadecadienoic acid preferably has the structure of the
polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence as shown in SEQ ID NO: 15661, 16662, 15663,
15664, 15665 and/or 15666 and/or the sequence of a consensus
sequence as indicated in Table IV, columns 5 or 7, line 180 and/or
the sequence of a polypeptide as indicated in Table II, columns 5
or 7, line 180 or of a functional homologue thereof as described
herein, or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, line 180 or its herein described
functional homologues and has the herein mentioned activity
conferring an increase in the hexadecadienoic acid, preferably
delta 7,10 hexadecadienoic acid level.
[6690] A protein having an activity conferring an increase in the
amount or level of the hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid preferably has the structure of the
polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence as shown in SEQ ID NO: 15706 and/or 15707 or
15689 and/or, 15690 or 15682, 15683, 15684 and/or, 15685 or 15658,
15659 and/or 15660 or 15677, 15678, 15679, 15680 and/or 15681
and/or the sequence of a consensus sequence as indicated in Table
IV, columns 5 or 7, lines 181 to 185 and/or the sequence of a
polypeptide as indicated in Table II, columns 5 or 7, lines 181 to
185 or of a functional homologue thereof as described herein, or of
a polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, lines 181 to 185 or its herein described functional
homologues and has the herein mentioned activity conferring an
increase in the hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid level.
[6691] A protein having an activity conferring an increase in the
amount or level of the 2-hydroxy-palmitic acid, preferably has the
structure of the polypeptide described herein. In a particular
embodiment, the polypeptides used in the process of the present
invention or the polypeptide of the present invention comprises the
sequence of a consensus sequence as shown in SEQ ID NO: 85367,
85368, 85369, 85370, 85371, 85372 or 85515, 85516, 85517, 85518
and/or the sequence of a consensus sequence as indicated in Table
IV, columns 5 or 7, lines 558 or 559 and/or the sequence of a
polypeptide as indicated in Table II, columns 5 or 7, lines 558 or
559 or of a functional homologue thereof as described herein, or of
a polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, lines 558 or 559 or its herein described functional
homologues and has the herein mentioned activity conferring an
increase in the 2-hydroxy-palmitic acid level.
[6692] A protein having an activity conferring an increase in the
amount or level of the C24:1 fatty acid, preferably C24:1 delta 15
fatty acid preferably has the structure of the polypeptide
described herein. In a particular embodiment, the polypeptides used
in the process of the present invention or the polypeptide of the
present invention comprises the sequence of a consensus sequence as
shown in SEQ ID NO: 85801, 85802, 85803 or 86104, 86105, 86106 or
86427, 86428, 86429 and/or the sequence of a consensus sequence as
indicated in Table IV, columns 5 or 7, lines 560 to 563 and/or the
sequence of a polypeptide as indicated in Table II, columns 5 or 7,
lines 560 to 563 or of a functional homologue thereof as described
herein, or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 560 to 563 or its herein described
functional homologues and has the herein mentioned activity
conferring an increase in the C24:1 fatty acid, preferably C24:1
fatty acid delta15 level.
[6693] [0050.0.15.15] For the purposes of the present invention,
the term "heptadecanoic acid", "2-hydroxy palmitic acid",
"2-hydroxy-tetracosenoic-acid", "hexadecadienoic acid", preferably
"delta 7,10 hexadecadienoic acid" and/or "hexadecatrienoic acid",
preferably "delta 7,10,13 hexadecatrienoic acid" and/or C24:1 fatty
acid also encompasses the corresponding salts, such as, for
example, the potassium or sodium salts of the above named fatty
acids or the salts of the above named fatty acids with amines such
as diethylamine or the esters the above named fatty acids.
[6694] [0051.0.15.15] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
fine chemical, i.e. an increased amount of the free chemical free
or bound, e.g fatty acid compositions. Depending on the choice of
the organism used for the process according to the present
invention, for example a microorganism or a plant, compositions or
mixtures of various fatty acids can be produced.
[6695] [0052.0.0.15] see [0052.0.0.0]
[6696] [0053.0.15.15] In one embodiment, the process of the present
invention comprises one or more of the following steps [6697] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or line 558, 559 and/or
line 560 to 563 having herein-mentioned the respective fine
chemical-increasing activity; [6698] b) stabilizing a mRNA
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185, or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
line 558, 559 and/or line 560 to 563, or of a mRNA encoding the
polypeptide of the present invention having herein-mentioned the
respective fine chemical increasing activity; [6699] c) increasing
the specific activity of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or line 558, 559 and/or line 560 to 563 or
decreasing the inhibitory regulation of the polypeptide of the
invention; [6700] d) generating or increasing the expression of an
endogenous or artificial transcription factor mediating the
expression of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention having herein-mentioned the
respective fine chemical increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185, and/or line 558, 559 and/or line 560 to
563 or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or line 558, 559 and/or
line 560 to 563; [6701] e) stimulating activity of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the present invention or a polypeptide of
the present invention having herein-mentioned the respective fine
chemical increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185, or its homologs activity, e.g. as indicated in Table
II, columns 5 or 7, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or line 558, 559
and/or line 560 to 563, by adding one or more exogenous inducing
factors to the organisms or parts thereof; [6702] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned the respective fine chemical increasing
activity, e.g. of a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185, or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or line 558, 559 and/or line 560 to
563; [6703] g) increasing the copy number of a gene conferring the
increased expression of a nucleic acid molecule encoding a
polypeptide encoded by the nucleic acid molecule of the invention
or the polypeptide of the invention having herein-mentioned the
respective fine chemical increasing activity, e.g. of a polypeptide
havingan an activity of a protein as indicated in Table II, column
3, lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185, or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
line 558, 559 and/or line 560 to 563; [6704] h) Increasing the
expression of the endogenous gene encoding the polypeptide of the
invention, e.g. a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185, or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or line 558, 559 and/or line 560 to 563
by adding positive expression or removing negative expression
elements, e.g. homologous recombination can be used to either
introduce positive regulatory elements like for plants the 35S
enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; [6705]
i) Modulating growth conditions of an organism in such a manner,
that the expression or activity of the gene encoding the protein of
the invention or the protein itself is enhanced for example
microorganisms or plants can be grown under a higher temperature
regime leading to an enhanced expression of heat shock proteins,
e.g. the heat shock protein of the invention, which can lead an
enhanced the fine chemical production; and/or [6706] j) selecting
of organisms with especially high activity of the proteins of the
invention from natural or from mutagenized resources and breeding
them into the target organisms, eg the elite crops.
[6707] [0054.0.15.15] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein according to Table II, column 3, lines 174 to
177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or line 558, 559 and/or
line 560 to 563.
[6708] [0055.0.0.15] to [0067.0.0.15] see [0055.0.0.0] to
[0067.0.0.0]
[6709] [0068.0.15.15] The mutation is introduced in such a way that
the production of the fatty acids is not adversely affected.
[6710] [0069.0.15.15] Less influence on the regulation of a gene or
its gene product is understood as meaning a reduced regulation of
the enzymatic activity leading to an increased specific or cellular
activity of the gene or its product. An increase of the enzymatic
activity is understood as meaning an enzymatic activity, which is
increased by at least 10%, advantageously at least 20, 30 or 40%,
especially advantageously by at least 50, 60 or 70% in comparison
with the starting organism. This leads to an increased productivity
of the desired fatty acid(s).
[6711] [0070.0.15.15] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below into an organism
alone or in combination with other genes, it is possible not only
to increase the biosynthetic flux towards the end product, but also
to increase, modify or create de novo an advantageous, preferably
novel metabolites composition in the organism, e.g. an advantageous
fatty acid composition comprising a higher content of (from a
viewpoint of nutritonal physiology limited) fatty acids, like
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid.
[6712] [0071.0.15.15] Preferably the composition comprises further
higher amounts of metabolites positively affecting or lower amounts
of metabolites negatively affecting the nutrition or health of
animals or humans provided with said compositions or organisms of
the invention or parts thereof. Likewise, the number or activity of
further genes which are required for the import or export of
nutrients or metabolites, including amino acids, fatty acids,
vitamins etc. or its precursors, required for the cell's
biosynthesis of the fine chemical may be increased so that the
concentration of necessary or relevant precursors, cofactors or
intermediates within the cell(s) or within the corresponding
storage compartments is increased. Owing to the increased or novel
generated activity of the polypeptide of the invention or owing to
the increased number of nucleic acid sequences of the invention
and/or to the modulation of further genes which are involved in the
biosynthesis of the fine chemical, e.g. by increasing the activity
of enzymes synthesizing precursors or by destroying the activity of
one or more genes which are involved in the breakdown of the fine
chemical, it is possible to increase the yield, production and/or
production efficiency of the fine chemical in the host organism,
such as plants or the microorganims.
[6713] [0072.0.15.15] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7, 10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid,
triglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid compounds like
other fatty acids such as palmitate, stearate, palmitoleate,
oleate, linoleate and/or linoleate or erucic acid and/or,
arachidonic acid.
[6714] [0073.0.15.15] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[6715] (k) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [6716] (l) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table II, columns 5 or 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
line 558, 559 and/or line 560 to 563 resp., or of a polypeptide
being encoded by the nucleic acid molecule of the present invention
and described below, i.e. conferring an increase of the respective
fine chemical in the organism, preferably in a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant, [6717] (m) growing the organism, preferably the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue or the plant under conditions which permit
the production of the fine chemical in the organism, preferably the
microorganism, the plant cell, the plant tissue or the plant; and
[6718] (n) if desired, recovering, optionally isolating, the free
and/or bound the fine chemical and, optionally further free and/or
bound fatty acids synthesized by the organism, the microorganism,
the non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[6719] [0074.0.15.15] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the fine chemical or the free and bound the fine
chemical but as option it is also possible to produce, recover and,
if desired isolate, other free or/and bound fatty acids.
[6720] [0075.0.0.15] to [0077.0.0.15] see [0075.0.0.0] to
[0077.0.0.0]
[6721] [0078.0.15.15] The organism such as microorganisms or plants
or the recovered, and if desired isolated, fine chemical can then
be processed further directly into foodstuffs or animal feeds or
for other applications, for example according to the disclosures
made in EP-A-0 568 608, EP-A-568 606, WO 2004/007732, WO 02/057465,
WO 01/02591, WO 2004/071467 or US 20020156254, which are expressly
incorporated herein by reference. The fermentation broth,
fermentation products, plants or plant products can be purified in
the customary manner by hydrolysis with strong bases, extraction
and crystallization or via thin layer chromatography and other
methods known to the person skilled in the art and described herein
below. Products of these different work-up procedures are fatty
acids or fatty acid compositions which still comprise fermentation
broth, plant particles and cell components in different amounts,
advantageously in the range of from 0 to 99% by weight, preferably
below 80% by weight, especially preferably between below 50% by
weight.
[6722] [0079.0.0.15] to [0084.0.0.15]: see [0079.0.0.0] to
[0084.0.0.0]
[6723] [0085.0.15.15] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [6724] a) the nucleic acid sequence as
shown in table I, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or line
558, 559 and/or line 560 to 563 or a derivative thereof, or [6725]
b) a genetic regulatory element, for example a promoter, which is
functionally linked to the nucleic acid sequence as shown table I,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or line 558,559 and/or
line 560 to 563 or a derivative thereof, or [6726] c) (a) and (b)
is/are not present in its/their natural genetic environment or
has/have been modified by means of genetic manipulation methods, it
being possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[6727] [0086.0.0.15] to [0087.0.0.15] see [0086.0.0.0] to
[0087.0.0.0]
[6728] [0088.0.15.15] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose fatty acid
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for animals is dependent on the abovementioned fatty acids and the
general amount of fatty acids as energy source in feed. After the
above mentioned protein activity has been increased or generated,
or after the expression of nucleic acid molecule or polypeptide
according to the invention has been generated or increased, the
transgenic plant generated thus is grown on or in a nutrient medium
or else in the soil and subsequently harvested.
[6729] [0089.0.0.15] to [0095.0.0.15] see [0089.0.0.0] to
[0095.0.0.0]
[6730] [0096.0.15.15] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptide for example a fatty acid
transporter protein or a compound, which functions as a sink for
the desired fatty acids in the organism is useful to increase the
production of the fine chemical (see Bao and Ohlrogge, Plant
Physiol. 1999 August; 120 (4): 1057-1062). Such fatty acid
transporter protein may serve as a link between the location of
fatty acid synthesis and the so called sink tissue, in which fatty
acids, triglycerides, oils and fats are stored.
[6731] [0097.0.0.15] see [0097.0.0.0] In may also be advantageous
to increase the content of the bound fine chemical.
[6732] [0098.0.15.15] In a preferred embodiment, the fine chemical
(heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid) is produced
in accordance with the invention and, if desired, is isolated. The
production of further fatty acids such as palmitoleic acid,
palmitic acid, stearic acid, oleic acid, linoleic acid, nervonic
acid and/or linolenic acid mixtures thereof or mixtures of other
fatty acids by the process according to the invention is
advantageous.
[6733] [0099.0.15.15] In the case of the fermentation of
microorganisms, the above mentioned fatty acids may accumulate in
the medium and/or the cells. If microorganisms are used in the
process according to the invention, the fermentation broth can be
processed after the cultivation. Depending on the requirement, all
or some of the biomass can be removed from the fermentation broth
by separation methods such as, for example, centrifugation,
filtration, decanting or a combination of these methods, or else
the biomass can be left in the fermentation broth. The fermentation
broth can subsequently be reduced, or concentrated, with the aid of
known methods such as, for example, rotary evaporator, thin-layer
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. Afterwards advantageously further compounds for
formulation can be added such as corn starch or silicates. This
concentrated fermentation broth advantageously together with
compounds for the formulation can subsequently be processed by
lyophilization, spray drying, spray granulation or by other
methods. Preferably the fatty acids or the fatty acid compositions
are isolated from the organisms, such as the microorganisms or
plants or the culture medium in or on which the organisms have been
grown, or from the organism and the culture medium, in the known
manner, for example via extraction, distillation, crystallization,
chromatography or a combination of these methods. These
purification methods can be used alone or in combination with the
aforementioned methods such as the separation and/or concentration
methods.
[6734] [0100.0.15.15] Transgenic plants which comprise the fatty
acids such as saturated or polyunsaturated fatty acids synthesized
in the process according to the invention can advantageously be
marketed directly without there being any need for the oils, lipids
or fatty acids synthesized to be isolated. Plants for the process
according to the invention are listed as meaning intact plants and
all plant parts, plant organs or plant parts such as leaf, stem,
seeds, root, tubers, anthers, fibers, root hairs, stalks, embryos,
calli, cotelydons, petioles, harvested material, plant tissue,
reproductive tissue and cell cultures which are derived from the
actual transgenic plant and/or can be used for bringing about the
transgenic plant. In this context, the seed comprises all parts of
the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue. However, the fine chemical produced
in the process according to the invention can also be isolated from
the organisms, advantageously plants, in the form of their oils,
fats, lipids and/or free fatty acids. Fatty acids produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts, preferably the plant
seeds. To increase the efficiency of oil extraction it is
beneficial to clean, to temper and if necessary to hull and to
flake the plant material especially the seeds. In this context, the
oils, fats, lipids and/or free fatty acids can be obtained by what
is known as cold beating or cold pressing without applying heat. To
allow for greater ease of disruption of the plant parts,
specifically the seeds, they are previously comminuted, steamed or
roasted. The seeds, which have been pretreated in this manner can
subsequently be pressed or extracted with solvents such as warm
hexane. The solvent is subsequently removed. In the case of
microorganisms, the latter are, after harvesting, for example
extracted directly without further processing steps or else, after
disruption, extracted via various methods with which the skilled
worker is familiar. In this manner, more than 96% of the compounds
produced in the process can be isolated. Thereafter, the resulting
products are processed further, i.e. degummed and/or refined. In
this process, substances such as the plant mucilages and suspended
matter are first removed. What is known as desliming can be
affected enzymatically or, for example, chemico-physically by
addition of acid such as phosphoric acid. Thereafter optionally,
the free fatty acids are removed by treatment with a base like
alkali, for example aqueous KOH or NaOH, or acid hydrolysis,
advantageously in the presence of an alcohol such as methanol or
ethanol, or via enzymatic cleavage, and isolated via, for example,
phase separation and subsequent acidification via, for example,
H.sub.2SO.sub.4. The fatty acids can also be liberated directly
without the above-described processing step. If desired the
resulting product can be washed thoroughly with water to remove
traces of soap and the alkali remaining in the product and then
dried. To remove the pigment remaining in the product, the products
can be subjected to bleaching, for example using filler's earth or
active charcoal. At the end, the product can be deodorized, for
example using steam distillation under vacuum. These chemically
pure fatty acids or fatty acid compositions are advantageous for
applications in the food industry sector, the cosmetic sector and
especially the pharmacological industry sector.
[6735] [0101.0.15.15] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[6736] [0102.0.15.15] Fatty acids can for example be detected
advantageously via GC separation methods. The unambiguous detection
for the presence of fatty acid products can be obtained by
analyzing recombinant organisms using analytical standard methods:
GC, GC-MS or TLC, as described on several occasions by Christie and
the references therein (1997, in: Advances on Lipid Methodology,
Fourth Edition: Christie, Oily Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfah ren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). One
example is the analysis of fatty acids via FAME and GC-MS or TLC
(abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer chromatography.
The material to be analyzed can be disrupted by sonication,
grinding in a glass mill, liquid nitrogen and grinding or via other
applicable methods. After disruption, the material must be
centrifuged. The sediment is resuspended in distilled water, heated
for 10 minutes at 100.degree. C., cooled on ice and recentrifuged,
followed by extraction for one hour at 90.degree. C. in 0.5 M
sulfuric acid in methanol with 2% dimethoxypropane, which leads to
hydrolyzed oil and lipid compounds, which give transmethylated
lipids. These fatty acid methyl esters are extracted in petroleum
ether and finally subjected to a GC analysis using a capillary
column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 .mu.m, 0.32
mm) at a temperature gradient of between 170.degree. C. and
240.degree. C. for 20 minutes and 5 minutes at 240.degree. C. The
identity of the resulting fatty acid methyl esters must be defined
using standards which are available from commercial sources (i.e.
Sigma).
[6737] [0102.1.15.15] An other analytical method is described by
Summit et al, (Proceedings of the Ocean Drilling Program,
Scientific Results Volume 169, 2000). The fatty acid methyl esters
are analyzed by capillary gas chromatography with flame ionization
detection. Various mono- and polyunsaturated fatty acids can
further be individually distinguished and quantified in one sample
without prior separation by semi-selective HSQC (heteronuclear
single quantum coherence)-NMR (Willker et al., Magn. Reson. Chem.
36, S79-S84 (1998)).
[6738] Fatty acid compositions, preferably of monogalactosyl
diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) can be
investigated using HPLC/ESI-MS combined with in-source (or cone
voltage) fragmentation, negative-ion electrospray ionization (ESI)
mass spectrometry interfaced with high performance liquid
chromatography (HPLC) (Kim et al., Bull. Korean Chem. Soc. 2003,
Vol. 24, No. 8).
[6739] [0103.0.15.15] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [6740]
a) nucleic acid molecule encoding, preferably at least the mature
form, of the polypeptide shown in Table II, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or line 558, 559 and/or line 560 to 563 or a
fragment thereof, which confers an increase in the amount of the
fine chemical in an organism or a part thereof; [6741] b) nucleic
acid molecule comprising, preferably at least the mature form, of
the nucleic acid molecule shown in Table IA or IB, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or line 558, 559 and/or line 560 to
563; [6742] c) nucleic acid molecule whose sequence can be deduced
from a polypeptide sequence encoded by a nucleic acid molecule of
(a) or (b) as result of the degeneracy of the genetic code and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [6743] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the fine chemical in an organism or a part thereof; [6744] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under under stringent hybridisation conditions and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [6745] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c) and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [6746] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
and conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [6747] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers shown in Table III, column 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or line 558, 559 and/or line 560 to 563 and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [6748] i) nucleic acid molecule encoding a
polypeptide which is isolated, e.g. from an expression library,
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (h), preferably to
(a) to (c), and and conferring an increase in the amount of the
fine chemical in an organism or a part thereof; [6749] j) nucleic
acid molecule which encodes a polypeptide comprising the consensus
sequence shown in Table IV, columns 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
line 558, 559 and/or line 560 to 563 and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[6750] k) nucleic acid molecule comprising one or more of the
nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of the polypeptide shown in Table II,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or line 558, 559 and/or
line 560 to 563 and conferring an increase in the amount of the
fine chemical in an organism or a part thereof; and [6751] l)
nucleic acid molecule which is obtainable by screening a suitable
library under stringent conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; or which comprises a
sequence which is complementary thereto.
[6752] [0104.0.15.15] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence shown in Table
IA or IB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or line 558,
559 and/or line 560 to 563 by one or more nucleotides or does not
consist of the sequence shown in Table IA or IB, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or line 558, 559 and/or line 560 to
563. In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence shown in Table IA or IB, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or line 558, 559 and/or line 560 to
563. In another embodiment, the nucleic acid molecule does not
encode a polypeptide of the sequence shown in Table II, columns 5
or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or line
180 and/or lines 181 to 185 and/or line 558, 559 and/or line 560 to
563.
[6753] [0105.0.0.15] to [0107.0.0.15] see [0105.0.0.0] to
[0107.0.0.0]
[6754] [0108.0.15.15] Nucleic acid molecules with the sequence
shown in Table IA or IB, columns 5 or 7, lines 174 to 177 and/or
line 178 and/or line 179 and/or line 180 and/or lines 181 to 185,
nucleic acid molecules which are derived from the amino acid
sequences shown in Table II, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or line 558, 559 and/or line 560 to 563 or from
polypeptides comprising the consensus sequence shown in Table IV,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185, and/or line 558, 559
and/or line 560 to 563 or their derivatives or homologues encoding
polypeptides with the enzymatic or biological activity of a
polypeptide as indicated in Table IA or IB, column 3, 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or line 558, 559 and/or line 560 to 563 resp.,
or e.g. conferring a increase in the respective fine chemical after
increasing its expression or activity are advantageously increased
in the process according to the invention.
[6755] [0109.0.15.15] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the fatty acids produced in the process according to
the invention.
[6756] [0110.0.0.15] see [0110.0.0.0]
[6757] [0111.0.0.15] see [0111.0.0.0]
[6758] [0112.0.15.15] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table IA or IB, column 3, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or line 558, 559 and/or line 560 to 563 resp.,
or having the sequence of a polypeptide as indicated in Table II,
columns 5 and 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or line 558, 559 and/or
line 560 to 563 resp., and conferring an increase of the respective
fine chemical.
[6759] [0113.0.0.15] to [0120.0.0.15] see [0113.0.0.0] to
[0120.0.0.0]
[6760] [0121.0.15.15] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or line 558, 559 and/or
line 560 to 563 resp., or the functional homologues thereof as
described herein, preferably conferring above-mentioned activity,
i.e.
[6761] conferring an increase in the level of heptadecanoic acid
after increasing the activity of the polypeptide sequences
indicated in Table II, columns 5 or 7, lines 174 to 177;
[6762] or conferring increase in the level of 2-hydroxy palmitic
acid after increasing the activity of the polypeptide sequences
indicated in Table II, columns 5 or 7, line 178 or 558, 559;
[6763] or conferring increase in the level of
2-hydroxy-tetracosenoic-acid after increasing the activity of the
polypeptide sequences indicated in Table II, columns 5 or 7, line
179;
[6764] or conferring increase in the level of hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid after increasing the
activity of the polypeptide sequences indicated in Table II,
columns 5 or 7, line 180;
[6765] or conferring increase in the level of hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid after
increasing the activity of the polypeptide sequences indicated in
Table II, columns 5 or 7, lines 181 to 185.
[6766] or conferring increase in the level of C24:1 fatty acid,
preferably C24:1 delta 15 fatty acid after increasing the activity
of the polypeptide sequences indicated in Table II, columns 5 or 7,
lines 560 to 563.
[6767] [0122.0.0.15] to [0127.0.0.15] see [0122.0.0.0] to
[0127.0.0.0]
[6768] [0128.0.15.15] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, columns 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., by means of polymerase chain reaction can be generated
on the basis of a sequence shown herein, for example the sequence
as indicated in Table IA or IB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., or the
sequences derived from sequences as indicated in Table II, columns
5 or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or
line 180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines
560 to 563 resp.
[6769] [0129.0.15.15] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequences indicated in Table IV,
columns 7, lines 174 to 177 and/or and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., are derived from said alignments.
[6770] [0130.0.15.15] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of fatty
acids, e.g. of heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid after increasing
its expression or activity of the protein comprising said
fragment.
[6771] [0131.0.0.15] to [0138.0.0.15] see [0131.0.0.0] to
[0138.0.0.0]
[6772] [0139.0.15.15] Polypeptides having above-mentioned activity,
i.e. conferring an increase of the respective fine chemical level,
derived from other organisms, can be encoded by other DNA sequences
which hybridize to a sequences indicated in Table IA or IB, columns
5 or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or
line 180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines
560 to 563 resp., preferably Table I B, columns 5 or 7, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp.,
under relaxed hybridization conditions and which code on expression
for peptides having the heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid resp., increasing activity.
[6773] [0140.0.0.15] to [0146.0.0.15]: see [0140.0.0.0] to
[0146.0.0.0]
[6774] [0147.0.15.15] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
IA or IB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp., preferably Table B, columns 5 or
7, lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., is one which is sufficiently complementary to one of
said nucleotide sequences s such that it can hybridize to one of
said nucleotide sequences thereby forming a stable duplex.
Preferably, the hybridisation is performed under stringent
hybridization conditions. However, a complement of one of the
herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[6775] [0148.0.15.15] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence shown in Table IA or IB, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., or a portion thereof and preferably has above mentioned
activity, in particular having a heptadecanoic acid and/or
2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homologs.
[6776] [0149.0.15.15] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table IA or IB, columns 5 or
7, lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., preferably Table I B, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., or a
portion thereof and encodes a protein having above-mentioned
activity, e.g. conferring an increase of the respective fine
chemical, e.g. of heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7, 10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and and/or
24:1 fatty acid, preferably C24:1 delta 15 fatty acid optionally
the activity of a protein indicated in Table Ila or IIB, column 5,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp.
[6777] [00149.1.15.15] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table IA
or IB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., preferably Table I B, columns 5 or
7, lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., has further one or more of the activities annotated or
known for the a protein as indicated in Table IIA or IIB, column 3,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp.
[6778] [0150.0.15.15] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences shown in Table IA or IB, columns 5 or 7, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp.,
preferably Table I B, columns 5 or 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., for example a
fragment which can be used as a probe or primer or a fragment
encoding a biologically active portion of the polypeptide of the
present invention or of a polypeptide used in the process of the
present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid if its activity is increased. The nucleotide
sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., in Table IA or IB, columns 5 or
7, lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., preferably Table I B, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., an
anti-sense sequence of one of the sequences, e.g., set forth in
Table IA or IB, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., preferably Table I B,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., or naturally occurring mutants
thereof. Primers based on a nucleotide of invention can be used in
PCR reactions to clone homologues of the polypeptide of the
invention or of the polypeptide used in the process of the
invention, e.g. as the primers described in the examples of the
present invention, e.g. as shown in the examples. A PCR with the
primers shown in table III, column 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., will result in a
fragment of a polynucleotide sequence as indicated in Table IA or
IB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp.
[6779] [0151.0.0.15] see [0151.0.0.0]
[6780] [0152.0.15.15] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table IIA or IB, columns 5 or 7, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp.,
such that the protein or portion thereof maintains the ability to
participate in the respective fine chemical production, in
particular an activity increasing the level of amino acids, in
particular of heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid as mentioned above
or as described in the examples in plants or microorganisms is
comprised.
[6781] [0153.0.15.15] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table IA or IB, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp., such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table IIA or IIB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., has for
example an activity of a polypeptide indicated in Table IIA or IIB,
column 3, lines 174 to 177 and/or line 178 and/or line 179 and/or
line 180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines
560 to 563 resp.
[6782] [0154.0.15.15] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp., and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[6783] [0155.0.0.15] to [0156.0.0.15]: see [0155.0.0.0] to
[0156.0.0.0]
[6784] [0157.0.15.15] The invention further relates to nucleic acid
molecules that differ from one of a nucleotide sequences as
indicated in Table IA or IB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides encoded by the sequences as indicated in Table
IV, columns 5 or 7, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., or of the polypeptide as indicated
in Table IIA or IIB, columns 5 or 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., or their functional
homologues. Advantageously, the nucleic acid molecule of the
invention comprises, or in an other embodiment has, a nucleotide
sequence encoding a protein comprising, or in an other embodiment
having, a consensus sequences as indicated in Table IV, columns 5
or 7, lines 174 to 177 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp., or
of the polypeptide as as indicated in Table IIA or IIB, columns 5
or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or line
180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560
to 563 resp., or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
IV, columns 5 or 7, lines 174 to 177 and/or line 179 and/or line
180 and/or lines 181 to 185 resp., or of a polypeptide as indicated
in Table IIA or IIB, columns 5 or 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., or the functional
homologues thereof. However, in a preferred embodiment, the nucleic
acid molecule of the present invention does not consist of a
sequence as indicated in Table IA or IB, columns 5 or 7, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp.,
preferably of Table I A, columns 5 or 7, lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563. Preferably the nucleic acid
molecule of the invention is a functional homologue or identical to
a nucleic acid molecule indicated in Table I B, columns 5 or 7,
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563.
[6785] [0158.0.0.15] to [0160.0.0.15]: see [0158.0.0.0] to
[0160.0.0.0]
[6786] [0161.0.15.15] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table IA or IB, columns 5 or 7, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp. The
nucleic acid molecule is preferably at least 20, 30, 50, 100, 250
or more nucleotides in length.
[6787] [0162.0.0.15] see [0162.0.0.0]
[6788] [0163.0.15.15] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table IA or IB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp.,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[6789] [0164.0.0.15] see [0164.0.0.0]
[6790] [0165.0.15.15] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table IA or IB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., preferably
Table I B columns, 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp.
[6791] [0166.0.0.15] to [0167.0.0.15] see [0166.0.0.0] to
[0167.0.0.0]
[6792] [0168.0.15.15] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or line
180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560
to 563 resp., preferably Table II B, columns 5 or 7, lines 174 to
177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp., yet
retain said activity described herein. The nucleic acid molecule
can comprise a nucleotide sequence encoding a polypeptide, wherein
the polypeptide comprises an amino acid sequence at least about 50%
identical to an amino acid sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., preferably Table II B, columns 5 or
7, lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp., preferably Table II B, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., more
preferably at least about 70% identical to one of the sequences as
indicated in Table IIA or IIB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., preferably
Table II B, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp., even more preferably at least
about 80%, 90%, or 95% homologous to a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., preferably Table II
B, columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp. and most preferably at least about
96%, 97%, 98%, or 99% identical to the sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., preferably Table II
B, columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp.
[6793] [0169.0.0.15] to [0172.0.0.15] see [0169.0.0.0] to
[0172.0.0.0]
[6794] [0173.0.15.15] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 13930 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 13930 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[6795] [0174.0.0.15] see [0174.0.0.0]
[6796] [0175.0.15.15] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 13931 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 13931 by the above program algorithm with the
above parameter set, has a 80% homology.
[6797] [0176.0.15.15] Functional equivalents derived from one of
the polypeptides as indicated in Table IIA or IIB, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., according to the invention by substitution, insertion or
deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at
least 55%, 60%, 65% or 70% by preference at least 80%, especially
preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very
especially preferably at least 95%, 97%, 98% or 99% homology with
one of the polypeptides as indicated in Table IIA or IIB, columns 5
or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or line
180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560
to 563 resp., according to the invention and are distinguished by
essentially the same properties as a polypeptide as indicated in
Table IIA or IIB, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp.
[6798] [0177.0.15.15] Functional equivalents derived from a nucleic
acid sequence as indicated in Table IA or IB, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp., according to the invention by substitution, insertion or
deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at
least 55%, 60%, 65% or 70% by preference at least 80%, especially
preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very
especially preferably at least 95%, 97%, 98% or 99% homology with
one of a polypeptides as indicated in Table IIA or IIB, columns 5
or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or line
180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560
to 563 resp., according to the invention and encode polypeptides
having essentially the same properties as a polypeptide as
indicated in Table IIA or IIB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp.
[6799] [0178.0.0.15] see [0178.0.0.0]
[6800] [0179.0.15.15] A nucleic acid molecule encoding a homologous
to a protein sequence of as indicated in Table IIA or IIB, columns
5 or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or
line 180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines
560 to 563 resp., preferably Table II B, columns 5 or 7, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp., can
be created by introducing one or more nucleotide substitutions,
additions or deletions into a nucleotide sequence of the nucleic
acid molecule of the present invention, in particular as indicated
in Table IA or IB, columns 5 or 7, Hines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., such that one or more
amino acid substitutions, additions or deletions are introduced
into the encoded protein. Mutations can be introduced into the
encoding sequences of a sequences as indicated in Table IA or IB,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
[6801] [0180.0.0.15] to [0183.0.0.15] see [0180.0.0.0] to
[0183.0.0.0]
[6802] [0184.0.15.15] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table IA or IB, columns 5 or
7, lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., preferably Table I B, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., or of the
nucleic acid sequences derived from a sequences as indicated in
Table IIA or IIB, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., comprise also allelic
variants with at least approximately 30%, 35%, 40% or 45% homology,
by preference at least approximately 50%, 60% or 70%, more
preferably at least approximately 90%, 91%, 92%, 93%, 94% or 95%
and even more preferably at least approximately 96%, 97%, 98%, 99%
or more homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
IA or IB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp., preferably Table I B, columns 5
or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or line
180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560
to 563 resp. or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[6803] [0185.0.15.15] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table IA or IB, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., preferably Table I B,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp. In one embodiment, it is preferred
that the nucleic acid molecule comprises as little as possible
other nucleotide sequences not shown in any one of sequences as
indicated in Table IA or IB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., preferably
Table I B, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp. In one embodiment, the nucleic
acid molecule comprises less than 500, 400, 300, 200, 100, 90, 80,
70, 60, 50 or 40 further nucleotides. In a further embodiment, the
nucleic acid molecule comprises less than 30, 20 or 10 further
nucleotides. In one embodiment, a nucleic acid molecule used in the
process of the invention is identical to a sequences as indicated
in Table IA or IB, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., preferably Table I B,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp.
[6804] [0186.0.15.15] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encode a
polypeptide comprising a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., preferably Table II B, columns 5 or
7, lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp. In one embodiment, the nucleic acid molecule encodes less
than 150, 130, 100, 80, 60, 50, 40 or 30 further amino acids. In a
further embodiment, the encoded polypeptide comprises less than 20,
15, 10, 9, 8, 7, 6 or 5 further amino acids. In one embodiment, the
encoded polypeptide used in the process of the invention is
identical to the sequences as indicated in Table IIA or IIB,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., preferably Table II B columns, 5 or
7, lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp.
[6805] [0187.0.15.15] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or line
180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560
to 563 resp., preferably Table II B, columns 5 or 7, lines 174 to
177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp.,
comprises less than 100 further nucleotides. In a further
embodiment, said nucleic acid molecule comprises less than 30
further nucleotides. In one embodiment, the nucleic acid molecule
used in the process is identical to a coding sequence encoding a
sequences as indicated in Table IIA or IIB, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp., preferably Table II B, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp.
[6806] [0188.0.15.15] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
IIA or IIB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp., preferably compared to a
sequence as indicated in Table IIA or IIB, column 3 and 5, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp., and expressed under identical conditions.
[6807] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, column 7, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp.
[6808] [0189.0.15.15] Homologues of a sequences as indicated in
Table IA or IB, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., or of a derived
sequences as indicated in Table IIA or IIB, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp., also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives which comprise
noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[6809] [0190.0.0.15] to [0203.0.0.15] see [0192.0.0.0] to
[0203.0.0.0]
[6810] [0204.0.15.15] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [6811] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp.; or a fragment thereof conferring an increase in the amount
of the respective fine chemical, i.e. heptadecanoic acid and/or
2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid in an organism or a part thereof; [6812] b)
nucleic acid molecule comprising, preferably at least the mature
form, of a nucleic acid molecule as indicated in Table IA or IB,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., or a fragment thereof conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [6813] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
fine chemical in an organism or a part thereof; [6814] d) nucleic
acid molecule encoding a polypeptide whose sequence has at least
50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [6815] e) nucleic acid molecule which hybridizes with
a nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [6816] f)
nucleic acid molecule encoding a polypeptide, the polypeptide being
derived by substituting, deleting and/or adding one or more amino
acids of the amino acid sequence of the polypeptide encoded by the
nucleic acid molecules (a) to (d), preferably to (a) to (c), and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [6817] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [6818] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying a cDNA library or a genomic library using primers or
primer pairs as indicated in Table III, column 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., and
conferring an increase in the amount of the respective fine
chemical, i.e. heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid in an organism or
a part thereof; [6819] i) nucleic acid molecule encoding a
polypeptide which is isolated, e.g. from a expression library, with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (g), preferably to (a)
to (c) and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [6820] j) nucleic acid
molecule which encodes a polypeptide comprising a consensus
sequence as indicated in Table IV, column 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., and
conferring an increase in the amount of the respective fine
chemical i.e. heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid in an organism or
a part thereof; [6821] k) nucleic acid molecule encoding the amino
acid sequence of a polypeptide encoding a domaine of a polypeptide
as indicated in Table IIA or IIB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., and
conferring an increase in the amount of the respective fine
chemical i.e. heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid in an organism or
a part thereof; and [6822] l) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of a nucleic acid molecule as indicated in Table IA or
IB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., or a nucleic acid molecule encoding,
preferably at least the mature form of, the polypeptide as
indicated in Table IIA or IIB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; whereby, preferably, the
nucleic acid molecule according to (a) to (l) distinguishes over a
sequence depicted in as indicated in Table I A, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., by one or more nucleotides. In one embodiment, the
nucleic acid molecule of the invention does not consist of a
sequence as indicated in Table I A or IB, columns 5 or 7, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp. In
an other embodiment, the nucleic acid molecule of the present
invention is at least 30 identical and less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to a sequence indicated in Table I A
or IB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp. In a further embodiment the nucleic
acid molecule does not encode a polypeptide sequence as indicated
in Table II A or II B, columns 5 or 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp. Accordingly, in one
embodiment, the nucleic acid molecule of the present invention
encodes in one embodiment a polypeptide which differs at least in
one or more amino acids from a polypeptide indicated in Table II A
or II B, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp. In another embodiment, a nucleic
acid molecule indicated in Table I A or I B, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp., does not encode a protein of a sequence indicated in Table
II A or II B, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp. Accordingly, in one
embodiment, the protein encoded by a sequences of a nucleic acid
according to (a) to (l) does not consist of a sequence as indicated
in Table II A or II B, columns 5 or 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp. In a further
embodiment, the protein of the present invention is at least 30%
identical to a protein sequence indicated in Table II A or II B,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., and less than 100%, preferably less
than 99.999%, 99.99% or 99.9%, more preferably less than 99%, 985,
97%, 96% or 95% identical to a sequence as indicated in Table I A
or I B, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp.
[6823] [0205.0.0.15] to [0206.0.0.15]: see [0205.0.0.0] to
[0206.0.0.0]
[6824] [0207.0.15.15] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes are genes of the fatty acid metabolism, amino
acid metabolism, of glycolysis, of the tricarboxylic acid
metabolism, of triacylglycerol or lipid, preferably
glycerophospholipids, sphingolipids and/or galactolipids
biosynthesis or their combinations. As described herein, regulator
sequences or factors can have a positive effect on preferably the
gene expression of the genes introduced, thus increasing it. Thus,
an enhancement of the regulator elements may advantageously take
place at the transcriptional level by using strong transcription
signals such as promoters and/or enhancers. In addition, however,
an enhancement of translation is also possible, for example by
increasing mRNA stability or by inserting a translation enhancer
sequence.
[6825] [0208.0.0.15] to [0226.0.0.15] see [0208.0.0.0] to
[0226.0.0.0]
[6826] [0227.0.15.15] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[6827] In addition to the sequence mentioned in Table IA or IB,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the fatty acid biosynthetic pathway such as for
palmitate, stearate, palmitoleate, oleate, linoleate and/or
linolenate or of the of triacylglycerol or lipid, preferably
glycerophospholipids, sphingolipids and/or galactolipids
biosynthetic pathway is expressed in the organisms such as plants
or microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the amino acids desired since, for example, feedback
regulations no longer exist to the same extent or not at all. In
addition it might be advantageously to combine the sequences shown
in Table IA or IB, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., with genes which
generally support or enhances to growth or yield of the target
organism, for example genes which lead to faster growth rate of
microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[6828] [0228.0.15.15] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the fatty acid
metabolism, in particular in fatty acid synthesis.
[6829] [0229.0.15.15] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the saturated, poly unsaturated
fatty acid biosynthesis such as desaturases like
.DELTA.-4-desaturases, .DELTA.-5-desaturases,
.DELTA.-6-desaturases, .DELTA.-8-desaturases,
.DELTA.-9-desaturases, .DELTA.-12-desaturases,
.DELTA.-17-desaturases, .omega.-3-desaturases, elongases like
.DELTA.-5-elongases, .DELTA.-6-elongases, 9-elongases,
acyl-CoA-dehydrogenases, acyl-ACP-desaturases,
acyl-ACP-thioesterases, fatty acid acyl-transferases, acyl-CoA
lysophospholipid-acyltransferases, acyl-CoA carboxylases, fatty
acid synthases, fatty acid hydroxylases, acyl-CoA oxydases,
acetylenases, lipoxygenases, triacyl-lipases etc. as described in
WO 98/46765, WO 98/46763, WO 98/46764, WO 99/64616, WO 00/20603, WO
00/20602, WO 00/40705, US 20040172682, US 20020156254, U.S. Pat.
No. 6,677,145 US 20040053379 or US 20030101486. These genes lead to
an increased synthesis of the essential fatty acids.
[6830] [0229.1.15.15] Further nucleic acid sequences which can be
expressed in combination with the sequences used in the process
and/or the abovementioned biosynthesis genes are the sequences
encoding further genes of the saturated, unsaturated,
polyunsaturated and/or hydroxylated fatty acid biosynthesis such as
the FA2H gene encoding a fatty acid 2-hydroxylase (Alderson et al.,
J. Biol. Chem. Vol. 279, No. 47, Issue of November 19, pp.
48562-48568, 2004), .omega.-3-desaturases encoded by FAD2 and FAD3
(US 20030221217), delta-12 fatty acid desaturase, delta-15 fatty
acid desaturase as disclosed by Okuley, et al., Plant Cell
6:147-158 (1994), Lightner et al., WO94/11516, Yadav, N., et al.,
Plant Physiol., 103:467-476 (1993), WO 93/11245 and Arondel, V. et
al., Science, 258:1353-1355 (1992), US 20040083503.
[6831] [0230.0.0.15] see [0230.0.0.0]
[6832] [0231.0.15.15] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid degrading protein is attenuated, in particular
by reducing the rate of expression of the corresponding gene.
[6833] [0232.0.0.15] to [0276.0.0.15] see [0232.0.0.0] to
[0276.0.0.0]
[6834] [0277.0.15.15] The fatty acids produced can be isolated from
the organism by methods with which the skilled worker is familiar.
For example via extraction, salt precipitation and/or different
chromatography methods. The process according to the invention can
be conducted batchwise, semibatchwise or continuously. The fine
chemical produced in the process according to the invention can be
isolated as mentioned above from the organisms, advantageously
plants, in the form of their oils, fats, lipids and/or free fatty
acids. Fatty acids produced by this process can be obtained by
harvesting the organisms, either from the crop in which they grow,
or from the field. This can be done via pressing or extraction of
the plant parts, preferably the plant seeds. Hexane is preferably
used as solvent in the process, in which more than 96% of the
compounds produced in the process can be isolated. Thereafter, the
resulting products are processed further, i.e. degummed, refined,
bleached and/or deodorized.
[6835] [0278.0.0.15] to [0282.0.0.15] see [0278.0.0.0] to
[0282.0.0.0]
[6836] [0283.0.15.15] Moreover, a native polypeptide conferring the
increase of the fine chemical in an organism or part thereof can be
isolated from cells (e.g., endothelial cells), for example using
the antibody of the present invention as described below, in
particular, an antibody against a protein as indicated in Table IIA
or IIB, column 3, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp. e.g. an antibody against a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp., which can be produced by standard techniques utilizing
polypeptides comprising or consisting of above mentioned sequences,
e.g. the polypeptide of the present invention or fragment thereof.
Preferred are monoclonal antibodies.
[6837] [0284.0.0.15] see [0284.0.0.0]
[6838] [0285.0.15.15] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp., or as coded by a nucleic acid
molecule as indicated in Table IA or IB, columns 5 or 7, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp., or
functional homologues thereof.
[6839] [0286.0.15.15] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., and in one another embodiment, the
present invention relates to a polypeptide comprising or consisting
of a consensus sequence as indicated in Table IV, column 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp., whereby 20 or less, preferably 15 or 10, preferably 9, 8, 7,
or 6, more preferred 5 or 4, even more preferred 3, even more
preferred 2, even more preferred 1, most preferred 0 of the amino
acids positions indicated can be replaced by any amino acid, or, in
an further embodiment, can be replaced and/or absent. In one
embodiment, the present invention relates to the method of the
present invention comprising a polypeptide or to a polypeptide
comprising more than one consensus sequences (of an individual
line) as indicated in Table IV, column 7, lines 174 to 177 and/or
line 178 and/or line 179 and/or line 180 and/or lines 181 to 185
and/or lines 558, 559 and/or lines 560 to 563 resp.
[6840] [0287.0.0.15] to [0290.0.0.15] see [0287.0.0.0] to
[0290.0.0.0]
[6841] [0291.0.15.15] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[6842] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table II A or II B,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., by one or more amino acids. In one
embodiment, polypeptide distinguishes form a sequence as indicated
in Table II A or II B, columns 5 or 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., by more than 5, 6, 7,
8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or 30
amino acids, even more preferred are more than 40, 50, or 60 amino
acids and, preferably, the sequence of the polypeptide of the
invention distinguishes from a sequence as indicated in Table II A
or II B, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp., by not more than 80% or 70% of
the amino acids, preferably not more than 60% or 50%, more
preferred not more than 40% or 30%, even more preferred not more
than 20% or 10%. In an other embodiment, said polypeptide of the
invention does not consist of a sequence as indicated in Table II A
or II B, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp.
[6843] [0292.0.0.15] see [0292.0.0.0]
[6844] [0293.0.15.15] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the process
of the invention. In one embodiment, the polypeptide of the
invention has a sequence which distinguishes from a sequence as
indicated in Table II A or II B, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., by one or more amino
acids. In an other embodiment, said polypeptide of the invention
does not consist of the sequence as indicated in Table II A or II
B, columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp. In a further embodiment, said
polypeptide of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical. In one embodiment, said polypeptide
does not consist of the sequence encoded by a nucleic acid
molecules as indicated in Table I A or I B, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp.
[6845] [0294.0.15.15] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., which distinguishes
over a sequence as indicated in Table II A or II B, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., by one or more amino acids, preferably by more than 5,
6, 7, 8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or
30 amino acids, even more preferred are more than 40, 50, or 60
amino acids but even more preferred by less than 70% of the amino
acids, more preferred by less than 50%, even more preferred my less
than 30% or 25%, more preferred are 20% or 15%, even more preferred
are less than 10%.
[6846] [0295.0.0.15] to [0297.0.0.15] see [0295.0.0.0] to
[0297.0.0.0]
[6847] [00297.1.15.15] Non-polypeptide of the
invention-chemicalsare e.g. polypeptides having not the activity of
a polypeptide indicated in Table IIA or IIB, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp.
[6848] [0298.0.15.15] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., such that
the protein or portion thereof maintains the ability to confer the
activity of the present invention. The portion of the protein is
preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp.
[6849] [0299.0.15.15] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences as shown in Table IIA or IIB, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence of Table IA or IB,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., or which is homologous thereto, as
defined above.
[6850] [0300.0.15.15] Accordingly the polypeptide of the present
invention can vary from the sequences shown in Table IIA or IIB,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., in amino acid sequence due to
natural variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprise an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence shown in table II A or II B, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp.
[6851] [0301.0.0.15] see [0301.0.0.0]
[6852] [0302.0.15.15] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., the amino acid sequence shown in Table IIA
or IIB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp., or the amino acid sequence of a
protein homologous thereto, which include fewer amino acids than a
full length polypeptide of the present invention or used in the
process of the present invention or the full length protein which
is homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[6853] [0303.0.0.15] see [0303.0.0.0]
[6854] [0304.0.15.15] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention, may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
IIA or IIB, column 3, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[6855] [0305.0.0.15] to [0308.0.0.15] see [0305.0.0.0] to
[0308.0.0.0]
[6856] [0309.0.15.15] In one embodiment, an reference to a "protein
(=polypeptide)" of the invention or as indicated in Table IIA or
IIB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., refers to a polypeptide having an
amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table IIA or IIB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., refers to
a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous a polypeptide of the
invention, preferably which is not substantially homologous to a as
indicated in Table IIA or IIB, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., e.g., a
protein which does not confer the activity described herein or
annotated or known for as indicated in Table IIA or IIB, column 3,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., and which is derived from the same or a different
organism. In one embodiment, a "non-polypeptide of the invention"
or "other polypeptide" not being indicated in Table IIA or IIB,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., does not confer an increase of the
respective fine chemical in an organism or part thereof.
[6857] [0310.0.0.15] to [0334.0.0.15] see [0310.0.0.0] to
[0334.0.0.0]
[6858] [0335.0.15.15] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of the nucleic acid sequences of the table IA or IB, columns 5 or
7, lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., and/or homologs thereof. As described inter alia in WO
99/32619, dsRNAi approaches are clearly superior to traditional
antisense approaches. The invention therefore furthermore relates
to double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived therefrom), bring about altered
metabolic activity by the reduction in the expression of the
nucleic acid sequences of the Table IA or IB, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558, 559 and/or lines 560 to 563
resp., and/or homologs thereof. In a double-stranded RNA molecule
for reducing the expression of an protein encoded by a nucleic acid
sequence of one of the Table IA orr IB, columns 5 or 7, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp.,
and/or homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[6859] [0336.0.0.15] to [0342.0.0.15] see [0336.0.0.0] to
[0342.0.0.0]
[6860] [0343.0.15.15] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence of one of the sequences shown in Table IA or IB, columns 5
or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or line
180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560
to 563 resp., or its homolog is not necessarily required in order
to bring about effective reduction in the expression. The advantage
is, accordingly, that the method is tolerant with regard to
sequence deviations as may be present as a consequence of genetic
mutations, polymorphisms or evolutionary divergences. Thus, for
example, using the dsRNA, which has been generated starting from a
sequence of one of sequences shown in Table IA or IB, columns 5 or
7, lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 resp., or homologs thereof of the one organism, may be used to
suppress the corresponding expression in another organism.
[6861] [0344.0.0.15] to [0361.0.0.15] see [0344.0.0.0] to
[0361.0.0.0]
[6862] [0362.0.15.15] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table IIA or IIB, columns 5 or 7, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp. Due to the above
mentioned activity the fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. In one embodiment, transgenic for a polypeptide having an
activity of a polypeptide as indicated in Table IIA or IIB, columns
5 or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or
line 180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines
560 to 563 resp., means herein that due to modulation or
manipulation of the genome, an activity as annotated for a
polypeptide as indicated in Table IIA or IIB, column 3, lines 174
to 177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558, 559 and/or lines 560 to 563 resp.,
e.g. having a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 174 to 177 and/or line 178 and/or line 179 and/or line
180 and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560
to 563 resp., is increased in a cell or an organism or a part
thereof. Examples are described above in context with the process
of the invention.
[6863] [0363.0.0.15] see [0363.0.0.0]
[6864] [0364.0.15.15] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a polypeptide of the invention as indicated in Table
IIA or IIB, column 3 and/or 5, lines 174 to 177 and/or line 178
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558, 559 and/or lines 560 to 563 resp., with the
corresponding protein-encoding sequence as indicated in Table IA or
IB, column 3 and/or 5, lines 174 to 177 and/or line 178 and/or line
179 and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp.,
[6865] [0365.0.0.15] to [0373.0.0.15] see [0365.0.0.0] to
[0373.0.0.0]
[6866] [0374.0.15.15] Transgenic plants comprising the fatty acids
synthesized in the process according to the invention can be
marketed directly without isolation of the compounds synthesized.
In the process according to the invention, plants are understood as
meaning all plant parts, plant organs such as leaf, stalk, root,
tubers or seeds or propagation material or harvested material or
the intact plant. In this context, the seed encompasses all parts
of the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue. The fatty acids produced in the
process according to the invention may, however, also be isolated
from the plant in the form of their free fatty acids or bound in or
to compounds. Fatty acids produced by this process can be harvested
by harvesting the organisms either from the culture in which they
grow or from the field. This can be done via expressing, grinding
and/or extraction, salt precipitation and/or ion-exchange
chromatography or other chromatographic methods of the plant parts,
preferably the plant seeds, plant fruits, plant tubers and the
like.
[6867] [0375.0.0.15] to [0376.0.0.15] see [0375.0.0.0] to
[0376.0.0.0]
[6868] [0377.0.15.15] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced fatty acid and/or fatty acid composition or the produced
the fine chemical is isolated.
[6869] [0378.0.15.15] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the fatty acids
produced in the process can be isolated. The resulting fatty acids
can, if appropriate, subsequently be further purified, if desired
mixed with other active ingredients such as vitamins, amino acids,
carbohydrates, antibiotics and the like, and, if appropriate,
formulated.
[6870] [0379.0.15.15] In one embodiment, the amino acid, in
particular, heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid resp., is a
mixture comprising of one or more the respective fine chemicals. In
one embodiment, the respective fine chemical means here amino acid,
in particular heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid. In one
embodiment, amino acid means here a mixture of the respective fine
chemicals.
[6871] [0380.0.15.15] The fatty acids obtained in the process are
suitable as starting material for the synthesis of further products
of value. For example, they can be used in combination with each
other or alone for the production of pharmaceuticals, foodstuffs,
animal feeds or cosmetics. Accordingly, the present invention
relates a method for the production of a pharmaceuticals, food
stuff, animal feeds, nutrients or cosmetics comprising the steps of
the process according to the invention, including the isolation of
the fatty acid composition produced or the fine chemical produced
if desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the fatty acids produced in the
process or of the transgenic organisms in animal feeds, foodstuffs,
medicines, food supplements, cosmetics or pharmaceuticals.
[6872] [0380.1.15.15] The fatty acids obtained in the process are
further suitable in purified form as internal standard in
quantification of fatty acids. They can be also used as starting
compound in the synthesis of further fatty acids, by elongation,
saturation or desaturation resp., hydroxylation or synthesis of
glycerides or lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids.
[6873] [0381.0.0.15] to [0382.0.0.15] see [0381.0.0.0] to
[0382.0.0.0]
[6874] [0383.0.15.15] For preparing fatty acid compound-containing
fine chemicals, in particular the fine chemical, it is possible to
use as fatty acid source organic compounds such as, for example,
oils, fats and/or lipids comprising fatty acids such as fatty acids
having a carbon back bone between C.sub.10- to C.sub.16-carbon
atoms and/or small organic acids such acetic acid, propionic acid
or butanoic acid as precursor compounds.
[6875] [0384.0.0.15] see [0384.0.0.0]
[6876] [0385.0.15.15] The fermentation broths obtained in this way,
containing in particular heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid in mixtures with other lipids, fats and/or
oils, normally have a dry matter content of from 7.5 to 25% by
weight. Sugar-limited fermentation is additionally advantageous, at
least at the end, but especially over at least 30% of the
fermentation time. This means that the concentration of utilizable
sugar in the fermentation medium is kept at, or reduced to, 0 to 3
g/l during this time. The fermentation broth is then processed
further. Depending on requirements, the biomass can be removed
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation or a combination of these
methods, from the fermentation broth or left completely in it. The
fermentation broth can then be thickened or concentrated by known
methods, such as, for example, with the aid of a rotary evaporator,
thin-film evaporator, falling film evaporator, by reverse osmosis
or by nanofiltration. This concentrated fermentation broth can then
be worked up by freeze-drying, spray drying, spray granulation or
by other processes.
[6877] [0386.0.15.15] However, it is also possible to purify the
fatty acid produced further. For this purpose, the
product-containing composition is subjected for example to a thin
layer chromatography on silica gel plates or to a chromatography
such as a Florisil column (Bouhours J. F., J. Chromatrogr. 1979,
169, 462), in which case the desired product or the impurities are
retained wholly or partly on the chromatography resin. These
chromatography steps can be repeated if necessary, using the same
or different chromatography resins. The skilled worker is familiar
with the choice of suitable chromatography resins and their most
effective use. An alternative method to purify the fatty acids is
for example crystallization in the presence of urea. These methods
can be combined with each other.
[6878] [0387.0.0.15] to [0392.0.0.15] see [0387.0.0.0] to
[0392.0.0.0]
[6879] [0393.0.15.15] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [6880] (m) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [6881] (n) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence shown in table
IA or IB, preferably table I B, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558, 559 and/or lines 560 to 563 resp., and,
optionally, isolating the full length cDNA clone or complete
genomic clone; [6882] (o) introducing the candidate nucleic acid
molecules in host cells, preferably in a plant cell or a
microorganism, appropriate for producing the fine chemical; [6883]
(p) expressing the identified nucleic acid molecules in the host
cells; [6884] (q) assaying the fine chemical level in the host
cells; and [6885] (r) identifying the nucleic acid molecule and its
gene product which expression confers an increase in the fine
chemical level in the host cell after expression compared to the
wild type.
[6886] [0394.0.0.15] to [0398.0.0.15] see [0394.0.0.0] to
[0398.0.0.0]
[6887] [0399.0.15.15] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increased the fine chemical production in a plant or
microorganism, comprising the steps:
(g) culturing a cell or tissue or microorganism or maintaining a
plant expressing the polypeptide according to the invention or a
nucleic acid molecule encoding said polypeptide and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and the polypeptide of the present invention or used
in the process of the invention; and (h) identifying if the
compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout
system.
[6888] The screen for a gene product or an agonist conferring an
increase in the fine chemical production can be performed by growth
of an organism for example a microorganism in the presence of
growth reducing amounts of an inhibitor of the synthesis of the
fine chemical. Better growth, eg higher dividing rate or high dry
mass in comparison to the control under such conditions would
identify a gene or gene product or an agonist conferring an
increase in fine chemical production.
[6889] One can think to screen for increased fine chemical
production by for example resistance to drugs blocking fatty
synthesis and looking whether this effect is YDR513W dependent eg
comparing near identical organisms with low and high YDR513W
activity.
[6890] [00399.1.15.15] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the fine
chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table IIA
or IIB, columns 5 or 7, lines 174 to 177 and/or line 178 and/or
line 179 and/or line 180 and/or lines 181 to 185 and/or lines 558,
559 and/or lines 560 to 563 resp., or a homolog thereof, e.g.
comparing the phenotype of nearly identical organisms with low and
high activity of a protein as indicated in Table IIA or IIB,
columns 5 or 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558, 559
and/or lines 560 to 563 resp., after incubation with the drug.
[6891] [0400.0.0.15] to [0416.0.0.15] see [0400.0.0.0] to
[0416.0.0.0]
[6892] [0417.0.15.15] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the fatty acid production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect an organism such as a
microorganism or a plant against inhibitors, which block the fatty
acid, in particular the fine chemical, synthesis in said organism.
Examples of inhibitors or herbicides blocking the fatty acid
synthesis in organism such as microorganism or plants are for
example cerulenin, Thiolactomycin, Diazoborines or Triclosan, which
inhibit the fatty acids (beta-ketoacyl thioester synthetase
inhibitors) and sterol biosynthesis inhibitors,
aryloxyphenoxypropionates such as diclofop, fenoxaprop, haloxyfop,
fluazifop or quizalofop or cyclohexanediones such as clethodim or
sethoxydim
[(2-[1-{ethoxyimino}butyl]-542-{ethylthio}propyl]-3-hydroxy-2-cyclohexen--
1-one], which inhibit the plant acetyl-coenzyme A carboxylase or
thiocarbamates such as butylate, EPTC [=S-ethyl
dipropylcarbamothioat] or vernolate.
[6893] [0418.0.0.15] to [0423.0.0.15] see [0418.0.0.0] to
[0423.0.0.0]
[6894] [0424.0.15.15] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the fine chemical or of the fine
chemical and one or more other fatty acids, in particular
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid mixtures thereof
or mixtures of other fatty acids. Accordingly, the nucleic acid of
the invention, or the nucleic acid molecule identified with the
method of the present invention or the complement sequences
thereof, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the
antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the fine
chemical in a organism or part thereof, e.g. in a cell.
[6895] [0425.0.0.15] to [0430.0.0.15] see [0425.0.0.0] to
[0430.0.0.0]
[0431.0.15.15] Example 1
Cloning SEQ ID NO: 13930 in Escherichia coli
[6896] [0432.0.15.15] SEQ ID NO: 13930 was cloned into the plasmids
pBR322 (Sutcliffe, J. G. (1979) Proc. Natl Acad. Sci. USA, 75:
3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol. 134:
1141-1156); plasmids of the pBS series (pBSSK+, pBSSK- and others;
Stratagene, LaJolla, USA) or cosmids such as SuperCosi (Stratagene,
LaJolla, USA) or Lorist6 (Gibson, T. J. Rosenthal, A., and
Waterson, R. H. (1987) Gene 53: 283-286) for expression in E. coli
using known, well-established procedures (see, for example,
Sambrook, J. et al. (1989) "Molecular Cloning: A Laboratory
Manual". Cold Spring Harbor Laboratory Press or Ausubel, F. M. et
al. (1994) "Current Protocols in Molecular Biology", John Wiley
& Sons).
[6897] [0433.0.0.15] to [0434.0.0.15] see [0433.0.0.0] to
[0434.0.0.0]
[0435.0.15.15] Example 3
In-Vivo and In-Vitro Mutagenesis
[6898] [0436.0.15.15] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of fatty acids can be
carried out by passing a plasmid DNA (or another vector DNA)
containing the desired nucleic acid sequence or nucleic acid
sequences through E. coli and other microorganisms (for example
Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are
not capable of maintaining the integrity of its genetic
information. Usual mutator strains have mutations in the genes for
the DNA repair system [for example mutHLS, mutD, mutT and the like;
for comparison, see Rupp, W. D. (1996) DNA repair mechanisms in
Escherichia coli and Salmonella, pp. 2277-2294, ASM: Washington].
The skilled worker knows these strains. The use of these strains is
illustrated for example in Greener, A. and Callahan, M. (1994)
Strategies 7; 32-34.
[6899] In-vitro mutation methods such as increasing the spontaneous
mutation rates by chemical or physical treatment are well known to
the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[6900] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[6901] Positive mutation events can be selected by screening the
organisms for the production of the desired fine chemical.
[0437.0.15.15] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpine
[6902] [0438.0.15.15] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology, VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[6903] [0439.0.15.15] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255269) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[6904] [0440.0.15.15] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002,) teaches a homolog recombination system dependent
on rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this
system for homologous recombination are vectors derived from the
Ylp series. Plasmid vectors derived for example from the
2.mu.-Vector are known by the skilled worker and used for the
expression in yeasts. Other preferred vectors are for example
pART1, pCHY21 or pEVP11 as they have been described by McLeod et
al. (EMBO J. 1987, 6:729-736) and Hoffman et al. (Genes Dev. 5,
1991: 561-571.) or Russell et al. (J. Biol. Chem. 258, 1983:
143-149.). Other beneficial yeast vectors are plasmids of the REP,
REP-X, pYZ or RIP series.
[6905] [0441.0.0.15] see [0441.0.0.0]
[6906] [0442.0.15.15] The observations of the acivity of a mutated,
or transgenic, protein in a transformed host cell are based on the
fact that the protein is expressed in a similar manner and in a
similar quantity as the wild-type protein. A suitable method for
determining the transcription quantity of the mutant, or
transgenic, gene (a sign for the amount of mRNA which is available
for the translation of the gene product) is to carry out a Northern
blot (see, for example, Ausubel et al., (1988) Current Protocols in
Molecular Biology, Wiley: New York), where a primer which is
designed in such a way that it binds to the gene of interest is
provided with a detectable marker (usually a radioactive or
chemiluminescent marker) so that, when the total RNA of a culture
of the organism is extracted, separated on a gel, applied to a
stable matrix and incubated with this probe, the binding and
quantity of the binding of the probe indicates the presence and
also the amount of mRNA for this gene. Another method is a
quantitative PCR. This information detects the extent to which the
gene has been transcribed. Total cell RNA can be isolated for
example from yeasts or E. coli by a variety of methods, which are
known in the art, for example with the Ambion kit according to the
instructions of the manufacturer or as described in Edgington et
al., Promega Notes Magazine Number 41, 1993, p. 14.
[6907] [0443.0.0.15] see [0443.0.0.0]
[0444.0.15.15] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[6908] [0445.0.15.15] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[6909] [0446.0.0.15] to [0450.0.0.15] see [0446.0.0.0] to
[0450.0.0.0]
[6910] [0451.0.15.15] If genetically modified clones are examined,
an unmodified control clone, or a control clone, which contains the
basic plasmid without insertion, should also be included in the
tests. If a transgenic sequence is expressed, a control clone
should advantageously again be included in these tests. The medium
is advantageously inoculated to an OD600 of 0.5 to 1.5 using cells
which have been grown on agar plates, such as CM plates (10 g/l
glucose, 2.5 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast
extract, 5 g/l meat extract, 22 g/l agar, pH value 6.8 established
with 2M NaOH), which have been incubated at 30.degree. C. The media
are inoculated for example by addition of a liquid preculture of
seed organism such as E. coli or S. cerevisiae.
[6911] [0452.0.0.15] to [0453.0.0.15] see [0452.0.0.0] to
[0453.0.0.0]
[0454.0.15.15] Example 8
Analysis of the Effect of the Nucleic Acid Molecule on the
Production of the Fatty Acids
[6912] [0455.0.15.15] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a fatty acid) can be determined by growing the
modified microorganisms or the modified plant under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the elevated production of
desired product (i.e. of the lipids or a fatty acid). These
analytical techniques are known to the skilled worker and comprise
spectroscopy, thin-layer chromatography, various types of staining
methods, enzymatic and microbiological methods and analytical
chromatography such as high-performance liquid chromatography (see,
for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2,
p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.,
(1987) "Applications of HPLC in Biochemistry" in: Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et
al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery
and purification", p. 469-714, VCH: Weinheim; Better, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[6913] In addition to the abovementioned processes, plant lipids
are extracted from plant material as described by Cahoon et al.
(1999) Proc. Natl. Acad. Sci. USA 96 (22): 12935-12940 and Browse
et al. (1986) Analytic Biochemistry 152:141-145. The qualitative
and quantitative analysis of lipids or fatty acids is described by
Christie, William W., Advances in Lipid Methodology, Ayr/Scotland:
Oily Press (Oily Press Lipid Library; 2); Christie, William W., Gas
Chromatography and Lipids. A Practical Guide--Ayr, Scotland: Oily
Press, 1989, Repr. 1992, IX, 307 pp. (Oily Press Lipid Library; 1);
"Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952)--16
(1977) under the title: Progress in the Chemistry of Fats and Other
Lipids CODEN.
[6914] [0456.0.0.15] see [0456.0.0.0] In addition to the
determination of the fermentation end product, other components of
the metabolic pathways which are used for the production of the
desired compound, such as intermediates and by-products, may also
be analyzed in order to determine the total productivity of the
organism, the yield and/or production efficiency of the compound.
The analytical methods encompass determining the amounts of
nutrients in the medium (for example sugars, hydrocarbons, nitrogen
sources, phosphate and other ions), determining biomass composition
and growth, analyzing the production of ordinary metabolites from
biosynthetic pathways and measuring gases generated during the
fermentation. Standard methods for these are described in Applied
Microbial Physiology; A Practical Approach, P. M. Rhodes and P. F.
Stanbury, Ed. IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN:
0199635773) and the references cited therein.
[0457.0.15.15] Example 9
Purification of the Fatty Acid
[6915] [0458.0.15.15] One example is the analysis of fatty acids
(abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid
chromatography/mass spectrometry; TAG, triacylglycerol; TLC,
thin-layer chromatography).
[6916] The unambiguous detection for the presence of fatty acid
products can be obtained by analyzing recombinant organisms using
analytical standard methods: GC, GC-MS or TLC, as described on
several occasions by Christie and the references therein (1997, in:
Advances on Lipid Methodology, Fourth Edition: Christie, Oily
Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353).
[6917] The total fatty acids produced in the organism for example
in yeasts used in the inventive process can be analysed for example
according to the following procedure:
[6918] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods. After
disruption, the material must be centrifuged (1000.times.g, 10
min., 4.degree. C.) and washed once with 100 mM NaHCO.sub.3, pH 8.0
to remove residual medium and fatty acids. For preparation of the
fatty acid methyl esters (FAMES) the sediment is resuspended in
distilled water, heated for 10 minutes at 100.degree. C., cooled on
ice and recentrifuged, followed by extraction for one hour at
90.degree. C. in 0.5 M sulfuric acid in methanol with 2%
dimethoxypropane, which leads to hydrolyzed oil and lipid
compounds, which give transmethylated lipids.
[6919] The FAMES are then extracted twice with 2 ml petrolether,
washed once with 100 mM NaHCO.sub.3, pH 8.0 and once with distilled
water and dried with Na.sub.2SO.sub.4. The organic solvent can be
evaporated under a stream of Argon and the FAMES were dissolved in
50 .mu.l of petrolether. The samples can be separated on a ZEBRON
ZB-Wax capillary column (30 m, 0.32 mm, 0.25 .mu.m; Phenomenex) in
a Hewlett Packard 6850 gas chromatograph with a flame ionisation
detector. The oven temperature is programmed from 70.degree. C. (1
min. hold) to 200.degree. C. at a rate of 20.degree. C./min., then
to 250.degree. C. (5 min. hold) at a rate of 5.degree. C./min and
finally to 260.degree. C. at a rate of 5.degree. C./min. Nitrogen
is used as carrier gas (4.5 ml/min. at 70.degree. C.). The identity
of the resulting fatty acid methyl esters can be identified by
comparison with retention times of FAME standards, which are
available from commercial sources (i.e. Sigma).
[6920] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[6921] This is followed by heating at 100.degree. C. for 10 minutes
and, after cooling on ice, by resedimentation. The cell sediment is
hydrolyzed for one hour at 90.degree. C. with 1 M methanolic
sulfuric acid and 2% dimethoxypropane, and the lipids are
transmethylated. The resulting fatty acid methyl esters (FAMEs) are
extracted in petroleum ether. The extracted FAMEs are analyzed by
gas liquid chromatography using a capillary column (Chrompack, WCOT
Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and a temperature
gradient of from 170.degree. C. to 240.degree. C. in 20 minutes and
5 minutes at 240.degree. C. The identity of the fatty acid methyl
esters is confirmed by comparison with corresponding FAME standards
(Sigma). The identity and position of the double bond can be
analyzed further by suitable chemical derivatization of the FAME
mixtures, for example to give 4,4-dimethoxyoxazoline derivatives
(Christie, 1998) by means of GC-MS.
[6922] The methodology is described for example in Napier and
Michaelson, 2001, Lipids. 36(8):761-766; Sayanova et al., 2001,
Journal of Experimental Botany. 52(360):1581-1585, Sperling et al.,
2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al.,
1998, FEBS Letters. 439(3):215-218.
[6923] [0459.0.15.15] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously the fatty acids can be further purified with a
so-called RTHPLC. As eluent different an acetonitrile/water or
chloroform/acetonitrile mixtures are advantageously is used. For
example canola oil can be separated said HPLC method using an
RP-18-column (ET 250/3 Nucleosil 120-5 C.sub.18 Macherey and Nagel,
Duren, Germany). A chloroform/acetonitrile mixture (v/v 30:70) is
used as eluent. The flow rate is beneficial 0.8 ml/min. For the
analysis of the fatty acids an
[6924] ELSD detector (evaporative light-scattering detector) is
used. MPLC, dry-flash chromatography or thin layer chromatography
are other beneficial chromatography methods for the purification of
fatty acids. If necessary, these chromatography steps may be
repeated, using identical or other chromatography resins. The
skilled worker is familiar with the selection of suitable
chromatography resin and the most effective use for a particular
molecule to be purified.
[6925] [0460.0.15.15] In addition depending on the produced fine
chemical purification is also possible with cristalisation or
destilation. Both methods are well known to a person skilled in the
art.
[0461.0.15.15] Example 10
Cloning SEQ ID NO: 13930 for the Expression in Plants
[6926] [0462.0.0.15] see [0462.0.0.0]
[6927] [0463.0.15.15] SEQ ID NO: 13930 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[6928] [0464.0.15.15] The composition for the protocol of the Pfu
Turbo DNA polymerase was as follows: 1.times.PCR buffer
(Stratagene), 0.2 mM of each dNTP, 100 ng genomic
[6929] DNA of Saccharomyces cerevisiae (strain S288C; Research
Genetics, Inc., now Invitrogen) or Escherichia coli (strain MG1655;
E. coli Genetic Stock Center), 50 .mu.mol forward primer, 50
.mu.mol reverse primer, 2.5 u Pfu Turbo DNA polymerase. The
amplification cycles were as follows:
[6930] [0465.0.15.15] 1 cycle of 3 minutes at 94-95.degree. C.,
followed by 25-36 cycles of in each case 1 minute at 95.degree. C.
or 30 seconds at 94.degree. C., 45 seconds at 50.degree. C., 30
seconds at 50.degree. C. or 30 seconds at 55.degree. C. and 210-480
seconds at 72.degree. C., followed by 1 cycle of 8 minutes at
72.degree. C., then 4.degree. C.
[6931] [0466.0.0.15] see [0466.0.0.0]
[6932] [0467.0.15.15] The following primer sequences were selected
for the gene SEQ ID NO: 13930:
TABLE-US-00049 i) forward primer (SEQ ID NO: 13950) atgaactcta
ttttagacag aaatgtt ii) reverse primer (SEQ ID NO: 13951) ttatttttgg
tcttgtttca aagtgtc
or were selected for the genes described in Table III, column 5,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558, 559 and/or lines 560 to
563 as described in Table III, column 7, lines 174 to 177 and/or
line 178 and/or line 179 and/or line 180 and/or lines 181 to 185
and/or lines 558, 559 and/or lines 560 to 563.
[6933] [0468.0.0.15] to [0479.0.0.15] see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.15.15] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 13930
[6934] [0481.0.0.15] to [0513.0.0.15] see [0481.0.0.0] to
[0513.0.0.0]
[6935] [0514.0.15.15] As an alternative, the fatty acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55).
[6936] The results of the different plant analyses can be seen from
the table, which follows:
TABLE-US-00050 TABLE 1 ORF Metabolite Method Min Max YDR513W
Margaric Acid (C17:0) GC 1,24 1,97 YER156C Margaric Acid (C17:0) GC
1,20 1,49 YLR255C Margaric Acid (C17:0) GC 1,20 1,27 b1829 Margaric
Acid (C17:0) GC 1,20 2,33 YER173W 2-Hydroxy-Palmitic Acid GC 1,26
4,94 YFR042W 2-Hydroxy-tetracosenoic acid GC 1,28 1,56 YOR317W
Hexadeca-dienoic acid GC 1,19 1,69 YGL205W Hexadecatrienoic Acid
(C16:3) GC 1,14 1,16 YIL150C Hexadecatrienoic Acid (C16:3) GC 3,24
3,24 YKL132C Hexadecatrienoic Acid (C16:3) GC 1,13 1,56 YOR344C
Hexadecatrienoic Acid (C16:3) GC 1,12 1,20 b3430 Hexadecatrienoic
Acid (C16:3) GC 1,12 1,20 b0057 C24:1 fatty acid GC 1.22 1.37 b0161
2-Hydroxy-palmitic acid GC 1.21 1.48 b0758 2-Hydroxy-palmitic acid
GC 1.19 1.38 b1097 C24:1 fatty acid GC 1.23 1.41 b2078 C24:1 fatty
acid GC 1.23 1.41 b3231 C24:1 fatty acid GC 1.23 1.48
[6937] [0515.0.15.15] Column 2 shows the fatty acid analyzed.
Columns 4 and 5 shows the ratio of the analyzed fatty acid between
the transgenic plants and the wild type; Increase of the
metabolites: Max: maximal x-fold (normalised to wild type)-Min:
minimal x-fold (normalised to wild type). Decrease of the
metabolites: Max: maximal x-fold (normalised to wild type) (minimal
decrease), Min: minimal x-fold (normalised to wild type) (maximal
decrease). Column 3 indicates the analytical method.
[6938] [0516.0.0.15] see [0516.0.0.0]
[0517.0.15.15] Example 14a
Engineering Ryegrass Plants by Over-Expressing YOR344C from
Saccharomyces cerevisiae or Homologs of YOR344C from Other
Organisms
[6939] [0518.0.0.15] to [0524.0.0.15] see [0518.0.0.0] to
[0524.0.0.0]
[0525.0.15.15] Example 14b
Engineering Soybean Plants by Over-Expressing YOR344C from
Saccharomyces cerevisiae or Homologs of YOR344C from Other
Organisms
[6940] [0526.0.0.15] to [0529.0.0.15] see [0526.0.0.0] to
[0529.0.0.0]
[0530.0.15.15] Example 14c
Engineering Corn Plants by Over-Expressing YOR344C from
Saccharomyces cerevisiae or Homologs of YOR344C from Other
Organisms
[6941] [0531.0.0.15] to [0533.0.0.15] see [0531.0.0.0] to
[0533.0.0.0]
[0534.0.15.15] Example 14d
Engineering Wheat Plants by Over-Expressing YOR344C from
Saccharomyces cerevisiae or Homologs of YOR344C from Other
Organisms
[6942] [0535.0.0.15] to [0537.0.0.15] see [0535.0.0.0] to
[0537.0.0.0]
[0538.0.15.15] Example 14e
Engineering Rapeseed/Canola Plants by Over-Expressing YOR344C from
Saccharomyces cerevisiae or Homologs of YOR344C from Other
Organisms
[6943] [0539.0.0.15] to [0542.0.0.15] see [0539.0.0.0] to
[0542.0.0.0]
[0543.0.15.15] Example 14f
Engineering Alfalfa Plants by Over-Expressing YOR344C from
Saccharomyces cerevisiae or Homologs of YOR344C from Other
Organisms
[6944] [0544.0.0.15] to [0547.0.0.15] see [0544.0.0.0] to
[0547.0.0.0]
[0548.0.15.15] Example 14 g
Engineering Alfalfa Plants by Over-Expressing YOR344C from
Saccharomyces cerevisiae or Homologs of YOR344C from Other
Organisms
[6945] [0549.0.0.15] to [0552.0.0.15] see [0549.0.0.0] to
[0552.0.0.0]
[0552.1.15.15]: Example 15
Metabolite Profiling Info from Zea mays
[6946] Zea mays plants were engineered, grown and analyzed as
described in Example 14c. The results of the different Zea mays
plants analysed can be seen from Table 2 which follows:
TABLE-US-00051 TABLE 2 ORF Metabolite Min Max b1829 Heptadecanoic
acid 1.23 1.71
[6947] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in heptadecanoic acid in
genetically modified corn plants expressing the Escherichia coli
nucleic acid sequence b1829 resp.
[6948] In case the activity of the Escherichia coli K12 protein
b1829 or a protein with an activity being defined as a heat shock
protein or its homolog, is increased in corn plants, preferably, an
increase of the fine chemical heptadecanoic acid between 23% and
71% is conferred.
[6949] [00552.2.0.15] see [00552.2.0.0]
[6950] [0553.0.15.15] [6951] 1. A process for the production of
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid, which comprises
(a) increasing or generating the activity of a protein as indicated
in Table IIA or IIB, columns 5 or 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558 to 559 and/or 560 to 563 resp., or a functional
equivalent thereof in a non-human organism, or in one or more parts
thereof; and (b) growing the organism under conditions which permit
the production of heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10, 13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid in said organism.
[6952] 2. A process for the production of heptadecanoic acid and/or
2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid, comprising the increasing or generating in an
organism or a part thereof the expression of at least one nucleic
acid molecule comprising a nucleic acid molecule selected from the
group consisting of: [6953] a) nucleic acid molecule encoding of
the polypeptide shown in table IIA or IIB, columns 5 or 7, lines
174 to 177 and/or line 178 and/or line 179 and/or line 180 and/or
lines 181 to 185 and/or lines 558 to 559 and/or 560 to 563 resp.,
or a fragment thereof, which confers an increase in the amount of
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid in an organism or
a part thereof; [6954] b) nucleic acid molecule comprising of the
nucleic acid molecule shown in table IA or IB, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558 to 559 and/or 560 to 563
resp; [6955] c) nucleic acid molecule whose sequence can be deduced
from a polypeptide sequence encoded by a nucleic acid molecule of
(a) or (b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of heptadecanoic acid and/or
2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid in an organism or a part thereof; [6956] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid in an organism or a part thereof; [6957] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under under stringent hybridisation conditions and
conferring an increase in the amount of heptadecanoic acid and/or
2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid in an organism or a part thereof; [6958] f)
nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers as shown in table
III, column 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558 to 559
and/or 560 to 563 resp., and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [6959] g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid in an organism or a part thereof; [6960] h)
nucleic acid molecule encoding a polypeptide comprising the
consensus sequence shown in table IV, column 7, lines 174 to 177
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558 to 559 and/or 560 to 563 resp., and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; and [6961] i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof. or
comprising a sequence which is complementary thereto. [6962] 3. The
process of claim 1 or 2, comprising recovering of the free or bound
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid. [6963] 4. The
process of any one of claims 1 to 3, comprising the following
steps: [6964] (a) selecting an organism or a part thereof
expressing a polypeptide encoded by the nucleic acid molecule
characterized in claim 2; [6965] (b) mutagenizing the selected
organism or the part thereof; [6966] (c) comparing the activity or
the expression level of said polypeptide in the mutagenized
organism or the part thereof with the activity or the expression of
said polypeptide of the selected organisms or the part thereof;
[6967] (d) selecting the mutated organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
[6968] (e) optionally, growing and cultivating the organisms or the
parts thereof; and [6969] (f) recovering, and optionally isolating,
the free or bound heptadecanoic acid and/or 2-hydroxy palmitic acid
and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid produced by the
selected mutated organisms or parts thereof. [6970] 5. The process
of any one of claims 1 to 4, wherein the activity of said protein
or the expression of said nucleic acid molecule is increased or
generated transiently or stably. [6971] 6. An isolated nucleic acid
molecule comprising a nucleic acid molecule selected from the group
consisting of: [6972] a) nucleic acid molecule encoding of the
polypeptide shown in table IIA or IIB, columns 5 or 7, lines 174 to
177 and/or line 178 and/or line 179 and/or line 180 and/or lines
181 to 185 and/or lines 558 to 559 and/or 560 to 563 resp., or a
fragment thereof, which confers an increase in the amount of
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid in an organism or
a part thereof; [6973] b) nucleic acid molecule comprising of the
nucleic acid molecule shown in table IA or IB, columns 5 or 7,
lines 174 to 177 and/or line 178 and/or line 179 and/or line 180
and/or lines 181 to 185 and/or lines 558 to 559 and/or 560 to 563
resp.; [6974] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of heptadecanoic acid
and/or 2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid
and/or hexadecadienoic acid, preferably delta 7,10 hexadecadienoic
acid and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid in an organism or a part thereof; [6975] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid in an organism or a part thereof; [6976] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of heptadecanoic acid and/or
2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid in an organism or a part thereof; [6977] f)
nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers as shown in table
III, column 7, lines 174 to 177 and/or line 178 and/or line 179
and/or line 180 and/or lines 181 to 185 and/or lines 558 to 559
and/or 560 to 563 resp., and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [6978] g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid in an organism or a part thereof; [6979] h)
nucleic acid molecule encoding a polypeptide comprising the
consensus sequence shown in table IV, column 7, lines 174 to 177
and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558 to 559 and/or 560 to 563 resp., and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; and [6980] i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof.
[6981] whereby the nucleic acid molecule distinguishes over the
sequence as shown in table I A, columns 5 or 7, lines 174 to 177
and/or line 178 and/or line 179 and/or line 180 and/or lines 181 to
185 and/or lines 558 to 563 resp., by one or more nucleotides.
[6982] 7. A nucleic acid construct which confers the expression of
the nucleic acid molecule of claim 6, comprising one or more
regulatory elements. [6983] 8. A vector comprising the nucleic acid
molecule as claimed in claim 6 or the nucleic acid construct of
claim 7. [6984] 9. The vector as claimed in claim 8, wherein the
nucleic acid molecule is in operable linkage with regulatory
sequences for the expression in a prokaryotic or eukaryotic, or in
a prokaryotic and eukaryotic, host. [6985] 10. A host cell, which
has been transformed stably or transiently with the vector as
claimed in claim 8 or 9 or the nucleic acid molecule as claimed in
claim 6 or the nucleic acid construct of claim 7 or produced as
described in claim any one of claims 2 to 5. [6986] 11. The host
cell of claim 10, which is a transgenic host cell. [6987] 12. The
host cell of claim 10 or 11, which is a plant cell, an animal cell,
a microorganism, or a yeast cell, a fungus cell, a prokaryotic
cell, an eukaryotic cell or an archaebacterium. [6988] 13. A
process for producing a polypeptide, wherein the polypeptide is
expressed in a host cell as claimed in any one of claims 10 to 12.
[6989] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over the sequence as
shown in table II A, columns 5 or 7, lines 174 to 177 and/or line
178 and/or line 179 and/or line 180 and/or lines 181 to 185 and/or
lines 558 to 563 resp., by one or more amino acids [6990] 15. An
antibody, which binds specifically to the polypeptide as claimed in
claim 14. [6991] 16. A plant tissue, propagation material,
harvested material or a plant comprising the host cell as claimed
in claim 12 which is plant cell or an Agrobacterium. [6992] 17. A
method for screening for agonists and antagonists of the activity
of a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of heptadecanoic acid and/or
2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid in an organism or a part thereof comprising:
[6993] (a) contacting cells, tissues, plants or microorganisms
which express the a polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid in an organism or
a part thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide;
[6994] (b) assaying the palmitic acid level or the polypeptide
expression level in the cell, tissue, plant or microorganism or the
media the cell, tissue, plant or microorganisms is cultured or
maintained in; and [6995] (c) identifying a agonist or antagonist
by comparing the measured palmitic acid level or polypeptide
expression level with a standard palmitic acid or polypeptide
expression level measured in the absence of said candidate compound
or a sample comprising said plurality of compounds, whereby an
increased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an agonist and
a decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[6996] 18. A process for the identification of a compound
conferring increased heptadecanoic acid and/or 2-hydroxy palmitic
acid and/or 2-hydroxy-tetracosenoic-acid and/or hexadecadienoic
acid, preferably delta 7,10 hexadecadienoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid and/or 24:1 fatty acid, preferably C24:1 delta 15 fatty acid
production in a plant or microorganism, comprising the steps:
[6997] (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid in an organism or
a part thereof and a readout system capable of interacting with the
polypeptide under suitable conditions which permit the interaction
of the polypeptide with dais readout system in the presence of a
compound or a sample comprising a plurality of compounds and
capable of providing a detectable signal in response to the binding
of a compound to said polypeptide under conditions which permit the
expression of said readout system and of the polypeptide encoded by
the nucleic acid molecule of claim 6 conferring an increase in the
amount of heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid in an organism or
a part thereof; [6998] (b) identifying if the compound is an
effective agonist by detecting the presence or absence or increase
of a signal produced by said readout system. [6999] 19. A method
for the identification of a gene product conferring an increase in
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid production in a
cell, comprising the following steps: [7000] (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid after expression
with the nucleic acid molecule of claim 6; [7001] (b) identifying
the nucleic acid molecules, which hybridise under relaxed stringent
conditions with the nucleic acid molecule of claim 6; [7002] (c)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid; [7003] (d) expressing the identified nucleic
acid molecules in the host cells; [7004] (e) assaying the
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid level and/or
24:1 fatty acid, preferably C24:1 delta 15 fatty acid in the host
cells; and [7005] (f) identifying nucleic acid molecule and its
gene product which expression confers an increase in the
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid level in the host
cell in the host cell after expression compared to the wild type.
[7006] 20. A method for the identification of a gene product
conferring an increase in heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid production in a cell, comprising the following
steps: [7007] (a) identifying in a data bank nucleic acid molecules
of an organism; which can contain a candidate gene encoding a gene
product conferring an increase in the heptadecanoic acid and/or
2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10, 13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid amount or level in an organism or a part
thereof after expression, and which are at least 20% homolog to the
nucleic acid molecule of claim 6; [7008] (b) introducing the
candidate nucleic acid molecules in host cells appropriate for
producing heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid; [7009] (c)
expressing the identified nucleic acid molecules in the host cells;
[7010] (d) assaying the heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid level in the host cells; and [7011] (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the heptadecanoic acid and/or
2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid level in the host cell after expression
compared to the wild type. [7012] 21. A method for the production
of an agricultural composition comprising the steps of the method
of any one of claims 17 to 20 and formulating the compound
identified in any one of claims 17 to 20 in a form acceptable for
an application in agriculture. [7013] 22. A composition comprising
the nucleic acid molecule of claim 6, the polypeptide of claim 14,
the nucleic acid construct of claim 7, the vector of any one of
claim 8 or 9, an antagonist or agonist identified according to
claim 17, the compound of claim 18, the gene product of claim 19 or
20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. [7014] 23. Use of the nucleic acid molecule as
claimed in claim 6 for the identification of a nucleic acid
molecule conferring an increase of heptadecanoic acid and/or
2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid and/or
hexadecadienoic acid, preferably delta 7,10 hexadecadienoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid after expression. [7015] 24. Use of the
polypeptide of claim 14 or the nucleic acid construct claim 7 or
the gene product identified according to the method of claim 19 or
20 for identifying compounds capable of conferring a modulation of
heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid levels in an
organism. [7016] 25. Food or feed composition comprising the
nucleic acid molecule of claim 6, the polypeptide of claim 14, the
nucleic acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20. [7017] 26. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the protection of a plant against
a heptadecanoic acid and/or 2-hydroxy palmitic acid and/or
2-hydroxy-tetracosenoic-acid and/or hexadecadienoic acid,
preferably delta 7,10 hexadecadienoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or 24:1
fatty acid, preferably C24:1 delta 15 fatty acid synthesis
inhibiting herbicide.
[7018] [0554.0.0.15] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[7019] [0000.0.0.16] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[7020] [0001.0.0.16] see [0001.0.0.0]
[7021] [0002.0.16.16] The discovery in Arabidopsis of citramalic
acid (Fiehn et al. 2000 Nature Biotechnology 18, 1157-1161) a
potential precursor of pyruvic acid and acetate suggests a novel
aspect of carbon metabolism and furthermore suggests the existence
of a tricarboxylic acid cycle bypass previously found only in
bacteria.
[7022] The malic acid oxaloacetate shuttle is characteristic for
plant cells. It transports redox equivalents intracellularly.
[7023] Malic acid is not only a central metabolite in intermediary
flow of carbon in organisms. In higher plants, vacuolar malic acid
accumulation, and hence, transtonoplast malic acid transport, also
plays a paramount role in many physiological functions. These
include adjustment of osmotic and turgor potentials in extension
growth and movements of stomata and pulvini, pH-regulation, e.g.
during nitrate reduction, and others (for review, see Luttge et al,
Plant Physiol, 124(2000),1335-1348).
[7024] Osawa and Matsumoto, Plant Physiol, 126(2001), 411-420
discuss the involvement malic acid in aluminium resistance in
plants.
[7025] Malic acid is a common constituent of all plants, and its
formation is controlled by an enzyme (protein catalyst) called
malic acid dehydrogenase (MDH).
[7026] Malic acid occupies a central role in plant metabolism. Its
importance in plant mineral nutrition is reflected by the role it
plays in symbiotic nitrogen fixation, phosphorus acquisition, and
aluminum tolerance.
[7027] During phosphorus deficiency, malic acid is frequently
secreted from roots to release unavailable forms of phosphorus.
[7028] In nitrogen-fixing root nodules, malic acid is the primary
substrate for bacteroid respiration, thus fueling nitrogenase.
[7029] Pyruvic acid is a naturally occurring component in plants
and vegetables and in the body, where it is inherently involved in
metabolism, the process whereby energy is produced. Pyruvic acid
represents the final step in the metabolism of glucose or
starch.
[7030] Increased pyruvic acid production in yeast strains is known
(WO 04/099425).
[7031] Glyceric acid is an improtan precursor in the anabolism of
amino acids, in particular for Serin and Glycin. Further, the
energy level of a cell may be depend on the level of glyceric acid
found. Glycerate and Glycerate-3-phophate form a shuttle for the
transportation of energy equivalents, e.g. during photorespiration
between glycosomes and peroxisomes.
[7032] Succinic acid is an intermediate of the citric acid cycle
(and the glyoxylate cycle) produced by action of the enzyme
succinvl-CoA synthetase on succinyl-CoA. Succinic acid is converted
to fumaric acid by action of the enzyme succinic acid dehydrogenase
(with formation of FADH2).
[7033] Trihydroxibutanoic acid, trihydroxybutyric acid lacton can
be regarded as hypothetic precusors of pheromone like metabolites
in plants.
[7034] Due to these interesting physiological roles and
agrobiotechnological potential of citramalic acid, glyceric acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid there is a need
to identify the genes of enzymes and other proteins involved in
citramalic acid, glyceric acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid metabolism, and to generate mutants or transgenic plant lines
with which to modify the citramalic acid, glyceric acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid or trihydroxybutanoic acid content.
[7035] [0003.0.0.16] to [0007.0.0.16] -/-
[7036] [0008.0.0.16] One way to increase the productive capacity of
biosynthesis is to apply recombinant DNA technology. Thus, it would
be desirable to produce glyceric acid, citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid or trihydroxybutanoic acid in plants. That type of production
permits control over quality, quantity and selection of the most
suitable and efficient producer organisms. The latter is especially
important for commercial production economics and therefore
availability to consumers. In addition it is desirable to produce
glyceric acid, citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid in plants in order to increase plant productivity and
resistance against biotic and abiotic stress as discussed
before.
[7037] Methods of recombinant DNA technology have been used for
some years to improve the production of fine chemicals in
microorganisms and plants by amplifying individual biosynthesis
genes and investigating the effect on production of fine chemicals.
It is for example reported, that the xanthophyll astaxanthin could
be produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Agrobacterium
tumefaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. Those results indicated
that about 75 percent of the carotenoids found in the flower of the
transformed plant contained a keto group.
[7038] [0009.0.16.16] Thus, it would be advantageous if an algae,
plant or other microorganism were available which produce large
amounts glyceric acid, citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid or
trihydroxybutanoic acid. The invention discussed hereinafter
relates in some embodiments to such transformed prokaryotic or
eukaryotic microorganisms.
[7039] It would also be advantageous if plants were available whose
roots, leaves, stem, fruits or flowers produced large amounts of
glyceric acid, citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid. The invention discussed hereinafter relates in some
embodiments to such transformed plants.
[7040] [0010.0.16.16] Therefore improving the quality of foodstuffs
and animal feeds is an important task of the food-and-feed
industry. This is necessary since, for example glyceric acid,
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid or trihydroxybutanoic acid, as
mentioned above, which occur in plants and some microorganisms are
limited with regard to the supply of mammals.
[7041] Especially advantageous for the quality of foodstuffs and
animal feeds is as balanced as possible a specific glyceric acid,
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid or trihydroxybutanoic acid profile in
the diet since an excess of glyceric acid, citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid or trihydroxybutanoic acid above a specific concentration in
the food has a positive effect. A further increase in quality is
only possible via addition of further glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid, which are
limiting.
[7042] [0011.0.16.16] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid in a balanced
manner to suit the organism.
[7043] [0012.0.16.16] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
proteins which participate in the biosynthesis of glyceric acid,
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid and/or trihydroxybutanoic acid and
make it possible to produce them specifically on an industrial
scale without unwanted by-products forming. In the selection of
genes for biosynthesis two characteristics above all are
particularly important. On the one hand, there is as ever a need
for improved processes for obtaining the highest possible contents
of glyceric acid, citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid; on the other hand as less as possible
by-products should be produced in the production process.
[7044] [0013.0.0.16] see [0013.0.0.0]
[7045] [0014.0.16.16] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid. Accordingly,
in the present invention, the term "the fine chemical" as used
herein relates to a glyceric acid, citramalic acid, fumaric acid,
malic acid, pyruvic acid, succinic acid, trihydroxybutyric acid
and/or trihydroxybutanoic acid. Further, the term "the fine
chemicals" as used herein also relates to fine chemicals comprising
glyceric acid, citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid.
[7046] [0015.0.16.16] In one embodiment, the term "the fine
chemical" or "the respective fine chemical" means at least one
chemical compound with glyceric acid, citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid and/or trihydroxybutanoic acid activity.
[7047] In one embodiment, the term "the fine chemical" or "the
respective fine chemical" means a glyceric acid.
[7048] In one embodiment, the term "the fine chemical" or "the
respective fine chemical" means a citramalic acid. In one
embodiment, the term "the fine chemical" or "the respective fine
chemical" means fumaric acid. In one embodiment, the term "the fine
chemical" or "the respective fine chemical" means malic acid. In
one embodiment, the term "the fine chemical" or "the respective
fine chemical" means pyruvic acid. In one embodiment, the term "the
fine chemical" or "the respective fine chemical" means succinic
acid. In one embodiment, the term "the fine chemical" or "the
respective fine chemical" means trihydroxybutyric acid. In one
embodiment, the term "the fine chemical" or "the respective fine
chemical" means trihydroxybutanoic acid depending on the context in
which the term is used. Throughout the specification the term "the
fine chemical" or "the respective fine chemical" means glyceric
acid, citramalic acid, fumaric acid, malic acid, pyruvic acid,
succinic acid, trihydroxybutyraic acid and/or trihydroxybutanoic
acid, its salts, ester, thioester or in free form or bound to other
compounds such sugars or sugar polymers, like glucoside, e.g.
diglucoside. In particular it is known to the skilled that anionic
compounds as acids are present in an equilibrium of the acid and
its salts according to the pH present in the respective compartment
of the cell or organism and the pK of the acid. Thus, the term "the
fine chemical", the term "the respective fine chemical", the term
"acid" or the use of a demonination referring to a neutralized
anionic compound respectivley relates the anionic form as well as
the neutralised status of that compound.
[7049] Thus, citramalic acid relates also to citramalate, fumaric
acid also relates to fumarate, malic acid also relates to malate,
pyruvic acid also relates to pyruvate, succinic acid relates to
succinate, trihydroxybuyraic acid relates to trihydroxybutyrate or
trihydroxybutanoic acid relates also to trihydroxybutanoic.
[7050] In one embodiment, the term "the fine chemical" and the term
"the respective fine chemical" mean at least one chemical compound
with an activity of the above mentioned fine chemical
[7051] [0016.0.16.16] Accordingly, the present invention relates to
a process comprising [7052] (a) increasing or generating the
activity of one or more YMR241 W, YJL099W, YJL055W, YGR007W,
YCR059C, YCL032W, YBR184W, YBR084W, b4139, b1676, b1611, b0695,
b0730, b1896, b2699, b4063, YBL015W, YFR007W, YJL072C, YKL132C,
YOR044W, YPR024W or YPR138C protein(s) or of a protein having the
sequence of a polypeptide encoded by a nucleic acid molecule
indicated in Table I, columns 5 or 7, lines 190 to 226 or lines 564
to 594 in a non-human organism in one or more parts thereof; and
[7053] (b) growing the organism under conditions which permit the
production of the fine chemical, thus glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid in said
organism.
[7054] Accordingly, the present invention relates to a process
comprising. [7055] (a) increasing or generating the activity of one
or more proteins having the activity of a protein indicated in
Table II, column 3, lines 190 to 226 or lines 564 to 594, resp. or
having the sequence of a polypeptide encoded by a nucleic acid
molecule indicated in Table I, column 5 or 7, lines 190 to 226 or
lines 564 to 594, resp. in a non-human organism in one or more
parts thereof; and growing the organism under conditions which
permit the production of the fine chemical, thus, glyceric acid
referring to line 572 to 576, citramalic acid referring to line 190
and/or 564, fumaric acid referring to line 191 to 205, and/or 565
to 571, malic acid referring to line 206 to 217 and/or 577 to 583,
pyruvic acid referring to line 220 or 584, succinic acid referring
to line 221 to 224 and/or 585 to 591, trihydroxybutyric acid
referring to line 218 or 219 and/or trihydroxybutanoic acid
referring to 225 and 226 and/or 592 to 594, in said organism.
[7056] [0016.1.16.16] Accordingly, the term "the fine chemical"
means "citramalic acid" in relation to all sequences listed in
Table I, line 190 and/or 564 or homologs thereof. Accordingly, the
term "the fine chemical" means "fumaric acid" in relation to the
sequences listed in Table I, line 191 to 205, and/or 565 to 571, or
homologs thereof. Accordingly, the term "the fine chemical" means
"malic acid" in relation to the sequences listed in table I, line
206 to 217 and/or 577 to 583, or homologs thereof. Accordingly, the
term "the fine chemical" means "trihydroxybutyric acid" in relation
to the sequences listed in Table I, line 218 or 219 or homologs
thereof. Accordingly, the term "the fine chemical" means "pyruvic
acid" in relation to the sequences listed in Table I, line 220 or
584, or homologs thereof. Accordingly, the term "the fine chemical"
means "succinic acid" in relation to the sequences listed in Table
I, line 221 to 224 and/or 585 to 591, or homologs thereof and means
"trihydroxybutanoic acid" in relation to the sequences listed in
Table I, line 225 and 226 and/or 592 to 594, or homologs thereof.
Accordingly, the term "the fine chemical" means "glyceric acid" in
relation to the sequences listed in Table I, line 572 to 576, or
homologs thereof. Accordingly, the term "the fine chemical" can
mean "glyceric acid", "citramalic acid", "fumaric acid", "malic
acid", "trihydroxybutyric acid", "pyruvic acid", "succinic acid" or
"trihydroxybutanoic acid" owing to circumstances and the context.
In order to illustrate that the meaning of the term "the respective
fine chemical" means "glyceric acid", "citramalic acid" or "fumaric
acid" or "malic acid" or "trihydroxybutyric acid" or "pyruvic acid"
or "succinic acid" or "trihydroxybutanoic acid" owing to the
sequences listed in the context the term "the respective fine
chemical" is also used.
[7057] In one embodiment, the method of the present invention
confers the increase of the content of more than one of the
respective fine chemicals, i.e. of "one or more of the organic
acids: "glyceric acid", "citramalic acid", "fumaric acid", "malic
acid", "trihydroxybutyric acid", "pyruvic acid", "succinic acid"
and/or "trihydroxybutanoic acid.
[7058] [0017.0.0.16] to [0018.0.0.16]: see [0017.0.0.0] to
[0018.0.0.0]
[7059] [0019.0.16.16] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the respective fine chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table II, column 3, lines 190 to 226 or lines 564 to
594 or encoded by nucleic acid molecule indicated in Table I,
columns 5 or 7, lines 190 to 226 or lines 564 to 594.
[7060] [0020.0.16.16] Surprisingly it was found, that the
transgenic expression of the Saccharomyces cerevisiae protein as
indicated in Table I or II, column 4, e.g. YBL015W, YCR059C,
YFR007W, YJL055W, YJL099W, YMR241W, YPRO24W, YPR138C, YBR084W,
YBR184W, YCL032W, YGR007W, YJL072C, YKL132C or YOR044W or line 571,
582 to 583, 591 or as indicated in Table I or II, column or the
Escherichia coli K12 protein as indicated in Table I or II, column
4, e.g b1896, b0730, b1611, b2699, b4139, b1676, b0695, or b4063 or
lines 564 to 570, 572 to 581, 584 to 590, 592 to 594 in Arabidopsis
thaliana conferred an increase in the glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid ("the fine
chemical" or "the fine respective chemical") content in respect to
said proteins and their homologs as wells as the encoding nucleic
acid molecules, in particular as indicated in Table II, column 3,
lines 190 to 226 or lines 564 to 594 of the transformed plants.
[7061] [0021.0.0.16] see [0021.0.0.0]
[7062] [0022.0.16.16] The sequence of b1343 from Escherichia coli
K12 has been published in Blattner, Science 277(5331), 1453-1474,
1997, and its activity is being defined as a ATP-dependent RNA
helicase, stimulated by 23S rRNA. Accordingly, in one embodiment,
the process of the present invention comprises the use of a
"ATP-dependent RNA helicase, stimulated by 23S rRNA" from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of trihydroxybutanoic acid and/or
compositions containing trihydroxybutanoic acid, in particular for
increasing the amount of trihydroxybutanoic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a protein having an activity in rRNA processing or translation is
increased or generated, e.g. from E. coli or a homolog thereof.
Accordingly, in one embodiment, in the process of the present
invention the activity of a ATP-dependent RNA helicase, stimulated
by 23S rRNA or its homolog is increased for the production of the
fine chemical, meaning of trihydroxybutanoic acid, in particular
for increasing the amount of trihydroxybutanoic acid in free or
bound form in an organism or a part thereof, as mentioned.
[7063] The sequence of b3160 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative monooxygenase with
luciferase-like ATPase activity. Accordingly, in one embodiment,
the process of the present invention comprises the use of a
putative monooxygenase with luciferase-like ATPase activity from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of succinic acid, in particular for
increasing the amount of succinic acid and/or compositions
containing succinic acid, preferably succinic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a putative monooxygenase with luciferase-like ATPase activity is
increased or generated, e.g. from E. coli or a homolog thereof.
[7064] The sequence of b3231 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a 50S ribosomal subunit
protein L13. Accordingly, in one embodiment, the process of the
present invention comprises the use of a 50S ribosomal subunit
protein L13 from E. coli or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of pyruvic acid and/or
compositions containing pyruvic acid, in particular for increasing
the amount of pyruvic acid, preferably pyruvic acid in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a 50S ribosomal subunit protein L13 is increased or generated, e.g.
from E. coli or a homolog thereof.
[7065] The sequence of b3231 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a 50S ribosomal subunit
protein L13. Accordingly, in one embodiment, the process of the
present invention comprises the use of a 50S ribosomal subunit
protein L13 from E. coli or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of glyceric acid
and/or compositions containing glyceric acid, in particular for
increasing the amount of glyceric acid, preferably glyceric acid in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of a 50S ribosomal subunit protein L13 is increased or
generated, e.g. from E. coli or a homolog thereof.
[7066] The sequence of b3231 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a 50S ribosomal subunit
protein L13. Accordingly, in one embodiment, the process of the
present invention comprises the use of a 50S ribosomal subunit
protein L13 from E. coli or its homolog, e.g. as shown herein, for
the production of pyruvic acid and glyceric acid and/or
compositions containing pyruvic acid and glyceric acid, in
particular for increasing the amount of pyruvic acid and glyceric
acid, preferably pyruvic acid and glyceric acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a 50S ribosomal subunit protein L13 is increased or generated, e.g.
from E. coli or a homolog thereof.
[7067] The sequence of b1738 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a PEP-dependent
phosphotransferase. Accordingly, in one embodiment, the process of
the present invention comprises the use of a PEP-dependent
phosphotransferase from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
succinic acid and/or compositions comprising succinic acid, in
particular for increasing the amount of succinic acid, preferably
succinic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a PEP-dependent
phosphotransferase is increased or generated, e.g. from E. coli or
a homolog thereof.
[7068] The sequence of b1738 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a PEP-dependent
phosphotransferase. Accordingly, in one embodiment, the process of
the present invention comprises the use of a PEP-dependent
phosphotransferase from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of fumaric
acid and/or compositions containing fumaric acid, in particular for
increasing the amount of fumaric acid, preferably fumaric acid in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of a PEP-dependent phosphotransferase is increased or
generated, e.g. from E. coli or a homolog thereof.
[7069] The sequence of b1738 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a PEP-dependent
phosphotransferase. Accordingly, in one embodiment, the process of
the present invention comprises the use of a PEP-dependent
phosphotransferase from E. coli or its homolog, e.g. as shown
herein, for the production of fumaric acid and succinic acid and/or
compositions containing fumaric acid and succinic acid, in
particular for increasing the amount of fumaric acid and succinic
acid, preferably fumaric acid and succinic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a PEP-dependent phosphotransferase is increased or generated, e.g.
from E. coli or a homolog thereof.
[7070] The sequence of b0161 (Accession number NP.sub.--414703)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a periplasmic serine protease (heat shock protein).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a periplasmic serine protease from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of fumaric acid and/or compositions
containing fumaric acid, in particular for increasing the amount of
fumaric acid, preferably fumaric acid in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a periplasmic
serine protease is increased or generated, e.g. from E. coli or a
homolog thereof.
[7071] The sequence of b0161 (Accession number NP.sub.--414703)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a periplasmic serine protease (heat shock protein).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a periplasmic serine protease from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of malic acid and/or compositions
containing malic acid, in particular for increasing the amount of
malic acid, preferably malic acid in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a periplasmic
serine protease is increased or generated, e.g. from E. coli or a
homolog thereof.
[7072] The sequence of b0161 (Accession number NP.sub.--414703)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a periplasmic serine protease (heat shock protein).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a periplasmic serine protease from
E. coli or its homolog, e.g. as shown herein, for the production of
malic acid and fumaric acid and/or compositions containing malic
acid and fumaric acid, in particular for increasing the amount of
malic acid and fumaric acid, preferably malic acid and fumaric acid
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a periplasmic serine protease is
increased or generated, e.g. from E. coli or a homolog thereof.
[7073] The sequence of b1693 (Accession number NP.sub.--416208)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a 3-dehydroquinate dehydratase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a 3-dehydroquinate dehydratase protein from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glyceric acid and/or compositions containing
glyceric acid, in particular for increasing the amount of glyceric
acid, preferably glyceric acid in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a 3-dehydroquinate
dehydratase protein is increased or generated, e.g. from E. coli or
a homolog thereof.
[7074] The sequence of b1693 (Accession number NP.sub.--416208)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a 3-dehydroquinate dehydratase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a 3-dehydroquinate dehydratase protein from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of citramalic acid and/or compositions containing
citramalic acid, in particular for increasing the amount of
citramalic acid, preferably citramalic acid in free or bound form
in an organism or a part thereof, as mentioned. In one embodiment,
in the process of the present invention the activity of a
3-dehydroquinate dehydratase protein is increased or generated,
e.g. from E. coli or a homolog thereof.
[7075] The sequence of b1693 (Accession number NP.sub.--416208)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a 3-dehydroquinate dehydratase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a 3-dehydroquinate dehydratase protein from E. coli or its
homolog, e.g. as shown herein, for the production of citramalic
acid and glyceric acid and/or compositions containing citramalic
acid and glyceric acid, in particular for increasing the amount of
citramalic acid and glyceric acid, preferably citramalic acid and
glyceric acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a 3-dehydroquinate dehydratase
protein is increased or generated, e.g. from E. coli or a homolog
thereof.
[7076] The sequence of b0970 (Accession number NP.sub.--415490)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a glutamate receptor. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of Escherichia coli ybhL protein
superfamily, preferably a protein with the activity of a glutamate
receptor protein from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of
trihydroxybutanoic acid and/or compositions comprising
trihydroxybutanoic acid, in particular for increasing the amount of
trihydroxybutanoic acid, preferably trihydroxybutanoic acid in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a glutamate receptor is increased or generated, e.g.
from E. coli or a homolog thereof.
[7077] The sequence of b3169 (Accession number NP.sub.--417638)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a transcription termination-antitermination factor.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
Escherichia coli transcription factor nusA superfamily, preferably
a protein with the activity of a transcription
termination-antitermination factor from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of malic acid or compositions comprising malic acid, in
particular for increasing the amount of malic acid, preferably
malic acid in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a transcription
termination-antitermination factor is increased or generated, e.g.
from E. coli or a homolog thereof.
[7078] The sequence of b3169 (Accession number NP.sub.--417638)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a transcription termination-antitermination factor.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
Escherichia coli transcription factor nusA superfamily, preferably
a protein with the activity of a transcription
termination-antitermination factor from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of trihydroxybutanoic acid or compositions comprising
trihydroxybutanoic acid, in particular for increasing the amount of
trihydroxybutanoic acid, preferably trihydroxybutanoic acid in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of a transcription termination-antitermination factor is
increased or generated, e.g. from E. coli or a homolog thereof.
[7079] The sequence of b3169 (Accession number NP.sub.--417638)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a transcription termination-antitermination factor.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
Escherichia coli transcription factor nusA superfamily, preferably
a protein with the activity of a transcription
termination-antitermination factor from E. coli or its homolog,
e.g. as shown herein, for the production of malic acid and
trihydroxyutanoic acid or compositions comprising malic acid and
trihydroxybutanoic acid, in particular for increasing the amount of
malic acid and trihydroxybutanoic acid, preferably malic acid and
trihydroxybutanoic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a transcription
termination-antitermination factor is increased or generated, e.g.
from E. coli or a homolog thereof.
[7080] The sequence of b3116 (Accession number NP.sub.--417586)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a L-threonine/L-serine permease, anaerobically inducible
(HAAAP family). Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of threonine-serine permease superfamily, preferably a
protein with the activity of a L-threonine/L-serine permease,
anaerobically inducible (HAAAP family) from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of succinic acid and/or compositions comprising succinic
acid, in particular for increasing the amount of succinic acid,
preferably succinic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a L-threonine/L-serine
permease, anaerobically inducible (HAAAP family) is increased or
generated, e.g. from E. coli or a homolog thereof.
[7081] The sequence of b3116 (Accession number NP.sub.--417586)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a L-threonine/L-serine permease, anaerobically inducible
(HAAAP family). Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of threonine-serine permease superfamily, preferably a
protein with the activity of a L-threonine/L-serine permease,
anaerobically inducible (HAAAP family) from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of fumaric acid and/or compositions comprising fumaric
acid, in particular for increasing the amount of fumaric acid,
preferably fumaric acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a L-threonine/L-serine
permease, anaerobically inducible (HAAAP family) is increased or
generated, e.g. from E. coli or a homolog thereof.
[7082] succinic acid, in particular for increasing the amount of
maleicmalic acid, fumaric acid and/or succinic acid preferably
maleicmalic acid, fumaric acid and/or succinic acid in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a L-threonine/L-serine permease, anaerobically inducible (HAAAP
family) is increased or generated, e.g. from E. coli or a homolog
thereof.
[7083] The sequence of b3116 (Accession number NP.sub.--417586)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a L-threonine/L-serine permease, anaerobically inducible
(HAAAP family). Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of threonine-serine permease superfamily, preferably a
protein with the activity of a L-threonine/L-serine permease,
anaerobically inducible (HAAAP family) from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of malic acid and/or compositions comprising malic acid, in
particular for increasing the amount of malic acid, preferably
malic acid in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a L-threonine/L-serine permease,
anaerobically inducible (HAAAP family) is increased or generated,
e.g. from E. coli or a homolog thereof.
[7084] The sequence of b3116 (Accession number NP.sub.--417586)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a L-threonine/L-serine permease, anaerobically inducible
(HAAAP family). Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of threonine-serine permease superfamily, preferably a
protein with the activity of a L-threonine/L-serine permease,
anaerobically inducible (HAAAP family) from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of malic acid, fumaric acid and/or succinic acid and/or
compositions comprising malic acid, fumaric acid and/or succinic
acid, in particular for increasing the amount of malic acid,
fumaric acid and/or succinic acid preferably malic acid, fumaric
acid and/or succinic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a L-threonine/L-serine
permease, anaerobically inducible (HAAAP family) is increased or
generated, e.g. from E. coli or a homolog thereof.
[7085] The sequence of b0057 (Accession number NP.sub.--414599)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is not been
characterized yet. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of b0057 protein from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
glyceric acid, in particular for increasing the amount of glyceric
acid or compositions comprising glyceric acid, preferably glyceric
acid in free or bound form in an organism or a part thereof, as
mentioned.
[7086] The sequence of b4129 (Accession number NP.sub.--418553)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a lysine tRNA synthetase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a lysine tRNA synthetase protein from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of succinic acid and/or compositions containing succinic
acid, preferably succinic acid in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a lysine tRNA synthetase
protein is increased or generated, e.g. from E. coli or a homolog
thereof. In one embodiment, in the process of the present invention
the activity of a lysine tRNA synthetase is increased or generated,
e.g. from E. coli or a homolog thereof.
[7087] The sequence of b3129 (Accession number NP.sub.--417598)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a putative protease; htrA suppressor protein.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a putative protease; htrA suppressor
protein from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of glyceric acid and/or
compositions containing glyceric acid, in particular for increasing
the amount of glyceric acid, preferably glyceric acid in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a putative protease; htrA suppressor protein is increased or
generated, e.g. from E. coli or a homolog thereof.
[7088] The sequence of b3938 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a transcriptional repressor
for methionine biosynthesis. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein of
the metJ protein superfamily, in particular of a transcriptional
repressor for methionine biosynthesis from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of glyceric acid and/or compositions containing glyceric
acid, in particular for increasing the amount of glyceric acid,
preferably glyceric acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a transcriptional repressor
for methionine biosynthesis is increased or generated, e.g. from E.
coli or a homolog thereof.
[7089] The sequence of b2478 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a dihydrodipicolinate synthase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a dihydrodipicolinate synthase from
E. coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of malic acid and/or compositions
containing malic acid, in particular for increasing the amount of
malic acid in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the superfamily of dihydrodipicolinate
synthase, e.g. a protein involved in diaminopimelin acid pathway,
biosynthesis of lysine, C-compound and carbohydrate utilization,
and/or Entner-Doudoroff pathway, preferably of a
dihydrodipicolinate synthase is increased or generated, e.g. from
E. coli or a homolog thereof. Accordingly, in one embodiment, in
the process of the present invention the activity of a
dihydrodipicolinate synthase or its homolog is increased for the
production of the fine chemical, meaning of malic acid, in
particular for increasing the amount of malic acid in free or bound
form in an organism or a part thereof, as mentioned.
[7090] The sequence of b3172 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a argininosuccinic acid
synthetase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of a protein of the argininosuccinic acid synthase
superfamily, preferably a protein being involved in amino acid
biosynthesis, nitrogen and sulfur metabolism, biosynthesis of the
glutamate group (proline, hydroxyprolin, arginine, glutamine,
glutamate), degradation of amino acids of the glutamate group,
nitrogen and sulfur utilization, urea cycle, biosynthesis of
polyamines and creatine, biosynthesis of the aspartate family,
assimilation of ammonia, and/or biosynthesis of the glutamate
group, more preferred with the activity of a argininosuccinic acid
synthetase from E. coli or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of fumaric acid and/or
compositions comprising fumaric acid, in particular for increasing
the amount of fumaric acid, preferably fumaric acid in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a argininosuccinic acid synthetase is increased or generated, e.g.
from E. coli or a homolog thereof.
[7091] The sequence of b3172 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a argininosuccinic acid
synthetase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of argininosuccinic acid synthase superfamily, preferably
a protein being involved in amino acid biosynthesis, nitrogen and
sulfur metabolism, biosynthesis of the glutamate group (proline,
hydroxyprolin, arginine, glutamine, glutamate), degradation of
amino acids of the glutamate group, nitrogen and sulfur
utilization, urea cycle, biosynthesis of polyamines and creatine,
biosynthesis of the aspartate family, assimilation of ammonia,
and/or biosynthesis of the glutamate group, more preferred with the
activity of a argininosuccinic acid synthetase from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of malic acid and/or compositions comprising
malic acid, in particular for increasing the amount of malic acid,
preferably malic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a argininosuccinic acid
synthetase is increased or generated, e.g. from E. coli or a
homolog thereof.
[7092] The sequence of b3172 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a argininosuccinic acid
synthetase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of argininosuccinic acid synthase superfamily, preferably
a protein being involved in amino acid biosynthesis, nitrogen and
sulfur metabolism, biosynthesis of the glutamate group (proline,
hydroxyprolin, arginine, glutamine, glutamate), degradation of
amino acids of the glutamate group, nitrogen and sulfur
utilization, urea cycle, biosynthesis of polyamines and creatine,
biosynthesis of the aspartate family, assimilation of ammonia,
and/or biosynthesis of the glutamate group, more preferred with the
activity of a argininosuccinic acid synthetase from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of malic acid and fumaric acid and/or
compositions comprising malic acid and fumaric acid, in particular
for increasing the amount of malic acid and fumaric acid,
preferably malic acid and fumaric acid in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a argininosuccinic
acid synthetase is increased or generated, e.g. from E. coli or a
homolog thereof.
[7093] The sequence of b1961 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a DNA cytosine methylase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
protein of "site-specific methyltransferase (cytosine-specific)
EcoRII" superfamily, preferably a protein being involved in
nucleotide metabolism, metabolism of cyclic and unusual
nucleotides, TRANSCRIPTION, DNA repair, cell differentiation,
DEVELOPMENT (Systemic), and/or nucleic acid binding, more preferred
with the activity of a cytosine methylase from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of succinic acid and/or compositions comprising
succinic acid, in particular for increasing the amount of succinic
acid, preferably succinic acid in free or bound form, in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a cytosine
methylase is increased or generated, e.g. from E. coli or a homolog
thereof.
[7094] The sequence of b1961 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a DNA cytosine methylase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
protein of "site-specific methyltransferase (cytosine-specific)
EcoRII" superfamily, preferably a protein being involved in
nucleotide metabolism, metabolism of cyclic and unusual
nucleotides, TRANSCRIPTION, DNA repair, cell differentiation,
DEVELOPMENT (Systemic), and/or nucleic acid binding, more preferred
with the activity of a cytosine methylase from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of fumaric acid and/or compositions comprising
fumaric acid, in particular for increasing the amount of fumaric
acid, preferably fumaric acid in free or bound form, in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a cytosine methylase is
increased or generated, e.g. from E. coli or a homolog thereof.
[7095] The sequence of b1961 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a DNA cytosine methylase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
protein of "site-specific methyltransferase (cytosine-specific)
EcoRII" superfamily, preferably a protein being involved in
nucleotide metabolism, metabolism of cyclic and unusual
nucleotides, TRANSCRIPTION, DNA repair, cell differentiation,
DEVELOPMENT (Systemic), and/or nucleic acid binding, more preferred
with the activity of a cytosine methylase from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of fumaric acid and succinic acid and/or
compositions comprising fumaric acid and succinic acid, in
particular for increasing the amount of fumaric acid and succinic
acid, preferably fumaric acid and succinic acid in free or bound
form, in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a cytosine methylase is increased or generated, e.g. from E. coli
or a homolog thereof.
[7096] The sequence of b2599 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a bifunctional enzyme:
chorismate mutase P (N-terminal) and prephenate dehydratase
(C-terminal). Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of "pheA bifunctional enzyme, prephenate dehydratase
homology"-superfamily, preferably a protein being involved in
biosynthesis of the cysteine-aromatic group, amino acid
biosynthesis, degradation of amino acids of the cysteine-aromatic
group, and/or biosynthesis of vitamins, cofactors, and prosthetic
groups, more preferred with the activity of a bifunctional enzyme:
chorismate mutase P (N-terminal) and prephenate dehydratase
(C-terminal) from E. coli or its homolog, e.g.
[7097] as shown herein, for the production of the fine chemical,
meaning of succinic acid and/or compositions comprising succinic
acid, in particular for increasing the amount of succinic acid,
preferably succinic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a bifunctional enzyme:
chorismate mutase P (N-terminal) and prephenate dehydratase
(C-terminal) is increased or generated, e.g. from E. coli or a
homolog thereof.
[7098] The sequence of b4122 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a fumarase B (hydratase Class
I). Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
"iron-dependent hydratase, iron-dependent tartrate dehydratase
alpha chain homology"-superfamily, preferably with the activity of
a fumarase B (hydratase Class I) from E. coli or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
of fumaric acid and/or compositions comprising fumaric acid, in
particular for increasing the amount of fumaric acid, preferably
fumaric acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a fumarase B (hydratase Class I)
is increased or generated, e.g. from E. coli or a homolog
thereof.
[7099] The sequence of YCL038C from Saccharomyces cerevisiae has
been published in Dujon, B. et al., Nature 387 (6632 Suppl), 98-102
(1997), and its activity is being defined as a Autophagy-related
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product being involved in
other transport facilitators, preferably with the activity of a
Autophagy-related protein from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of succinic acid and/or compositions comprising
succinic acid, in particular for increasing the amount of succinic
acid, preferably succinic acid in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a Autophagy-related
protein is increased or generated, e.g. from Saccharomyces
cerevisiae or a homolog thereof.
[7100] The sequence of YCL038C. from Saccharomyces cerevisiae has
been published in Goffeau, A, Science 274 (5287), 546-547 (1996),
Oliver, S. G et al., Nature 357 (6373), 38-46 (1992), and its
activity is being defined as a Autophagy-related protein.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product being involved in
other transport facilitators, preferably with the activity of a
Autophagy-related protein from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of malic acid and/or compositions comprising
malic acid, in particular for increasing the amount of malic acid,
preferably malic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a Autophagy-related protein
is increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[7101] The sequence of YCL038C from Saccharomyces cerevisiae has
been published in Dujon, B. et al., Nature 387 (6632 Suppl), 98-102
(1997), and its activity is being defined as a Autophagy-related
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product being involved in
other transport facilitators, preferably with the activity of a
Autophagy-related protein from Saccharomyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of succinic acid and malic acid and/or
compositions comprising succinic acid and malic acid, in particular
for increasing the amount of succinic acid and malic acid,
preferably succinic acid and malic acid in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a
Autophagy-related protein is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[7102] The sequence of YCR012W from Saccharomyces cerevisiae has
been published in Goffeau, A., Science 274 (5287), 546-547 (1996),
and Dujon, B. et al., Nature 387 (6632 Suppl), 98-102 (1997), and
its activity is being defined as a 3-phosphoglycerate kinase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product of the
phosphoglycerate kinase-superfamily, preferably being involved in
C-compound and carbohydrate utilization, glycolysis and
gluconeogenesis, sugar, glucoside, polyol and carboxylate
catabolism, and/or ENERGY, more preferred with the activity of a
3-phosphoglycerate kinase from
[7103] Saccharomyces cerevisiae or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of fumaric
acid and/or compositions comprising fumaric acid, in particular for
increasing the amount of fumaric acid, preferably fumaric acid in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of a 3-phosphoglycerate kinase is increased or generated,
e.g. from Saccharomyces cerevisiae or a homolog thereof.
[7104] The sequence of YOR168W from Saccharomyces cerevisiae has
been published in Dujon, B. et al., Nature 387 (6632 Suppl), 98-102
(1997), and Goffeau, A, Science 274 (5287), 546-547 (1996), and its
activity is being defined as a glutamine-tRNA ligase. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product of the "human glutamine-tRNA ligase,
glutamine-tRNA ligase homology"-superfamily, preferably being
involved in aminoacyl-tRNA-synthetases, nucleotide binding,
translation, and/or PROTEIN SYNTHESIS, more preferred with the
activity of a glutamine-tRNA ligase from Saccharomyces cerevisiae
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of malic acid and/or compositions comprising
malic acid, in particular for increasing the amount of malic acid,
preferably malic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a glutamine-tRNA ligase is
increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[7105] The sequence of b1896 from Escherichia coli K12 has been
published in Blattner, F. R. et al., Science 277 (5331), 1453-1474
(1997) and it is being defined as a protein with
trehalose-6-phosphate synthase activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein b1896 from Escherichia coli K12 or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of citramalic
acid, fumaric acid, malic acid, succinic acid, trihydroxybutyric
acid and/or trihydroxybutanoic acid, preferably in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the trehalose-6-phosphate synthase b1896 is increased.
[7106] The sequence of YBL015W from Saccharomyces cerevisiae has
been published in Goffeau, A. et al., Science 274 (5287), 546-547
(1996) and it is being defined as a protein having acetyl-coA
hydrolase activity. Accordingly, in one embodiment, the process of
the present invention comprises the use of a protein having said
activity, for the production of the respective fine chemical, in
particular for increasing the amount of fumaric acid and/or malic
acid, preferably in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention said acetyl-coA hydrolase activity YBL015W is
increased.
[7107] The sequence of YCR059C from Saccharomyces cerevisiae has
been published in Goffeau, A. et al. Science 274 (5287), 546-547
(1996) and its activity is being defined as an unclassified
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of said unclassified protein from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, in particular for
increasing the amount of fumaric acid, preferably in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the unclassified protein YCR059Cis increased.
[7108] The sequence of YFR007W from Saccharomyces cerevisiae has
been published in Goffeau, A. et al. Science 274 (5287), 546-547
(1996) and its activity is being defined as an unclassified
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of said unclassified protein from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, in particular for
increasing the amount of fumaric acid preferably in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the unclassified protein YFR007W is increased.
[7109] The sequence of YJL055W from Saccharomyces cerevisiae has
been published in Goffeau, A. et al. Science 274 (5287), 546-547
(1996) and its activity is being defined as a
lysine-decarboxylase-like protein. Accordingly, in one embodiment,
the process of the present invention comprises the use of said
protein YJL055W from Saccharomyces cerevisiae or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of fumaric acid,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein YJL055W is increased.
[7110] The sequence of YJL099W from Saccharomyces cerevisiae has
been published in Goffeau, A. et al. Science 274 (5287), 546-547
(1996) and its activity is being defined as a protein involved in
chitin biosynthesis. Accordingly, in one embodiment, the process of
the present invention comprises the use of said protein involved in
chitin biosynthesis from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of fumaric acid
and/or malic acid, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of the protein YJL099W is
increased.
[7111] The sequence of YMR241W from Saccharomyces cerevisiae has
been published in Bowman, S. Nature 387 (6632 Suppl), 90-93 (1997)
and its activity is being defined as a mitochondrial DNA
replication protein having yeast suppressor gene activity (abf2).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein having suppressor gene
activity from Saccharomyces cerevisiae or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
in particular for increasing the amount of fumaric acid, preferably
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of said protein YMR241W is increased.
[7112] The sequence of YPRO24W from Saccharomyces cerevisiae has
been published in Bussey, H. et al. Nature 387 (6632 Suppl),
103-105 (1997) and its activity is being defined as a protein
having mitochondrial ATPase activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of an ATPase from Saccharomyces cerevisiae or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
in particular for increasing the amount of fumaric acid, preferably
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of the protein YPRO24W is increased.
[7113] The sequence of YPR138C from Saccharomyces cerevisiae has
been published in Bussey, H. et al. Nature 387 (6632 Suppl),
103-105 (1997) and its activity is being defined as a protein
having ammonia transporter activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein having ammonia transporter activity from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of fumaric acid and/or malic acid, preferably in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the protein YPR138C is increased.
[7114] The sequence of b0730 from Escherichia coli K12 has been
published in Buck D. et al. Biochem. J. 260:737-747(1989) and its
activity is being defined as a protein having fatty acyl responsive
regulator and/or "transcriptional regulator of the succinyl-CoA
synthase operon" function. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
b0730 having fatty acyl responsive regulator function from
Escherichia coli K12 or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, in particular for
increasing the amount of fumaric acid, malic acid and/or succinic
acid, preferably in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of the protein b0730 is
increased.
[7115] The sequence of b1611 from Escherichia coli K12 has been
published in Blattner, F. R. et al. Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a protein having
fumarase C or fumaric acid hydratase class II activity.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein having fumarase C or
fumaric acid hydratase class II activity from Escherichia coli K12
or its homolog, e.g. as shown herein, for the production of the
respective fine chemical, in particular for increasing the amount
of fumaric acid, preferably in free or bound form in an organism or
a part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of the protein b1611 is
increased.
[7116] The sequence of b2699 from Escherichia coli K12 has been
published in Blattner, F. R. et al. Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a protein having DNA
strand exchange and recombination activity (RecA). Accordingly, in
one embodiment, the process of the present invention comprises the
use of a protein b2699 having RecA activity from Escherichia coli
K12 or its homolog, e.g. as shown herein, for the production of the
respective fine chemical, in particular for increasing the amount
of fumaric acid, trihydroxybutyric acid and/or trihydroxybutanoic
acid, preferably in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of the protein b2699 is
increased.
[7117] The sequence of b4139 from Escherichia coli K12 has been
published in Blattner, F. R. et al. Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a protein having
aspartate ammonia-lyase (aspartase) activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein having aspartate ammonia-lyase (aspartase) activity
from Escherichia coli K12 or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, in particular for
increasing the amount of fumaric acid, preferably in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the protein b4139 is increased.
[7118] The sequence of b1676 from Escherichia coli K12 has been
published in Blattner, F. R. et al. Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a protein having
pyruvic acid kinase I (formerly F) activity and is stimulated by
fructose. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein having pyruvic
acid kinase I (formerly F) activity from Escherichia coli K12 or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, in particular for increasing the amount
of fumaric acid, preferably in free or bound form in an organism or
a part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of the protein b1676 is
increased.
[7119] The sequence of b4063 from Escherichia coli K12 has been
published in Blattner, F. R. et al. Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a protein having
redox-sensing activator function of soxS and transcriptional
activator function for superoxide response. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein having redox-sensing activator function of soxS and
transcriptional activator function for superoxide response from
Escherichia coli K12 or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, in particular for
increasing the amount of fumaric acid and/or malic acid, preferably
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of the protein b4063 is increased.
[7120] The sequence of YBR084W from Saccharomyces cerevisiae has
been published in Goffeau, A. et al., Science 274 (5287), 546-547
(1996) and its activity is being defined as a protein having
mitochondrial Cl-tetrahydrofolate synthase activity. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a protein having mitochondrial Cl-tetrahydrofolate
synthase activity from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of malic acid,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein YBR084W is increased.
[7121] The sequence of YBR184W from Saccharomyces cerevisiae has
been published in Goffeau, A. et al., Science 274 (5287), 546-547
(1996) and its activity is being defined as an unclassified
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of an unclassified protein YBR184W from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, in particular for
increasing the amount of malic acid, preferably in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the unclassified protein YBR184W is increased.
[7122] The sequence of YCL032W from Saccharomyces cerevisiae has
been published in Goffeau, A. et al., Science 274 (5287), 546-547
(1996) and its activity is being defined as a protein involved in
the pheromone signal transduction pathway. Accordingly, in one
embodiment, the process of the present invention comprises the use
of the protein YCL032W involved in the pheromone signal
transduction pathway from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of malic acid
and/or succinic acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein
YCL032W is increased.
[7123] The sequence of YGR007W from Saccharomyces cerevisiae has
been published in Tettelin, H. et al., Nature 387 (6632 Suppl),
81-84 (1997) and its activity is being defined as a protein having
choline phosphate cytidylyltransferase (also called
phosphoethanolamine cytidylyltransferase or phosphocholine
cytidylyltransferase) activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
having choline phosphate cytidylyltransferase activity from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, in particular for
increasing the amount of malic acid, preferably in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the protein YGR007W is increased.
[7124] The sequence of YJL072C from Saccharomyces cerevisiae has
been published in Goffeau, A. Science 274 (5287), 546-547 (1996)
and its function is being defined as a protein being the subunit of
the GINS complex required for chromosomal DNA replication.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein YJL072C from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of malic acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein
YJL072C is increased.
[7125] The sequence of YKL132C from Saccharomyces cerevisiae has
been published in Goffeau, A. et al., Science 274 (5287), 546-547
(1996) and its activity is being defined as a protein having
folyl-polyglutamate synthetase activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein YKL132C from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of malic acid,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the YKL132C protein is increased.
[7126] The sequence of b0695 from Escherichia coli K12 has been
published in Blattner, F. R. et al., Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a protein having
histidine kinase activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
b0695 from Escherichia coli K12 or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, in
particular for increasing the amount of pyruvic acid, preferably in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of the protein b0695 is increased.
[7127] The sequence of YOR044W from Saccharomyces cerevisiae has
been published in Dujon, B. et al., Nature 387 (6632 Suppl), 98-102
(1997) and its activity is being defined as an unclassified
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein YOR044W from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of succinic acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein
YOR044W is increased.
[7128] [0023.0.16.16] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[7129] Further, in the present invention, the term "homologue"
relates to the sequence of an organism having the highest sequence
homology to the herein mentioned or listed sequences of all
expressed sequences of said organism.
[7130] However, the person skilled in the art knows, that,
preferably, the homologue has said the--fine-chemical-increasing
activity and, if known, the same biological function or activity in
the organism as at least one of the protein(s) indicated in Table
I, Column 3, lines 190 to 226 or lines 564 to 594, e.g. having the
sequence of a polypeptide encoded by a nucleic acid molecule
comprising the sequence indicated in indicated in Table I, Column 5
or 7, lines 190 to 226 or lines 564 to 594.
[7131] In one embodiment, the homolog of any one of the
polypeptides indicated in Table II, column 3, line 190 or 564 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms preferably citramalic
acid.
[7132] In one embodiment, the homolog of the polypeptides indicated
in Table II, column 3, lines 191 to 205 or lines 565 to 571 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms preferably fumaric
acid.
[7133] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 206 to 217 or
lines 577 to 583 is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of malic acid in the organisms.
[7134] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 218 to 219 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
lacton of trihydroxybutyric acid in the organisms.
[7135] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 220 or line 584
is a homolog having the same or a similar activity. In particular
an increase of activity confers an increase in the content of
pyruvic acid in the organisms.
[7136] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 221 to 224 or
lines 585 to 591 is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of succinic acid in the organisms.
[7137] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 225 to 226 or
lines 592 to 594 is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of trihydroxybutanoic acid in the organisms.
[7138] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 572 to 576 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of glyceric
acid in the organisms.
[7139] [0023.1.0.16] Homologs of the polypeptides indicated in
Table II, column 3, lines 190 to 226 or lines 564 to 594 may be the
polypeptides encoded by the nucleic acid molecules indicated in
Table I, column 7, lines 190 to 226 or lines 564 to 594 or may be
the polypeptides indicated in Table II, column 7, lines 190 to 226
or lines 564 to 594.
[7140] Homologs of the polypeptides indicated in Table II, column
3, lines 190 or 564 may be the polypeptides encoded by the nucleic
acid molecules indicated in Table I, column 7, line 190 or 564,
respectively or may be the polypeptides indicated in Table II,
column 7, lines 190 or 564, having citramalic acid content- and/or
amount-increasing activity.
[7141] Homologs of the polypeptide indicated in Table II, column 3,
lines 191 to 205 or lines 565 to 571 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 191 to 205 or lines 565 to 571, respectively or may be the
polypeptides indicated in Table II, column 7, lines 191 to 205 or
lines 565 to 571, having a fumaric acid content- and/or
amount-increasing activity.
[7142] Homologs of the polypeptide indicated in Table II, column 3,
lines 206 to 217 or lines 577 to 583 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 206 to 217 or lines 577 to 583, respectively or may be the
polypeptides indicated in Table II, column 7, lines 206 to 217 or
lines 577 to 583, having a malic acid content- and/or
amount-increasing activity.
[7143] Homologs of the polypeptide indicated in Table II, column 3,
lines 218 and 219 may be the polypeptides encoded by the nucleic
acid molecules indicated in Table I, column 7, lines 218 and 219,
respectively or may be the polypeptides indicated in Table II,
column 7, lines 218 and 219, having a trihydroxybuyrate content-
and/or amount-increasing activity.
[7144] Homologs of the polypeptide indicated in Table II, column 3,
line 220 or line 584 may be the polypeptides encoded by the nucleic
acid molecules indicated in Table I, column 7, line 220 or line
584, respectively or may be the polypeptides indicated in Table II,
column 7, line 220 or line 584, having a pyruvic acid content-
and/or amount-increasing activity.
[7145] Homologs of the polypeptide indicated in Table II, column 3,
lines 221 to 224 or lines 585 to 591 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 221 to 224 or lines 585 to 591, respectively or may be the
polypeptides indicated in Table II, column 7, lines 221 to 224 or
lines 585 to 591, having a succinic acid content- and/or
amount-increasing activity.
[7146] Homologs of the polypeptide indicated in Table II, column 3,
lines 225 and 226 or lines 592 to 594 may be the polypeptides
encoded by the nucleic acid molecules indicated in
[7147] Table I, column 7, lines 225 and 226 or lines 592 to 594,
respectively or may be the polypeptides indicated in Table II,
column 7, lines 225 and 226 or lines 592 to 594, having a
trihydroxybutanoic acid content- and/or amount-increasing
activity.
[7148] [0024.0.0.16] see [0024.0.0.0]
[7149] [0025.0.16.16] In accordance with the invention, a protein
or polypeptide has the "activity of a protein of the invention",
e.g. the activity of a protein indicated in Table II, column 3,
lines 190 to 226 or lines 564 to 594 if its de novo activity, or
its increased expression directly or indirectly leads to an
increased glyceric acid, citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid level, resp., in the organism or a part
thereof, preferably in a cell of said organism. In a preferred
embodiment, the protein or polypeptide has the above-mentioned
additional activities of a protein indicated in Table II, column 3,
lines 190 to 226 or lines 564 to 594. Throughout the specification
the activity or preferably the biological activity of such a
protein or polypeptide or an nucleic acid molecule or sequence
encoding such protein or polypeptide is identical or similar if it
still has the biological or enzymatic activity of any one of the
proteins indicated in Table II, column 3, lines 190 to 226 or lines
564 to 594, or which has at least 10% of the original enzymatic
activity, preferably 20%, particularly preferably 30%, most
particularly preferably 40% in comparison to any one of the
proteins indicated in Table II, column 3, line 190 to 226 or lines
564 to 594.
[7150] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[7151] [0025.1.0.16] and [0025.2.0.16] see [0025.1.0.0] and
[0025.2.0.0]
[7152] [0025.3.16.16] In one embodiment, the polypeptide of the
invention or used in the method of the invention confers said
activity, e.g. the increase of the respective fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[7153] [0026.0.0.16] to [0033.0.0.16]: see [0026.0.0.0] to
[0033.0.0.0]
[7154] [0034.0.16.16] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention or used in
the process of the invention, e.g. as result of an increase in the
level of the nucleic acid molecule of the present invention or an
increase of the specific activity of the polypeptide of the
invention or used in the process of the invention. E.g., it differs
by or in the expression level or activity of an protein having the
activity of a protein as indicated in Table II, column 3, lines 190
to 226 or lines 564 to 594 or being encoded by a nucleic acid
molecule indicated in Table I, column 5, lines 190 to 226 or lines
564 to 594 or its homologs, e.g. as indicated in Table I, column 7,
lines 190 to 226 or lines 564 to 594, its biochemical or genetic
causes. It therefore shows the increased amount of the respective
fine chemical.
[7155] [0035.0.0.16] to [0044.0.0.16]: see [0035.0.0.0] to
[0044.0.0.0]
[7156] [0045.0.16.16] In one embodiment the activity of the
Escherichia coli K12 protein b0057 or its homologs, as indicated in
Table II, columns 5 or 7, line 572, e.g. a protein with an activity
as defined in [0022.0.16.16], is increased, conferring preferably,
the increase of the fine chemical, preferably of glyceric acid
between 35% and 57%, or more.
[7157] In one embodiment the activity of the Escherichia coli K12
protein b0161 or its homologs, as indicated in Table II, columns 5
or 7, line 565 for fumaric acid or line 577 for malic acid, e.g. a
protein with an activity as defined in [0022.0.16.16], is
increased, conferring preferably, the increase of the fine
chemical, preferably of fumaric acid and/or of malic acid,
preferably of fumaric acid between 64% and 151% and/or of malic
acid between 64% and 229%, or more.
[7158] In one embodiment the activity of the Escherichia coli K12
protein b0970 or its homologs, as indicated in Table II, columns 5
or 7, line 592, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, conferring preferably, the increase
of the fine chemical, preferably of trihydroxybutanoic acid between
34% and 80%, or more.
[7159] In one embodiment the activity of the Escherichia coli K12
protein b1343 or its homologs, as indicated in Table II, columns 5
or 7, line 593, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, conferring preferably, the increase
of the fine chemical, preferably of trihydroxybutanoic acid between
33% and 108%, or more.
[7160] In one embodiment the activity of the Escherichia coli K12
protein b1693 or its homologs, as indicated in Table II, columns 5
or 7, line 564 for citramalic acid or line 573 for glyceric acid,
e.g. a protein with an activity as defined in [0022.0.16.16], is
increased, conferring preferably, the increase of the fine
chemical, preferably of glyceric acid and/or of citramalic acid,
preferably of glyceric acid between 33% and 52% and/or of
citramalic acid between 48% and 105%, or more.
[7161] In one embodiment the activity of the Escherichia coli K12
protein b1738 or its homologs, as indicated in Table II, columns 5
or 7, line 566 for fumaric acid or line 585 for succinic acid, e.g.
a protein with an activity as defined in [0022.0.16.16], is
increased, conferring preferably, the increase of the fine
chemical, preferably of succinic acid and/or of fumaric acid,
preferably of succinic acid between 49% and 95% and/or of fumaric
acid between 53% and 149%, or more.
[7162] In one embodiment the activity of the Escherichia coli K12
protein b1961 or its homologs, as indicated in Table II, columns 5
or 7, line 567 for fumaric acid or line 586 for succinic acid, e.g.
a protein with an activity as defined in [0022.0.16.16], is
increased, conferring preferably, the increase of the fine
chemical, preferably of succinic acid and/or of fumaric acid,
preferably of succinic acid between 29% and 54% and/or of fumaric
acid between 76% and 105%, or more.
[7163] In one embodiment the activity of the Escherichia coli K12
protein b2478 or its homologs, as indicated in Table II, columns 5
or 7, line 578, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, conferring preferably, the increase
of the fine chemical, preferably of malic acid between 51% and
124%, or more.
[7164] In one embodiment the activity of the Escherichia coli K12
protein b2599 or its homologs, as indicated in Table II, columns 5
or 7, line 587, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, conferring preferably, the increase
of the fine chemical, preferably of succinic acid between 33% and
156%, or more.
[7165] In one embodiment the activity of the Escherichia coli K12
protein b3116 or its homologs, as indicated in Table II, columns 5
or 7, line 588 for succinic acid or line 568 for fumaric acid or
line 579 for malic acid, e.g. a protein with an activity as defined
in [0022.0.16.16], is increased, conferring preferably, the
increase of the fine chemical, preferably of succinic acid, and/or
of fumaric acid, and/or of malic acid between, preferably of
succinic acid between 31% and 42%, and/or of fumaric acid between
63% and 99%, and/or of malic acid between 60% and 212%, or
more.
[7166] In one embodiment the activity of the Escherichia coli K12
protein b3129 or its homologs, as indicated in Table II, columns 5
or 7, line 574, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, conferring preferably, the increase
of the fine chemical, preferably of glyceric acid between 30% and
35%, or more.
[7167] In one embodiment the activity of the Escherichia coli K12
protein b3160 or its homologs, as indicated in Table II, columns 5
or 7, line 589, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, conferring preferably, the increase
of the fine chemical, preferably of succinic acid between 33% and
90%, or more.
[7168] In one embodiment the activity of the Escherichia coli K12
protein b3169 or its homologs, as indicated in Table II, columns 5
or 7, line 580 for malic acid or 594 for trihydroxybutanoic acid,
e.g. a protein with an activity as defined in [0022.0.16.16], is
increased, conferring preferably, the increase of the fine
chemical, preferably of malic acid and/or of trihydroxybutanoic
acid, preferably of malic acid between 132% and 143% and/or of
trihydroxybutanoic acid between 60% and 88%, or more.
[7169] In one embodiment the activity of the Escherichia coli K12
protein b3172 or its homologs, as indicated in Table II, columns 5
or 7, line 581 for malic acid or 569 for fumaric acid, e.g. a
protein with an activity as defined in [0022.0.16.16], is
increased, conferring preferably, the increase of the fine
chemical, preferably of fumaric acid and/or of malic acid,
preferably of fumaric acid between 63% and 325% and/or of malic
acid between 64% and 408%, or more.
[7170] In one embodiment the activity of the Escherichia coli K12
protein b3231 or its homologs, as indicated in Table II, columns 5
or 7, line 575 for glyceric acid or line 584 for pyruvic acid, e.g.
a protein with an activity as defined in [0022.0.16.16], is
increased, conferring preferably, the increase of the fine
chemical, preferably of pyruvic acid and/or of glyceric acid,
preferably of pyruvic acid between 43% and 46% and/or of glyceric
acid between 21% and 34%, or more.
[7171] In one embodiment the activity of the Escherichia coli K12
protein b3938 or its homologs, as indicated in Table II, columns 5
or 7, line 576, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, conferring preferably, the increase
of the fine chemical, preferably of glyceric acid between 37% and
40%, or more.
[7172] In one embodiment the activity of the Escherichia coli K12
protein b4122 or its homologs, as indicated in Table II, columns 5
or 7, line 570, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, conferring preferably, the increase
of the fine chemical, preferably of fumaric acid between 108% and
430%, or more.
[7173] In one embodiment the activity of the Escherichia coli K12
protein b4129 or its homologs, as indicated in Table II, columns 5
or 7, line 590, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, conferring preferably, the increase
of the fine chemical, preferably of succinic acid between 40% and
84%, or more.
[7174] In one embodiment the activity of the Saccharomyces
cerevisiae protein YCL038C or its homologs, as indicated in Table
II, columns 5 or 7, line 591 for succinic acid or line 582 for
malic acid, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, conferring preferably, the increase
of the fine chemical, preferably of succinic acid and/or of malic
acid, preferably of succinic acid between 47% and 91% and/or of
malic acid between 87% and 123%, or more.
[7175] In one embodiment the activity of the Saccharomyces
cerevisiae protein YCR012W or its homologs, as indicated in Table
II, columns 5 or 7, line 571, e.g. a protein with an activity as
defined in [0022.0.16.16], is increased, conferring preferably, the
increase of the fine chemical, preferably of fumaric acid between
62% and 93%, or more.
[7176] In one embodiment the activity of the Saccharomyces
cerevisiae protein YOR168W or its homologs, as indicated in Table
II, columns 5 or 7, line 583, e.g. a protein with an activity as
defined in [0022.0.16.16], is increased, conferring preferably, the
increase of the fine chemical, preferably of malic acid between 56%
and 562%, or more.
[7177] In case the activity of the Escherichia coli K12 protein
b1896 or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 190 is increased, in one embodiment the increase of the
respective fine chemical, preferably of citramalic acid between 63
and 99% or more is conferred.
[7178] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YBL015W or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 191 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of fumaric acid between 50% and 126% or more is conferred.
[7179] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YCR059C or its homologs e.g. a protein as
indicated in Table II, columns 5 or 7, line 192, is increased,
preferably, in one embodiment the increase of the respective fine
chemical, preferably of fumaric acid between 71% and 183% or more
is conferred.
[7180] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YFR007W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 193, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of fumaric acid between 69% and 156% or more
is conferred.
[7181] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YJL055W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 194, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of fumaric acid between 72% and 561% or more
is conferred.
[7182] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YJL099W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 195, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of fumaric acid between 98% and 734% or more
is conferred.
[7183] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YMR241W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 196, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of fumaric acid between 75% and 368% or more
is conferred.
[7184] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YPR024W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 197, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of fumaric acid between 97% and 374% or more
is conferred.
[7185] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YPR138C or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 198, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of fumaric acid between 61% and 327% or more
is conferred.
[7186] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0730 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 199, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of fumaric acid between 75% and 838% or more is
conferred.
[7187] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1611 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 200, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of fumaric acid between 106% and 328% or more is
conferred.
[7188] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2699 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 201, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of fumaric acid between 65% and 802% or more is
conferred.
[7189] In one embodiment, in case the activity of the Escherichia
coli K12 protein b4139 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 202, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of fumaric acid between 118% and 525% or more is
conferred.
[7190] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1676 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 203, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of fumaric acid between 108% and 430% or more is
conferred.
[7191] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 204, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of fumaric acid between 232% and 320% or more is
conferred.
[7192] In one embodiment, in case the activity of the Escherichia
coli K12 protein b4063 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 205, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of fumaric acid between 64% and 308% or more is
conferred.
[7193] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YBL015W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 206, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of malic acid between 45% and 86% or more is
conferred.
[7194] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YBR084W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 207, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of malic acid between 107% and 448% or more is
conferred.
[7195] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YBR184W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 208, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of malic acid between 67% and 213% or more is
conferred.
[7196] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YCL032W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 209, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of malic acid between 87% and 123% or more is
conferred.
[7197] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YGR007W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 210, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of malic acid between 55% and 90% or more is
conferred.
[7198] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YJL072C or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 211, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of malic acid between 49% and 249% or more is
conferred.
[7199] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YJL099W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 212, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of malic acid between 94% and 797% or more is
conferred.
[7200] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YKL132C or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 213, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of malic acid between 109% and 213% or more is
conferred.
[7201] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YPR138C or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 214, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of malic acid between 72% and 359% or more is
conferred.
[7202] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0730 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 215, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of malic acid between 96% and 672% or more is
conferred.
[7203] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 216, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of malic acid between 149% and 319% or more is
conferred.
[7204] In one embodiment, in case the activity of the Escherichia
coli K12 protein b4063 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 217, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of malic acid between 78% and 93% or more is
conferred.
[7205] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2699 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 218, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of trihydroxybutyric acid between 48% and 215% or more
is conferred.
[7206] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 219, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of trihydroxybutyric acid between 34% and 96% or more is
conferred.
[7207] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0695 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 220, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of pyruvic acid between 61% and 155% or more is
conferred.
[7208] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YCL032W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 221, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of succinic acid between 47% and 91% or more
is conferred.
[7209] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YOR044W or its homologs, e.g. a protein as
indicated in Table II, columns 5 or 7, line 222, is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of succinic acid between 45% and 265% or more
is conferred.
[7210] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0730 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 223, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of succinic acid between 51% and 332% or more is
conferred.
[7211] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 224, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of succinic acid between 55% and 110% or more is
conferred.
[7212] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2699 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 225, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of trihydroxybutanoic acid between 82% and 281% or more
is conferred.
[7213] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs, e.g. a protein as indicated
in Table II, columns 5 or 7, line 226, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of trihydroxybutanoic acid between 60% and 161% or more
is conferred.
[7214] [0046.0.16.16] In case the activity of a protein as
disclosed in [0016.0.16.16] or its homologs, as indicated in Table
I, columns 5 or 7, line 190, e.g. a protein with an activity as
defined in [0022.0.16.16], is increased, preferably, an increase of
the respective fine chemical, preferably of citramalic acid and of
an other organic acid is conferred.
[7215] In case the activity of a protein as disclosed in
[0016.0.16.16] or its homologs, as indicated in Table I, columns 5
or 7, lines 191 to 205, e.g. a protein with an activity as defined
in [0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of fumaric acid and of an
other organic acid is conferred.
[7216] In case the activity of a protein as disclosed in
[0016.0.16.16] or its homologs, as indicated in Table I, columns 5
or 7, lines 206 to 217, e.g. a protein with an activity as defined
in [0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of malic acid and of an other
organic acid is conferred.
[7217] In case the activity of a protein as disclosed in
[0016.0.16.16] or its homologs, as indicated in Table I, columns 5
or 7, lines 218 or 219, e.g. a protein with an activity as defined
in [0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of lacton of trihydroxybutyric
acid and of an other organic acid is conferred.
[7218] In case the activity of a protein as disclosed in
[0016.0.16.16] or its homologs, as indicated in Table I, columns 5
or 7, line 220, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of pyruvic acid and of an
other organic acid is conferred.
[7219] In case the activity of a protein as disclosed in
[0016.0.16.16] or its homologs, as indicated in Table I, columns 5
or 7, lines 221 to 224, e.g. a protein with an activity as defined
in [0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of succinic acid and of an
other organic acid is conferred.
[7220] In case the activity of a protein as disclosed in
[0016.0.16.16] or its homologs, as indicated in Table I, columns 5
or 7, lines 225 to 226, e.g. a protein with an activity as defined
in [0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of trihydroxybutanoic acid and
of an other organic acid is conferred.
[7221] In case the activity of a Escherichia coli K12 protein b0057
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 572, e.g. a protein with an activity
as defined in [0022.0.16.16], is increased, preferably, an increase
of the respective fine chemical, preferably of glyceric acid and of
an other organic acid is conferred.
[7222] In case the activity of a Escherichia coli K12 protein b0161
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 565 for fumaric acid and/or line 577
for malic acid, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of fumaric acid and/or malic
acid, and of an other organic acid is conferred.
[7223] In case the activity of a Escherichia coli K12 protein b0970
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 592, e.g. a protein with an activity
as defined in [0022.0.16.16], is increased, preferably, an increase
of the respective fine chemical, preferably of trihydroxybutanoic
acid, and of an other organic acid is conferred.
[7224] In case the activity of a Escherichia coli K12 protein b1343
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 593, e.g. a protein with an activity
as defined in [0022.0.16.16], is increased, preferably, an increase
of the respective fine chemical, preferably of trihydroxybutanoic
acid, and of an other organic acid is conferred.
[7225] In case the activity of a Escherichia coli K12 protein b1693
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 573 for glyceric acid and/or line 564
for citramalic acid, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of glyceric acid and/or
citramalic acid, and of an other organic acid is conferred.
[7226] In case the activity of a Escherichia coli K12 protein b1738
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 566 for fumaric acid and/or line 585
for succinic acid, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of fumaric acid and/or
succinic acid, and of an other organic acid is conferred.
[7227] In case the activity of a Escherichia coli K12 protein b1961
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 567 for fumaric acid and/or line 586
for succinic acid, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of fumaric acid and/or
succinic acid, and of an other organic acid is conferred.
[7228] In case the activity of a Escherichia coli K12 protein b2478
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 578, e.g. a protein with an activity
as defined in [0022.0.16.16], is increased, preferably, an increase
of the respective fine chemical, preferably of malic acid and of an
other organic acid is conferred.
[7229] In case the activity of a Escherichia coli K12 protein b2599
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 587, e.g. a protein with an activity
as defined in [0022.0.16.16], is increased, preferably, an increase
of the respective fine chemical, preferably of succinic acid, and
of an other organic acid is conferred.
[7230] In case the activity of a Escherichia coli K12 protein b3116
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 588 for succinic acid, and/or line
568 for fumaric acid and/or line 579 for malic acid, e.g. a protein
with an activity as defined in [0022.0.16.16], is increased,
preferably, an increase of the respective fine chemical, preferably
of succinic acid, fumaric acid and/or malic acid, and of an other
organic acid is conferred.
[7231] In case the activity of a Escherichia coli K12 protein b3129
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 574, e.g. a protein with an activity
as defined in [0022.0.16.16], is increased, preferably, an increase
of the respective fine chemical, preferably of glyceric acid, and
of an other organic acid is conferred.
[7232] In case the activity of a Escherichia coli K12 protein b3160
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 589, e.g. a protein with an activity
as defined in [0022.0.16.16], is increased, preferably, an increase
of the respective fine chemical, preferably of succinic acid, and
of an other organic acid is conferred.
[7233] In case the activity of a Escherichia coli K12 protein b3169
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 594 for trihydroxabutanoic acid
and/or line 580 for malic acid, e.g. a protein with an activity as
defined in [0022.0.16.16], is increased, preferably, an increase of
the respective fine chemical, preferably of trihydroxybutanoic acid
and/or malic acid, and of an other organic acid is conferred.
[7234] In case the activity of a Escherichia coli K12 protein b3172
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 569 for fumaric acid and/or line 581
for malic acid, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of fumaric acid and/or malic
acid, and of an other organic acid is conferred.
[7235] In case the activity of a Escherichia coli K12 protein b3231
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 575 for glyceric acid and/or line 584
for pyruvic acid, e.g. a protein with an activity as defined in
[0022.0.16.16], is increased, preferably, an increase of the
respective fine chemical, preferably of glyceric acid and/or
pyruvic acid, and of an other organic acid is conferred.
[7236] In case the activity of a Escherichia coli K12 protein b3938
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 576, e.g. a protein with an activity
as defined in [0022.0.16.16], is increased, preferably, an increase
of the respective fine chemical, preferably of glyceric acid, and
of an other organic acid is conferred.
[7237] In case the activity of a Escherichia coli K12 protein b4122
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 570, e.g. a protein with an activity
as defined in [0022.0.16.16], is increased, preferably, an increase
of the respective fine chemical, preferably of fumaric acid, and of
an other organic acid is conferred.
[7238] In case the activity of a Escherichia coli K12 protein b4129
as disclosed in [0016.0.16.16] or its homologs, as indicated in
Table I, columns 5 or 7, line 590, e.g. a protein with an activity
as defined in [0022.0.16.16], is increased, preferably, an increase
of the respective fine chemical, preferably of succinic acid, and
of an other organic acid is conferred.
[7239] In case the activity of a Saccharomyces cerevisiae protein
YCL038C as disclosed in [0016.0.16.16] or its homologs, as
indicated in Table I, columns 5 or 7, line 591 for succinic acid
and/or line 582 for malic acid, e.g. a protein with an activity as
defined in [0022.0.16.16], is increased, preferably, an increase of
the respective fine chemical, preferably of succinic acid and/or
malic acid, and of an other organic acid is conferred.
[7240] In case the activity of a Saccharomyces cerevisiae protein
YCR012W as disclosed in [0016.0.16.16] or its homologs, as
indicated in Table I, columns 5 or 7, line 571, e.g. a protein with
an activity as defined in [0022.0.16.16], is increased, preferably,
an increase of the respective fine chemical, preferably of fumaric
acid, and of an other organic acid is conferred.
[7241] In case the activity of a Saccharomyces cerevisiae protein
YOR168W as disclosed in [0016.0.16.16] or its homologs, as
indicated in Table I, columns 5 or 7, line 583, e.g. a protein with
an activity as defined in [0022.0.16.16], is increased, preferably,
an increase of the respective fine chemical, preferably of malic
acid, and of an other organic acid is conferred.
[7242] [0047.0.0.16] to [0048.0.0.16]: see [0047.0.0.0] to
[0048.0.0.0]
[7243] [0049.0.16.16] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
citramalic acid preferably has the structure of the polypeptide
described herein. In a particular embodiment, the polypeptides used
in the process of the present invention or the polypeptide of the
present invention comprises the sequence of a consensus sequence as
indicated in Table IV, column 7, lines 190 or line 564 or of a
polypeptide as indicated in Table II, columns 5 or 7, line 190 or
line 564 or of a functional homologue thereof as described herein,
or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, line 190 or line 564 or its herein
described functional homologues and has the herein mentioned
activity conferring an increase in the citramalic acid level.
[7244] A protein having an activity conferring an increase in the
amount or level of the fumaric acid preferably has the structure of
the polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence as indicated in Table IV, column 7, lines 191 to
205 or lines 565 to 571 or of a polypeptide as indicated in Table
II, columns 5 or 7, lines 191 to 205 or lines 565 to 571 or of a
functional homologue thereof as described herein, or of a
polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, lines 191 to 205 or lines 565 to 571 or its herein
described functional homologues and has the herein mentioned
activity conferring an increase in the fumaric acid level.
[7245] A protein having an activity conferring an increase in the
amount or level of the glyceric acid preferably has the structure
of the polypeptide described herein. In a particular embodiment,
the polypeptides used in the process of the present invention or
the polypeptide of the present invention comprises the sequence of
a consensus sequence as indicated in Table IV, column 7, lines 572
to 576 or of a polypeptide as indicated in Table II, columns 5 or
7, lines 572 to 576 or of a functional homologue thereof as
described herein, or of a polypeptide encoded by the nucleic acid
molecule characterized herein or the nucleic acid molecule
according to the invention, for example by a nucleic acid molecule
as indicated in Table I, columns 5 or 7, lines 572 to 576 or its
herein described functional homologues and has the herein mentioned
activity conferring an increase in the glyceric acid level.
[7246] A protein having an activity conferring an increase in the
amount or level of the respective fine chemical malic acid
preferably has the structure of the polypeptide described herein.
In a particular embodiment, the polypeptides used in the process of
the present invention or the polypeptide of the present invention
comprises the sequence of a consensus sequence as indicated in
Table IV, column 7, lines 206 to 217 or lines 577 to 583 or of a
polypeptide as indicated in Table II, columns 5 or 7, lines 577 to
583 or of a functional homologue thereof as described herein, or of
a polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, line 206 to 217 or lines 577 to 583 or its herein described
functional homologues and has the herein mentioned activity
conferring an increase in the malic acidlevel.
[7247] A protein having an activity conferring an increase in the
amount or level of the respective fine chemical trihydroxybutyric
acid preferably has the structure of the polypeptide described
herein. In a particular embodiment, the polypeptides used in the
process of the present invention or the polypeptide of the present
invention comprises the sequence of a consensus sequence as
indicated in Table IV, column 7, line 218 or 219, or of a
polypeptide as indicated in Table II, columns 5 or 7, line 218 or
219 or of a functional homologue thereof as described herein, or of
a polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, line 218 or 219 or its herein described functional
homologues and has the herein mentioned activity conferring an
increase in the trihydroxybutyric acid level.
[7248] A protein having an activity conferring an increase in the
amount or level of the respective fine chemical pyruvic acid
preferably has the structure of the polypeptide described herein.
In a particular embodiment, the polypeptides used in the process of
the present invention or the polypeptide of the present invention
comprises the sequence of a consensus sequence as indicated in
Table IV, column 7, line 220 or line 584 or of a polypeptide as
indicated in Table II, columns 5 or 7, line 220 or line 584 or of a
functional homologue thereof as described herein, or of a
polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, line 220 or line 584 or its herein described functional
homologues and has the herein mentioned activity conferring an
increase in the pyruvic acid level.
[7249] A protein having an activity conferring an increase in the
amount or level of the respective fine chemical succinic acid
preferably has the structure of the polypeptide described herein.
In a particular embodiment, the polypeptides used in the process of
the present invention or the polypeptide of the present invention
comprises the sequence of a consensus sequence as indicated in
Table IV, column 7, lines 221 to 224 or lines 585 to 591 or of a
polypeptide as indicated in Table II, columns 5 or 7, lines 221 to
224 or lines 585 to 591 or of a functional homologue thereof as
described herein, or of a polypeptide encoded by the nucleic acid
molecule characterized herein or the nucleic acid molecule
according to the invention, for example by a nucleic acid molecule
as indicated in Table I, columns 5 or 7, lines 221 to 224 or lines
585 to 591 or its herein described functional homologues and has
the herein mentioned activity conferring an increase in the
succinic acid level.
[7250] A protein having an activity conferring an increase in the
amount or level of the respective fine chemical trihydroxybutanoic
acid preferably has the structure of the polypeptide described
herein. In a particular embodiment, the polypeptides used in the
process of the present invention or the polypeptide of the present
invention comprises the sequence of a consensus sequence as
indicated in Table IV, column 7, lines 225 to 226 or lines 592 to
594 or of a polypeptide as indicated in Table II, columns 5 or 7,
lines 225 to 226 or lines 592 to 594 or of a functional homologue
thereof as described herein, or of a polypeptide encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by a nucleic acid
molecule as indicated in Table I, columns 5 or 7, line 225 to 226
or lines 592 to 594 or its herein described functional homologues
and has the herein mentioned activity conferring an increase in the
trihydroxybutanoic acid level.
[7251] [0050.0.16.16] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of glyceric acid, citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid, resp., or their ester, or glucoside
thereof, e.g the diglucoside thereof.
[7252] [0051.0.16.16] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g. compositions comprising glyceric acid,
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid and/or trihydroxybutanoic acid.
Depending on the choice of the organism used for the process
according to the present invention, for example a microorganism or
a plant, compositions or mixtures of organic acids or there salts,
e.g. glyceric acid, citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid can be produced.
[7253] [0052.0.0.16] see [0052.0.0.0]
[7254] [0053.0.16.16] In one embodiment, the process of the present
invention comprises one or more of the following steps [7255] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or
used in the process of the invention or of the polypeptid of the
invention or used in the process of the invention, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 190 to 226 or lines 564 to 594 or its homologs,
e.g. as indicated in Table II, columns 5 or 7, lines 190 to 226 or
lines 564 to 594, activity having herein-mentioned the respective
fine chemical increasing activity; [7256] b) stabilizing a mRNA
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or used in the process of
the invention, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 190 to 226 or
lines 564 to 594 or its homologs activity, e.g. as indicated in
Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594, or
of a mRNA encoding the polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity;
[7257] c) increasing the specific activity of a protein conferring
the increased expression of a protein encoded by the nucleic acid
molecule of the invention or used in the process of the invention
or of the polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 190 to 226 or lines 564 to 594 or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, or decreasing the inhibitory
regulation of the polypeptide of the invention or used in the
process of the invention; [7258] d) generating or increasing the
expression of an endogenous or artificial transcription factor
mediating the expression of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or used in the process of the invention or of the
polypeptide of the invention or used in the process of the
invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 190 to 226 or
lines 564 to 594 or its homologs activity, e.g. as indicated in
Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594;
[7259] e) stimulating activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 190 to 226 or
lines 564 to 594 or its homologs activity, e.g. as indicated in
Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594, by
adding one or more exogenous inducing factors to the organism or
parts thereof; [7260] f) expressing a transgenic gene encoding a
protein conferring the increased expression of a polypeptide
encoded by the nucleic acid molecule of the present invention or a
polypeptide of the present invention, having herein-mentioned the
respective fine chemical increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 190 to 226 or lines 564 to 594 or its homologs activity, e.g.
as indicated in Table II, columns 5 or 7, lines 190 to 226 or lines
564 to 594, [7261] g) increasing the copy number of a gene
conferring the increased expression of a nucleic acid molecule
encoding a polypeptide encoded by the nucleic acid molecule of the
invention or used in the process of the invention or the
polypeptide of the invention or used in the process of the
invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 190 to 226 or
lines 564 to 594 or its homologs, e.g. as indicated in Table II,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, activity.
[7262] h) Increasing the expression of the endogenous gene encoding
the polypeptide of the invention or used in the process of the
invention, e.g. a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 190 to 226 or lines 564 to
594 or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, by adding
positive expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention or used in the process of the invention, the expression
of which is thereby enhanced; [7263] i) Modulating growth
conditions of an organism in such a manner, that the expression or
activity of the gene encoding the protein of the invention or used
in the process of the invention or the protein itself is enhanced
for example microorganisms or plants can be grown for example under
a higher temperature regime leading to an enhanced expression of
heat shock proteins, which can lead to an enhanced respective fine
chemical production, and/or [7264] j) selecting of organisms with
especially high activity of the proteins of the invention or used
in the process of the invention from natural or from mutagenized
resources and breeding them into the target organisms, e.g. the
elite crops.
[7265] [0054.0.16.16] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table II, columns 3 or 5,
lines 190 to 226 or lines 564 to 594, resp., or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 190
to 226 or lines 564 to 594, resp.
[7266] [0055.0.0.16] to [0067.0.0.16]: see [0055.0.0.0] to
[0067.0.0.0]
[7267] [0068.0.16.16] The mutation is introduced in such a way that
the production of glyceric acid, citramalic acid, fumaric acid,
malic acid, pyruvic acid, succinic acid, trihydroxybutyric acid
and/or trihydroxybutanoic acid is not adversely affected.
[7268] [0069.0.0.16] see [0069.0.0.0]
[7269] [0070.0.16.16] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or used in the process of the invention
or the polypeptide of the invention or used in the process of the
invention, for example the nucleic acid construct mentioned below,
or encoding a protein of the invention or used in the process of
the invention into an organism alone or in combination with other
genes, it is possible not only to increase the biosynthetic flux
towards the end product, but also to increase, modify or create de
novo an advantageous, preferably novel metabolite composition in
the organism, e.g. an advantageous composition of citramalic acid,
glyceric acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid and/or trihydroxybutanoic acid or
their biochemical derivatives, e.g. comprising a higher content of
(from a viewpoint of nutritional physiology limited) glyceric acid,
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid and/or trihydroxybutanoic acid or
their derivatives.
[7270] [0071.0.0.16] see [0071.0.0.0]
[7271] [0072.0.16.16]
[7272] [0073.0.16.16] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
[7273] providing a non-human organism, preferably a microorganism,
a non-human animal, a plant or animal cell, a plant or animal
tissue or a plant; and
[7274] increasing an activity of a polypeptide of the invention or
used in the process of the invention or a homolog thereof, e.g. as
indicated in Table II, columns 5 or 7, lines 190 to 226 or lines
564 to 594, or of a polypeptide being encoded by the nucleic acid
molecule of the present invention and described below, e.g.
conferring an increase of the respective fine chemical in an
organism, preferably in a microorganism, a non-human animal, a
plant or animal cell, a plant or animal tissue or a plant, and
growing an organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a plant
under conditions which permit the production of the respective fine
chemical in the organism, preferably the microorganism, the plant
cell, the plant tissue or the plant; and
[7275] if desired, recovering, optionally isolating, the free
and/or bound the respective fine chemical synthesized by the
organism, the microorganism, the non-human animal, the plant or
animal cell, the plant or animal tissue or the plant.
[7276] [0074.0.16.16] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound respective fine chemical.
[7277] [0075.0.0.16] to [0077.0.0.16]: see [0075.0.0.0] to
[0077.0.0.0]
[7278] [0078.0.16.16] The organism such as microorganisms or plants
or the recovered, and if desired isolated, the respective fine
chemical can then be processed further directly into foodstuffs or
animal feeds or for other applications. The fermentation broth,
fermentation products, plants or plant products can be purified
with methods known to the person skilled in the art. Products of
these different work-up procedures are glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid or comprising
compositions of glyceric acid, citramalic acid, fumaric acid, malic
acid, pyruvic acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid still comprising fermentation broth, plant
particles and cell components in different amounts, advantageously
in the range of from 0 to 99% by weight, preferably below 80% by
weight, especially preferably below 50% by weight.
[7279] [0079.0.0.16] to [0084.0.0.16]: see [0079.0.0.0] to
[0084.0.0.0]
[7280] [0084.0.16.16] -/-
[7281] [0085.0.16.16] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [7282] a) a nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 190 to 226 or lines 564
to 594, or a derivative thereof, or [7283] b) a genetic regulatory
element, for example a promoter, which is functionally linked to
the nucleic acid sequence as indicated in Table I, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, or a derivative thereof, or
[7284] c) (a) and (b) is/are not present in its/their natural
genetic environment or has/have been modified by means of genetic
manipulation methods, it being possible for the modification to be,
by way of example, a substitution, addition, deletion, inversion or
insertion of one or more nucleotide. "Natural genetic environment"
means the natural chromosomal locus in the organism of origin or
the presence in a genomic library. In the case of a genomic
library, the natural, genetic environment of the nucleic acid
sequence is preferably at least partially still preserved. The
environment flanks the nucleic acid sequence at least on one side
and has a sequence length of at least 50 bp, preferably at least
500 bp, particularly preferably at least 1000 bp, very particularly
preferably at least 5000 bp.
[7285] [0086.0.0.16] to [0087.0.0.16]: see [0086.0.0.0] to
[0087.0.0.0]
[7286] [0088.0.16.16] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned fine
chemicals or the plants are more resistant to biotic and abiotic
stress and the yield is increased.
[7287] [0088.1.0.16] see [0088.1.0.0]
[7288] [0089.0.0.16] to [0094.0.0.16]: see [0089.0.0.0] to
[0094.0.0.0]
[7289] [0095.0.16.16] It may be advantageous to increase the pool
of glyceric acid, citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid in the transgenic organisms by the process
according to the invention in order to isolate high amounts of the
pure respective fine chemical and/or to obtain increased resistance
against biotic and abiotic stresses and to obtain higher yield.
[7290] [0096.0.16.16] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention or used in the
process of the invention together with the transformation of a
protein or polypeptid or a compound, which functions as a sink for
the desired fine chemical, for example in the organism, is useful
to increase the production of the respective fine chemical.
[7291] [0097.0.16.16] -/-
[7292] [0098.0.16.16] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated.
[7293] [0099.0.10.16] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[7294] [0100.0.16.16] Transgenic plants which comprise the fine
chemicals such as glyceric acid, citramalic acid, fumaric acid,
malic acid, pyruvic acid, succinic acid, trihydroxybutyric acid
and/or trihydroxybutanoic acid synthesized in the process according
to the invention can advantageously be marketed directly without
there being any need for the fine chemicals synthesized to be
isolated. Plants for the process according to the invention are
listed as meaning intact plants and all plant parts, plant organs
or plant parts such as leaf, stem, seeds, root, tubers, anthers,
fibers, root hairs, stalks, embryos, calli, cotelydons, petioles,
harvested material, plant tissue, reproductive tissue and cell
cultures which are derived from the actual transgenic plant and/or
can be used for bringing about the transgenic plant. In this
context, the seed comprises all parts of the seed such as the seed
coats, epidermal cells, seed cells, endosperm or embryonic
tissue.
[7295] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, as extracts, e.g. ether, alcohol,
or other organic solvents or water containing extract and/or free
fine chemicals. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pre-treated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[7296] Because glyceric acid, citramalic acid, fumaric acid, malic
acid, pyruvic acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid in microorganisms are localized
intracellular, their recovery essentially comes down to the
isolation of the biomass. Well-established approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Of the residual
hydrocarbon, adsorbed on the cells, has to be removed. Solvent
extraction or treatment with surfactants have been suggested for
this purpose.
[7297] [0101.0.10.16] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[7298] [0102.0.16.16] glyceric acid, citramalic acid, fumaric acid,
malic acid, pyruvic acid, succinic acid, trihydroxybutyric acid
and/or trihydroxybutanoic acid can for example be detected
advantageously via HPLC, LC or GC separation methods. The
unambiguous detection for the presence of glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid containing
products can be obtained by analyzing recombinant organisms using
analytical standard methods: LC, LC-MS, MS or TLC. The material to
be analyzed can be disrupted by sonication, grinding in a glass
mill, liquid nitrogen and grinding, cooking, or via other
applicable methods.
[7299] [0103.0.16.16] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [7300] a) nucleic acid molecule encoding, preferably
at least the mature form, of the polypeptide having a sequence as
indicated in Table II, columns 5 or 7, lines 190 to 226 or lines
564 to 594, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [7301] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table I, columns 5 or 7, lines 190 to 226 or lines
564 to 594, [7302] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [7303] d) nucleic
acid molecule encoding a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [7304] e) nucleic acid molecule which hybridizes with
a nucleic acid molecule of (a) to (c) under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [7305]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [7306] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[7307] h) nucleic acid molecule comprising a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers pairs having a
sequence as indicated in Table III, columns 7, lines 190 to 226 or
lines 564 to 594, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [7308]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [7309] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence having a
sequences as indicated in Table IV, columns 7, lines 190 to 226 or
lines 564 to 594 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [7310]
k) nucleic acid molecule comprising one or more of the nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domain of the polypeptide indicated in Table II, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; and [7311] l) nucleic acid molecule which is obtainable by
screening a suitable library under stringent conditions with a
probe comprising one of the sequences of the nucleic acid molecule
of (a) to (k), preferably to (a) to (c), or with a fragment of at
least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500
nt of the nucleic acid molecule characterized in (a) to (k),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
or which comprises a sequence which is complementary thereto.
[7312] [00103.1.18.16] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 190 to 226 or
lines 564 to 594, by one or more nucleotides. In one embodiment,
the nucleic acid molecule used in the process of the invention does
not consist of the sequence shown in indicated in Table I A,
columns 5 or 7, lines 190 to 226 or lines 564 to 594: In one
embodiment, the nucleic acid molecule used in the process of the
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I A, columns 5 or 7,
lines 190 to 226 or lines 564 to 594. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II A, columns 5 or 7, lines 190 to 226 or lines
564 to 594.
[7313] [00103.2.0.16] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, lines 190 to 226
or lines 564 to 594, by one or more nucleotides. In one embodiment,
the nucleic acid molecule used in the process of the invention does
not consist of the sequence shown in indicated in Table I B,
columns 5 or 7, lines 190 to 226 or lines 564 to 594: In one
embodiment, the nucleic acid molecule used in the process of the
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I B, columns 5 or 7,
lines 190 to 226 or lines 564 to 594. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II B, columns 5 or 7, lines 190 to 226 or lines
564 to 594.
[7314] [0104.0.16.16] In one embodiment, the nucleic acid molecule
used in the process of the present invention or used in the the
process of the invention distinguishes over the sequence indicated
in Table I, columns 5 or 7, lines 190 to 226 or lines 564 to 594 by
one or more nucleotides. In one embodiment, the nucleic acid
molecule of the invention or used in the process of the invention
does not consist of the sequence indicated in Table I, columns 5 or
7, lines 190 to 226 or lines 564 to 594. In one embodiment, the
nucleic acid molecule of the present invention is less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to the sequence indicated
in Table I, columns 5 or 7, lines 190 to 226 or lines 564 to 594.
In another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II, columns 5 or 7,
lines 190 to 226 or lines 564 to 594.
[7315] [0105.0.0.16] to [0107.0.0.16]: see [0105.0.0.0] to
[0107.0.0.0]
[7316] [0108.0.16.16] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 190 to 226 or lines 564
to 594, nucleic acid molecules which are derived from an amino acid
sequences as indicated in Table II, columns 5 or 7, lines 190 to
226 or lines 564 to 594 or from polypeptides comprising the
consensus sequence as indicated in Table IV, column 7, lines 190 to
226 or lines 564 to 594, or their derivatives or homologues
encoding polypeptides with the enzymatic or biological activity of
an activity of a polypeptide as indicated in Table II, column 3, 5
or 7, lines 190 to 226 or lines 564 to 594, e.g. conferring the
increase of the respective fine chemical, meaning glyceric acid,
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid and/or trihydroxybutanoic acid, resp.,
after increasing its expression or activity, are advantageously
increased in the process according to the invention.
[7317] [0109.0.16.16] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular glyceric
acid, citramalic acid, fumaric acid, malic acid, pyruvic acid,
succinic acid, trihydroxybutyric acid and/or trihydroxybutanoic
acid, produced in the process according to the invention.
[7318] [0110.0.0.16] see [0110.0.0.0]
[7319] [0111.0.0.16] see [0111.0.0.0]
[7320] [0112.0.16.16] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table II, column 3, lines 190 to
226 or lines 564 to 594 or having the sequence of a polypeptide as
indicated in Table II, columns 5 and 7, lines 190 to 226 or lines
564 to 594 and conferring an increase in the glyceric acid,
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid and/or trihydroxybutanoic acid
level.
[7321] [0113.0.0.16] to [0114.0.0.16]: see [0113.0.0.0] to
[0114.0.0.0]
[7322] [0115.0.0.16] see [0115.0.0.0]
[7323] [0116.0.0.16] to [0120.0.0.16] see [0116.0.0.0] to
[0120.0.0.0]
[7324] [0120.1.0.16]: -/-
[7325] [0121.0.16.16] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 190 to 226 or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a citramalic acid level increase after
increasing the activity of the polypeptide sequences indicated in
Table II, columns 5 or 7, line 190 or line 564 or conferring a
fumaric acid level increase after increasing the activity of the
polypeptide sequences indicated in Table II, columns 5 or 7, lines
191 to 205 or lines 565 to 571 or conferring a glyceric acid level
increase after increasing the activity of the polypeptide sequence
indicated in Table II, columns 5 or 7, lines 572 to 576, or
conferring a malic acid level increase after increasing the
activity of the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 206 to 217 or lines 577 to 583 or conferring
a trihydroxybutyric acid level increase after increasing the
activity of the polypeptide sequences indicated in Table II,
columns 5 or 7, line 218 or 219 or conferring a pyruvic acid level
increase after increasing the activity of the polypeptide sequences
indicated in Table II, columns 5 or 7, line 220 or 584 or
conferring a succinic acid level increase after increasing the
activity of the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 221 to 224 or lines 585 to 591 or conferring
a trihydroxybutanoic acid level increase after increasing the
activity of the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 225 and 226 or lines 592 to 594.
[7326] [0122.0.0.16] to [0127.0.0.16]: see [0122.0.0.0] to
[0127.0.0.0]
[7327] [0128.0.16.16] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, columns 7,
lines 190 to 226 or lines 564 to 594, by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table I, columns 5 or 7,
lines 190 to 226 or lines 564 to 594 or the sequences derived from
a sequence as indicated in Table II, columns 5 or 7, lines 190 to
226 or lines 564 to 594.
[7328] [0129.0.16.16] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention or used in the process of the invention, from which
conserved regions, and in turn, degenerate primers can be derived.
Conserved region for the polypeptide of the invention or used in
the process of the invention are indicated in the alignments shown
in the figures. Conserved regions are those, which show a very
little variation in the amino acid in one particular position of
several homologs from different origin. The consensus sequence
indicated in Table IV, columns 7, lines 190 to 226 or lines 564 to
594 is derived from said alignments.
[7329] [0130.0.16.16] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of glyceric
acid, citramalic acid, fumaric acid, malic acid, pyruvic acid,
succinic acid, trihydroxybutyric acid and/or trihydroxybutanoic
acid after increasing the expression or activity the protein
comprising said fragment.
[7330] [0131.0.0.16] to [0138.0.0.16]: see [0131.0.0.0] to
[0138.0.0.0]
[7331] [0139.0.16.16] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical level increase,
derived from other organisms, can be encoded by other DNA sequences
which hybridise to a sequence indicated in Table I, columns 5 or 7,
line 190 or line 564 for citramalic acid or indicated in Table I,
columns 5 or 7, lines 191 to 205 or 565 to 571 for fumaric acid or
indicated in Table I, columns 5 or 7, lines 206 to 217 or 577 to
583 for malic acid or indicated in Table I, columns 5 or 7, line
218 or 219 for trihydroxybutyric acid or indicated in Table I,
columns 5 or 7, line 220 or line 584 for pyruvic acid or indicated
in Table I, columns 5 or 7, lines 221 to 224 or lines 585 to 591
for succinic acid or indicated in Table I, columns 5 or 7, lines
225 or 226 or 592 to 594 for trihydroxybutanoic acid under relaxed
hybridization conditions and which code on expression for peptides
having the respective fine chemical, i.e. glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid, resp.,
increasing-activity.
[7332] [0140.0.0.16] to [0146.0.0.16]: see [0140.0.0.0] to
[0146.0.0.0]
[7333] [0147.0.16.16] Further, the nucleic acid molecule of the
invention or used in the process of the invention comprises a
nucleic acid molecule, which is a complement of one of the
nucleotide sequences of above mentioned nucleic acid molecules or a
portion thereof. A nucleic acid molecule which is complementary to
one of the nucleotide sequences indicated in Table I, columns 5 or
7, lines 190 to 226 or lines 564 to 594, preferably in Table I B,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, is one which
is sufficiently complementary to one of said nucleotide sequences
such that it can hybridise to one of said nucleotide sequences,
thereby forming a stable duplex. Preferably, the hybridisation is
performed under stringent hybridization conditions. However, a
complement of one of the herein disclosed sequences is preferably a
sequence complement thereto according to the base pairing of
nucleic acid molecules well known to the skilled person. For
example, the bases A and G undergo base pairing with the bases T
and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[7334] [0148.0.16.16] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines
190 to 226 or lines 564 to 594, preferably in Table I B, columns 5
or 7, lines 190 to 226 or lines 564 to 594, or a portion thereof
and preferably has above mentioned activity, in particular having a
glyceric acid, citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid increasing activity after increasing the
activity or an activity of a product of a gene encoding said
sequences or their homologs.
[7335] [0149.0.16.16] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, preferably in Table IB,
columns 7, lines 190 to 226 or lines 564 to 594, or a portion
thereof and encodes a protein having above-mentioned activity, e.g.
conferring a of glyceric acid, citramalic acid, fumaric acid, malic
acid, pyruvic acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid increase, resp., and optionally, the
activity of protein indicated in Table II, column 5, lines 190 to
226 or lines 564 to 594, preferably in Table II B, columns 7, lines
190 to 226 or lines 564 to 594.
[7336] [00149.1.0.16] Optionally, in one embodiment, the nucleotide
sequence, which hybridises to one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 190 to 226 or lines 564
to 594, preferably in Table I B, columns 7, lines 190 to 226 or
lines 564 to 594, has further one or more of the activities
annotated or known for a protein as indicated in Table II, column
3, lines 190 to 226 or lines 564 to 594, preferably in Table I B,
columns 7, lines 190 to 226 or lines 564 to 594.
[7337] [0150.0.16.16] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table I, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
preferably in Table I B, columns 7, lines 190 to 226 or lines 564
to 594 for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid, resp., if
its activity is increased. The nucleotide sequences determined from
the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table I, columns
5 or 7, lines 190 to 226 or lines 564 to 594, an anti-sense
sequence of one of the sequences, e.g., as indicated in Table I,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, or naturally
occurring mutants thereof. Primers based on a nucleotide of
invention can be used in PCR reactions to clone homologues of the
polypeptide of the invention or of the polypeptide used in the
process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primer pairs indicated in Table III, column 7, lines
190 to 226 or lines 564 to 594 will result in a fragment of a
polynucleotide sequence as indicated in Table I, columns 5 or 7,
lines 190 to 226 or lines 564 to 594 or its gene product. Preferred
is Table I B, columns 7, lines 190 to 226 or lines 564 to 594.
[7338] [0151.0.0.16]: see [0151.0.0.0]
[7339] [0152.0.16.16] The nucleic acid molecule of the invention or
used in the process of the invention encodes a polypeptide or
portion thereof which includes an amino acid sequence which is
sufficiently homologous to an amino acid sequence as indicated in
Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594 such
that the protein or portion thereof maintains the ability to
participate in the respective fine chemical production, in
particular a citramalic acid (line 190) or fumaric acid (lines 191
to 205 or 565 to 571) or malic acid (lines 206 to 217 or 577 to
583) or trihydroxybutyric acid (line 218 or 219) or pyruvic acid
(line 220 or line 584) or succinic acid (lines 221 to 224 or lines
585 to 591) or trihydroxybutanoic acid (line 225 or 226) increasing
activity as mentioned above or as described in the examples in
plants or microorganisms is comprised.
[7340] [0153.0.16.16] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 190
to 226 or lines 564 to 594 such that the protein or portion thereof
is able to participate in the increase of the respective fine
chemical production. In one embodiment, a protein or portion
thereof as indicated in Table II, columns 5 or 7, lines 190 to 226
or lines 564 to 594 has for example an activity of a polypeptide
indicated in Table II, column 3, lines 190 to 226 or lines 564 to
594.
[7341] [0154.0.16.16] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 190 to 226
or lines 564 to 594 and has above-mentioned activity, e.g.
conferring preferably the increase of the respective fine
chemical.
[7342] [0155.0.0.16] to [0156.0.0.16]: see [0155.0.0.0] to
[0156.0.0.0]
[7343] [0157.0.16.16] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences as
indicated in Table I, columns 5 or 7, lines 190 to 226 or lines 564
to 594 (and portions thereof) due to degeneracy of the genetic code
and thus encode a polypeptide of the present invention, in
particular a polypeptide having above mentioned activity, e.g.
conferring an increase in the respective fine chemical in a
organism, e.g. as polypeptides comprising the consensus sequence as
indicated in Table IV, columns 5 or 7, lines 190 to 226 or lines
564 to 594 or as polypeptides depicted in Table II, columns 5 or 7,
lines 190 to 226 or lines 564 to 594 or the functional homologues.
Advantageously, the nucleic acid molecule of the invention or used
in the process of the invention comprises, or in an other
embodiment has, a nucleotide sequence encoding a protein
comprising, or in an other embodiment having, an amino acid
sequence of a consensus sequences as indicated in Table IV, columns
5 or 7, lines 190 to 226 or lines 564 to 594 or of the polypeptide
as indicated in Table II, columns 5 or 7, lines 190 to 226 or lines
564 to 594, resp., or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
IV, columns 5 or 7, lines 190 to 226 or lines 564 to 594 or of a
polypeptide as indicated in Table II, columns 5 or 7, lines 190 to
226 or lines 564 to 594, or the functional homologues thereof.
However, in a preferred embodiment, the nucleic acid molecule of
the present invention does not consist of a sequence as indicated
in Table I, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
preferably in Table I A, columns 7, lines 190 to 226 or lines 564
to 594, resp. Preferably, the nucleic acid molecule of the
invention is a functional homologue or identical to a nucleic acid
molecule indicated in Table I B, columns 5 or 7, ines 190 to 226 or
lines 564 to 594.
[7344] [0158.0.0.16] to [0160.0.0.16]: see [0158.0.0.0] to
[0160.0.0.0]
[7345] [0161.0.16.16] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 190 to 226
or lines 564 to 594. The nucleic acid molecule is preferably at
least 20, 30, 50, 100, 250 or more nucleotides in length.
[7346] [0162.0.0.16] see [0162.0.0.0]
[7347] [0163.0.16.16] Preferably, a nucleic acid molecule of the
invention or used in the process of the invention that hybridizes
under stringent conditions to a sequence as indicated in Table I,
columns 5 or 7, lines 190 to 226 or lines 564 to 594 corresponds to
a naturally-occurring nucleic acid molecule of the invention. As
used herein, a "naturally-occurring" nucleic acid molecule refers
to an RNA or DNA molecule having a nucleotide sequence that occurs
in nature (e.g., encodes a natural protein). Preferably, the
nucleic acid molecule encodes a natural protein having
above-mentioned activity, e.g. conferring the increase of the
amount of the respective fine chemical in a organism or a part
thereof, e.g. a tissue, a cell, or a compartment of a cell, after
increasing the expression or activity thereof or the activity of a
protein of the invention or used in the process of the
invention.
[7348] [0164.0.0.16] see [0164.0.0.0]
[7349] [0165.0.16.16] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 190 to 226 or lines 564
to 594, resp.
[7350] [0166.0.0.16] to [0167.0.0.16]: see [0166.0.0.0] to
[0167.0.0.0]
[7351] [0168.0.16.16] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, preferably in Table II B,
columns 7, lines 190 to 226 or lines 564 to 594, resp., yet retain
said activity described herein. The nucleic acid molecule can
comprise a nucleotide sequence encoding a polypeptide, wherein the
polypeptide comprises an amino acid sequence at least about 50%
identical to an amino acid sequence as indicated in Table II,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, preferably in
Table II B, columns 7, lines 190 to 226 or lines 564 to 594, resp.,
and is capable of participation in the increase of production of
the respective fine chemical after increasing its activity, e.g.
its expression. Preferably, the protein encoded by the nucleic acid
molecule is at least about 60% identical to a sequence as indicated
in Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
preferably in Table II B, columns 7, lines 190 to 226 or lines 564
to 594, resp., more preferably at least about 70% identical to one
of the sequences as indicated in Table II, columns 5 or 7, lines
190 to 226 or lines 564 to 594, preferably in Table II B, columns
7, lines 190 to 226 or lines 564 to 594, resp., even more
preferably at least about 80%, 90%, 95% homologous to a sequence as
indicated in Table II, columns 5 or 7, lines 190 to 226 or lines
564 to 594, preferably in Table II B, columns 7, lines 190 to 226
or lines 564 to 594, resp., and most preferably at least about 96%,
97%, 98%, or 99% identical to the sequence as indicated in Table
II, columns 5 or 7, lines 190 to 226 or lines 564 to 594 preferably
in Table II B, columns 7, lines 190 to 226 or lines 564 to 594.
[7352] [0169.0.0.16] to [0172.0.0.16]: see [0169.0.0.0] to
[0172.0.0.0]
[7353] [0173.0.16.16] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 17198 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 17198 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[7354] [0174.0.0.16]: see [0174.0.0.0]
[7355] [0175.0.16.16] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 17199 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 17199 by the above program algorithm with the
above parameter set, has a 80% homology.
[7356] [0176.0.16.16] Functional equivalents derived from one of
the polypeptides as indicated in Table II, columns 5 or 7, lines
190 to 226 or lines 564 to 594, resp., according to the invention
by substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, resp.,
according to the invention and are distinguished by essentially the
same properties as a polypeptide as indicated in Table II, columns
5 or 7, lines 190 to 226 or lines 564 to 594, resp.
[7357] [0177.0.16.16] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 190 to
226 or lines 564 to 594, preferably in Table I B, columns 7, lines
190 to 226 or lines 564 to 594, resp., according to the invention
by substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, preferably in
Table II B, columns 7, lines 190 to 226 or lines 564 to 594, resp.,
according to the invention and encode polypeptides having
essentially the same properties as a polypeptide as indicated in
Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
preferably in Table II B, columns 7, lines 190 to 226 or lines 564
to 594, resp.
[7358] [0178.0.0.16] see [0178.0.0.0]
[7359] [0179.0.16.16] A nucleic acid molecule encoding a homologous
to a protein sequence as indicated in Table II, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, preferably in Table I B,
columns 7, lines 190 to 226 or lines 564 to 594, resp., can be
created by introducing one or more nucleotide substitutions,
additions or deletions into a nucleotide sequence of the nucleic
acid molecule of the present invention, in particular as indicated
in Table I, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
resp., such that one or more amino acid substitutions, additions or
deletions are introduced into the encoded protein. Mutations can be
introduced into the encoding sequences for example into sequences
of nucleic acid molecules as indicated in Table I, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, resp., by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[7360] [0180.0.0.16] to [0183.0.0.16]: see [0180.0.0.0] to
[0183.0.0.0]
[7361] [0184.0.16.16] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table I, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, preferably in Table I B,
columns 7, lines 190 to 226 or lines 564 to 594, resp., or of the
nucleic acid sequences derived from a sequences as indicated in
Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
preferably in Table II B, columns 7, lines 190 to 226 or lines 564
to 594, resp., comprise also allelic variants with at least
approximately 30%, 35%, 40% or 45% homology, by preference at least
approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
I, columns 5 or 7, lines 190 to 226 or lines 564 to 594, resp., or
from the derived nucleic acid sequences, the intention being,
however, that the enzyme activity or the biological activity of the
resulting proteins synthesized is advantageously retained or
increased.
[7362] [0185.0.16.16] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table I, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
preferably in Table I B, columns 7, lines 190 to 226 or lines 564
to 594, resp. In one embodiment, it is preferred that the nucleic
acid molecule comprises as little as possible other nucleotides not
shown in any one of sequences as indicated in Table I, columns 5 or
7, lines 190 to 226 or lines 564 to 594, preferably in Table I B,
columns 7, lines 190 to 226 or lines 564 to 594, resp. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequence as indicated in Table I, columns 5 or 7, lines 190 to 226
or lines 564 to 594, preferably in Table I B, columns 7, lines 190
to 226 or lines 564 to 594, resp.
[7363] [0186.0.16.16] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 190 to 226 or lines 564 to 594, preferably in Table
II B, columns 7, lines 190 to 226 or lines 564 to 594, resp. In one
embodiment, the nucleic acid molecule encodes less than 150, 130,
100, 80, 60, 50, 40 or 30 further amino acids. In a further
embodiment, the encoded polypeptide comprises less than 20, 15, 10,
9, 8, 7, 6 or 5 further amino acids. In one embodiment, the encoded
polypeptide used in the process of the invention is identical to
the sequences as indicated in Table II, columns 5 or 7, lines 190
to 226 or lines 564 to 594, preferably in Table II B, columns 7,
lines 190 to 226 or lines 564 to 594, resp.
[7364] [0187.0.16.16] In one embodiment, a nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, preferably in Table II B,
columns 7, lines 190 to 226 or lines 564 to 594, resp., comprises
less than 100 further nucleotides. In a further embodiment, said
nucleic acid molecule comprises less than 30 further nucleotides.
In one embodiment, the nucleic acid molecule used in the process is
identical to a coding sequence encoding a sequences as indicated in
Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
preferably in Table II B, columns 7, lines 190 to 226 or lines 564
to 594, resp.
[7365] [0188.0.16.16] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 190 to 226 or lines 564 to 594, resp.,
preferably compared to a sequence as indicated in preferably in
Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594, and
is expressed under identical conditions. In one embodiment, the
polypeptide of the invention is a homolog consisting of or
comprising the sequence as indicated in Table I B, columns 7, lines
190 to 226 or lines 564 to 594.
[7366] [0189.0.16.16] Homologues of a sequence as indicated in
Table I, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
resp., or of a derived sequences as indicated in Table II, columns
5 or 7, lines 190 to 226 or lines 564 to 594, resp., also mean
truncated sequences, cDNA, single-stranded DNA or RNA of the coding
and noncoding DNA sequence. Homologues of said sequences are also
understood as meaning derivatives, which comprise noncoding regions
such as, for example, UTRs, terminators, enhancers or promoter
variants. The promoters upstream of the nucleotide sequences stated
can be modified by one or more nucleotide substitution(s),
insertion(s) and/or deletion(s) without, however, interfering with
the functionality or activity either of the promoters, the open
reading frame (=ORF) or with the 3'-regulatory region such as
terminators or other 3' regulatory regions, which are far away from
the ORF. It is furthermore possible that the activity of the
promoters is increased by modification of their sequence, or that
they are replaced completely by more active promoters, even
promoters from heterologous organisms. Appropriate promoters are
known to the person skilled in the art and are mentioned herein
below.
[7367] [0190.0.0.16]: see [0190.0.0.0]
[7368] [0191.0.0.16] see [0191.0.0.0]
[7369] [0191.0.16.16] The organisms or part thereof produce
according to the herein mentioned process of the invention an
increased level of free and/or bound the respective fine chemical
compared to said control or selected organisms or parts
thereof.
[7370] [0192.0.0.16] to [0203.0.0.16]: see [0192.0.0.0] to
[0203.0.0.0]
[7371] [0204.0.16.16] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [7372] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, columns 5 or 7, lines 190 to
226 or lines 564 to 594, preferably in Table II B, columns 7, lines
190 to 226 or lines 564 to 594, resp.; or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, i.e. glyceric acid (lines 572 to 576), citramalic acid
(line 190 or 564) or fumaric acid (lines 191 to 205 or 565 to 571)
or malic acid (lines 206 to 217 or 577 to 583) or trihydroxybutyric
acid (line 218 or 219) or pyruvic acid (line 220 or line 584) or
succinic acid (lines 221 to 224 or lines 585 to 591) or
trihydroxybutanoic acid (lines 225 or 226 or 592 to 594), resp., in
an organism or a part thereof [7373] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 190 to 226
or lines 564 to 594, preferably in Table I B, columns 7, lines 190
to 226 or lines 564 to 594, resp., or a fragment thereof conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [7374] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [7375]
d) nucleic acid molecule encoding a polypeptide whose sequence has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [7376] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under stringent hybridisation conditions and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [7377] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [7378] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [7379]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
primers or primer pairs as indicated in Table III, columns 5 or 7,
lines 190 to 226 or lines 564 to 594 and conferring an increase in
the amount of the respective fine chemical, i.e. glyceric acid
(lines 572 to 576), citramalic acid (line 190 or 564) or fumaric
acid (lines 191 to 205 or 565 to 571) or malic acid (lines 206 to
217 or 577 to 583) or trihydroxybutyric acid (line 218 or 219) or
pyruvic acid (line 220 or line 584) or succinic acid (lines 221 to
224 or lines 585 to 591) or trihydroxybutanoic acid (lines 225 or
226 or 592 to 594), resp., in an organism or a part thereof; [7380]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from a expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (g), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [7381] j) nucleic acid molecule which
encodes a polypeptide comprising a consensus sequence as indicated
in Table IV, columns 7, lines 190 to 226 or lines 564 to 594 and
conferring an increase in the amount of the respective fine
chemical, i.e. glyceric acid (lines 572 to 576), citramalic acid
(line 190 or 564) or fumaric acid (lines 191 to 205 or 565 to 571)
or malic acid (lines 206 to 217 or 577 to 583) or trihydroxybutyric
acid (line 218 or 219) or pyruvic acid (line 220 or line 584) or
succinic acid (lines 221 to 224 or lines 585 to 591) or
trihydroxybutanoic acid (lines 225 or 226 or 592 to 594), resp., in
an organism or a part thereof; [7382] k) nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of a polypeptide as indicated in Table II, columns 5 or 7, lines
190 to 226 or lines 564 to 594, preferably in Table II B, columns
7, lines 190 to 226 or lines 564 to 594, resp., and conferring an
increase in the amount of the respective fine chemical, i.e.
glyceric acid (lines 572 to 576), citramalic acid (line 190 or 564)
or fumaric acid (lines 191 to 205 or 565 to 571) or malic acid
(lines 206 to 217 or 577 to 583) or trihydroxybutyric acid (line
218 or 219) or pyruvic acid (line 220 or line 584) or succinic acid
(lines 221 to 224 or lines 585 to 591) or trihydroxybutanoic acid
(lines 225 or 226 or 592 to 594), resp., in an organism or a part
thereof; and [7383] l) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, preferably in
Table I B, columns 7, lines 190 to 226 or lines 564 to 594, resp.,
or a nucleic acid molecule encoding, preferably at least the mature
form of, a polypeptide as indicated in Table II, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, resp., and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; or which encompasses a sequence which
is complementary thereto; whereby, preferably, the nucleic acid
molecule according to (a) to (l) distinguishes over a sequence as
indicated in Table IA, columns 5 or 7, lines 190 to 226 or lines
564 to 594, resp., by one or more nucleotides. In one embodiment,
the nucleic acid molecule of the invention does not consist of the
sequence as indicated in Table I A, columns 5 or 7, lines 190 to
226 or lines 564 to 594, resp. In an other embodiment, the nucleic
acid molecule of the present invention is at least 30% identical
and less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence as indicated in Table I A, columns 5 or 7, lines 190 to
226 or lines 564 to 594, resp. In a further embodiment the nucleic
acid molecule does not encode a polypeptide sequence as indicated
in Table II A, columns 5 or 7, lines 190 to 226 or lines 564 to
594, resp. Accordingly, in one embodiment, the nucleic acid
molecule of the present invention encodes in one embodiment a
polypeptide which differs at least in one or more amino acids from
a polypeptide indicated in Table II A, columns 5 or 7, lines 190 to
226 or lines 564 to 594 does not encode a protein of a sequence as
indicated in Table II A, columns 5 or 7, lines 190 to 226 or lines
564 to 594. Accordingly, in one embodiment, the protein encoded by
a sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table II A, columns 5 or 7,
lines 190 to 226 or lines 564 to 594. In a further embodiment, the
protein of the present invention is at least 30 identical to a
protein sequence indicated in Table II A, columns 5 or 7, lines 190
to 226 or lines 564 to 594 and less than 100%, preferably less than
99.999%, 99.99% or 99.9%, more preferably less than 99%, 985, 97%,
96% or 95% identical to a sequence as indicated in Table II A,
columns 5 or 7, lines 190 to 226 or lines 564 to 594.
[7384] In an other embodiment, however, the nucleic acid molecule
according to (a) to (l) distinguishes over a sequence as indicated
in Table I B, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
resp., by one or more nucleotides. In one embodiment, the nucleic
acid molecule of the invention does not consist of the sequence as
indicated in Table I B, columns 5 or 7, lines 190 to 226 or lines
564 to 594, resp. In an other embodiment, the nucleic acid molecule
of the present invention is at least 30 identical and less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence as
indicated in Table I B, columns 5 or 7, lines 190 to 226 or lines
564 to 594, resp. In a further embodiment the nucleic acid molecule
does not encode a polypeptide sequence as indicated in Table II B,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, resp.
Accordingly, in one embodiment, the nucleic acid molecule of the
present invention encodes in one embodiment a polypeptide which
differs at least in one or more amino acids from a polypeptide
indicated in Table II B, columns 5 or 7, lines 190 to 226 or lines
564 to 594 does not encode a protein of a sequence as indicated in
Table II B, columns 5 or 7, lines 190 to 226 or lines 564 to 594.
Accordingly, in one embodiment, the protein encoded by a sequences
of a nucleic acid according to (a) to (l) does not consist of a
sequence as indicated in Table II B, columns 5 or 7, lines 190 to
226 or lines 564 to 594. In a further embodiment, the protein of
the present invention is at least 30% identical to a protein
sequence indicated in Table II B, columns 5 or 7, lines 190 to 226
or lines 564 to 594 and less than 100%, preferably less than
99.999%, 99.99% or 99.9%, more preferably less than 99%, 98%, 97%,
96% or 95% identical to a sequence as indicated in Table II B,
columns 5 or 7, lines 190 to 226 or lines 564 to 594.
[7385] [0205.0.0.16] to [0206.0.0.16]: see [0205.0.0.0] to
[0206.0.0.0]
[7386] [0207.0.16.16] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the glutamic acid metabolism, the
phosphoenolpyruvate metabolism, the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[7387] [0208.0.0.16] to [0226.0.0.16]: see [0208.0.0.0] to
[0226.0.0.0]
[7388] [0227.0.16.16] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[7389] In addition to a sequence indicated in Table I, columns 5 or
7, lines 190 to 226 or lines 564 to 594 or its derivatives, it is
advantageous to express and/or mutate further genes in the
organisms. Especially advantageously, additionally at least one
further gene of the glutamic acid or phosphoenolpyruvate metabolic
pathway, is expressed in the organisms such as plants or
microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the fine chemicals desired since, for example,
feedback regulations no longer exist to the same extent or not at
all. In addition it might be advantageously to combine one or more
of the sequences indicated in Table I, columns 5 or 7, lines 190 to
226 or lines 564 to 594, resp., with genes which generally support
or enhances to growth or yield of the target organism, for example
genes which lead to faster growth rate of microorganisms or genes
which produces stress-, pathogen, or herbicide resistant
plants.
[7390] [0228.0.16.16] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins directly or indirectly involved
in the glutamic acid or phosphoenolpyruvate metabolism.
[7391] [0229.0.0.0] %
[7392] [0230.0.0.16] see [230.0.0.0]
[7393] [0231.0.16.16] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a glyceric acid, citramalic acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid degrading
protein is attenuated, in particular by reducing the rate of
expression of the corresponding gene.
[7394] [0232.0.0.16] to [0276.0.0.16]: see [0232.0.0.0] to
[0276.0.0.0]
[7395] [0277.0.16.16] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously.
[7396] [0278.0.0.16] to [0282.0.0.16]: see [0278.0.0.0] to
[0282.0.0.0]
[7397] [0283.0.16.16] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells, for example using the antibody
of the present invention as described below, e.g. an antibody
against a protein as indicated in Table II, column 3, lines 190 to
226 or lines 564 to 594, resp., or an antibody against a
polypeptide as indicated in Table II, columns 5 or 7, lines 190 to
226 or lines 564 to 594, resp., or an antigenic part thereof which
can be produced by standard techniques utilizing polypeptides
comprising or consisting of above mentioned sequences, e.g. the
polypeptid of the present invention or fragment thereof. Preferred
are monoclonal antibodies specifically binding to polypeptide as
indicated in Table II, columns 5 or 7, lines 190 to 226 or lines
564 to 594.
[7398] [0284.0.0.16] see [0284.0.0.0]
[7399] [0285.0.16.16] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 190 to 226 or lines 564 to 594, resp., or
as coded by a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, resp., or
functional homologues thereof.
[7400] [0286.0.16.16] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, columns 7, lines 190 to 226 or lines 564 to 594 and in
one another embodiment, the present invention relates to a
polypeptide comprising or consisting of a consensus sequence as
indicated in Table IV, columns 7, lines 190 to 226 or lines 564 to
594 whereby 20 or less, preferably 15 or 10, preferably 9, 8, 7, or
6, more preferred 5 or 4, even more preferred 3, even more
preferred 2, even more preferred 1, most preferred 0 of the amino
acids positions indicated can be replaced by any amino acid or, in
an further embodiment, can be replaced and/or absent. In one
embodiment, the present invention relates to the method of the
present invention comprising a poylpeptide or to a polypeptide
comprising more than one consensus sequences as indicated in Table
IV, column 7, ines 190 to 226 or lines 564 to 594.
[7401] [0287.0.0.16] to [0289.0.0.16]: see [0287.0.0.0] to
[0289.0.0.0]
[7402] [00290.0.16.16] The alignment was performed with the
Software AlignX (Sep. 25, 2002) a component of Vector NTI Suite
8.0, InforMax.TM., Invitrogen.TM. life science software, U.S. Main
Office, 7305 Executive Way, Frederick, Md. 21704, USA with the
following settings: For pair wise alignments: gap opening penalty:
10.0; gap extension penalty 0.1. For multiple alignments: Gap
opening penalty: 10.0; Gap extension penalty: 0.1; Gap separation
penalty range: 8; Residue substitution matrix: blosum62;
Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0.5;
Consensus calculation options: Residue fraction for consensus: 0.9.
Presettings were selected to allow also for the alignment of
conserved amino acids
[7403] [0291.0.16.16] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of
plant or microorganism specific consensus sequences.
[7404] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table II A or II B,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, resp., by one
or more amino acids. In one embodiment, polypeptide distinguishes
from a sequence as indicated in Table II A or II B, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, resp., by more than 5, 6, 7,
8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or 30
amino acids, even more preferred are more than 40, 50, or 60 amino
acids and, preferably, the sequence of the polypeptide of the
invention distinguishes from a sequence as indicated in Table II A
or II B, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
resp., by not more than 80% or 70% of the amino acids, preferably
not more than 60% or 50%, more preferred not more than 40% or 30%,
even more preferred not more than 20% or 10%. In an other
embodiment, said polypeptide of the invention does not consist of a
sequence as indicated in Table II A or II B, columns 5 or 7, lines
190 to 226 or lines 564 to 594.
[7405] [0292.0.0.16] see [0292.0.0.0]
[7406] [0293.0.16.16] In one embodiment, the invention relates to a
polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or by a nucleic acid molecule used in the process of the
invention. In one embodiment, the polypeptide of the invention has
a sequence which distinguishes from a sequence as indicated in
Table II A or II B, columns 5 or 7, lines 190 to 226 or lines 564
to 594, resp., by one or more amino acids. In an other embodiment,
said polypeptide of the invention does not consist of the sequence
as indicated in Table II A or II B, columns 5 or 7, lines 190 to
226 or lines 564 to 594, resp.
[7407] In a further embodiment, said polypeptide of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by a nucleic acid molecules as indicated in
Table I A or I B, columns 5 or 7, lines 190 to 226 or lines 564 to
594, resp.
[7408] [0294.0.16.16] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 190 to 226 or lines 564 to
594, resp., which distinguishes over a sequence as indicated in
Table II A or II B, columns 5 or 7, lines 190 to 226 or lines 564
to 594, resp., by one or more amino acids, preferably by more than
5, 6, 7, 8 or 9 amino acids, preferably by more than 10, 15, 20, 25
or 30 amino acids, even more preferred are more than 40, 50, or 60
amino acids but even more preferred by less than 70% of the amino
acids, more preferred by less than 50%, even more preferred my less
than 30% or 25%, more preferred are 20% or 15%, even more preferred
are less than 10%.
[7409] [0295.0.0.16] to [0296.0.0.16]: see [0295.0.0.0] to
[0296.0.0.0]
[7410] [0297.0.0.16] see [0297.0.0.0]
[7411] [00297.1.0.16] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity and/or amino acid
sequence of a polypeptide indicated in Table II, columns 3, 5 or 7,
lines 190 to 226 or lines 564 to 594
[7412] [0298.0.16.16] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table II, columns 5 or 7, lines 190 to 226 or lines
564 to 594, resp. The portion of the protein is preferably a
biologically active portion as described herein. Preferably, the
polypeptide used in the process of the invention has an amino acid
sequence identical to a sequence as indicated in Table II, columns
5 or 7, lines 190 to 226 or lines 564 to 594, resp.
[7413] [0299.0.16.16] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
190 to 226 or lines 564 to 594, resp. The preferred polypeptide of
the present invention preferably possesses at least one of the
activities according to the invention and described herein. A
preferred polypeptide of the present invention includes an amino
acid sequence encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions, to a nucleotide
sequence as indicated in Table I, columns 5 or 7, lines 190 to 226
or lines 564 to 594, resp., or which is homologous thereto, as
defined above.
[7414] [0300.0.16.16] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, resp., in
amino acid sequence due to natural variation or mutagenesis, as
described in detail herein. Accordingly, the polypeptide comprise
an amino acid sequence which is at least about 35%, 40%, 45%, 50%,
55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or
90, and more preferably at least about 91%, 92%, 93%, 94% or 95%,
and most preferably at least about 96%, 97%, 98%, 99% or more
homologous to an entire amino acid sequence of as indicated in
Table II A or B, columns 5 or 7, lines 190 to 226 or lines 564 to
594, resp.
[7415] [0301.0.0.16] see [0301.0.0.0]
[7416] [0302.0.16.16] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
resp., or the amino acid sequence of a protein homologous thereto,
which include fewer amino acids than a full length polypeptide of
the present invention or used in the process of the present
invention or the full length protein which is homologous to an
polypeptide of the present invention or used in the process of the
present invention depicted herein, and exhibit at least one
activity of polypeptide of the present invention or used in the
process of the present invention.
[7417] [0303.0.0.16] see [0303.0.0.0]
[7418] [0304.0.16.16] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 190 to 226 or lines 564 to 594 but having
differences in the sequence from said wild-type protein. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity in relation to the wild type protein.
[7419] [0305.0.0.16] to [0308.0.0.16]: see [0305.0.0.0] to
[0308.0.0.0]
[7420] [00306.1.0.0] In one embodiment, the compound is a
composition comprising the repective fine chemical, i.e. said
organic acids, or a recovered fine chemical, i.e. said organic acid
free or in protein-bound form.
[7421] [0309.0.16.16] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table II,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, resp., refers
to a polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas an "other polypeptide" not being indicated in
Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
resp., refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to
a polypeptide of the invention, preferably which is not
substantially homologous to a polypeptide as indicated in Table II,
columns 5 or 7, lines 190 to 226 or lines 564 to 594, resp., e.g.,
a protein which does not confer the activity described herein or
annotated or known for as indicated in Table II, column 3, lines
190 to 226 or lines 564 to 594, resp., and which is derived from
the same or a different organism. In one embodiment, an "other
polypeptide" not being indicated in Table II, columns 5 or 7, lines
190 to 226 or lines 564 to 594, resp., does not confer an increase
of the respective fine chemical in an organism or part thereof. In
one embodiment, a "non-polypeptide of the invention" or "other
polypeptide" not being indicated in Table II, columns 5 or 7, lines
190 to 226 or lines 564 to 594, resp., does not confer an increase
of the respective fine chemical in an organism or part thereof.
[7422] [0310.0.0.16] to [0334.0.0.16] but[0318.0.18.18]: see
[0310.0.0.0] to [0334.0.0.0]
[7423] [0318.0.18.18] In an especially preferred embodiment, the
polypeptide according to the invention furthermore also does not
have the sequences of those proteins which are encoded by a
sequences shown in table II, lines 190 to 226 or lines 564 to
594.
[7424] [0335.0.16.16] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table I, columns 5 or
7, lines 190 to 226 or lines 564 to 594, resp., and/or homologs
thereof. As described inter alia in WO 99/32619, dsRNAi approaches
are clearly superior to traditional antisense approaches. The
invention therefore furthermore relates to double-stranded RNA
molecules (dsRNA molecules) which, when introduced into an
organism, advantageously into a plant (or a cell, tissue, organ or
seed derived there from), bring about altered metabolic activity by
the reduction in the expression of a nucleic acid sequences as
indicated in Table I, columns 5 or 7, lines 190 to 226 or lines 564
to 594, resp., and/or homologs thereof. In a double-stranded RNA
molecule for reducing the expression of a protein encoded by a
nucleic acid sequence as indicated in Table I, columns 5 or 7,
lines 190 to 226 or lines 564 to 594, resp., and/or homologs
thereof, one of the two RNA strands is essentially identical to at
least part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[7425] [0336.0.0.16] to [0342.0.0.16]: see [0336.0.0.0] to
[0342.0.0.0]
[7426] [0343.0.16.16] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 190 to 226
or lines 564 to 594, resp., or its homolog is not necessarily
required in order to bring about effective reduction in the
expression. The advantage is, accordingly, that the method is
tolerant with regard to sequence deviations as may be present as a
consequence of genetic mutations, polymorphisms or evolutionary
divergences. Thus, for example, using the dsRNA, which has been
generated starting from a sequence as indicated in Table I, columns
5 or 7, lines 190 to 226 or lines 564 to 594, resp., or homologs
thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[7427] [0344.0.0.16] to [0361.0.0.16]: see [0344.0.0.0] to
[0361.0.0.0]
[7428] [0362.0.16.16] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
resp., e.g. encoding a polypeptide having protein activity, as
indicated in Table II, columns 3, lines 190 to 226 or lines 564 to
594, resp., Due to the abovementioned activity the respective fine
chemical content in a cell or an organism is increased. For
example, due to modulation or manipulation, the cellular activity
of the polypeptide of the invention or nucleic acid molecule of the
invention or the nucleic acid molecule or polypeptide used in the
method of the invention is increased, e.g. due to an increased
expression or specific activity of the subject matters of the
invention in a cell or an organism or a part thereof. Transgenic
for a polypeptide having an activity of a polypeptide as indicated
in Table II, columns 5 or 7, lines 190 to 226 or lines 564 to 594,
resp., means herein that due to modulation or manipulation of the
genome, an activity as annotated for a polypeptide as indicated in
Table II, column 3, lines 190 to 226 or lines 564 to 594, e.g.
having a sequence as indicated in Table II, columns 5 or 7, lines
190 to 226 or lines 564 to 594, resp., is increased in a cell or an
organism or a part thereof. Examples are described above in context
with the process of the invention.
[7429] [0363.0.0.16] see [0363.0.0.0]
[7430] [0364.0.16.16] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table II, column 3, lines 190 to 226 or lines 564 to
594, resp. with the corresponding protein-encoding sequence as
indicated in Table I, column 5, lines 190 to 226 or lines 564 to
594, resp., becomes a transgenic expression cassette when it is
modified by non-natural, synthetic "artificial" methods such as,
for example, mutagenization. Such methods have been described (U.S.
Pat. No. 5,565,350; WO 00/15815; also see above).
[7431] [0365.0.0.16] to [0373.0.0.16]: see [0365.0.0.0] to
[0373.0.0.0]
[7432] [0374.0.16.16] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid, in
particular the respective fine chemical, produced in the process
according to the invention may, however, also be isolated from the
plant in the form of their free glyceric acid, citramalic acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid, in
particular the free respective fine chemical, or bound in or to
compounds or moieties, like glucosides, e.g. diglucosides. The
respective fine chemical produced by this process can be harvested
by harvesting the organisms either from the culture in which they
grow or from the field.
[7433] This can be done via expressing, grinding and/or extraction,
salt precipitation and/or ion-exchange chromatography or other
chromatographic methods of the plant parts, preferably the plant
seeds, plant fruits, plant tubers and the like.
[7434] [0375.0.0.16] to [0376.0.0.16]: see [0375.0.0.0] to
[0376.0.0.0]
[7435] [0377.0.16.16] Accordingly, the present invention relates
also to a process whereby the produced glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid is
isolated.
[7436] [0378.0.16.16] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the glyceric
acid, citramalic acid, fumaric acid, malic acid, pyruvic acid,
succinic acid, trihydroxybutyric acid and/or trihydroxybutanoic
acid produced in the process can be isolated. The resulting
glyceric acid, citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid can, if appropriate, subsequently be
further purified, if desired mixed with other active ingredients
such as vitamins, amino acids, carbohydrates, antibiotics and the
like, and, if appropriate, formulated.
[7437] [0379.0.16.16] In one embodiment, gamma-aminobutyric acid
and shikimate are a mixture of the respective fine chemicals.
[7438] [0380.0.16.16] The glyceric acid, citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid and/or trihydroxybutanoic acid obtained in the process are
suitable as starting material for the synthesis of further products
of value. For example, they can be used in combination with each
other or alone for the production of pharmaceuticals, foodstuffs,
animal feeds or cosmetics. Accordingly, the present invention
relates a method for the production of pharmaceuticals, food stuff,
animal feeds, nutrients or cosmetics comprising the steps of the
process according to the invention, including the isolation of the
glyceric acid, citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid composition produced or the respective fine
chemical produced if desired and formulating the product with a
pharmaceutical acceptable carrier or formulating the product in a
form acceptable for an application in agriculture. A further
embodiment according to the invention is the use of the glyceric
acid, citramalic acid, fumaric acid, malic acid, pyruvic acid,
succinic acid, trihydroxybutyric acid and/or trihydroxybutanoic
acid produced in the process or of the transgenic organisms in
animal feeds, foodstuffs, medicines, food supplements, cosmetics or
pharmaceuticals or for the production of glyceric acid, citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid e.g. after
isolation of the respective fine chemical or without, e.g. in situ,
e.g. in the organism used for the process for the production of the
respective fine chemical.
[7439] [0381.0.0.16] to [0382.0.0.16]: see [0381.0.0.0] to
[0382.0.0.0]
[7440] [0383.0.16.16] -/-
[7441] [0384.0.0.16] see [0384.0.0.0]
[7442] [0385.0.16.16] The fermentation broths obtained in this way,
containing in particular glyceric acid, citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid and/or trihydroxybutanoic acid in mixtures with other organic
acids, amino acids, polypeptides or polysaccarides, normally have a
dry matter content of from 1 to 70% by weight, preferably 7.5 to
25% by weight. Sugar-limited fermentation is additionally
advantageous, e.g. at the end, for example over at least 30% of the
fermentation time. This means that the concentration of utilizable
sugar in the fermentation medium is kept at, or reduced to, 0 to 10
g/l, preferably to 0 to 3 g/I during this time. The fermentation
broth is then processed further. Depending on requirements, the
biomass can be removed or isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth or left completely in it.
[7443] The fermentation broth can then be thickened or concentrated
by known methods, such as, for example, with the aid of a rotary
evaporator, thin-film evaporator, falling film evaporator, by
reverse osmosis or by nanofiltration. This concentrated
fermentation broth can then be worked up by freeze-drying, spray
drying, spray granulation or by other processes.
[7444] [0386.0.16.16] Accordingly, it is possible to purify the
glyceric acid, citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid produced according to the invention
further. For this purpose, the product-containing composition is
subjected for example to separation via e.g. an open column
chromatography or HPLC in which case the desired product or the
impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use.
[7445] [0387.0.0.16] to [0392.0.0.16]: see [0387.0.0.0] to
[0392.0.0.0]
[7446] [0393.0.16.16] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [7447] a) contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [7448] b) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table I, columns 5 or 7, lines
190 to 226 or lines 564 to 594, preferably in Table I B, columns 5
or 7, ines 190 to 226 or lines 564 to 594, resp., and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[7449] c) introducing the candidate nucleic acid molecules in host
cells, preferably in a plant cell or a microorganism, appropriate
for producing the respective fine chemical; [7450] d) expressing
the identified nucleic acid molecules in the host cells; [7451] e)
assaying the respective fine chemical level in the host cells; and
[7452] f) identifying the nucleic acid molecule and its gene
product which expression confers an increase in the respective fine
chemical level in the host cell after expression compared to the
wild type.
[7453] [0394.0.0.16] to [0398.0.0.16]: see [0394.0.0.0] to
[0398.0.0.0]
[7454] [0399.0.16.16] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increase of the respective fine chemical production in a
plant or microorganism, comprising the steps:
[7455] culturing a cell or tissue or microorganism or maintaining a
plant expressing the polypeptide according to the invention or a
nucleic acid molecule encoding said polypeptide and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and the polypeptide of the present invention or used
in the process of the invention; and identifying if the compound is
an effective agonist by detecting the presence or absence or
increase of a signal produced by said readout system.
[7456] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in respective fine chemical
production.
[7457] [00399.1.0.16] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 190 to 226 or lines 564 to 594 or a homolog
thereof, e.g. comparing the phenotype of nearly identical organisms
with low and high activity of a protein as indicated in Table II,
columns 5 or 7, lines 190 to 226 or lines 564 to 594 after
incubation with the drug.
[7458] [0400.0.0.16] to [0415.0.0.16]: see [0400.0.0.0] to
[0415.0.0.0]
[7459] [0416.0.0.16] see [0416.0.0.0]
[7460] [0417.0.16.16] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the glyceric acid, citramalic acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect an organism such as a
microorganism or a plant against inhibitors, which block the
glyceric acid, citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid synthesis.
[7461] [0418.0.0.16] to [0423.0.0.16]: see [0418.0.0.0] to
[0423.0.0.0]
[7462] [0424.0.16.16] Accordingly, the nucleic acid of the
invention or used in the method of the invention, the polypeptide
of the invention or used in the method of the invention, the
nucleic acid construct of the invention, the organisms, the host
cell, the microorganisms, the plant, plant tissue, plant cell, or
the part thereof of the invention, the vector of the invention, the
agonist identified with the method of the invention, the nucleic
acid molecule identified with the method of the present invention,
can be used for the production of the respective fine chemical or
of the respective fine chemical and one or more other organic
acids. Accordingly, the nucleic acid of the invention, or the
nucleic acid molecule identified with the method of the present
invention or the complement sequences thereof, the polypeptide of
the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the respective fine chemical in a organism or part
thereof, e.g. in a cell.
[7463] [0425.0.0.16] to [0434.0.0.0]: see [0425.0.0.0] to
[0434.0.0.0]
[0435.0.16.16] Example 3
In-Vivo and In-Vitro Mutagenesis
[7464] [0436.0.16.16] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of glyceric acid,
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid and/or trihydroxybutanoic acid can be
carried out by passing a plasmid DNA (or another vector DNA)
containing the desired nucleic acid sequence or nucleic acid
sequences, e.g. the nucleic acid molecule of the invention or the
vector of the invention, through E. coli and other microorganisms
(for example Bacillus spp. or yeasts such as Saccharomyces
cerevisiae) which are not capable of maintaining the integrity of
its genetic information. Usual mutator strains have mutations in
the genes for the DNA repair system [for example mutHLS, mutD, mutT
and the like; for comparison, see Rupp, W. D. (1996) DNA repair
mechanisms in Escherichia coli and Salmonella, pp. 2277-2294, ASM:
Washington]. The skilled worker knows these strains. The use of
these strains is illustrated for example in Greener, A. and
Callahan, M. (1994) Strategies 7; 32-34.
[7465] In-vitro mutation methods such as increasing the spontaneous
mutation rates by chemical or physical treatment are well known to
the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widely used as
chemical agents for random in-vitro mutagenesis. The most common
physical method for mutagenesis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[7466] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[7467] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.16.16] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces Cerevisiae and
Mortierella alpina
[7468] [0438.0.16.16] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology", VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[7469] [0439.0.16.16] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255f) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[7470] [0440.0.16.16] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002,) teaches a homolog recombination system dependent
on rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this
system for homologous recombination are vectors derived from the
Ylp series. Plasmid vectors derived for example from the
2.mu.-Vector are known by the skilled worker and used for the
expression in yeasts. Other preferred vectors are for example
pART1, pCHY21 or pEVP11 as they have been described by McLeod et
al. (EMBO J. 1987, 6:729-736) and Hoffman et al. (Genes Dev. 5,
1991: :561-571.) or Russell et al. (J. Biol. Chem. 258, 1983:
143-149.). Other beneficial yeast vectors are plasmids of the REP,
REP-X, pYZ or RIP series.
[7471] [0441.0.0.16] to [0443.0.0.16] see [0441.0.0.0] to
[0443.0.0.0]
[0444.0.16.16] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[7472] [0445.0.16.16] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[7473] [0446.0.0.16] to [0450.0.0.16]: see [0446.0.0.0] to
[0450.0.0.0]
[7474] [0451.0.0.16] see [0451.0.5.5]
[7475] [0452.0.0.16] to [0453.0.0.16]: see [0452.0.0.0] to
[0453.0.0.0]
[7476] [0454.0.16.16] Analysis of the effect of the nucleic acid
molecule on the production of glyceric acid, citramalic acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid
[7477] [0455.0.16.16] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a glyceric acid, citramalic acid, fumaric acid,
malic acid, pyruvic acid, succinic acid, trihydroxybutyric acid
and/or trihydroxybutanoic acid) can be determined by growing the
modified microorganisms or the modified plant under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the elevated production of
desired product (i.e. of glyceric acid, citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid and/or trihydroxybutanoic acid). These analytical techniques
are known to the skilled worker and comprise spectroscopy,
thin-layer chromatography, various types of staining methods,
enzymatic and microbiological methods and analytical chromatography
such as high-performance liquid chromatography (see, for example,
Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, p. 89-90 and
p. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987)
"Applications of HPLC in Biochemistry" in: Laboratory Techniques in
Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993)
Biotechnology, Vol. 3, Chapter III: "Product recovery and
purification", p. 469-714, VCH: Weinheim; Belter, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[7478] [0456.0.0.16]: see [0456.0.0.0]
[0457.0.16.16] Example 9
Purification of Glyceric Acid, Citramalic Acid, Fumaric Acid, Malic
Acid, Pyruvic Acid, Succinic Acid, Trihydroxybutyric Acid or
Trihydroxybutanoic Acid
[7479] [0458.0.16.16] Abbreviations; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[7480] The unambiguous detection for the presence of glyceric acid,
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid or trihydroxybutanoic acid can be
obtained by analyzing recombinant organisms using analytical
standard methods: LC, LC-MSMS or TLC, as described. The total
amount produced in the organism for example in yeasts used in the
inventive process can be analysed for example according to the
following procedure:
[7481] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods.
[7482] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[7483] A typical sample pre-treatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography.
[7484] For analysis, solvent delivery and aliquot removal can be
accomplished with a robotic system comprising a single injector
valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000
W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of
50% potassium hydroxide hydro-ethanolic solution (4 water--1
ethanol) can be added to each vial, followed by the addition of 3
ml of octanol. The saponification treatment can be conducted at
room temperature with vials maintained on an IKA HS 501 horizontal
shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours
at 250 movements/minute, followed by a stationary phase of
approximately one hour.
[7485] Following saponification, the supernatant can be diluted
with 0.17 ml of methanol. The addition of methanol can be conducted
under pressure to ensure sample homogeneity. Using a 0.25 ml
syringe, a 0.1 ml aliquot can be removed and transferred to HPLC
vials for analysis.
[7486] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete
with a quaternary pump, vacuum degassing system, six-way injection
valve, temperature regulated autosampler, column oven and
Photodiode Array detector can be used [Agilent Technologies
available through Ultra Scientific Inc., 250 Smith Street, North
Kingstown, R.I.]. The column can be a Waters YMC30, 5-micron,
4.6.times.250 mm with a guard column of the same material [Waters,
34 Maple Street, Milford, Mass.]. The solvents for the mobile phase
can be 81 methanol: 4 water: 15 tetrahydrofuran (THF) stabilized
with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injections were
20 l. Separation can be isocratic at 30.degree. C. with a flow rate
of 1.7 ml/minute. The peak responses can be measured by absorbance
at 447 nm.
[7487] [0459.0.16.16] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the glyceric acid, citramalic acid, fumaric acid,
malic acid, pyruvic acid, succinic acid, trihydroxybutyric acid
and/or trihydroxybutanoic acid can be further purified with a
so-called RTHPLC. As eluent acetonitrile/water or
chloroform/acetonitrile mixtures can be used. If necessary, these
chromatography steps may be repeated, using identical or other
chromatography resins. The skilled worker is familiar with the
selection of suitable chromatography resin and the most effective
use for a particular molecule to be purified.
[7488] [0460.0.0.16] see [0460.0.0.0]
[0461.0.16.16] Example 10
Cloning SEQ ID NO: 17198, 17818, 16100, 17898, 15872, 15848, 15780,
18092, 18380, 17104, 16824, 17374, 16284, 16410, 17712, 16132,
16128, 16118, 16014, 17920, 17948, 17198, 17038 or 18088 or Others
as Indicated, for the Expression in Plants
[7489] [0462.0.0.16] see [0462.0.0.0]
[7490] [0463.0.16.16] SEQ ID NO: 17198, 17818, 16100, 17898, 15872,
15848, 15780, 18092, 18380, 17104, 16824, 17374, 16284, 16410,
17712, 16132, 16128, 16118, 16014, 17920, 17948, 17198, 17038 or
18088 or others as indicated, is amplified by PCR as described in
the protocol of the Pfu Turbo or DNA Herculase polymerase
(Stratagene).
[7491] [0464.0.0.0.16] to [0466.0.0.16]: see [0464.0.0.0] to
[0466.0.0.0]
[7492] [0466.1.0.16] In case the Herculase enzyme can be used for
the amplification, the PCR amplification cycles were as follows: 1
cycle of 2-3 minutes at 94.degree. C., followed by 25-30 cycles of
in each case 30 seconds at 94.degree. C., 30 seconds at
55-60.degree. C. and 5-10 minutes at 72.degree. C., followed by 1
cycle of 10 minutes at 72.degree. C., then 4.degree. C.
[7493] [0467.0.16.16] The following primer sequences were selected
for the gene SEQ ID NO: 17198:
TABLE-US-00052 i) forward primer (SEQ ID NO: 17372) atgagtcgtt
tagtcgtagt atcta ii) reverse primer (SEQ ID NO: 17373) ttacgcaagc
tttggaaagg tagc
[7494] The following primer sequences were selected for the gene
SEQ ID NO: 17818:
TABLE-US-00053 i) forward primer (SEQ ID NO: 17896) atgacaattt
ctaatttgtt aaagcag ii) reverse primer (SEQ ID NO: 17897) ctagtcaact
ggttcccagc tgt
[7495] The following primer sequences were selected for the gene
SEQ ID NO: 16100
TABLE-US-00054 i) forward primer (SEQ ID NO: 16116) atggatgacg
atcacgaaca gttg ii) reverse primer (SEQ ID NO: 16117) ttacgagtcg
aagccggccc t
[7496] The following primer sequences were selected for the gene
SEQ ID NO: 17898:
TABLE-US-00055 i) forward primer (SEQ ID NO: 17918) atggtcgata
cgcataaact agca ii) reverse primer (SEQ ID NO: 17919) ctagtcgttg
cggatatgga cga
[7497] The following primer sequences were selected for the gene
SEQ ID NO: 15872:
TABLE-US-00056 i) forward primer (SEQ ID NO: 16012) atgacaatgg
aaaaaaatgg aggtaa ii) reverse primer (SEQ ID NO: 16013) ttatttagca
caatcctcgt gagc
[7498] The following primer sequences were selected for the gene
SEQ ID NO: 15848:
TABLE-US-00057 i) forward primer (SEQ ID NO: 15870) atgaatttgt
tttggccatc ggaaa ii) reverse primer (SEQ ID NO: 15871) tcaaggttgt
actgtgtgat ctag
[7499] The following primer sequences were selected for the gene
SEQ ID NO: 15780:
TABLE-US-00058 i) forward primer (SEQ ID NO: 15846) atgccatcta
ccactaatac tgct ii) reverse primer (SEQ ID NO: 15847) ctaatgtttg
gcaactgggg tttc
[7500] The following primer sequences were selected for the gene
SEQ ID NO: 18092:
TABLE-US-00059 i) forward primer (SEQ ID NO: 18378) atgaacgttt
caaaaatact tgtgtc ii) reverse primer (SEQ ID NO: 18379) tcatgcattt
aacattgtag gaatttt
[7501] The following primer sequences were selected for the gene
SEQ ID NO: 18380:
TABLE-US-00060 i) forward primer (SEQ ID NO: 18640) atggctcggg
gtgacggaca t ii) reverse primer (SEQ ID NO: 18641) tcatgcttct
tttgcgtgat gcaat
[7502] The following primer sequences were selected for the gene
SEQ ID NO: 17104:
TABLE-US-00061 i) forward primer (SEQ ID NO: 17196) atgggacaca
agcccttata ccg ii) reverse primer (SEQ ID NO: 17197) ttatcgcgat
gattttcgct gcg
[7503] The following primer sequences were selected for the gene
SEQ ID NO: 16824:
TABLE-US-00062 i) forward primer (SEQ ID NO: 17036) atgaatacag
tacgcagcga aaa ii) reverse primer (SEQ ID NO: 17037) ttaacgcccg
gctttcatac tgc
[7504] The following primer sequences were selected for the gene
SEQ ID NO: 17374:
TABLE-US-00063 i) forward primer (SEQ ID NO: 17710) atggctatcg
acgaaaacaa acag ii) reverse primer (SEQ ID NO: 17711) ttaaaaatct
tcgttagttt ctgctac
[7505] The following primer sequences were selected for the gene
SEQ ID NO: 16284:
TABLE-US-00064 i) forward primer (SEQ ID NO: 16408) atgtgtttaa
agcaaatcat tggcag ii) reverse primer (SEQ ID NO: 16409) ttactgttcg
ctttcatcag tatag
[7506] The following primer sequences were selected for the gene
SEQ ID NO: 16410:
TABLE-US-00065 i) forward primer (SEQ ID NO: 16822) atgaaaaaga
ccaaaattgt ttgca ii) reverse primer (SEQ ID NO: 16823) ttacaggacg
tgaacagatg cgg
[7507] The following primer sequences were selected for the gene
SEQ ID NO: 17712:
TABLE-US-00066 i) forward primer (SEQ ID NO: 17816) atggaaaaga
aattaccccg catta ii) reverse primer (SEQ ID NO: 17817) ttagttttgt
tcatcttcca gcaag
[7508] The following primer sequences were selected for the gene
SEQ ID NO: 17818:
TABLE-US-00067 i) forward primer (SEQ ID NO: 17896) atgacaattt
ctaatttgtt aaagcag ii) reverse primer (SEQ ID NO: 17897) ctagtcaact
ggttcccagc tgt
[7509] The following primer sequences were selected for the gene
SEQ ID NO: 16132:
TABLE-US-00068 i) forward primer (SEQ ID NO: 16282) atgttgtcga
gactatcttt attgag ii) reverse primer (SEQ ID NO: 16283) ttaaaataga
ccttcaattt caccgt
[7510] The following primer sequences were selected for the gene
SEQ ID NO: 16128:
TABLE-US-00069 i) forward primer (SEQ ID NO: 16130) atgtaccaaa
ataatgtatt gaatgct ii) reverse primer (SEQ ID NO: 16131) tcaatagtgc
attaactctc ccatt
[7511] The following primer sequences were selected for the gene
SEQ ID NO: 16118:
TABLE-US-00070 i) forward primer (SEQ ID NO: 16126) atggaggacg
gtaaacaggc cat ii) reverse primer (SEQ ID NO: 16127) ttagagtctt
ccaccggggg tg
[7512] The following primer sequences were selected for the gene
SEQ ID NO: 16014:
TABLE-US-00071 i) forward primer (SEQ ID NO: 16098) atgacggtaa
acttagatcc ggata ii) reverse primer (SEQ ID NO: 16099) ttatatggac
attccctttt tttggt
[7513] The following primer sequences were selected for the gene
SEQ ID NO: 17920:
TABLE-US-00072 i) forward primer (SEQ ID NO: 17946) atgtcactac
cggcacattt gca ii) reverse primer (SEQ ID NO: 17947) ttaaatattg
aattcttctt catcatttt
[7514] The following primer sequences were selected for the gene
SEQ ID NO: 17948:
TABLE-US-00073 i) forward primer (SEQ ID NO: 18086) atggatgata
taagcggaag gcaaa ii) reverse primer (SEQ ID NO: 18087) ctatactggc
aagtgacagt tgtg
[7515] The following primer sequences were selected for the gene
SEQ ID NO: 17038:
TABLE-US-00074 i) forward primer (SEQ ID NO: 17102) atgaataacg
aacccttacg tccc ii) reverse primer (SEQ ID NO: 17103) ttacatatcc
tcatgaaatt cttcaagt
[7516] The following primer sequences were selected for the gene
SEQ ID NO: 16118:
TABLE-US-00075 i) forward primer (SEQ ID NO: 16126 ) atggaggacg
gtaaacaggc cat ii) reverse primer (SEQ ID NO: 16127) ttagagtctt
ccaccggggg tg
[7517] The following primer sequences were selected for the gene
SEQ ID NO: 18088:
TABLE-US-00076 i) forward primer (SEQ ID NO: 18090) atgatagagg
cgttggaaat agttc ii) reverse primer (SEQ ID NO: 18091) ttatatcgat
cctgcgatat aataac
[7518] Further primers for the cloning of the nucleic acids
indicated in table II, column 5 see table III, column 7, resp.,
[7519] [0468.0.16.16] to [0470.0.16.16]: see [0468.0.0.0] to
[0470.0.0.0]
[7520] [0470.1.16.16] The PCR-products, produced by Pfu Turbo DNA
polymerase, were phosphorylated using a T4 DNA polymerase using a
standard protocol (e.g. MBI Fermentas) and cloned into the
processed binary vector.
[7521] [0471.0.16.16] see [0471.0.0.0]
[7522] [0471.1.16.16] The DNA termini of the PCR-products, produced
by Herculase DNA polymerase, were blunted in a second synthesis
reaction using Pfu Turbo DNA polymerase. The composition for the
protocol of the blunting the DNA-termini was as follows: 0.2 mM
blunting dTTP and 1.25 u Pfu Turbo DNA polymerase. The reaction was
incubated at 72.degree. C. for 30 minutes. Then the PCR-products
were phosphorylated using a T4 DNA polymerase using a standard
protocol (e.g. MBI Fermentas) and cloned into the processed vector
as well.
[7523] [0472.0.16.16] to [0479.0.16.16]: see [0472.0.0.0] to
[0479.0.0.0]
[0480.0.16.16] Example 11
Generation of Transgenic Plants which Express SEQ ID NO:17198,
17818, 16100, 17898, 15872, 15848, 15780, 18092, 18380, 17104,
16824, 17374, 16284, 16410, 17712, 16132, 16128, 16118, 16014,
17920, 17948, 17198, 17038 or 18088 or as Indicated in Table I
Column 5
[7524] [0481.0.0.16] to [0513.0.0.16]: see [0481.0.0.0] to
[0513.0.0.0]
[7525] [0514.0.16.16] As an alternative, the organic acids can be
detected as described in Farre, E. et al., Plant Physiol, 2001,
Vol. 127, pp. 685-700.
[7526] The results of the different plant analyses can be seen from
the table 1 which follows:
TABLE-US-00077 TABLE 1 ORF Metabolite Method Min Max b1896
citramalic acid GC 1.63 1.99 YBL015W fumaric acid LC 1.50 2.26
YCR059C fumaric acid GC 1.71 2.83 YFR007W fumaric acid GC 1.69 2.56
YJL055W fumaric acid GC 1.72 6.61 YJL099W fumaric acid GC 1.98 8.34
YMR241W fumaric acid GC + LC 1.75 4.68 YPR024W fumaric acid GC 1.97
4.74 YPR138C fumaric acid LC 1.61 4.27 b0730 fumaric acid GC + LC
1.75 9.38 b1611 fumaric acid GC + LC 2.06 4.28 b2699 fumaric acid
GC 1.65 9.02 b4139 fumaric acid GC + LC 2.18 6.25 b1676 fumaric
acid GC 2.08 5.30 b1896 fumaric acid GC + LC 3.32 4.20 b4063
fumaric acid GC 1.64 4.08 YBL015W malic acid LC 1.45 1.86 YBR084W
malic acid GC 2.07 5.48 YBR184W malic acid LC 1.67 3.13 YCL032W
malic acid LC 1.87 2.23 YGR007W malic acid LC 1.55 1.90 YJL072C
malic acid GC 1.49 3.49 YJL099W malic acid GC 1.94 8.97 YKL132C
malic acid GC 2.09 3.13 YPR138C malic acid GC 1.72 4.59 b0730 malic
acid GC + LC 1.96 7.72 b1896 malic acid GC 2.49 4.19 b4063 malic
acid GC 1.78 1.93 b2699 trihydroxybutyric acid GC 1.48 3.15 b1896
trihydroxybutyric acid GC 1.34 1.96 b0695 pyruvic acid GC 1.61 2.55
YCL032W succinic acid LC 1.47 1.91 YOR044W succinic acid LC 1.45
3.65 b0730 succinic acid LC 1.51 4.32 b1896 succinic acid GC + LC
1.55 2.10 b2699 Trihydroxybutanoic acid GC 1.82 3.81 b1896
Trihydroxybutanoic acid GC 1.60 2.61 b1693 Citramalic acid GC 1.48
2.05 b0161 Fumaric acid LC 1.64 2.51 b1738 Fumaric acid LC 1.53
2.49 b1961 Fumaric acid GC + LC 1.76 2.05 b3116 Fumaric acid GC
1.63 1.99 b3172 Fumaric acid GC 1.63 4.25 b4122 Fumaric acid GC
2.08 5.30 YCR012W Fumaric acid LC 1.62 1.93 b0057 Glyceric acid GC
1.35 1.57 b1693 Glyceric acid GC 1.33 1.52 b3129 Glyceric acid GC
1.30 1.35 b3231 Glyceric acid GC 1.21 1.34 b3938 Glyceric acid GC
1.37 1.40 b0161 Malic acid GC 1.64 3.29 b2478 Malic acid GC + LC
1.51 2.24 b3116 Malic acid GC 1.60 3.12 b3169 Malic acid GC 2.32
2.43 b3172 Malic acid GC 1.64 5.08 YCL038C Malic acid LC 1.87 2.23
YOR168W Malic acid GC + LC 1.56 6.62 b3231 Pyruvic acid GC 1.43
1.46 b1738 Succinic acid LC 1.49 1.95 b1961 Succinic acid LC 1.29
1.54 b2599 Succinic acid GC + LC 1.33 2.56 b3116 Succinic acid LC
1.31 1.42 b3160 Succinic acid LC 1.33 1.90 b4129 Succinic acid LC
1.40 1.84 YCL038C Succinic acid LC 1.47 1.91 b0970
Trihydroxybutanoic acid GC 1.34 1.80 b1343 Trihydroxybutanoic acid
GC 1.33 2.08 b3169 Trihydroxybutanoic acid GC 1.60 1.88
[7527] Column 2 shows the organic acids citramalic acid, fumaric
acid, malic acid, trihydroxybutyric acid, trihydroxybutanoic acid,
succinic acid, pyruvic acid as analyzed. Columns 4 and 5 show the
ratio of the analyzed organic acid between the transgenic plants
and the wild type; Increase of the metabolites: Max: maximal x-fold
(normalised to wild type)-Min: minimal x-fold (normalised to wild
type). Decrease of the metabolites: Max: maximal x-fold (normalised
to wild type) minimal decrease), Min: minimal x-fold (normalised to
wild type) (minimal decrease), Min: minimal x-fold (normalised to
wild type) maximal decrease). Column 3 indicates the analytical
method.
[7528] [0515.0.0.16] to [0552.0.0.16]: see [0515.0.0.0] to
[0552.0.0.0] including [0530.1.0.0] to [0530.6.0.0] as well as
[0552.2.0.0]
[0552.1.16.16] Example 15
Metabolite Profiling Info from Zea mays
[7529] Zea mays plants were engineered, grown and analyzed as
described in Example 14c.
[7530] The results of the different Zea mays plants analysed can be
seen from Table 2 which follows:
TABLE-US-00078 TABLE 2 ORF Metabolite Min Max b2699 Fumaric acid
1.56 10.21 b2699 Trihydroxybutanoic acid 1.43 2.03 YJL055W Fumaric
acid 2.08 2.32 YMR241W Fumaric acid 1.93 19.59 b1611 Fumaric acid
1.40 1.88 YBRO84W Malic acid 1.73 11.39 b1896 Lacton of 1.59 2.01
Trihydroxybutyric acid b0970 Trihydroxybutanoic acid, 1.56 2.32
putative b3116 Fumaric acid 1.42 3.47 b3116 Malic acid 1.33 5.30
b3172 Malic acid 1.82 2.48 YBL015W Fumaric acid 1.39 2.69 YBL015W
Malic acid 1.31 2.59 YOR044W Succinic acid 1.54 4.33
[7531] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in organic acids in
genetically modified corn plants expressing the E. coli nucleic
acid sequence b2699, b1611, b1896, b0970, b3116 or b3172 or the
Saccaromyces cerevisiae nucleic acid sequence YJL055W, YMR241W,
YBR084W, YBL015W or YOR044W resp.
[7532] In one embodiment, the activity of the Escherichia coli K12
protein b2699 or its homologs, e.g. a protein having the activity
of a recombination protein recA, preferably being involved in DNA
recombination and DNA repair, pheromone response, mating-type
determination, sex-specific proteins, nucleotide binding, is
increased in corn plants, preferably, an increase of the fine
chemical fumaric acid between 56% and 921% is conferred and/or an
increase of the fine chemical Trihydroxybutanoic acid, between 43%
and 103% is conferred.
[7533] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs, e.g. a
trehalose-6-phosphate synthase, is increased in corn plants,
conferring an increase of the fine chemical preferably of Lacton of
Trihydroxybutyric acid between 59% and 101% or more.
[7534] In one embodiment, the activity of the Escherichia coli K12
protein b1611 or its homologs, e.g. a protein of the superfamily of
fumarate hydratase, preferably being involved in C-compound,
carbohydrate catabolism, tricarboxylic-acid pathway (citrate cycle,
Krebs cycle, TCA cycle), ENERGY, biosynthesis of aspartate,
degradation of aspartate, nitrogen and sulfur utilization,
degradation of amino acids of the aspartate group, biosynthesis of
the pyruvate family (alanine, isoleucine, leucine, valine) and
d-alanine, or regulation of nitrogen and sulphur utilization,
preferably being a fumarase C (fumarate hydratase Class II), is
increased in corn plants, preferably, conferring the increase of
fumaric acid between 40% and 88% or more.
[7535] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YJL055W or its homologs, e.g. a protein of the
superfamily of yeast conserved hypothetical protein YJL055w,
preferably being involved in biosynthesis of lysine, preferably
being a Lysine decarboxylase-like protein, is increased in corn
plants, preferably, conferring the increase of fumaric acid between
108% and 132% or more.
[7536] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YMR241W or its homologs, e.g. a protein of the
superfamily of ADP,ATP carrier protein, preferably being involved
in C-compound, carbohydrate transport, and/or mitochondrial
transport, preferably being a Mitochondrial DNA replication
protein, is increased in corn plants, preferably, conferring the
increase of fumaric acid between 93% and 1859% or more.
[7537] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YBR084W or its homologs, e.g. a protein of the
superfamily "C1-tetrahydrofolate synthase, formate-tetrahydrofolate
ligase homology, methylenetetrahydrofolate dehydrogenase (NAD+)
homology", preferably being involved in amino acid biosynthesis,
purine nucleotide metabolism, biosynthesis of vitamins, cofactors,
and prosthetic groups, C-compound and carbohydrate utilization,
tRNA modification, biosynthesis of histidine, degradation of
histidine, biosynthesis of the aspartate family, degradation of
amino acids of the aspartate group, nucleotide binding, and/or
nucleotide metabolism, preferably being a C1-tetrahydrofolate
synthase, is increased in corn plants, preferably, conferring the
increase of malic acid between 73% and 1039% or more.
[7538] In one embodiment, in case the activity of the E. coli
protein b0970 or its homologs, e.g. "the activity of a glutamate
receptor", is increased in corn plants, preferably, an increase of
the fine chemical Trihydroxybutanoic acid between 56% and 132% is
conferred.
[7539] In one embodiment, in case the activity of the E. coli
protein b3116 or its homologs, e.g. "the activity of a
L-threonine/L-serine permease, anaerobically inducible (HAAAP
family)", is increased in corn plants, preferably, an increase of
the fine chemical fumaric acid between 42% and 247% is conferred
and/or an increase of the fine chemical malic acid between 33% and
430% is conferred.
[7540] In one embodiment, in case the activity of the E. coli
protein b3172 or its homologs, e.g. "the activity of an
argininosuccinic acid synthetase", is increased in corn plants,
preferably, an increase of the fine chemical malic acid between 82%
and 148% is conferred.
[7541] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YBL015W or its homologs, e.g. "the activity of
an acetyl-coA hydrolase", is increased in corn plants, preferably,
an increase of the fine chemical fumaric acid between 39% and 169%
is conferred and/or an increase of the fine chemical malic acid
between 31% and 159% is conferred.
[7542] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YOR044W or its homologs, its activity has not
been characterized yet, is increased in corn plants, preferably, an
increase of the fine chemical succinic acid between 54% and 333% is
conferred.
[7543] [00552.2.0.16] see [00552.2.0.0]
[7544] [0553.0.16.16] [7545] 1. A process for the production of
glyceric acid. citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid, which comprises a) increasing or generating the activity of a
protein as indicated in Table II, columns 5 or 7, lines 190 to 226
or lines 564 to 594 or a functional equivalent thereof in a
non-human organism or in one or more parts thereof; and b) growing
the organism under conditions which permit the production of
glyceric acid. citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid in said organism. [7546] 2. A process for the production of
glyceric acid. citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid, comprising the increasing or generating in an organism or a
part thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [7547] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 190 to
226 or lines 564 to 594 or a fragment thereof, which confers an
increase in the amount of glyceric acid. citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid or trihydroxybutanoic acid in an organism or a part thereof;
[7548] b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 190 to 226
or lines 564 to 594; [7549] c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as a result of the degeneracy of the
genetic code and conferring an increase in the amount of glyceric
acid. citramalic acid, fumaric acid, malic acid, pyruvic acid,
succinic acid, trihydroxybutyric acid or trihydroxybutanoic acid in
an organism or a part thereof; [7550] d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of glyceric acid. citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid or
trihydroxybutanoic acid in an organism or a part thereof; [7551] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to [7552] (c) under stringent hybridisation conditions and
conferring an increase in the amount of glyceric acid. citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid in an organism or
a part thereof; [7553] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, columns 5 or 7,
lines 190 to 226 or lines 564 to 594 and conferring an increase in
the amount of glyceric acid. citramalic acid, fumaric acid, malic
acid, pyruvic acid, succinic acid, trihydroxybutyric acid or
trihydroxybutanoic acid in an organism or a part thereof; [7554] g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of glyceric acid. citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid or trihydroxybutanoic acid in an organism or a part thereof;
[7555] h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table IV, columns 5 or 7, lines 190 to
226 or lines 564 to 594 and conferring an increase in the amount of
glyceric acid. citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid in an organism or a part thereof; and [7556] i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of glyceric acid. citramalic acid, fumaric acid, malic
acid, pyruvic acid, succinic acid, trihydroxybutyric acid or
trihydroxybutanoic acid in an organism or a part thereof. [7557] or
comprising a sequence which is complementary thereto. [7558] 3. The
process of claim 1 or 2, comprising recovering of the free or bound
glyceric acid. citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid. [7559] 4. The process of any one of claims 1 to 3, comprising
the following steps: [7560] a) selecting an organism or a part
thereof expressing a polypeptide encoded by the nucleic acid
molecule characterized in claim 2; [7561] b) mutagenizing the
selected organism or the part thereof; [7562] c) comparing the
activity or the expression level of said polypeptide in the
mutagenized organism or the part thereof with the activity or the
expression of said polypeptide of the selected organisms or the
part thereof; [7563] d) selecting the mutated organisms or parts
thereof, which comprise an increased activity or expression level
of said polypeptide compared to the selected organism or the part
thereof; [7564] e) optionally, growing and cultivating the
organisms or the parts thereof; and [7565] f) recovering, and
optionally isolating, the free or bound glyceric acid. citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid produced by the
selected mutated organisms or parts thereof. [7566] 5. The process
of any one of claims 1 to 4, wherein the activity of said protein
or the expression of said nucleic acid molecule is increased or
generated transiently or stably. [7567] 6. An isolated nucleic acid
molecule comprising a nucleic acid molecule selected from the group
consisting of: [7568] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 190 to
226 or lines 564 to 594 or a fragment thereof, which confers an
increase in the amount of glyceric acid. citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid or trihydroxybutanoic acid in an organism or a part thereof;
[7569] b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 90 to 226
or lines 564 to 594; [7570] c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as a result of the degeneracy of the
genetic code and conferring an increase in the amount of glyceric
acid. citramalic acid, fumaric acid, malic acid, pyruvic acid,
succinic acid, trihydroxybutyric acid or trihydroxybutanoic acid in
an organism or a part thereof; [7571] d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of glyceric acid. citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid or
trihydroxybutanoic acid in an organism or a part thereof; [7572] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of glyceric acid. citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid in an organism or
a part thereof; [7573] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, column 7, lines
190 to 226 or lines 564 to 594 and conferring an increase in the
amount of glyceric acid. citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid or
trihydroxybutanoic acid in an organism or a part thereof; [7574] g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of glyceric acid. citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid or trihydroxybutanoic acid in an organism or a part thereof;
[7575] h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table IV, column 7, lines 190 to 226 or
lines 564 to 594 and conferring an increase in the amount of in an
organism or a part thereof; and [7576] i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of glyceric acid. citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid or
trihydroxybutanoic acid in an organism or a part thereof. [7577]
whereby the nucleic acid molecule distinguishes over the sequence
as indicated in Table I A, columns 5 or 7, lines 190 to 226 or
lines 564 to 594 by one or more nucleotides. [7578] 7. A nucleic
acid construct which confers the expression of the nucleic acid
molecule of claim 6, comprising one or more regulatory elements.
[7579] 8. A vector comprising the nucleic acid molecule as claimed
in claim 6 or the nucleic acid construct of claim 7. [7580] 9. The
vector as claimed in claim 8, wherein the nucleic acid molecule is
in operable linkage with regulatory sequences for the expression in
a prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [7581] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [7582] 11. The host cell of claim 10, which is a
transgenic host cell. [7583] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [7584] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [7585] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table II A, columns 5
or 7, lines 190 to 226 or lines 564 to 594 by one or more amino
acids. [7586] 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. [7587] 16. A plant tissue,
propagation material, harvested material or a plant comprising the
host cell as claimed in claim 12 which is plant cell or an
Agrobacterium. [7588] 17. A method for screening for agonists and
antagonists of the activity of a polypeptide encoded by the nucleic
acid molecule of claim 6 conferring an increase in the amount of
glyceric acid. citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid in an organism or a part thereof comprising: (a) contacting
cells, tissues, plants or microorganisms which express the a
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of glyceric acid. citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid in an organism or
a part thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; (b) assaying the glyceric acid. citramalic acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and (c) identifying a
agonist or antagonist by comparing the measured glyceric acid.
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid or trihydroxybutanoic acid level or
polypeptide expression level with a standard glyceric acid.
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid or trihydroxybutanoic acid or
polypeptide expression level measured in the absence of said
candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. [7589] 18. A process for the
identification of a compound conferring increased glyceric acid.
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid or trihydroxybutanoic acid production
in a plant or microorganism, comprising the steps: (a) culturing a
plant cell or tissue or microorganism or maintaining a plant
expressing the polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of glyceric acid.
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid or trihydroxybutanoic acid in an
organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of glyceric acid. citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid in an organism or
a part thereof; (b) identifying if the compound is an effective
agonist by detecting the presence or absence or increase of a
signal produced by said readout system.
[7590] 19. A method for the identification of a gene product
conferring an increase in glyceric acid. citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid or trihydroxybutanoic acid production in a cell, comprising
the following steps: (a) contacting the nucleic acid molecules of a
sample, which can contain a candidate gene encoding a gene product
conferring an increase in glyceric acid. citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid or trihydroxybutanoic acid after expression with the nucleic
acid molecule of claim 6; (b) identifying the nucleic acid
molecules, which hybridise under relaxed stringent conditions with
the nucleic acid molecule of claim 6; (c) introducing the candidate
nucleic acid molecules in host cells appropriate for producing
glyceric acid. citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid; (d) expressing the identified nucleic acid molecules in the
host cells; (e) assaying the glyceric acid. citramalic acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid level in the host
cells; and (f) identifying nucleic acid molecule and its gene
product which expression confers an increase in the glyceric acid.
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid or trihydroxybutanoic acid level in
the host cell in the host cell after expression compared to the
wild type. [7591] 20. A method for the identification of a gene
product conferring an increase in glyceric acid. citramalic acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid production in a
cell, comprising the following steps: [7592] (a) identifying in a
data bank nucleic acid molecules of an organism; which can contain
a candidate gene encoding a gene product conferring an increase in
the glyceric acid. citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid or
trihydroxybutanoic acid amount or level in an organism or a part
thereof after expression, and which are at least 20% homolog to the
nucleic acid molecule of claim 6; [7593] (b) introducing the
candidate nucleic acid molecules in host cells appropriate for
producing glyceric acid. citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid or
trihydroxybutanoic acid; [7594] (c) expressing the identified
nucleic acid molecules in the host cells; [7595] (d) assaying the
glyceric acid. citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid level in the host cells; and [7596] (e) identifying nucleic
acid molecule and its gene product which expression confers an
increase in the glyceric acid. citramalic acid, fumaric acid, malic
acid, pyruvic acid, succinic acid, trihydroxybutyric acid or
trihydroxybutanoic acid level in the host cell after expression
compared to the wild type. [7597] 21. A method for the production
of an agricultural composition comprising the steps of the method
of any one of claims 17 to 20 and formulating the compound
identified in any one of claims 17 to 20 in a form acceptable for
an application in agriculture. [7598] 22. A composition comprising
the nucleic acid molecule of claim 6, the polypeptide of claim 14,
the nucleic acid construct of claim 7, the vector of any one of
claim 8 or 9, an antagonist or agonist identified according to
claim 17, the compound of claim 18, the gene product of claim 19 or
20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. [7599] 23. Use of the nucleic acid molecule as
claimed in claim 6 for the identification of a nucleic acid
molecule conferring an increase of glyceric acid. citramalic acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid after expression.
[7600] 24. Use of the polypeptide of claim 14 or the nucleic acid
construct claim 7 or the gene product identified according to the
method of claim 19 or 20 for identifying compounds capable of
conferring a modulation of glyceric acid. citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid or trihydroxybutanoic acid levels in an organism. [7601] 25.
Agrochemical, pharmaceutical, food or feed composition comprising
the nucleic acid molecule of claim 6, the polypeptide of claim 14,
the nucleic acid construct of claim 7, the vector of claim 8 or 9,
the antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20. [7602] 26. The method of any one of claims 1 to 5, the nucleic
acid molecule of claim 6, the polypeptide of claim 14, the nucleic
acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20, wherein the fine chemical is glyceric acid. citramalic acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid.
[7603] [0554.0.0.16] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[7604] [0000.0.0.17] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[7605] [0001.0.0.17] see [0001.0.0.0]
[7606] [0002.0.17.17] Gamma-aminobutyric acid is used to enhance
growth of specified plants, prevent development of powdery mildew
on grapes, and suppress certain other plant diseases. Humans and
animals normally ingest and metabolize gamma-aminobutyric acid in
variable amounts. Gamma-aminobutyric acid was registered (licensed
for sale) as growth enhancing pesticidal active ingredient in 1998.
Gamma-aminobutyric acid is an important signal which helps to
regulate mineral availability in plants. Minerals support the
biochemical pathways governing growth and reproduction as well as
the pathways that direct plant's response to a variety of biotic
and abiotic stresses. Mineral needs are especially high during
times of stress and at certain stages of plant growth.
Gamma-aminobutyric acid levels in plants naturally increase at
these times.
[7607] Gamma-Aminobutyric acid (GABA), a nonprotein amino acid, is
often accumulated in plants following environmental stimuli that
can also cause ethylene production. Exogenous GABA causes up to a
14-fold increase in the ethylene production rate after about 12 h.
GABA causes increases in ACC synthase mRNA accumulation, ACC
levels, ACC oxidase mRNA levels and in vitro ACC oxidase activity.
Possible roles of GABA as a signal transducer are suggested, see
Plant Physiol. 115(1):129-35(1997)
[7608] Gamma-aminobutyric acid (GABA), a four-carbon non-protein
amino acid, is a significant component of the free amino acid pool
in most prokaryotic and eukaryotic organisms. In plants, stress
initiates a signal-transduction pathway, in which increased
cytosolic Ca.sup.2+ activates Ca.sup.2+/calmodulin-dependent
glutamate decarboxylase activity and GABA synthesis. Elevated
H.sup.+ and substrate levels can also stimulate glutamate
decarboxylase activity. GABA accumulation probably is mediated
primarily by glutamate decarboxylase. Experimental evidence
supports the involvement of GABA synthesis in pH regulation,
nitrogen storage, plant development and defence, as well as a
compatible osmolyte and an alternative pathway for glutamate
utilization, see Trends Plant Sci. 4(11):446-452(1999).
[7609] Gamma-aminobutyric acid enhances nutrient uptake by roots
and leaves so that plant nutrient levels are higher than those
achieved by using nutrients alone. When plants are stressed and
nutrient uptake is limited, it is believed that gamma-aminobutyric
acid facilitates nutrient utilization, thereby enhancing growth
during stress.
[7610] Rapid GABA accumulation in response to wounding may play a
role in plant defense against insects (Ramputh and Brown, Plant
Physiol. 111(1996): 1349-1352). The development of gamma
aminobutyrate (GABA) as a potential control agent in
plant-invertebrate pest systems has been reviewed in She1p et al.,
Canadien Journal of Botany (2003) 81, 11, 1045-1048. The authors
describe that available evidence indicates that GABA accumulation
in plants in response to biotic and abiotic stresses is mediated
via the activation of glutamate decarboxylase. More applied
research, based on the fact that GABA acts as an inhibitory
neurotransmitter in invertebrate pests, indicates that ingested
GABA disrupts nerve functioning and causes damage to oblique-banded
leaf roller larvae, and that walking or herbivory by tobacco
budworm and oblique-banded leaf roller larvae stimulate GABA
accumulation in soybean and tobacco, respectively. In addition,
elevated levels of endogenous GABA in genetically engineered
tobacco deter feeding by tobacco budworm larvae and infestation by
the northern root-knot nematode. Therefore the author concluded
that genetically engineered crop species having high GABA-producing
potential may be an alternative strategy to chemical pesticides for
the management of invertebrate pests.
[7611] During angiosperm reproduction, pollen grains form a tube
that navigates through female tissues to the micropyle, delivering
sperm to the egg. In vitro, GABA stimulates pollen tube growth. The
Arabidopsis POP2 gene encodes a transaminase that degrades GABA and
contributes to the formation of a gradient leading up to the
micropyle, see Cell. 114(1):47-59(2003).
[7612] Due to these interesting physiological roles and
agrobiotechnological potential of GABA there is a need to identify
the genes of enzymes and other proteins involved in GABA
metabolism, and to generate mutants or transgenic plant lines with
which to modify the GABA content in plants.
[7613] Shikimic acid is found in various plants. It has two
functional groups in the same molecule, hydroxyl groups and a
carboxylic acid group which are optically active. They can yield
various kinds of esters and salts. It belongs to the class of
cyclitols, which means it is a hydroxylated cycloalkane containing
at least three hydroxy groups, each attached to a different ring
carbon atom. The term "shikimic acid" or "shikimate" relates to the
anionic form as well as the neutralised status of that
compound.
[7614] A key intermediate in synthesis of virtually all aromatic
compounds in the cells is shikimic acid. These include
phenylalanine, tyrosine, tryptophan, p-aminobenzoic acid, and
p-hvdroxvbenzoic acid.
[7615] Glyphosate (N-phosphonomethylglycine) is a non-selective,
broad spectrum herbicide that is symplastically translocated to the
meristems of growing plants. It causes shikimate accumulation
through inhibition of the chloroplast localized EPSP synthase
(5-enolpyruvylshikimate-3-phosphate synthase; EPSPs) [EC 2.5.1.19]
(Amrhein et al, 1980, Plant Physiol. 66: 830-834).
[7616] The starting product of the biosynthesis of most phenolic
compounds is shikimate. Phenols are acidic due to the
dissociability of their --OH group. They are rather reactive
compounds and as long as no steric inhibition due to additional
side chains occurs, they form hydrogen bonds. Consequently, many
flavonoids have intramolecular bonds. Another important feature is
their ability to form chelate complexes with metals. Also, they are
easily oxidized and, if so, form polymers (dark aggregates). The
darkening of cut or dying plant parts is caused by this reaction.
They have usually an inhibiting effect on plant growth. Among the
phenylpropanol derivatives of lower molecular weight are a number
of scents like the coumarins, cinnamic acid, sinapinic acid, the
coniferyl alcohols and others. These substances and their
derivatives are at the same time intermediates of the biosynthesis
of lignin.
[7617] The shikimate pathway links metabolism of carbohydrates to
biosynthesis of aromatic compounds. In a sequence of seven
metabolic steps, phosphoenolpyruvate and erythrose 4-phosphate are
converted to chorismate, the precursor of the aromatic amino acids
and many aromatic secondary metabolites. All pathway intermediates
can also be considered branch point compounds that may serve as
substrates for other metabolic pathways. The shikimate pathway is
found only in microorganisms and plants, never in animals. All
enzymes of this pathway have been obtained in pure form from
prokaryotic and eukaryotic sources and their respective DNAs have
been characterized from several organisms. The cDNAs of higher
plants encode proteins with amino terminal signal sequences for
plastid import, suggesting that plastids are the exclusive locale
for chorismate biosynthesis. In microorganisms, the shikimate
pathway is regulated by feedback inhibition and by repression of
the first enzyme. In higher plants, no physiological feedback
inhibitor has been identified, suggesting that pathway regulation
may occur exclusively at the genetic level. This difference between
microorganisms and plants is reflected in the unusually large
variation in the primary structures of the respective first
enzymes. Several of the pathway enzymes occur in isoenzymic forms
whose expression varies with changing environmental conditions and,
within the plant, from organ to organ. The penultimate enzyme of
the pathway is the sole target for the herbicide glyphosate.
Glyphosate-tolerant transgenic plants are at the core of novel weed
control systems for several crop plants (Annual Review of Plant
Physiology and Plant Molecular Biology 50(1999): 473-503). The term
"shikimic acid" or "shikimate" relates to the anionic form as well
as the neutralised status of that compound.
[7618] Natural products derived from shikimic acid range in
complexity from the very simple, such as vanillin (used primarily
as a flavoring agent), salicylic acid (the precursor of aspirin),
lawsone (a naphthoquinone used in some sunscreens), and scopletin
(a coumarin once used as a uterine sedative), to the more complex,
such as the lignan lactone podophyllotoxin. Podophyllotoxin is
basically a dimer incorporating two phenylpropanoid (a nine-carbon
unit derived from shikimic acid) units. Podophyllotoxin was first
isolated from Podophyllum peltatum, also known as mayapple or
American mandrake, a plant which has a long history of use as a
cathartic and purgative. Podophyllotoxin has been used to treat
warts, and is a mitotic inhibitor which shows antineoplastic
activity. Etoposide, in particular, is used to treat forms of lung
cancer, testicular cancer, and acute lymphocytic leukemia.
[7619] Furthermore shikimic acid is a important basic substance for
the or the production of pharmacyticals. For example the synthesis
of Oseltamivir, which is known under ist trading name Tamiflu is
synthesized from shikimic acid in a complex synthesis process.
[7620] Putrescine is synthesized by healthy living cells by the
action of ornithin decarboxylase, is one of the simplest polyamines
and appears to be a growth factor necessary for cell division.
[7621] Experimental evidence indicate that polyamines may be
involved in growth, differentiation or morphogenesis, stress and
senescence in plants (Evans and Malmberg, 1989).
[7622] [0003.0.0.17] %
[7623] [0004.0.0.17] %
[7624] [0005.0.0.17] %
[7625] [0006.0.0.17] %
[7626] [0007.0.0.17] %
[7627] [0008.0.17.17] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce gamma-aminobutyric acid or shikimate
in plants. That type of production permits control over quality,
quantity and selection of the most suitable and efficient producer
organisms. The latter is especially important for commercial
production economics and therefore availability to consumers. In
addition it is desireable to produce gamma-aminobutyric acid or
shikimate in plants in order to increase plant productivity and
resistance against biotic and abiotic stress as discussed before.
The term "shikimic acid" or "shikimate" relates to the anionic form
as well as the neutralised status of that compound.
[7628] Methods of recombinant DNA technology have been used for
some years to improve the production of fine chemicals in
microorganisms and plants by amplifying individual biosynthesis
genes and investigating the effect on production of fine chemicals.
It is for example reported, that the xanthophyll astaxanthin could
be produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. Those results indicated
that about 75 percent of the carotenoids found in the flower of the
transformed plant contained a keto group.
[7629] [0009.0.17.17] Thus, it would be advantageous if an algae,
plant or other microorganism were available which produce large
amounts gamma-aminobutyric acid or shikimate or putrescine. The
invention discussed hereinafter relates in some embodiments to such
transformed prokaryotic or eukaryotic microorganisms.
[7630] It would also be advantageous if plants were available whose
roots, leaves, stem, fruits or flowers produced large amounts of
gamma-aminobutyric acid or shikimate or putrescine. The invention
discussed hereinafter relates in some embodiments to such
transformed plants.
[7631] [0010.0.17.17] Therefore improving the quality of foodstuffs
and animal feeds is an important task of the food-and-feed
industry. This is necessary since, for example gamma-aminobutyric
acid or shikimate, as mentioned above, which occur in plants and
some microorganisms are limited with regard to the supply of
mammals. Especially advantageous for the quality of foodstuffs and
animal feeds is as balanced as possible a specific
gamma-aminobutyric acid or shikimate profile in the diet since an
excess of gamma-aminobutyric acid or shikimate above a specific
concentration in the food has a positive effect. A further increase
in quality is only possible via addition of further
gamma-aminobutyric acid or shikimate, which are limiting.
[7632] [0011.0.17.17] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add gamma-aminobutyric acid or
shikimate in a balanced manner to suit the organism.
[7633] [0012.0.17.17] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
proteins which participate in the biosynthesis of
gamma-aminobutyric acid or shikimate or putrescine and make it
possible to produce them specifically on an industrial scale
without unwanted byproducts forming. In the selection of genes for
biosynthesis two characteristics above all are particularly
important. On the one hand, there is as ever a need for improved
processes for obtaining the highest possible contents of
gamma-aminobutyric acid, putrescine and shikimate; on the other
hand as less as possible byproducts should be produced in the
production process.
[7634] [0013.0.0.17] see [0013.0.0.0]
[7635] [0014.0.17.17] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a gamma-aminobutyric acid or
shikimate or putrescine. Accordingly, in the present invention, the
term "the fine chemical" as used herein relates to a
gamma-aminobutyric acid or shikimate or putrescine. Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising gamma-aminobutyric acid or shikimate or
putrescine.
[7636] [0015.0.17.17] In one embodiment, the term "the fine
chemical" or "the respective fine chemical" means at least one
chemical compound with gamma-aminobutyric acid or shikimate or
putrescine activity.
[7637] In one embodiment, the term "the fine chemical" means a
gamma-aminobutyric acid. In one embodiment, the term "the fine
chemical" means shikimate depending on the context in which the
term is used. In one embodiment, the term "the fine chemical" means
putrescine depending on the context in which the term is used.
Throughout the specification the term "the fine chemical" means
gamma-aminobutyric acid or shikimate or putrescine, its salts,
ester, thioester or in free form or bound to other compounds such
sugars or sugarpolymers, like glucoside, e.g. diglucoside.
[7638] [0016.0.17.17] Accordingly, the present invention relates to
a process comprising [7639] (a) increasing or generating the
activity of one or more YER156C, YFR042W, YOR084W, YLR375W, b1693,
b0651, b0847 or b 2965 protein(s) in a non-human organism in one or
more parts thereof; and [7640] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
gamma-aminobutyric acid, putrescine or shikimate in said
organism.
[7641] Accordingly, the present invention relates to a process
comprising. [7642] (a) increasing or generating the activity of one
or more proteins having the activity of a protein indicated in
Table IIA or IIB, column 3, lines 227 to 230, 595 to 598, resp. or
having the sequence of a polypeptide encoded by a nucleic acid
molecule indicated in Table IA or IB, column 5 or 7, lines 227 to
230, 595 to 598, resp. in a non-human organism in one or more parts
thereof; and growing the organism under conditions which permit the
production of the fine chemical, thus, gamma-aminobutyric acid,
putrescine or shikimate, in said organism.
[7643] [0016.1.17.17] Accordingly, the term "the fine chemical"
means "gamma-aminobutyric acid" in relation to all sequences listed
in Table IA or IB, lines 227 to 229, 595 and 596 or homologs
thereof and means "shikimate" in relation to the sequence listed in
Table IA or IB, line 230 and 597 or homologs thereof and means
"putrescine" in relation to the sequence listed in Table IA or IB,
line 598. Accordingly, the term "the fine chemical" can mean
"gamma-aminobutyric acid" or "shikimate" or "putrescine", owing to
circumstances and the context. In order to illustrate that the
meaning of the term "the respective fine chemical" means
"gamma-aminobutyric acid" or "shikimate" or "putrescine" owing to
the sequences listed in the context the term "the respective fine
chemical" is also used.
[7644] [0017.0.0.17] to [0018.0.0.17]: see [0017.0.0.0] to
[0018.0.0.0]
[7645] [0019.0.17.17] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the respective fine chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table IIA or IIB, column 3, lines 227 to 230, 595 to
598 or encoded by nucleic acid molecule indicated in Table IA or
IB, columns 5 or 7, lines 227 to 230, 595 to 598.
[7646] [0020.0.17.17] Surprisingly it was found, that the
transgenic expression of the Saccharomyces cerevisiae proteins
YER156C, YFR042W, YOR084W, YLR375W, and/or the Escherichia coli
proteins b1693, b0651, b0847 or b 2965 in Arabidopsis thaliana
conferred an increase in the gamma-aminobutyric acid or shikimate
or putrescine ("the fine chemical" or "the fine respective
chemical") content in respect to said proteins and their homologs
as well as the encoding nucleic acid molecules, in particular as
indicated in Table IA or IB and Table IIA or IIB, column 3, lines
227 to 230, 595 to 598 of the transformed plants.
[7647] [0021.0.0.17] see [0021.0.0.0]
[7648] [0022.0.17.17] The sequence of YER156C from Saccharomyces
cerevisiae has been published in Dietrich, F. S et al, Nature 387
(6632 Suppl), 78-81 (1997), and its activity is being defined as an
unclassified protein. Accordingly, in one embodiment, the process
of the present invention comprises the use of a protein YER156C
from Saccharomyces cerevisiae or its homolog, e.g. as shown herein,
for the production of the respective fine chemical, in particular
for increasing the amount of gamma-aminobutyric acid, preferably in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of the unspecified protein YER156C is increased.
[7649] The sequence of YFR042W from Saccharomyces cerevisiae has
been published in Goffeau, A. et al., Science 274 (5287), 546-547
(1996) and its activity is being defined as a protein required for
cell viability. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein having said
activity, for the production of the respective fine chemical, in
particular for increasing the amount of gamma-aminobutyric acid,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention said activity required for cell viability is
increased.
[7650] The sequence of YOR084W from Saccharomyces cerevisiae has
been published in Dujon, B. et al, Nature 387 (6632 Suppl), 98-102
(1997) and its activity is being defined as a putative lipase of
the peroxisomal martrix. Accordingly, in one embodiment, the
process of the present invention comprises the use of a lipase
protein of the peroxisomal martrix from Saccharomyces cerevisiae or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, in particular for increasing the amount
of gamma-aminobutyric acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention said activity, e.g. the activity
of the lipase protein of the peroxisomal matrix is increased.
[7651] The sequence of YLR375W from Saccharomyces cerevisiae has
been published in Johnston, M., Nature 387 (6632 Suppl), 87-90
(1997) and its activity is being defined as protein involved in
pre-tRNA splicing and in uptake of branched-chain amino acids.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein with pre-tRNA splicing and
in uptake of branched-chain amino acids activity from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of shikimate preferably in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention said activity, e.g. the activity of a
protein involved in pre-tRNA splicing and in uptake of
branched-chain amino acids is increased.
[7652] The sequence of b0651 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a pyrimidine specific
nucleoside hydrolase. Accordingly, in one embodiment, the process
of the present invention comprises the use of a protein with
pyrimidine specific nucleoside hydrolase activity from Escherichia
coli K12 or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of gamma-aminobutyric acid preferably in free or bound form
in an organism or a part thereof, as mentioned. In one embodiment,
in the process of the present invention said activity, e.g. the
activity of a protein with pyrimidine specific nucleoside hydrolase
activity, is increased.
[7653] The sequence of b0847 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a putative transport protein.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein with putative transport
protein activity from Escherichia coli K12 or its homolog, e.g. as
shown herein, for the production of the respective fine chemical,
in particular for increasing the amount of gamma-aminobutyric acid
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention said activity, e.g. the activity of a transport protein
b0847 is increased.
[7654] The sequence of b1693 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a 3-dehydroquinate
dehydratase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a 3-dehydroquinate
dehydratase protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
shikimate or linoleic acid and/or tryglycerides, lipids, oils
and/or fats containing shikimate, linoleic acid, in particular for
increasing the amount of shikimate, linoleic acid and/or
tryglycerides, lipids, oils and/or fats containing shikimate,
linoleic acid, preferably linoleic acid in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a 3-dehydroquinate
dehydratase protein is increased or generated, e.g. from E. coli or
a homolog thereof.
[7655] The sequence of b 2965 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474,1997,
and its activity is being defined as an ornithine decarboxylase
isozyme. Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein with ornithine
decarboxylase isozyme activity from Escherichia coli K12 or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, in particular for increasing the amount of
putrescine preferably in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention said activity, e.g. the activity of a protein
with ornithine decarboxylase activity is increased.
[7656] [0023.0.17.17] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[7657] In one embodiment, the homolog of any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 227 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms preferably
gamma-aminobutyric acid.
[7658] In one embodiment, the homolog of the polypeptides indicated
in Table IIA or IIB, column 3, line 228 is a homolog having the
same or a similar activity. In particular an increase of activity
confers an increase in the content of the respective fine chemical
in the organisms preferably gamma-aminobutyric acid.
[7659] In one embodiment, the homolog of the polypeptides indicated
in Table IIA or IIB, column 3, line 229 is a homolog having the
same or a similar activity. In particular an increase of activity
confers an increase in the content of the respective fine chemical
in the organisms preferably gamma-aminobutyric acid.
[7660] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 230 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of
shikimate in the organisms.
[7661] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 595 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of
gamma-aminobutyric acid in the organisms.
[7662] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 596 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of
gamma-aminobutyric acid in the organisms.
[7663] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 597 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of
shikimate in the organisms.
[7664] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 598 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of
putrescine in the organisms.
[7665] [0023.1.0.17] Homologs of the polypeptides indicated in
Table IIA or IIB, column 3, lines 227 to 230, 595 to 598 may be the
polypeptides encoded by the nucleic acid molecules indicated in
Table IA or IB, column 7, lines 227 to 230, 595 to 598 or may be
the polypeptides indicated in Table IIA or IIB, column 7, lines 227
to 230, 595 to 598.
[7666] Homologs of the polypeptides indicated in Table IIA or IIB,
column 3, lines 227 to 229, 595, 596 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table IA or IB,
column 7, lines 227 to 229, 595, 596, respectively or may be the
polypeptides indicated in Table IIA or IIB, column 7, lines 227 to
229, 595, 596, having a gamma-aminobutyric acid content and/or
amount increasing activity.
[7667] Homologs of the polypeptide indicated in Table IIA or IIB,
column 3, line 230 and 597 may be the polypeptides encoded by the
nucleic acid molecules indicated in Table IA or IB, column 7, line
230 and 597, respectively or may be the polypeptides indicated in
Table IIA or IIB, column 7, line 230 and 597, having a shikimate
content and/or amount increasing activity.
[7668] Homologs of the polypeptide indicated in Table IIA or IIB,
column 3, line 598 may be the polypeptides encoded by the nucleic
acid molecules indicated in Table IA or IB, column 7, line 598,
respectively or may be the polypeptides indicated in Table IIA or
IIB, column 7, line 598 having a putrescine content and/or amount
increasing activity.
[7669] [0024.0.0.17] see [0024.0.0.0]
[7670] [0025.0.17.17] In accordance with the invention, a protein
or polypeptide has the "activity of a protein of the invention",
e.g. the activity of a protein indicated in Table IIA or IIB,
column 3, lines 227 to 229 or line 230 or lines 595 to 598 if its
de novo activity, or its increased expression directly or
indirectly leads to an increased gamma-aminobutyric acid or
shikimate or putrescine level, resp., in the organism or a part
thereof, preferably in a cell of said organism. In a preferred
embodiment, the protein or polypeptide has the above-mentioned
additional activities of a protein indicated in Table IIA or IIB,
column 3, lines 227 to 229 or line 230 or lines 595 to 598.
Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or a nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of any one of the proteins indicated in Table IIA or IIB,
column 3, lines 227 to 229 or line 230 or lines 595 to 598, or
which has at least 10% of the original enzymatic activity,
preferably 20%, particularly preferably 30%, most particularly
preferably 40% in comparison to any one of the proteins indicated
in Table IIA or IIB, column 3, line 227 to 230, 595 to 598 of
Saccharomyces cerevisiae.
[7671] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table IA
or IB, column 4 and is expressed in an organism, which is
evolutionary distant to the origin organism. For example origin and
expressing organism are derived from different families, orders,
classes or phylums whereas origin and the organism indicated in
Table IA or IB, column 4 are derived from the same families,
orders, classes or phylums.
[7672] [0025.1.0.17] see [0025.1.0.0]
[7673] [0026.0.0.17] to [0033.0.0.17]: see [0026.0.0.0] to
[0033.0.0.0]
[7674] [0034.0.17.17] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of an protein having the activity of a
protein as indicated in Table IIA or IIB, column 3, lines 227 to
229 or line 230 or lines 595 to 598 or being encoded by a nucleic
acid molecule indicated in Table IA or IB, column 5, lines 227 to
229 or line 230 or 595 to 598 or its homologs, e.g. as indicated in
Table IA or IB, column 7, lines 227 to 229 or line 230 or lines 595
to 598, its biochemical or genetic causes. It therefore shows the
increased amount of the respective fine chemical.
[7675] [0035.0.0.17] to [0038.0.0.17]: see [0035.0.0.0] to
[0038.0.0.0]
[7676] [0039.0.0.17]: see [0039.0.0.0]
[7677] [0040.0.0.17] to [0044.0.0.17]: see [0040.0.0.0] to
[0044.0.0.0]
[7678] [0045.0.17.17] In case the activity of the Saccharomyces
cerevisae protein YER156C or its homologs, e.g. as indicated in
Table IIA or IIB, columns 5 or 7, line 227, is increased, in one
embodiment the increase of the respective fine chemical, preferably
of gamma-aminobutyric acid between 68% and 1029% or more is
conferred.
[7679] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YFR042W or its homologs, e.g. as indicated in
Table IIA or IIB, columns 5 or 7, line 228 is increased,
preferably, in one embodiment the increase of the respective fine
chemical, preferably of gamma-aminobutyric acid between 91% and
110% or more is conferred.
[7680] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YOR084W or its homologs e.g. a protein of a
putative lipase of the peroxisomal matrix as indicated in Table IIA
or IIB, columns 5 or 7, line 229, is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of gamma-aminobutyric acid between 71% and 459% or more is
conferred.
[7681] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YLR375W or its homologs, e.g. a protein involved
in pre-tRNA splicing and in uptake of branched-chain amino acids as
indicated in Table IIA or IIB, columns 5 or 7, line 230, is
increased, preferably, in one embodiment an increase of the
respective fine chemical, preferably of shikimate between 14% and
26% or more is conferred.
[7682] In one embodiment, in case the activity of the Escherichia
coli protein b 0651 or its homologs, e.g. a protein with pyrimidine
specific nucleoside hydrolase activity as indicated in Table IIA or
IIB, columns 5 or 7, line 595, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of gamma-aminobutyric acid between 53% and 206% or more is
conferred.
[7683] In one embodiment, in case the activity of the Escherichia
coli protein b 0847 or its homologs, e.g. a protein with putative
transport protein activity as indicated in Table IIA or IIB,
columns 5 or 7, line 596, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of gamma-aminobutyric acid between 85% and 187% or more is
conferred.
[7684] In one embodiment, in case the activity of the Escherichia
coli protein b 1693 or its homologs, e.g. a protein with
3-dehydroquinate dehydratase activity as indicated in Table IIA or
IIB, columns 5 or 7, line 597, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of shikimate between 15% and 31% or more is conferred.
[7685] In one embodiment, in case the activity of the Escherichia
coli protein b 2965 or its homologs, e.g. a protein with ornithine
decarboxylase activity as indicated in Table IIA or IIB, columns 5
or 7, line 598, is increased, preferably, in one embodiment an
increase of the respective fine chemical, preferably of putrescine
between 111% and 1693% or more is conferred.
[7686] [0046.0.17.17] %
[7687] [0047.0.0.17] to [0048.0.0.17]: see [0047.0.0.0] to
[0048.0.0.0]
[7688] [0049.0.17.17] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
gamma-aminobutyric acid preferably has the structure of the
polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence as shown in SEQ ID NO: 15722, 15723, 15724,
15725, 15726, 15727 or 15778, 15779, 91412, 91413, 91414, 91525,
91526, 91527, 91528, 91529 or as indicated in Table IV, column 7,
lines 227, 228, 229, 595 and 596 or of a functional homologue
thereof as described herein, or of a polypeptide encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by a nucleic acid
molecule as indicated in Table IA or IB, columns 5 or 7, lines 227,
228, 229, 595 and 596 or its herein described functional homologues
and has the herein mentioned activity conferring an increase in the
gamma-aminobutyric acid level.
[7689] A protein having an activity conferring an increase in the
amount or level of the shikimate preferably has the structure of
the polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence as shown in SEQ ID NO: 15768, 15769, 15770,
15771, 91630, 91631, 91632, 91633, 91634 e.g. as indicated in Table
IV, column 7, line 230, 597 or of a polypeptide as indicated in
Table IIA or IIB, columns 5 or 7, line 230, 597 or of a functional
homologue thereof as described herein, or of a polypeptide encoded
by the nucleic acid molecule characterized herein or the nucleic
acid molecule according to the invention, for example by a nucleic
acid molecule as indicated in Table IA or IB, columns 5 or 7, line
230, 597 or its herein described functional homologues and has the
herein mentioned activity conferring an increase in the shikimate
level.
[7690] A protein having an activity conferring an increase in the
amount or level of the putrescine preferably has the structure of
the polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence as shown in SEQ ID NO: 91831, 91832, 91833,
91834, 91835, 91836 e.g. as indicated in Table IV, column 7, line
598 or of a polypeptide as indicated in Table IIA or IIB, columns 5
or 7, line 598 or of a functional homologue thereof as described
herein, or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table IA or IB, columns 5 or 7, line 598 or its herein described
functional homologues and has the herein mentioned activity
conferring an increase in the putrescine level.
[7691] [0050.0.17.17] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of gamma-aminobutyric acid or shikimate, resp., or their
ester, or glucoside thereof, e.g the diglucoside thereof.
[7692] [0051.0.17.17] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g compositions comprising
gamma-aminobutyric acid or shikimate or putrescine. Depending on
the choice of the organism used for the process according to the
present invention, for example a microorganism or a plant,
compositions or mixtures of gamma-aminobutyric acid or shikimate or
putrescine can be produced.
[7693] [0052.0.0.17] see [0052.0.0.0]
[7694] [0053.0.17.17] In one embodiment, the process of the present
invention comprises one or more of the following steps [7695] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention, e.g. of a polypeptide having an
activity of a protein as indicated in Table IIA or IIB, column 3,
lines 227 to 229 or line 230 or lines 595 to 598 or its homologs,
e.g. as indicated in Table IIA or IIB, columns 5 or 7, lines 227 to
229 or line 230 or lines 595 to 598, activity having
herein-mentioned the respective fine chemical increasing activity;
[7696] b) stabilizing a mRNA conferring the increased expression of
a protein encoded by the nucleic acid molecule of the invention,
e.g. of a polypeptide having an activity of a protein as indicated
in Table IIA or IIB, column 3, lines 227 to 229 or line 230 or
lines 595 to 598 or its homologs activity, e.g. as indicated in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, or of a mRNA encoding the polypeptide of the
present invention having herein-mentioned the respective fine
chemical increasing activity; [7697] c) increasing the specific
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the present invention having herein-mentioned
the respective fine chemical increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
IIA or IIB, column 3, lines 227 to 229 or line 230 or lines 595 to
598 or its homologs activity, e.g. as indicated in Table IIA or
IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598, or decreasing the inhibitory regulation of the polypeptide of
the invention; [7698] d) generating or increasing the expression of
an endogenous or artificial transcription factor mediating the
expression of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention having herein-mentioned the
respective fine chemical increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table IIA or IIB,
column 3, lines 227 to 229 or line 230 or lines 595 to 598 or its
homologs activity, e.g. as indicated in Table IIA or IIB, columns 5
or 7, lines 227 to 229 or line 230 or lines 595 to 598; [7699] e)
stimulating activity of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
present invention or a polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table IIA or IIB, column 3, lines 227 to 229 or line 230 or
lines 595 to 598 or its homologs activity, e.g. as indicated in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, by adding one or more exogenous inducing factors
to the organism or parts thereof; [7700] f) expressing a transgenic
gene encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table IIA or IIB, column 3, lines 227 to 229 or line 230 or
lines 595 to 598 or its homologs activity, e.g. as indicated in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, and/or [7701] g) increasing the copy number of a
gene conferring the increased expression of a nucleic acid molecule
encoding a polypeptide encoded by the nucleic acid molecule of the
invention or the polypeptide of the invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table IIA or IIB, column 3, lines 227 to 229 or line 230 or
lines 595 to 598 or its homologs, e.g. as indicated in Table IIA or
IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598, activity. [7702] h) Increasing the expression of the
endogenous gene encoding the polypeptide of the invention, e.g. a
polypeptide having an activity of a protein as indicated in Table
IIA or IIB, column 3, lines 227 to 229 or line 230 or lines 595 to
598 or its homologs activity, e.g. as indicated in Table IIA or
IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598, by adding positive expression or removing negative expression
elements, e.g. homologous recombination can be used to either
introduce positive regulatory elements like for plants the 35S
enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[7703] i) Modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead to an enhanced respective fine
chemical production. [7704] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, e.g. the elite crops.
[7705] [0054.0.17.17] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table IIA or IIB, columns 3
or 5, lines 227 to 229 or line 230 or lines 595 to 598, resp., or
its homologs activity, e.g. as indicated in Table IIA or IIB,
columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to 598,
resp.
[7706] [0055.0.0.17] to [0067.0.0.17]: see [0055.0.0.0] to
[0067.0.0.0]
[7707] [0068.0.17.17] The mutation is introduced in such a way that
the production of gamma-aminobutyric acid or shikimate or
putrescine is not adversely affected.
[7708] [0069.0.0.17] see [0069.0.0.0]
[7709] [0070.0.17.17] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolite composition in the organism, e.g. an advantageous
composition ofgamma-aminobutyric acid and shikimate or their
biochemical derivatives, e.g. comprising a higher content of (from
a viewpoint of nutritional physiology limited) gamma-aminobutyric
acid and shikimate or putrescine or their derivatives.
[7710] [0071.0.0.17] see [0071.0.0.0]
[7711] [0072.0.17.17] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to shikimate their biochemical derivatives like
chorismate, prephenate, anthranilate, phenylpyruvate,
phenylalanine, 4-hydroxyphenylbutyrate, tyrosine, tryptophan,
vanillin, salicylic acid, lawsone or scopletin.
[7712] [0073.0.17.17] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
[7713] providing a non-human organism, preferably a microorganism,
a non-human animal, a plant or animal cell, a plant or animal
tissue or a plant;
[7714] increasing an activity of a polypeptide of the invention or
a homolog thereof, e.g. as indicated in Table IIA or IIB, columns 5
or 7, lines 227 to 229 or line 230 or lines 595 to 598, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the respective fine chemical in an organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, growing an organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant under conditions which permit the
production of the respective fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and if desired, recovering, optionally isolating, the
free and/or bound the respective fine chemical synthesized by the
organism, the microorganism, the non-human animal, the plant or
animal cell, the plant or animal tissue or the plant.
[7715] [0074.0.17.17] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound respective fine chemical.
[7716] [0075.0.0.17] to [0077.0.0.17]: see [0075.0.0.0] to
[0077.0.0.0]
[7717] [0078.0.17.17] The organism such as microorganisms or plants
or the recovered, and if desired isolated, the respective fine
chemical can then be processed further directly into foodstuffs or
animal feeds or for other applications. The fermentation broth,
fermentation products, plants or plant products can be purified
with methods known to the person skilled in the art. Products of
these different work-up procedures are gamma-aminobutyric acid or
shikimate or putrescine or comprising compositions of gamma-butyric
acid or shikimate or putrescine still comprising fermentation
broth, plant particles and cell components in different amounts,
advantageously in the range of from 0 to 99% by weight, preferably
below 80% by weight, especially preferably below 50% by weight.
[7718] [0079.0.0.17] to [0084.0.0.17]: see [0079.0.0.0] to
[0084.0.0.0]
[7719] [0084.0.17.17] %
[7720] [0085.0.17.17] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [7721] a) a nucleic acid sequence as
indicated in Table IA or IB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598, or a derivative thereof, or [7722] b)
a genetic regulatory element, for example a promoter, which is
functionally linked to the nucleic acid sequence as indicated in
Table IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, or a derivative thereof, or [7723] c) (a) and (b)
is/are not present in its/their natural genetic environment or
has/have been modified by means of genetic manipulation methods, it
being possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[7724] [0086.0.0.17] to [0087.0.0.17]: see [0086.0.0.0] to
[0087.0.0.0]
[7725] [0088.0.17.17] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned fine
chemicals or the plants are more resistant to biotic and abiotic
stress and the yield is increased.
[7726] [0088.1.0.17] see [0088.1.0.0]
[7727] [0089.0.0.17] to [0090.0.0.17]: see [0089.0.0.0] to
[0090.0.0.0]
[7728] [0091.0.0.17] see [0091.0.0.0]
[7729] [0092.0.0.17] to [0094.0.0.17]: see [0092.0.0.0] to
[0094.0.0.0]
[7730] [0095.0.17.17] It may be advantageous to increase the pool
of gamma-aminobutyric acid or shikimate or putrescine in the
transgenic organisms by the process according to the invention in
order to isolate high amounts of the pure respective fine chemical
and/or to obtain increased resistance against biotic and abiotic
stresses and to obtain higher yield.
[7731] [0096.0.17.17] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example in
the organism, is useful to increase the production of the
respective fine chemical.
[7732] [0097.0.17.17] %
[7733] [0098.0.17.17] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated.
[7734] [0099.0.10.17] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[7735] [0100.0.17.17] Transgenic plants which comprise the fine
chemicals such as gamma-aminobutyric acid or shikimate or
putrescine synthesized in the process according to the invention
can advantageously be marketed directly without there being any
need for the fine chemicals synthesized to be isolated. Plants for
the process according to the invention are listed as meaning intact
plants and all plant parts, plant organs or plant parts such as
leaf, stem, seeds, root, tubers, anthers, fibers, root hairs,
stalks, embryos, calli, cotelydons, petioles, harvested material,
plant tissue, reproductive tissue and cell cultures which are
derived from the actual transgenic plant and/or can be used for
bringing about the transgenic plant. In this context, the seed
comprises all parts of the seed such as the seed coats, epidermal
cells, seed cells, endosperm or embryonic tissue.
[7736] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, as extracts, e.g. ether, alcohol,
or other organic solvents or water containing extract and/or free
fine chemicals. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pretreated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[7737] Because gamma-aminobutyric acid or shikimate or putrescine
in microorganisms are localized intracellular, their recovery
essentially comes down to the isolation of the biomass.
Well-established approaches for the harvesting of cells include
filtration, centrifugation and coagulation/flocculation as
described herein. Of the residual hydrocarbon, adsorbed on the
cells, has to be removed. Solvent extraction or treatment with
surfactants have been suggested for this purpose.
[7738] [0101.0.10.17] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[7739] [0102.0.17.17] Gamma-aminobutyric acid or shikimate or
putrescine can for example be detected advantageously via HPLC, LC
or GC separation methods. The unambiguous detection for the
presence of gamma-aminobutyric acid or shikimate containing or
putrescine products can be obtained by analyzing recombinant
organisms using analytical standard methods: LC, LC-MS, MS or TLC).
The material to be analyzed can be disrupted by sonication,
grinding in a glass mill, liquid nitrogen and grinding, cooking, or
via other applicable methods.
[7740] [0103.0.17.17] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [7741] a) nucleic acid molecule encoding, preferably
at least the mature form, of the polypeptide having a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598 or a fragment thereof, which confers
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [7742] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule having a sequence as indicated in Table IA or IB, columns
5 or 7, lines 227 to 229 or line 230 or lines 595 to 598, [7743] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [7744] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[7745] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [7746]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [7747] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [7748] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers pairs
having a sequence as indicated in Table III, columns 7, lines 227
to 229 or line 230 or lines 595 to 598, and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [7749] i) nucleic acid molecule encoding a
polypeptide which is isolated, e.g. from an expression library,
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (h), preferably to
(a) to (c), and and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [7750]
j) nucleic acid molecule which encodes a polypeptide comprising the
consensus sequence having a sequences as indicated in Table IV,
columns 7, lines 227 to 229 or line 230 or lines 595 to 598 and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [7751] k) nucleic acid
molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of the polypeptide indicated in Table IIA or IIB, columns 5 or 7,
lines 227 to 229 or line 230 or lines 595 to 598, and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; and [7752] l) nucleic acid molecule
which is obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably to (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; or which comprises a sequence which is complementary
thereto.
[7753] [0104.0.17.17] In one embodiment, the nucleic acid molecule
used in the process of the present invention distinguishes over the
sequence indicated in Table IA or IB, columns 5 or 7, lines 227 to
229 or line 230 or lines 595 to 598 by one or more nucleotides. In
one embodiment, the nucleic acid molecule used in the process of
the invention does not consist of the sequence indicated in Table
IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595
to 598. In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence indicated in Table IA or IB, columns 5 or
7, lines 227 to 229 or line 230 or lines 595 to 598. In another
embodiment, the nucleic acid molecule does not encode a polypeptide
of a sequence indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598.
[7754] [0105.0.0.17] to [0107.0.0.17]: see [0105.0.0.0] to
[0107.0.0.0]
[7755] [0108.0.17.17] Nucleic acid molecules with the sequence as
indicated in Table IA or IB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598, nucleic acid molecules which are
derived from an amino acid sequences as indicated in Table IIA or
IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598 or from polypeptides comprising the consensus sequence as
indicated in Table IV, column 7, lines 227 to 229 or lines 230 or
lines 595 to 598, or their derivatives or homologues encoding
polypeptides with the enzymatic or biological activity of an
activity of a polypeptide as indicated in Table IIA or IIB, column
3, 5 or 7, lines 227 to 229 or line 230 or lines 595 to 598, e.g.
conferring the increase of the respective fine chemical, meaning
gamma-aminobutyric acid or shikimate or putrescine, resp., after
increasing its expression or activity, are advantageously increased
in the process according to the invention.
[7756] [0109.0.17.17] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular
gamma-aminobutyric acid or shikimate or putrescine, produced in the
process according to the invention.
[7757] [0110.0.0.17] see [0110.0.0.0]
[7758] [0111.0.0.17] see [0111.0.0.0]
[7759] [0112.0.17.17] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table IIA or IIB, column 3, lines
227 to 229 or line 230 or lines 595 to 598 or having the sequence
of a polypeptide as indicated in Table IIA or IIB, columns 5 and 7,
lines 227 to 229 or line 230 or lines 595 to 598 and conferring an
increase in the gamma-aminobutyric acid or shikimate or putrescine
level.
[7760] [0113.0.0.17] to [0114.0.0.17]: see [0113.0.0.0] to
[0114.0.0.0]
[7761] [0115.0.0.17] see [0115.0.0.0]
[7762] [0116.0.0.17] to [0120.0.0.17] see [0116.0.0.0] to
[0120.0.0.0]
[7763] [0120.1.0.17]: %
[7764] [0121.0.17.17] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table IIA or
IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598 or the functional homologues thereof as described herein,
preferably conferring above-mentioned activity, i.e. conferring a
gamma-aminobutyric acid level increase after increasing the
activity of the polypeptide sequences indicated in Table IIA or
IIB, columns 5 or 7, lines 227 to 229, 595 or 596 or conferring a
shikimate level increase after increasing the activity of the
polypeptide sequences indicated in Table IIA or IIB, columns 5 or
7, lines 230 or 597 or conferring a putrescin level increase after
increasing the activity of the polypeptide sequences indicated in
Table IIA or IIB, columns 5 or 7, lines 598.
[7765] [0122.0.0.17] to [0127.0.0.17]: see [0122.0.0.0] to
[0127.0.0.0]
[7766] [0128.0.17.17] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, columns 7,
lines 227 to 229 or line 230 or lines 595 to 598, by means of
polymerase chain reaction can be generated on the basis of a
sequence shown herein, for example the sequence as indicated in
Table IA or IB, columns 5 or 7, lines 227 to 229, resp. or line 230
or lines 595 to 598, resp. or the sequences derived from a
sequences as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229, resp. or line 230 or lines 595 to 598, resp.
[7767] [0129.0.17.17] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence indicated in Table IV,
columns 7, lines 227 and 228 or line 230 or lines 595 to 598 is
derived from said alignments.
[7768] [0130.0.17.17] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g.
[7769] conferring the increase of gamma-aminobutyric acid or
shikimate after increasing the expression or activity the protein
comprising said fragment.
[7770] [0131.0.0.17] to [0138.0.0.17]: see [0131.0.0.0] to
[0138.0.0.0]
[7771] [0139.0.17.17] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical level increase,
derived from other organisms, can be encoded by other DNA sequences
which hybridise to a sequence indicated in Table IA or IB, columns
5 or 7, lines 227 to 229, 595, 596 for gamma-aminobutyric acid or
indicated in Table IA or IB, columns 5 or 7 lines 230, 597 for
shikimate or indicated in Table IA or IB, columns 5 or 7, line 598
for putrescine under relaxed hybridization conditions and which
code on expression for peptides having the respective fine
chemical, i.e. gamma-aminobutyric acid or shikimate or putrescine,
resp., increasing-activity.
[7772] [0140.0.0.17] to [0146.0.0.17]: see [0140.0.0.0] to
[0146.0.0.0]
[7773] [0147.0.17.17] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595
to 598 is one which is sufficiently complementary to one of said
nucleotide sequences such that it can hybridise to one of said
nucleotide sequences, thereby forming a stable duplex. Preferably,
the hybridisation is performed under stringent hybridization
conditions. However, a complement of one of the herein disclosed
sequences is preferably a sequence complement thereto according to
the base pairing of nucleic acid molecules well known to the
skilled person. For example, the bases A and G undergo base pairing
with the bases T and U or C, resp. and visa versa. Modifications of
the bases can influence the base-pairing partner.
[7774] [0148.0.17.17] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table IA or IB, columns 5 or 7,
lines 227 to 229 or line 230 or lines 595 to 598, or a portion
thereof and preferably has above mentioned activity, in particular
having a gamma-aminobutyric acid or shikimate or putrescine
increasing activity after increasing the activity or an activity of
a product of a gene encoding said sequences or their homologs.
[7775] [0149.0.17.17] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table IA or IB, columns 5 or
7, lines 227 to 229 or line 230 or lines 595 to 598, or a portion
thereof and encodes a protein having above-mentioned activity, e.g.
conferring a of gamma-aminobutyric acid or shikimate or putrescine
increase, resp., and optionally, the activity of protein indicated
in Table IIA or IIB, column 5, lines 227 to 229 or line 230 or
lines 595 to 598.
[7776] [00149.1.0.17] Optionally, in one embodiment, the nucleotide
sequence, which hybridises to one of the nucleotide sequences
indicated in Table IA or IB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598 has further one or more of the
activities annotated or known for a protein as indicated in Table
IIA or IIB, column 3, lines 227 to 229 or line 230 or lines 595 to
598.
[7777] [0150.0.17.17] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table IA or IB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598, for example a fragment
which can be used as a probe or primer or a fragment encoding a
biologically active portion of the polypeptide of the present
invention or of a polypeptide used in the process of the present
invention, i.e. having above-mentioned activity, e.g. conferring an
increase of gamma-aminobutyric acid or shikimate or putrescine,
resp., if its activity is increased. The nucleotide sequences
determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table IA or IB,
columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to 598,
an anti-sense sequence of one of the sequences, e.g., as indicated
in Table IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table III, column 7, lines 227 to 229 or line 230 or lines 595 to
598 will result in a fragment of a polynucleotide sequence as
indicated in Table IA or IB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598 or its gene product.
[7778] [0151.0.0.17]: see [0151.0.0.0]
[7779] [0152.0.17.17] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598 such that the protein or
portion thereof maintains the ability to participate in the
respective fine chemical production, in particular a
gamma-aminobutyric acid (lines 227 to 229, 595, 596) or shikimate
(lines 230, 597) or putrescine (line 598) increasing activity as
mentioned above or as described in the examples in plants or
microorganisms is comprised.
[7780] [0153.0.17.17] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II A or IIB, columns 5 or 7,
lines 227 to 229 or line 230 or lines 595 to 598 such that the
protein or portion thereof is able to participate in the increase
of the respective fine chemical production. In one embodiment, a
protein or portion thereof as indicated in Table IIA or IIB,
columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to 598
has for example an activity of a polypeptide indicated in Table IIA
or IIB, column 3, lines 227 to 229 or line 230 or lines 595 to
598.
[7781] [0154.0.17.17] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598 and has above-mentioned
activity, e.g. conferring preferably the increase of the respective
fine chemical.
[7782] [0155.0.0.17] to [0156.0.0.17]: see [0155.0.0.0] to
[0156.0.0.0]
[7783] [0157.0.17.17] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences as
indicated in Table IA or IB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598 (and portions thereof) due to
degeneracy of the genetic code and thus encode a polypeptide of the
present invention, in particular a polypeptide having above
mentioned activity, e.g. conferring an increase in the respective
fine chemical in a organism, e.g. as polypeptides comprising the
sequence as indicated in Table IV, columns 5 or 7, lines 227 to 229
or line 230 or lines 595 to 598 or as polypeptides depicted in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598 or the functional homologues. Advantageously, the
nucleic acid molecule of the invention comprises, or in an other
embodiment has, a nucleotide sequence encoding a protein
comprising, or in an other embodiment having, an amino acid
sequence of a consensus sequences as indicated in Table IV, columns
5 or 7, lines 227 to 229 or line 230 or lines 595 to 598 or of the
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598, resp., or the
functional homologues. In a still further embodiment, the nucleic
acid molecule of the invention encodes a full length protein which
is substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table IV, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598 or of a polypeptide as
indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598 or the functional homologues. However,
in a preferred embodiment, the nucleic acid molecule of the present
invention does not consist of a sequence as indicated in Table IA
or IB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598, resp.
[7784] [0158.0.0.17] to [0160.0.0.17]: see [0158.0.0.0] to
[0160.0.0.0]
[7785] [0161.0.17.17] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table IA or IB, columns 5 or 7, lines 227
to 229 or line 230 or lines 595 to 598. The nucleic acid molecule
is preferably at least 20, 30, 50, 100, 250 or more nucleotides in
length.
[7786] [0162.0.0.17] see [0162.0.0.0]
[7787] [0163.0.17.17] Preferably, a nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table IA or IB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598 corresponds to a naturally-occurring
nucleic acid molecule of the invention. As used herein, a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA
molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein). Preferably, the nucleic acid molecule
encodes a natural protein having above-mentioned activity, e.g.
conferring the increase of the amount of the respective fine
chemical in a organism or a part thereof, e.g. a tissue, a cell, or
a compartment of a cell, after increasing the expression or
activity thereof or the activity of a protein of the invention or
used in the process of the invention.
[7788] [0164.0.0.17] see [0164.0.0.0]
[7789] [0165.0.17.17] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table IA or IB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598, resp.
[7790] [0166.0.0.17] to [0167.0.0.17]: see [0166.0.0.0] to
[0167.0.0.0]
[7791] [0168.0.17.17] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 227 to 229 or line 230 or lines 595 to 598, resp., yet
retain said activity described herein. The nucleic acid molecule
can comprise a nucleotide sequence encoding a polypeptide, wherein
the polypeptide comprises an amino acid sequence at least about 50%
identical to an amino acid sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598, resp., and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598, resp., more preferably
at least about 70% identical to one of the sequences as indicated
in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230
or lines 595 to 598, resp., even more preferably at least about
80%, 90%, 95% homologous to a sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598, resp., and most preferably at least about 96%, 97%, 98%, or
99% identical to the sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598.
[7792] [0169.0.0.17] to [0172.0.0.17]: see [0169.0.0.0] to
[0172.0.0.0]
[7793] [0173.0.17.17] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 15720 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 15720 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[7794] [0174.0.0.17]: see [0174.0.0.0]
[7795] [0175.0.17.17] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 15721 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 15721 by the above program algorithm with the
above parameter set, has a 80% homology.
[7796] [0176.0.17.17] Functional equivalents derived from one of
the polypeptides as indicated in Table IIA or IIB, columns 5 or 7,
lines 227 to 229 or line 230 or lines 595 to 598, resp., according
to the invention by substitution, insertion or deletion have at
least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65%
or 70% by preference at least 80%, especially preferably at least
85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at
least 95%, 97%, 98% or 99% homology with one of the polypeptides as
indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598, resp., according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598, resp.
[7797] [0177.0.17.17] Functional equivalents derived from a nucleic
acid sequence as indicated in Table IA or IB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598, resp., according to the
invention by substitution, insertion or deletion have at least 30%,
35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65% or 70% by
preference at least 80%, especially preferably at least 85% or 90%,
91%, 92%, 93% or 94%, very especially preferably at least 95%, 97%,
98% or 99% homology with one of the polypeptides as indicated in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, resp., according to the invention and encode
polypeptides having essentially the same properties as a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598, resp.
[7798] [0178.0.0.17] see [0178.0.0.0]
[7799] [0179.0.17.17] A nucleic acid molecule encoding a homologous
to a protein sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 227 to 229 or line 230 or lines 595 to 598, resp., can
be created by introducing one or more nucleotide substitutions,
additions or deletions into a nucleotide sequence of the nucleic
acid molecule of the present invention, in particular as indicated
in Table IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, resp., such that one or more amino acid
substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced into the encoding
sequences as indicated in Table IA or IB, columns 5 or 7, lines 227
to 229 or line 230 or lines 595 to 598, resp., by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[7800] [0180.0.0.17] to [0183.0.0.17]: see [0180.0.0.0] to
[0183.0.0.0]
[7801] [0184.0.17.17] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table IA or IB, columns 5 or
7, lines 227 to 229 or line 230 or lines 595 to 598, resp., or of
the nucleic acid sequences derived from a sequences as indicated in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, resp., comprise also allelic variants with at
least approximately 30%, 35%, 40% or 45% homology, by preference at
least approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595
to 598, resp., or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[7802] [0185.0.17.17] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, resp. In one embodiment, it is preferred that the
nucleic acid molecule comprises as little as possible other
nucleotides not shown in any one of sequences as indicated in Table
IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595
to 598, resp. In one embodiment, the nucleic acid molecule
comprises less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or
40 further nucleotides. In a further embodiment, the nucleic acid
molecule comprises less than 30, 20 or 10 further nucleotides. In
one embodiment, a nucleic acid molecule used in the process of the
invention is identical to a sequence as indicated in Table IA or
IB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598, resp.
[7803] [0186.0.17.17] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to 598,
resp. In one embodiment, the nucleic acid molecule encodes less
than 150, 130, 100, 80, 60, 50, 40 or 30 further amino acids. In a
further embodiment, the encoded polypeptide comprises less than 20,
15, 10, 9, 8, 7, 6 or 5 further amino acids. In one embodiment, the
encoded polypeptide used in the process of the invention is
identical to the sequences as indicated in Table IIA or IIB,
columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to 598,
resp.
[7804] [0187.0.17.17] In one embodiment, a nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II A or IIB, columns
5 or 7, lines 227 to 229 or line 230 or lines 595 to 598, resp.,
comprises less than 100 further nucleotides. In a further
embodiment, said nucleic acid molecule comprises less than 30
further nucleotides. In one embodiment, the nucleic acid molecule
used in the process is identical to a coding sequence encoding a
sequences as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598, resp.
[7805] [0188.0.17.17] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines
595 to 598, resp., and is expressed under identical conditions.
[7806] [0189.0.17.17] Homologues of a sequences as indicated in
Table IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, resp., or of a derived sequences as indicated in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, resp., also mean truncated sequences, cDNA,
single-stranded DNA or RNA of the coding and noncoding DNA
sequence. Homologues of said sequences are also understood as
meaning derivatives, which comprise noncoding regions such as, for
example, UTRs, terminators, enhancers or promoter variants. The
promoters upstream of the nucleotide sequences stated can be
modified by one or more nucleotide substitution(s), insertion(s)
and/or deletion(s) without, however, interfering with the
functionality or activity either of the promoters, the open reading
frame (=ORF) or with the 3'-regulatory region such as terminators
or other 3' regulatory regions, which are far away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[7807] [0190.0.0.17]: see [0190.0.0.0]
[7808] [0191.0.17.17] The organisms or part thereof produce
according to the herein mentioned process of the invention an
increased level of free and/or bound the respective fine chemical
compared to said control or selected organisms or parts
thereof.
[7809] [0192.0.0.17] to [0203.0.0.17]: see [0192.0.0.0] to
[0203.0.0.0]
[7810] [0204.0.17.17] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [7811] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598, resp.; or a fragment
thereof conferring an increase in the amount of the respective fine
chemical, i.e. gamma-aminobutyric acid (lines 227 to 229, 595, 596)
or shikimate (lines 230, 597) or putrescine (line 598), resp., in
an organism or a part thereof [7812] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule as indicated in Table IA or IB, columns 5 or 7, lines 227
to 229 or line 230 or lines 595, 596 or line 597 or line 598,
resp., or a fragment thereof conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[7813] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [7814] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [7815] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[7816] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[7817] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [7818] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table III, columns 5 or 7, lines 227 to 229 or line
230 or line 595, 596 or line 597 or line 598 and conferring an
increase in the amount of the respective fine chemical, i.e.
gamma-aminobutyric acid (lines 227 to 229, 595, 596) or shikimate
(line 230, 597) or putrescine (line 598), resp., in an organism or
a part thereof; [7819] i) nucleic acid molecule encoding a
polypeptide which is isolated, e.g. from a expression library, with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (g), preferably to (a)
to (c) and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [7820] j) nucleic
acid molecule which encodes a polypeptide comprising a consensus
sequence as indicated in Table IV, columns 5 or 7, lines 227 to 229
or line 230 and conferring an increase in the amount of the
respective fine chemical, i.e. gamma-aminobutyric acid (lines 227
to 229, 595, 596) or shikimate (line 230, 597) or putrescine (line
598), resp., in an organism or a part thereof; [7821] k) nucleic
acid molecule encoding the amino acid sequence of a polypeptide
encoding a domaine of a polypeptide as indicated in Table IIA or
IIB, columns 5 or 7, lines 227 to 229 or line 230, resp., and
conferring an increase in the amount of the respective fine
chemical, i.e. gamma-aminobutyric acid (lines 227 to 229, 595, 596)
or shikimate (line 230, 597) or putrescine (line 598), resp., in an
organism or a part thereof; and [7822] l) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (h) or of a nucleic acid molecule as indicated in Table
IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595
to 598, resp., or a nucleic acid molecule encoding, preferably at
least the mature form of, a polypeptide as indicated in Table IIA
or IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595
to 598, resp., and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
encompasses a sequence which is complementary thereto; whereby,
preferably, the nucleic acid molecule according to (a) to (l)
distinguishes over a sequence as indicated in Table IA or IB,
columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to 598,
resp., by one or more nucleotides. In one embodiment, the nucleic
acid molecule of the invention does not consist of the sequence as
indicated in Table I A or IB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598, resp. In an other embodiment, the
nucleic acid molecule of the present invention is at least 30
identical and less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence as indicated in Table IA or IB, columns 5
or 7, lines 227 to 229 or line 230 or lines 595 to 598, resp. In a
further embodiment the nucleic acid molecule does not encode a
polypeptide sequence as indicated in Table IIA or IIB, columns 5 or
7, lines 227 to 229 or line 230 or lines 595 to 598, resp.
Accordingly, in one embodiment, the nucleic acid molecule of the
present invention encodes in one embodiment a polypeptide which
differs at least in one or more amino acids from a polypeptide
indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598 does not encode a protein of a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598. Accordingly, in one
embodiment, the protein encoded by a sequences of a nucleic acid
according to (a) to (l) does not consist of a sequence as indicated
in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230
or lines 595 to 598. In a further embodiment, the protein of the
present invention is at least 30 identical to a protein sequence
indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598 and less than 100%, preferably less
than 99.999%, 99.99% or 99.9%, more preferably less than 99%, 98%,
97%, 96% or 95% identical to a sequence as indicated in Table IIA
or IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595
to 598.
[7823] [0205.0.0.17] to [0206.0.0.17]: see [0205.0.0.0] to
[0206.0.0.0]
[7824] [0207.0.17.17] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the glutamic acid metabolism, the
phosphoenolpyruvate metabolism, the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[7825] [0208.0.0.17] to [0226.0.0.17]: see [0208.0.0.0] to
[0226.0.0.0]
[7826] [0227.0.17.17] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[7827] In addition to a sequence indicated in Table IA or IB,
columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to 598 or
its derivatives, it is advantageous to express and/or mutate
further genes in the organisms. Especially advantageously,
additionally at least one further gene of the glutamic acid or
phosphoenolpyruvate metabolic pathway, is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the fine
chemicals desired since, for example, feedback regulations no
longer exist to the same extent or not at all. In addition it might
be advantageously to combine one or more of the sequences indicated
in Table IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, resp., with genes which generally support or
enhances to growth or yield of the target organisms, for example
genes which lead to faster growth rate of microorganisms or genes
which produces stress-, pathogen, or herbicide resistant
plants.
[7828] [0228.0.17.17] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins directly or indirectly involved
in the glutamic acid or phosphoenolpyruvate metabolism.
[7829] [0229.0.17.17] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences indicated
in Table IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598 used in the process and/or the abovementioned
biosynthesis genes are the sequences encoding further genes of the
aromatic amino acid pathway, such as tryptophan, phenylalanine or
tyrosine. These genes can lead to an increased synthesis of the
essential amino acids tryptophan, phenylalanine or tyrosine.
[7830] [0230.0.0.17] see [230.0.0.0]
[7831] [0231.0.17.17] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a gamma-aminobutyric acid or
shikimate degrading protein is attenuated, in particular by
reducing the rate of expression of the corresponding gene. A person
skilled in the art knows for example, that the inhibition of an
enzyme or a gene of the biosynthesis of the essential aromatic
amino acids tryptophan, tyrosine or phenylalanine may increase the
amount of shikimate accumulating in an organism, in particular in
plants. Furthermore the inhibition of tryptophan, phenylalanine or
tyrosine degrading enzymes also may result in an increased
shikimate accumulation in the organism.
[7832] [0232.0.0.17] to [0276.0.0.17]: see [0232.0.0.0] to
[0276.0.0.0]
[7833] [0277.0.17.17] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously.
[7834] [0278.0.0.17] to [0282.0.0.17]: see [0278.0.0.0] to
[0282.0.0.0]
[7835] [0283.0.17.17] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells, for example using the antibody
of the present invention as described below, e.g. an antibody
against a protein as indicated in Table IIA or IIB, column 3, lines
227 to 229 or line 230 or lines 595 to 598, resp., or an antibody
against a polypeptide as indicated in Table II A or IIB, columns 5
or 7, lines 227 to 229 or line 230 or lines 595 to 598, resp.,
which can be produced by standard techniques utilizing the
polypeptid of the present invention or fragment thereof. Preferred
are monoclonal antibodies.
[7836] [0284.0.0.17] see [0284.0.0.0]
[7837] [0285.0.17.17] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines
595 to 598, resp., or as coded by a nucleic acid molecule as
indicated in Table IA or IB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598, resp., or functional homologues
thereof.
[7838] [0286.0.17.17] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 227 to 229 or line 230 or lines 595 to
598 and in one another embodiment, the present invention relates to
a polypeptide comprising or consisting of a consensus sequence as
indicated in Table
[7839] IV, column 7, lines 227 to 229 or line 230 or lines 595 to
598 whereby 20 or less, preferably 15 or 10, preferably 9, 8, 7, or
6, more preferred 5 or 4, even more preferred 3, even more
preferred 2, even more preferred 1, most preferred 0 of the amino
acids positions indicated can be replaced by any amino acid or, in
an further embodiment, can be replaced and/or absent. In one
embodiment, the present invention relates to the method of the
present invention comprising a polypeptide or to a polypeptide
comprising more than one consensus sequences (of an individual
line) as indicated in Table IV, column 7, lines 227 to 230 and/or
595 to 598, resp.
[7840] [0287.0.0.17] to [0289.0.0.17]: see [0287.0.0.0] to
[0289.0.0.0]
[7841] [00290.0.17.17] The alignment was performed with the
Software AlignX (Sep. 25, 2002) a component of Vector NTI Suite
8.0, InforMax.TM., Invitrogen.TM. life science software, U.S. Main
Office, 7305 Executive Way, Frederick, Md. 21704, USA with the
following settings: For pairwise alignments: gap opening penalty:
10.0; gap extension penalty 0.1. For multiple alignments: Gap
opening penalty: 10.0; Gap extension penalty: 0.1; Gap separation
penalty range: 8; Residue substitution matrix: blosum62;
Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0.5;
Consensus calculation options: Residue fraction for consensus: 0.9.
Presettings were selected to allow also for the alignment of
conserved amino acids
[7842] [0291.0.17.17] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[7843] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to 598,
resp., by one or more amino acids. In one embodiment, polypeptide
distinguishes from a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to 598,
resp., by more than 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, even more preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598, resp., by not more than 80% or 70% of
the amino acids, preferably not more than 60% or 50%, more
preferred not more than 40% or 30%, even more preferred not more
than 20% or 10%. In an other embodiment, said polypeptide of the
invention does not consist of a sequence as indicated in Table IIA
or IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595
to 598.
[7844] [0292.0.0.17] see [0292.0.0.0]
[7845] [0293.0.17.17] In one embodiment, the invention relates to a
polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or by a nucleic acid molecule used in the process of the
invention. In one embodiment, the polypeptide of the invention has
a sequence which distinguishes from a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, resp., by one or more amino acids. In an other
embodiment, said polypeptide of the invention does not consist of
the sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 227 to 229 or line 230 or lines 595 to 598, resp. In a
further embodiment, said polypeptide of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical. In one
embodiment, said polypeptide does not consist of the sequence
encoded by a nucleic acid molecules as indicated in Table IA or IB,
columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to 598,
resp.
[7846] [0294.0.17.17] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table IIA or IIB, column 3, lines 227 to 229 or line
230 or lines 595 to 598, resp., which distinguishes over a sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229
or line 230 or lines 595 to 598, resp., by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[7847] [0295.0.0.17] to [0296.0.0.17]: see [0295.0.0.0] to
[0296.0.0.0]
[7848] [0297.0.0.17] see [0297.0.0.0]
[7849] [00297.1.0.17] %
[7850] [0298.0.17.17] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229
or line 230 or lines 595 to 598, resp. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, resp.
[7851] [0299.0.17.17] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table IA or IB, columns 5 or 7,
lines 227 to 229 or line 230 or lines 595 to 598, resp. The
preferred polypeptide of the present invention preferably possesses
at least one of the activities according to the invention and
described herein. A preferred polypeptide of the present invention
includes an amino acid sequence encoded by a nucleotide sequence
which hybridizes, preferably hybridizes under stringent conditions,
to a nucleotide sequence as indicated in Table IA or IB, columns 5
or 7, lines 227 to 229 or line 230 or lines 595 to 598, resp., or
which is homologous thereto, as defined above.
[7852] [0300.0.17.17] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598, resp., in amino acid sequence due to natural variation or
mutagenesis, as described in detail herein. Accordingly, the
polypeptide comprise an amino acid sequence which is at least about
35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about
75%, 80%, 85% or 90, and more preferably at least about 91%, 92%,
93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%,
99% or more homologous to an entire amino acid sequence of as
indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598, resp.
[7853] [0301.0.0.17] see [0301.0.0.0]
[7854] [0302.0.17.17] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, resp., or the amino acid sequence of a protein
homologous thereto, which include fewer amino acids than a full
length polypeptide of the present invention or used in the process
of the present invention or the full length protein which is
homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[7855] [0303.0.0.17] see [0303.0.0.0]
[7856] [0304.0.17.17] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
IIA or IIB, column 3, lines 227 to 229 or line 230 or lines 595 to
598 but having differences in the sequence from said wild-type
protein. These proteins may be improved in efficiency or activity,
may be present in greater numbers in the cell than is usual, or may
be decreased in efficiency or activity in relation to the wild type
protein.
[7857] [0305.0.0.17] to [0308.0.0.17]: see [0305.0.0.0] to
[0308.0.0.0]
[7858] [0309.0.17.17] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table IIA or
IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598, resp., refers to a polypeptide having an amino acid sequence
corresponding to the polypeptide of the invention or used in the
process of the invention, whereas an "other polypeptide" not being
indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598, resp., refers to a polypeptide having
an amino acid sequence corresponding to a protein which is not
substantially homologous to a polypeptide of the invention,
preferably which is not substantially homologous to a polypeptide
as indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229
or line 230 or lines 595 to 598, resp., e.g., a protein which does
not confer the activity described herein or annotated or known for
as indicated in Table IIA or IIB, column 3, lines 227 to 229 or
line 230 or lines 595 to 598, resp., and which is derived from the
same or a different organism. In one embodiment, an "other
polypeptide" not being indicated in Table IIA or IIB, columns 5 or
7, lines 227 to 229 or line 230 or lines 595 to 598, resp., does
not confer an increase of the respective fine chemical in an
organism or part thereof.
[7859] [0310.0.0.17] to [0334.0.0.17]: see [0310.0.0.0] to
[0334.0.0.0] [0335.0.17.17] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table IA or IB, columns
5 or 7, lines 227 to 229 or line 230 or lines 595 to 598, resp.,
and/or homologs thereof. As described inter alia in WO 99/32619,
dsRNAi approaches are clearly superior to traditional antisense
approaches. The invention therefore furthermore relates to
double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived there from), bring about
altered metabolic activity by the reduction in the expression of a
nucleic acid sequences as indicated in Table IA or IB, columns 5 or
7, lines 227 to 229 or line 230 or lines 595 to 598, resp., and/or
homologs thereof. In a double-stranded RNA molecule for reducing
the expression of aprotein encoded by a nucleic acid sequence as
indicated in Table IA or IB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598, resp., and/or homologs thereof, one
of the two RNA strands is essentially identical to at least part of
a nucleic acid sequence, and the respective other RNA strand is
essentially identical to at least part of the complementary strand
of a nucleic acid sequence.
[7860] [0336.0.0.17] to [0342.0.0.17]: see [0336.0.0.0] to
[0342.0.0.0]
[7861] [0343.0.17.17] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table IA or IB, columns 5 or 7, lines 227
to 229 or line 230 or lines 595 to 598, resp., or its homolog is
not necessarily required in order to bring about effective
reduction in the expression. The advantage is, accordingly, that
the method is tolerant with regard to sequence deviations as may be
present as a consequence of genetic mutations, polymorphisms or
evolutionary divergences. Thus, for example, using the dsRNA, which
has been generated starting from a sequence as indicated in Table
IA or IB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595
to 598, resp., or homologs thereof of the one organism, may be used
to suppress the corresponding expression in another organism.
[7862] [0344.0.0.17] to [0350.0.0.17]: see [0344.0.0.0] to
[0350.0.0.0]
[7863] [0351.0.0.17] to [0361.0.0.17]: see [0351.0.0.0] to
[0361.0.0.0]
[7864] [0362.0.17.17] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598, resp., e.g. encoding a polypeptide having protein
activity, as indicated in Table IIA or IIB, columns 3, lines 227 to
229 or line 230 or lines 595 to 598, resp. Due to the
abovementioned activity the respective fine chemical content in a
cell or an organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. Transgenic for a polypeptide having an activity of a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598, resp., means herein
that due to modulation or manipulation of the genome, an activity
as annotated for a polypeptide as indicated in Table IIA or IIB,
column 3, lines 227 to 229 or line 230 or lines 595 to 598, e.g.
having a sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 227 to 229 or line 230 or lines 595 to 598, resp., is
increased in a cell or an organism or a part thereof. Examples are
described above in context with the process of the invention.
[7865] [0363.0.0.17] see [0363.0.0.0]
[7866] [0364.0.17.17] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table IIA or IIB, column 3, lines 227 to 229 or line
230 or lines 595 to 598, resp. with the corresponding
protein-encoding sequence as indicated in Table IA or IB, column 5,
lines 227 to 229 or line 230 or lines 595 to 598, resp., becomes a
transgenic expression cassette when it is modified by non-natural,
synthetic "artificial" methods such as, for example,
mutagenization. Such methods have been described (U.S. Pat. No.
5,565,350; WO 00/15815; also see above).
[7867] [0365.0.0.17] to [0373.0.0.17]: see [0365.0.0.0] to
[0373.0.0.0]
[7868] [0374.0.17.17] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. gamma-aminobutyric acid or
shikimate, in particular the respective fine chemical, produced in
the process according to the invention may, however, also be
isolated from the plant in the form of their free
gamma-aminobutyric acid or shikimate, in particular the free
respective fine chemical, or bound in or to compounds or moieties,
like glucosides, e.g. diglucosides. The respective fine chemical
produced by this process can be harvested by harvesting the
organisms either from the culture in which they grow or from the
field. This can be done via expressing, grinding and/or extraction,
salt precipitation and/or ion-exchange chromatography or other
chromatographic methods of the plant parts, preferably the plant
seeds, plant fruits, plant tubers and the like.
[7869] [0375.0.0.17] to [0376.0.0.17]: see [0375.0.0.0] to
[0376.0.0.0]
[7870] [0377.0.17.17] Accordingly, the present invention relates
also to a process whereby the produced gamma-aminobutyric acid or
shikimate is isolated.
[7871] [0378.0.17.17] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the
gamma-aminobutyric acid or shikimate produced in the process can be
isolated. The resulting gamma-aminobutyric acid or shikimate can,
if appropriate, subsequently be further purified, if desired mixed
with other active ingredients such as vitamins, amino acids,
carbohydrates, antibiotics and the like, and, if appropriate,
formulated.
[7872] [0379.0.17.17] In one embodiment, gamma-aminobutyric acid
and shikimate are a mixture of the respective fine chemicals.
[7873] [0380.0.17.17] The gamma-aminobutyric acid or shikimate
obtained in the process are suitable as starting material for the
synthesis of further products of value. For example, they can be
used in combination with each other or alone for the production of
pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the gamma-aminobutyric acid
or shikimate composition produced or the respective fine chemical
produced if desired and formulating the product with a
pharmaceutical acceptable carrier or formulating the product in a
form acceptable for an application in agriculture. A further
embodiment according to the invention is the use of the
gamma-aminobutyric acid or shikimate produced in the process or of
the transgenic organisms in animal feeds, foodstuffs, medicines,
food supplements, cosmetics or pharmaceuticals or for the
production of gamma-aminobutyric acid or shikimate e.g. after
isolation of the respective fine chemical or without, e.g. in situ,
e.g in the organism used for the process for the production of the
respective fine chemical.
[7874] [0381.0.0.17] to [0382.0.0.17]: see [0381.0.0.0] to
[0382.0.0.0] [0383.0.17.17]
[7875] [0384.0.0.17] see [0384.0.0.0]
[7876] [0385.0.17.17] The fermentation broths obtained in this way,
containing in particular gamma-aminobutyric acid or shikimate in
mixtures with other organic acids, aminoacids, polypeptides or
polysaccarides, normally have a dry matter content of from 1 to 70%
by weight, preferably 7.5 to 25% by weight. Sugar-limited
fermentation is additionally advantageous, e.g. at the end, for
example over at least 30% of the fermentation time. This means that
the concentration of utilizable sugar in the fermentation medium is
kept at, or reduced to, 0 to 10 g/l, preferably to 0 to 3 g/I
during this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed or isolated
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation, coagulation/flocculation
or a combination of these methods, from the fermentation broth or
left completely in it.
[7877] The fermentation broth can then be thickened or concentrated
by known methods, such as, for example, with the aid of a rotary
evaporator, thin-film evaporator, falling film evaporator, by
reverse osmosis or by nanofiltration. This concentrated
fermentation broth can then be worked up by freeze-drying, spray
drying, spray granulation or by other processes.
[7878] [0386.0.17.17] Accordingly, it is possible to purify the
gamma-aminobutyric acid or shikimate produced according to the
invention further. For this purpose, the product-containing
compositions subjected for example to separation via e.g. an open
column chromatography or HPLC in which case the desired product or
the impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use.
[7879] [0387.0.0.17] to [0392.0.0.17]: see [0387.0.0.0] to
[0392.0.0.0]
[7880] [0393.0.17.17] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [7881] a. contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [7882] b. identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table IA or IB, columns 5 or 7,
lines 227 to 229 or line 230 or lines 595 to 598, resp., and,
optionally, isolating the full length cDNA clone or complete
genomic clone; [7883] c. introducing the candidate nucleic acid
molecules in host cells, preferably in a plant cell or a
microorganism, appropriate for producing the respective fine
chemical; [7884] d. expressing the identified nucleic acid
molecules in the host cells; [7885] e. assaying the respective fine
chemical level in the host cells; and [7886] f. identifying the
nucleic acid molecule and its gene product which expression confers
an increase in the respective fine chemical level in the host cell
after expression compared to the wild type.
[7887] [0394.0.0.17] to [0398.0.0.17]: see [0394.0.0.0] to
[0398.0.0.0]
[7888] [0399.0.17.17] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increase of the respective fine chemical production in a
plant or microorganism, comprising the steps:
[7889] culturing a cell or tissue or microorganism or maintaining a
plant expressing the polypeptide according to the invention or a
nucleic acid molecule encoding said polypeptide and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and the polypeptide of the present invention or used
in the process of the invention; and
[7890] identifying if the compound is an effective agonist by
detecting the presence or absence or increase of a signal produced
by said readout system.
[7891] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in respective fine chemical
production.
[7892] [00399.1.0.17] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table IIA
or IIB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595
to 598 or a homolog thereof, e.g. comparing the phenotype of nearly
identical organisms with low and high activity of a protein as
indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598 after incubation with the drug.
[7893] [0400.0.0.17] to [0415.0.0.17]: see [0400.0.0.0] to
[0415.0.0.0]
[7894] [0416.0.0.17] see [0416.0.0.0]
[7895] [0417.0.17.17] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the gamma-aminobutyric acid or shikimate
or putrescine biosynthesis pathways. In particular, the
overexpression of the polypeptide of the present invention may
protect an organism such as a microorganism or a plant against
inhibitors, which block the gamma-aminobutyric acid or shikimate or
putrescine synthesis.
[7896] [0418.0.0.17] to [0423.0.0.17]: see [0418.0.0.0] to
[0423.0.0.0]
[7897] [0424.0.17.17] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other organic acids.
Accordingly, the nucleic acid of the invention, or the nucleic acid
molecule identified with the method of the present invention or the
complement sequences thereof, the polypeptide of the invention, the
nucleic acid construct of the invention, the organisms, the host
cell, the microorganisms, the plant, plant tissue, plant cell, or
the part thereof of the invention, the vector of the invention, the
antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the respective
fine chemical in a organism or part thereof, e.g. in a cell.
[7898] [0425.0.0.17] to [0434.0.0.0]: see [0425.0.0.0] to
[0434.0.0.0]
[0435.0.17.17] Example 3
In-Vivo and In-Vitro Mutagenesis
[7899] [0436.0.17.17] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of gamma-aminobutyric acid
or shikimate or putrescine can be carried out by passing a plasmid
DNA (or another vector DNA) containing the desired nucleic acid
sequence or nucleic acid sequences, e.g. the nucleic acid molecule
of the invention or the vector of the invention, through E. coli
and other microorganisms (for example Bacillus spp. or yeasts such
as Saccharomyces cerevisiae) which are not capable of maintaining
the integrity of its genetic information. Usual mutator strains
have mutations in the genes for the DNA repair system [for example
mutHLS, mutD, mutT and the like; for comparison, see Rupp, W. D.
(1996) DNA repair mechanisms in Escherichia coli and Salmonella,
pp. 2277-2294, ASM: Washington]. The skilled worker knows these
strains. The use of these strains is illustrated for example in
Greener, A. and Callahan, M. (1994) Strategies 7; 32-34.
[7900] In-vitro mutation methods such as increasing the spontaneous
mutation rates by chemical or physical treatment are well known to
the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[7901] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[7902] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.17.17] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces Cerevisiae and
Mortierella alpina
[7903] [0438.0.17.17] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology", VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[7904] [0439.0.17.17] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255f) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[7905] [0440.0.17.17] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002,) teaches a homolog recombination system dependent
on rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this
system for homologous recombination are vectors derived from the
Ylp series. Plasmid vectors derived for example from the
2.mu.-Vector are known by the skilled worker and used for the
expression in yeasts. Other preferred vectors are for example
pART1, pCHY21 or pEVP11 as they have been described by McLeod et
al. (EMBO J. 1987, 6:729-736) and Hoffman et al. (Genes Dev. 5,
1991: :561-571.) or Russell et al. (J. Biol. Chem. 258, 1983:
143-149.). Other beneficial yeast vectors are plasmids of the REP,
REP-X, pYZ or RIP series.
[7906] [0441.0.0.17] see [0441.0.0.0]
[7907] [0442.0.0.17] see [0442.0.0.0]
[7908] [0443.0.0.17] see [0443.0.0.0]
[0444.0.17.17] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[7909] [0445.0.17.17] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[7910] [0446.0.0.17] to [0450.0.0.17]: see [0446.0.0.0] to
[0450.0.0.0]
[7911] [0451.0.0.17] see [0451.0.5.5]
[7912] [0452.0.0.17] to [0453.0.0.17]: see [0452.0.0.0] to
[0453.0.0.0]
[7913] [0454.0.17.17] Analysis of the effect of the nucleic acid
molecule on the production of gamma-aminobutyric acid or shikimate
or putrescine
[7914] [0455.0.17.17] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a gamma-aminobutyric acid or shikimate or
putrescine) can be determined by growing the modified
microorganisms or the modified plant under suitable conditions
(such as those described above) and analyzing the medium and/or the
cellular components for the elevated production of desired product
(i.e. of gamma-aminobutyric acid or shikimate or putrescine). These
analytical techniques are known to the skilled worker and comprise
spectroscopy, thin-layer chromatography, various types of staining
methods, enzymatic and microbiological methods and analytical
chromatography such as high-performance liquid chromatography (see,
for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2,
p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.,
(1987) "Applications of HPLC in Biochemistry" in: Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et
al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery
and purification", p. 469-714, VCH: Weinheim; Better, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[7915] [0456.0.0.17]: see [0456.0.0.0]
[0457.0.17.17] Example 9
Purification of Gamma-Aminobutyric Acid or Shikimate or
Putrescine
[7916] [0458.0.17.17] Abbreviations; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[7917] The unambiguous detection for the presence of
gamma-aminobutyric acid or shikimate or putrescine can be obtained
by analyzing recombinant organisms using analytical standard
methods: LC, LC-MSMS or TLC, as described. The total amount
produced in the organism for example in yeasts used in the
inventive process can be analysed for example according to the
following procedure:
[7918] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods.
[7919] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[7920] A typical sample pretreatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography.
[7921] For analysis, solvent delivery and aliquot removal can be
accomplished with a robotic system comprising a single injector
valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000
W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of
50% potassium hydroxide hydro-ethanolic solution (4 water--1
ethanol) can be added to each vial, followed by the addition of 3
ml of octanol. The saponification treatment can be conducted at
room temperature with vials maintained on an IKA HS 501 horizontal
shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours
at 250 movements/minute, followed by a stationary phase of
approximately one hour.
[7922] Following saponification, the supernatant can be diluted
with 0.17 ml of methanol. The addition of methanol can be conducted
under pressure to ensure sample homogeneity. Using a 0.25 ml
syringe, a 0.1 ml aliquot can be removed and transferred to HPLC
vials for analysis.
[7923] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete
with a quaternary pump, vacuum degassing system, six-way injection
valve, temperature regulated autosampler, column oven and
Photodiode Array detector can be used [Agilent Technologies
available through Ultra Scientific Inc., 250 Smith Street, North
Kingstown, R.I.]. The column can be a Waters YMC30, 5-micron,
4.6.times.250 mm with a guard column of the same material [Waters,
34 Maple Street, Milford, Mass.]. The solvents for the mobile phase
can be 81 methanol: 4 water: 15 tetrahydrofuran (THF) stabilized
with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injections were
20 l. Separation can be isocratic at 30.degree. C. with a flow rate
of 1.7 ml/minute. The peak responses can be measured by absorbance
at 447 nm.
[7924] [0459.0.17.17] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the gamma-aminobutyric acid or shikimate or
putrescine can be further purified with a so-called RTHPLC. As
eluent acetonitrile/water or chloroform/acetonitrile mixtures can
be used. If necessary, these chromatography steps may be repeated,
using identical or other chromatography resins. The skilled worker
is familiar with the selection of suitable chromatography resin and
the most effective use for a particular molecule to be
purified.
[7925] [0460.0.0.17] see [0460.0.0.0]
[0461.0.17.17] Example 10
Cloning SEQ ID NO: 15720, 15762, 15708, 15712, 91094, 91415, 91530
or 91635 for the Expression in Plants
[7926] [0462.0.0.17] see [0462.0.0.0]
[7927] [0463.0.17.17] SEQ ID NO: 15720, 15762, 15708,15712, 91094,
91415, 91530 or 91635 is amplified by PCR as described in the
protocol of the Pfu Turbo or DNA Herculase polymerase
(Stratagene).
[7928] [0464.0.0.0.17] to [0466.0.0.17]: see [0464.0.0.0] to
[0466.0.0.0]
[7929] [0466.1.0.17] In case the Herculase enzyme can be used for
the amplification, the PCR amplification cycles were as follows: 1
cycle of 2-3 minutes at 94.degree. C., followed by 25-30 cycles of
in each case 30 seconds at 94.degree. C., 30 seconds at
55-60.degree. C. and 5-10 minutes at 72.degree. C., followed by 1
cycle of 10 minutes at 72.degree. C., then 4.degree. C.
[7930] [0467.0.17.17] The following primer sequences were selected
for the gene SEQ ID NO: 15720:
TABLE-US-00079 i) forward primer (SEQ ID NO: 15760) atgaatagcg
taaaaagagt aaagct ii) reverse primer (SEQ ID NO: 15761) ctaggccaaa
gacatcttag cca
[7931] The following primer sequences were selected for the gene
SEQ ID NO: 15762:
TABLE-US-00080 i) forward primer (SEQ ID NO: 15766) atggcaggta
tcaagttgac gcat ii) reverse primer (SEQ ID NO: 15767) tcattttgtt
aatagttttt tgtatgct
[7932] The following primer sequences were selected for the gene
SEQ ID NO: 15708
TABLE-US-00081 i) forward primer (SEQ ID NO: 15710) atggaacaga
acaggttcaa gaaag ii) reverse primer (SEQ ID NO: 15711) ttacagtttt
tgtttagtcg ttttaac
[7933] The following primer sequences were selected for the gene
SEQ ID NO: 15712:
TABLE-US-00082 i) forward primer (SEQ ID NO: 15718) atgagtaatg
caaacaatag tgctat ii) reverse primer (SEQ ID NO: 15719) tcaatggtat
ttatagccgc attgt
[7934] The primer sequences were selected for the genes described
in Table III, column 5, lines 227 to 230 or lines 595 to 598 and as
described in Table III, column 7, lines 227 to 230 or lines 595 to
598.
[7935] [0468.0.17.17] to [0470.0.17.17]: see [0468.0.0.0] to
[0470.0.0.0]
[7936] [0470.1.17.17] The PCR-products, produced by Pfu Turbo DNA
polymerase, were phosphorylated using a T4 DNA polymerase using a
standard protocol (e.g. MBI Fermentas) and cloned into the
processed binary vector.
[7937] [0471.0.17.17] see [0471.0.0.0]
[7938] [0471.1.17.17] The DNA termini of the PCR-products, produced
by Herculase DNA polymerase, were blunted in a second synthesis
reaction using Pfu Turbo DNA polymerase. The composition for the
protocol of the blunting the DNA-termini was as follows: 0.2 mM
blunting dTTP and 1.25 u Pfu Turbo DNA polymerase. The reaction was
incubated at 72.degree. C. for 30 minutes. Then the PCR-products
were phosphorylated using a T4 DNA polymerase using a standard
protocol (e.g. MBI Fermentas) and cloned into the processed vector
as well.
[7939] [0472.0.17.17] to [0479.0.17.17]: see [0472.0.0.0] to
[0479.0.0.0]
[0480.0.17.17] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 15720,
15762, 15708,15712, 91094, 91415, 91530 or 91635
[7940] [0481.0.0.17] to [0513.0.0.17]: see [0481.0.0.0] to
[0513.0.0.0]
[7941] [0514.0.17.17] As an alternative, gamma-aminobutyric acid
can be detected as described in Haak and Reineke, Antimicrob.
Agents Chemother. 19(3): 493(1981)
[7942] As an alternative, shikimate can be detected as described in
Gould and Erickson, J Antibiot 41(5), 688-9 (1988).
[7943] As an alternative, putrescine can be detected as described
in Endo Y., Anal Biochem. 89(1):235-46(1978).
[7944] The results of the different plant analyses can be seen from
the table 1 which follows:
TABLE-US-00083 TABLE 1 ORF metabolite method min max YER156C
gamma-Aminobutyric Acid GC 1,68 11,29 YFR042W gamma-Aminobutyric
Acid GC 1,91 2,10 YOR084W gamma-Aminobutyric Acid GC 1,71 5,59
YLR375W Shikimate GC 1,14 1,26 b0651 gamma-Aminobutyric acid GC
1.53 3.06 (GABA) b1693 Shikimic Acid GC 1.15 1.31 b2965 Putrescine
GC 2.11 17.93 gamma-Aminobutyric acid b0847 (GABA) GC 1.85 2.87
[7945] [0515.0.0.17] to [0552.0.0.17]: see [0515.0.0.0] to
[0552.0.0.0]
[0552.1.17.17]: Example 15
Metabolite Profiling Info from Zea mays
[7946] Zea mays plants were engineered, grown and analyzed as
described in Example 14c.
[7947] The results of the different Zea mays plants analysed can be
seen from Table 2 which follows:
TABLE-US-00084 TABLE 2 ORF Metabolite Min Max YLR375W Shikimate
1.59 5.97
[7948] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in shikimate in
genetically modified corn plants expressing the Saccharomyces
cerevisiae nucleic acid sequence YLR375W.
[7949] In case the activity of the Saccharomyces cerevisiae protein
YLR375W or a protein with an activity being involved in pre-tRNA
splicing and in uptake of branched-chain amino acids or its
homolog, is increased in corn plants, preferably, an increase of
the fine chemical shikimate between 59% and 497% is conferred.
[7950] [00552.2.0.17]: see [00552.2.0.0]
[7951] [0553.0.17.17] [7952] 1. A process for the production of
gamma-aminobutyric acid or shikimate or putrescine, which comprises
a) increasing or generating the activity of a protein as indicated
in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230
or lines 595 to 598 or a functional equivalent thereof in a
non-human organism or in one or more parts thereof; and b) growing
the organism under conditions which permit the production of
gamma-aminobutyric acid or shikimate or putrescine in said
organism. [7953] 2. A process for the production of
gamma-aminobutyric acid or shikimate or putrescine, comprising the
increasing or generating in an organism or a part thereof the
expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [7954]
a) nucleic acid molecule encoding of a polypeptide as indicated in
Table IIA or IIB, columns 5 or 7, lines 227 to 229 or line 230 or
lines 595 to 598 or a fragment thereof, which confers an increase
in the amount of gamma-aminobutyric acid or shikimate or putrescine
in an organism or a part thereof; [7955] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table IA or
IB, columns 5 or 7, lines 227 to 229 or line 230 or lines 595 to
598; [7956] c) nucleic acid molecule whose sequence can be deduced
from a polypeptide sequence encoded by a nucleic acid molecule of
(a) or (b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of gamma-aminobutyric acid or
shikimate or putrescine in an organism or a part thereof; [7957] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of gamma-aminobutyric acid or shikimate
or putrescine in an organism or a part thereof; [7958] e) nucleic
acid molecule which hybridizes with a nucleic acid molecule of (a)
to (c) under under stringent hybridisation conditions and
conferring an increase in the amount of gamma-aminobutyric acid or
shikimate or putrescine in an organism or a part thereof; [7959] f)
nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table III, columns 5 or 7, lines 227 to 229 or line
230 or lines 595 to 598 and conferring an increase in the amount of
gamma-aminobutyric acid or shikimate or putrescine in an organism
or a part thereof; [7960] g) nucleic acid molecule encoding a
polypeptide which is isolated with the aid of monoclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (f) and conferring an increase in the amount of
gamma-aminobutyric acid or shikimate or putrescine in an organism
or a part thereof; [7961] h) nucleic acid molecule encoding a
polypeptide comprising a consensus as indicated in Table IV, column
7, lines 227 to 229 or line 230 or lines 595 to 598 and conferring
an increase in the amount of gamma-aminobutyric acid or shikimate
or putrescine in an organism or a part thereof; and [7962] i)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment thereof having at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (k) and
conferring an increase in the amount of gamma-aminobutyric acid or
shikimate or putrescine in an organism or a part thereof. [7963] or
comprising a sequence which is complementary thereto. [7964] 3. The
process of claim 1 or 2, comprising recovering of the free or bound
gamma-aminobutyric acid or shikimate or putrescine. [7965] 4. The
process of any one of claims 1 to 3, comprising the following
steps: [7966] a) selecting an organism or a part thereof expressing
a polypeptide encoded by the nucleic acid molecule characterized in
claim 2; [7967] b) mutagenizing the selected organism or the part
thereof; [7968] c) comparing the activity or the expression level
of said polypeptide in the mutagenized organism or the part thereof
with the activity or the expression of said polypeptide of the
selected organisms or the part thereof; [7969] d) selecting the
mutated organisms or parts thereof, which comprise an increased
activity or expression level of said polypeptide compared to the
selected organism or the part thereof; [7970] e) optionally,
growing and cultivating the organisms or the parts thereof; and
[7971] f) recovering, and optionally isolating, the free or bound
gamma-aminobutyric acid or shikimate or putrescine produced by the
selected mutated organisms or parts thereof. [7972] 5. The process
of any one of claims 1 to 4, wherein the activity of said protein
or the expression of said nucleic acid molecule is increased or
generated transiently or stably. [7973] 6. An isolated nucleic acid
molecule comprising a nucleic acid molecule selected from the group
consisting of: [7974] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
227 to 229 or line 230 or lines 595 to 598 or a fragment thereof,
which confers an increase in the amount of gamma-aminobutyric acid
or shikimate or putrescine in an organism or a part thereof; [7975]
b) nucleic acid molecule comprising of a nucleic acid molecule as
indicated in Table IA or IB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598; [7976] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as a result of the degeneracy
of the genetic code and conferring an increase in the amount of
gamma-aminobutyric acid or shikimate or putrescine in an organism
or a part thereof; [7977] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of
gamma-aminobutyric acid or shikimate or putrescine in an organism
or a part thereof; [7978] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under stringent
hybridisation conditions and conferring an increase in the amount
of gamma-aminobutyric acid or shikimate or putrescine in an
organism or a part thereof; [7979] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table III, column
7, lines 227 to 229 or line 230 or lines 595 to 598 and conferring
an increase in the amount of gamma-aminobutyric acid or shikimate
or putrescine in an organism or a part thereof; [7980] g) nucleic
acid molecule encoding a polypeptide which is isolated with the aid
of monoclonal antibodies against a polypeptide encoded by one of
the nucleic acid molecules of (a) to (f) and conferring an increase
in the amount of gamma-aminobutyric acid or shikimate or putrescine
in an organism or a part thereof; [7981] h) nucleic acid molecule
encoding a polypeptide comprising a consensus as indicated in Table
IV, column 7, lines 227 to 229 or line 230 or lines 595 to 598 and
conferring an increase in the amount of gamma-aminobutyric acid or
shikimate or putrescine in an organism or a part thereof; and
[7982] i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of gamma-aminobutyric
acid or shikimate or putrescine in an organism or a part thereof.
[7983] whereby the nucleic acid molecule distinguishes over the
sequence as indicated in Table IA or IB, columns 5 or 7, lines 227
to 229 or line 230 or lines 595 to 598 by one or more nucleotides.
[7984] 7. A nucleic acid construct which confers the expression of
the nucleic acid molecule of claim 6, comprising one or more
regulatory elements. [7985] 8. A vector comprising the nucleic acid
molecule as claimed in claim 6 or the nucleic acid construct of
claim 7. [7986] 9. The vector as claimed in claim 8, wherein the
nucleic acid molecule is in operable linkage with regulatory
sequences for the expression in a prokaryotic or eukaryotic, or in
a prokaryotic and eukaryotic, host. [7987] 10. A host cell, which
has been transformed stably or transiently with the vector as
claimed in claim 8 or 9 or the nucleic acid molecule as claimed in
claim 6 or the nucleic acid construct of claim 7 or produced as
described in claim any one of claims 2 to 5. [7988] 11. The host
cell of claim 10, which is a transgenic host cell. [7989] 12. The
host cell of claim 10 or 11, which is a plant cell, an animal cell,
a microorganism, or a yeast cell, a fungus cell, a prokaryotic
cell, an eukaryotic cell or an archaebacterium. [7990] 13. A
process for producing a polypeptide, wherein the polypeptide is
expressed in a host cell as claimed in any one of claims 10 to 12.
[7991] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 227 to 229 or
line 230 or lines 595 to 598 by one or more amino acids. [7992] 15.
An antibody, which binds specifically to the polypeptide as claimed
in claim 14. [7993] 16. A plant tissue, propagation material,
harvested material or a plant comprising the host cell as claimed
in claim 12 which is plant cell or an Agrobacterium. [7994] 17. A
method for screening for agonists and antagonists of the activity
of a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of gamma-aminobutyric acid or
shikimate or putrescine in an organism or a part thereof
comprising: (a) contacting cells, tissues, plants or microorganisms
which express the a polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
gamma-aminobutyric acid or shikimate or putrescine in an organism
or a part thereof with a candidate compound or a sample comprising
a plurality of compounds under conditions which permit the
expression the polypeptide; (b) assaying the gamma-aminobutyric
acid or shikimate or putrescine level or the polypeptide expression
level in the cell, tissue, plant or microorganism or the media the
cell, tissue, plant or microorganisms is cultured or maintained in;
and (c) identifying a agonist or antagonist by comparing the
measured gamma-aminobutyric acid or shikimate or putrescine level
or polypeptide expression level with a standard gamma-aminobutyric
acid or shikimate or putrescine or polypeptide expression level
measured in the absence of said candidate compound or a sample
comprising said plurality of compounds, whereby an increased level
over the standard indicates that the compound or the sample
comprising said plurality of compounds is an agonist and a
decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[7995] 18. A process for the identification of a compound
conferring increased gamma-aminobutyric acid or shikimate or
putrescine production in a plant or microorganism, comprising the
steps: (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of gamma-aminobutyric acid or shikimate or putrescine in an
organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of gamma-aminobutyric acid or
shikimate or putrescine in an organism or a part thereof; (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [7996] 19. A method for the identification of a
gene product conferring an increase in gamma-aminobutyric acid or
shikimate or putrescine production in a cell, comprising the
following steps: (a) contacting the nucleic acid molecules of a
sample, which can contain a candidate gene encoding a gene product
conferring an increase in gamma-aminobutyric acid or shikimate or
putrescine after expression with the nucleic acid molecule of claim
6; (b) identifying the nucleic acid molecules, which hybridise
under relaxed stringent conditions with the nucleic acid molecule
of claim 6; (c) introducing the candidate nucleic acid molecules in
host cells appropriate for producing gamma-aminobutyric acid or
shikimate or putrescine; (d) expressing the identified nucleic acid
molecules in the host cells; (e) assaying the gamma-aminobutyric
acid or shikimate or putrescine level in the host cells; and (f)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the gamma-aminobutyric acid or
shikimate or putrescine level in the host cell in the host cell
after expression compared to the wild type. [7997] 20. A method for
the identification of a gene product conferring an increase in
gamma-aminobutyric acid or shikimate or putrescine production in a
cell, comprising the following steps: [7998] (a) identifying in a
data bank nucleic acid molecules of an organism; which can contain
a candidate gene encoding a gene product conferring an increase in
the gamma-aminobutyric acid or shikimate or putrescine amount or
level in an organism or a part thereof after expression, and which
are at least 20% homolog to the nucleic acid molecule of claim 6;
[7999] (b) introducing the candidate nucleic acid molecules in host
cells appropriate for producing gamma-aminobutyric acid or
shikimate or putrescine; [8000] (c) expressing the identified
nucleic acid molecules in the host cells; [8001] (d) assaying the
gamma-aminobutyric acid or shikimate or putrescine level in the
host cells; and [8002] (e) identifying nucleic acid molecule and
its gene product which expression confers an increase in the
gamma-aminobutyric acid or shikimate or putrescine level in the
host cell after expression compared to the wild type.
[8003] 21. A method for the production of an agricultural
composition comprising the steps of the method of any one of claims
17 to 20 and formulating the compound identified in any one of
claims 17 to 20 in a form acceptable for an application in
agriculture. [8004] 22. A composition comprising the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of any one of claim 8 or 9, an
antagonist or agonist identified according to claim 17, the
compound of claim 18, the gene product of claim 19 or 20, the
antibody of claim 15, and optionally an agricultural acceptable
carrier. [8005] 23. Use of the nucleic acid molecule as claimed in
claim 6 for the identification of a nucleic acid molecule
conferring an increase of gamma-aminobutyric acid or shikimate or
putrescine after expression. [8006] 24. Use of the polypeptide of
claim 14 or the nucleic acid construct claim 7 or the gene product
identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of
gamma-aminobutyric acid or shikimate or putrescine levels in an
organism. [8007] 25. Agrochemical, pharmaceutical, food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. [8008] 26. The method of any one of
claims 1 to 5, the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20, wherein the fine chemical is
gamma-aminobutyric acid or shikimate or putrescine.
[8009] [0554.0.0.17] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[8010] [0000.0.0.18] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[8011] [0001.0.18.18] The present invention relates to a process
for the production of the fine chemical in a microorganism, a plant
cell, a plant, a plant tissue or in one or more parts thereof. The
invention furthermore relates to nucleic acid molecules,
polypeptides, nucleic acid constructs, vectors, antisense
molecules, antibodies, host cells, plant tissue, propagation
material, harvested material, plants, microorganisms as well as
agricultural compositions and to their use.
[8012] [0002.0.18.18] Coenzymes are molecules that cooperate in the
catalytic action of an enzyme. Like enzymes, coenzymes are not
irreversibly changed during catalysis; they are either unmodified
or regenerated. Each kind of coenzyme has a particular chemical
function. Coenzymes may either be attached by covalent bonds to a
particular enzyme or exist freely in solution, but in either case
they participate intimately in the chemical reactions catalyzed by
the enzyme.
[8013] [0003.0.18.18] Coenzyme Q10 (CoQ 10) or ubiquinone is
essentially a vitamin or vitamin-like substance. Disagreements on
nomenclature notwithstanding, vitamins are defined as organic
compounds essential in minute amounts for normal body function
acting as coenzymes or precursors to coenzymes. Coenzyme Q10 or
CoQ10 belongs to a family of substances called ubiquinones.
Ubiquinones, also known as coenzymes Q and mitoquinones, are
lipophilic, water-insoluble substances involved in electron
transport and energy production in mitochondria. The basic
structure of ubiquinones consists of a benzoquinone "head" and a
terpinoid "tail." The "head" structure participates in the redox
activity of the electron transport chain. The major difference
among the various coenzymes Q is in the number of isoprenoid units
(5-carbon structures) in the "tail." Coenzymes Q contain one to 12
isoprenoid units in the "tail"; 10 isoprenoid units are common in
animals. Coenzymes Q occur in the majority of aerobic organisms,
from bacteria to plants and animals. Two numbering systems exist
for designation of the number of isoprenoid units in the terpinoid
"tail": coenzyme Qn and coenzyme Q(x). N refers to the number of
isoprenoid side chains, and x refers to the number of carbons in
the terpinoid "tail" and can be any multiple of five. Thus,
coenzyme Q10 refers to a coenzyme Q having 10 isoprenoid units in
the "tail." Since each isoprenoid unit has five carbons, coenzyme
Q10 can also be designated coenzyme Q(50). The structures of
coenzymes Q are analogous to those of vitamin K2.
[8014] Coenzyme Q10 is also known as Coenzyme Q(50), CoQ10,
CoQ(50), ubiquinone (50), ubiquinol-10 and ubidecarerone.
[8015] They are present naturally in foods and sometimes are also
synthesized in the body. CoQ10 likewise is found in small amounts
in a wide variety of foods and is synthesized in all tissues. The
biosynthesis of CoQ10 from the amino acid tyrosine is a multistage
process requiring at least eight vitamins and several trace
elements. Coenzymes are cofactors upon which the comparatively
large and complex enzymes absolutely depend for their function.
Coenzyme Q10 is the coenzyme for at least three mitochondrial
enzymes (complexes I, II and III) as well as enzymes in other parts
of the cell. Mitochondrial enzymes of the oxidative phosphorylation
pathway are essential for the production of the high-energy
phosphate, adenosine triphosphate (ATP), upon which all cellular
functions depend. The electron and proton transfer functions of the
quinone ring are of fundamental importance to all life forms;
ubiquinone in the mitochondria of animals, plastoquinone in the
chloroplast of plants, and menaquinone in bacteria. The term
"bioenergetics" has been used to describe the field of biochemistry
looking specifically at cellular energy production. In the related
field of free radical chemistry, CoQ10 has been studied in its
reduced form as a potent antioxidant. The bioenergetics and free
radical chemistry of CoQ10 are reviewed in Gian Paolo Littarru's
book, Energy and Defense, published in 1994. The precise chemical
structure of CoQ10 is 2,3 dimethoxy-5 methyl-6 decaprenyl
benzoquinone
[8016] [0004.0.18.18] Discovered in 1957, CoQ-10 is also called
ubiquinone because it belongs to a class of compounds called
quinones, and because it's ubiquitous in living organisms,
especially in the heart, liver, and kidneys. It plays a crucial
role in producing energy in cells. And it acts as a powerful
antioxidant, meaning that it helps neutralize cell-damaging
molecules called free radicals. Manufactured by all cells in the
body, CoQ-10 is also found in small amounts in foods, notably meat
and fish. By the mid-1970's, the industrial technology to produce
pure CoQ10 in quantities sufficient for larger clinical trials was
established. Principally CoQ10 can be isolated from microorganisms
or plants or algae; in particular mitochondria are a common source
for CoQ10. Alternatively, they are obtained advantageously from
animals or fish.
[8017] [0005.0.18.18] Since the actions of supplemental CoQ10 have
yet to be clarified, the mechanism of these actions is a matter of
speculation. However, much is known about the biochemistry of
CoQ10. CoQ10 is an essential cofactor in the mitochondrial electron
transport chain, where it accepts electrons from complex I and II,
an activity that is vital for the production of ATP. CoQ10 has
antioxidant activity in mitochondria and cellular membranes,
protecting against peroxidation of lipid membranes. It also
inhibits the oxidation of LDL-cholesterol. LDL-cholesterol
oxidation is believed to play a significant role in the
pathogenesis of atherosclerosis. CoQ10 is biosynthesized in the
body and shares a common synthetic pathway with cholesterol.
[8018] CoQ10 levels decrease with aging in humans. Why this occurs
is not known but may be due to decreased synthesis and/or increased
lipid peroxidation which occurs with aging. Significantly decreased
levels of CoQ10 have been noted in a wide variety of diseases in
both animal and human studies. CoQ10 deficiency may be caused by
insufficient dietary CoQ10, impairment in CoQ10 biosynthesis,
excessive utilization of CoQ10 by the body, or any combination of
the three. Decreased dietary intake is presumed in chronic
malnutrition and cachexia.
[8019] [0006.0.18.18] The relative contribution of CoQ10
biosynthesis versus dietary CoQ10 is under investigation. Karl
Folkers takes the position that the dominant source of CoQ10 in man
is biosynthesis. This complex, 17 step process, requiring at least
seven vitamins (vitamin B2--riboflavin, vitamin B3--niacinamide,
vitamin B6, folic acid, vitamin B12, vitamin C, and pantothenic
acid) and several trace elements, is, by its nature, highly
vulnerable. Karl Folkers argues that suboptimal nutrient intake in
man is almost universal and that there is subsequent secondary
impairment in CoQ10 biosynthesis. This would mean that average or
"normal" levels of CoQ10 are really suboptimal and the very low
levels observed in advanced disease states represent only the tip
of a deficiency "ice berg".
[8020] Supplemental CoQ10 may have cardioprotective, cytoprotective
and neuroprotective activities. There are claims that it has
positive effects in cancer, muscular dystrophy and immune
dysfunction. Similarly, it may inhibit obesity or enhance athletic
performance.
[8021] [0007.0.18.18] HMG-CoA reductase inhibitors used to treat
elevated blood cholesterol levels by blocking cholesterol
biosynthesis also block CoQ10 biosynthesis. The resulting lowering
of blood CoQ10 level is due to the partially shared biosynthetic
pathway of CoQ10 and cholesterol. In patients with heart failure
this is more than a laboratory observation. It has a significant
harmful effect which can be negated by oral CoQ10
supplementation.
[8022] Increased body consumption of CoQ10 is the presumed cause of
low blood CoQ10 levels seen in excessive exertion, hypermetabolism,
and acute shock states. It is likely that all three mechanisms
(insufficient dietary CoQ10, impaired CoQ10 biosynthesis, and
excessive utilization of CoQ10) are operable to varying degrees in
most cases of observed CoQ10 deficiency.
[8023] [0008.0.18.18] In nature, Coenzymes Q0 to Q9 are found as
well. E.g. Coenzyme Q9 is a derivative of CoQ10 found e.g. in the
chloroplast of plants. Coenzyme Q9 has a shorter aliphatic group
bound to the ring structure. Due to the high structural homology of
Coenzymes Q0 to Q9 are expected to provide the same or very similar
activities as CoQ10 in cells or organisms. However, Matsura et al.,
Biochim Biophys Acta, 1992, 1123(3) pp. 309-15 concluded from their
study that CoQ9 constantly acts as a potential antioxidant in
hepatocytes whereas CoQ10 manly exhibit its antioxidant activity in
cells containing CoQ10 as the predominate CoQ homolog. Coenzyme Q10
is actual a very common ingredient in different types of cosmetics,
due to its protective role against radicals and its predicted
function in skin tautening.
[8024] [0009.0.18.18] Thus, Coenzymes, in particular CoQ10 or CoQ9
can be used in a lot of different applications, for example in
cosmetics, pharmaceuticals and in feed and food.
[8025] [0010.0.18.18] Therefore improving the productivity of said
Coenzymes and improving the quality of cosmetics, pharmaceuticals,
foodstuffs and animal feeds, in particular of nutrition
supplements, is an important task of the different industries.
[8026] [0011.0.18.18] To ensure a high productivity of said
Coenzymes in plants or microorganism, it is necessary to manipulate
the natural biosynthesis of said Coenzymes in said organisms.
[8027] [0012.0.18.18] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
regulators which participate in the biosynthesis of said Coenzymes
and make it possible to produce certain said Coenzymes specifically
on an industrial scale without unwanted byproducts forming. In the
selection of genes for biosynthesis two characteristics above all
are particularly important. On the one hand, there is as ever a
need for improved processes for obtaining the highest possible
contents of said Coenzymes on the other hand as less as possible
byproducts should be produced in the production process.
[8028] [0013.0.0.18] It was now found that this object is achieved
by providing the process according to the invention described
herein and the embodiments characterized in the claims.
[8029] [0014.0.18.18] It was found that the overexpression of the
nucleic acid molecules characterized herein confers an increase in
the content of Coenzyme Q10 or Coenzyme Q9 in plants. Accordingly,
in a first embodiment, the invention relates to a process for the
production of Coenzyme Q10 and/or Coenzyme Q9. Accordingly, in a
further embodiment, the invention relates to a process for the
production of a fine chemical, whereby the fine chemical is
Coenzyme Q10 and/or Coenzyme Q9. Accordingly, in the present
invention, the term "the fine chemical" as used herein relates to
"Coenzyme Q10 and/or Coenzyme Q9". Further, in another embodiment
the term "the fine chemicals" as used herein also relates to
compositions of the fine chemicals comprising Coenzyme Q10 and/or
Coenzyme Q9.
[8030] [0015.0.18.18] In one embodiment, the term "the respective
fine chemicals" means Coenzyme Q10 and/or Coenzyme Q9, depending on
the context. For example, the increase of the gene product of a
gene with the Gene/ORF Locus name YPR138C, YBR184W, b2699, or b1829
or its homologs as mentioned confer the increase of the level of
coenzyme CoQ10 in plants or parts thereof. For example, the
increase of the gene product of a gene with the Gene/ORF Locus name
YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644,
b3605, b2699, b0730, or b0175 or its homologs as mentioned confers
the increase of the level of coenzymes CoQ9 in plants or parts
thereof. For example, the increase of the gene product of a gene
with the ORF name b2699 from E. coli or its homologs confer the
increase of the level of coenzymes CoQ9 and CoQ10 in plants or
parts thereof. For example, the increase of a sequence as indicated
in the Tables, lines 231 to 234 confers the increase of the level
of coenzyme CoQ10 in plants or parts thereof. For example, the
increase of a sequence as indicated in the Tables, lines 235 to 242
and/or 599 to 602 confers the increase of the level of coenzyme Q9
in plants or parts thereof. Accordingly, in one embodiment, the
term "the respective fine chemicals" means Coenzyme Q10 and
Coenzyme Q9. In one embodiment, the term "the respective fine
chemical" means Coenzyme Q10 or Coenzyme Q9. In a further
embodiment, the term "the respective fine chemical" means Coenzyme
Q10 and/or Coenzyme Q9 and their salts, ester, or thioester or
Coenzyme Q10 and/or Coenzyme Q9 in free form or bound to
protein(s), e.g. enzyme(s), or peptide(s), e.g. polypeptide(s) or
to membranes or parts thereof, e.g. in the form of oils or waxes or
in compositions with lipids, oils, fats or lipid mixture, as well
as Coenzyme Q10 and/or Coenzyme Q9 in its reduced or oxidized form.
In a preferred embodiment, the term "the respective fine chemical"
means Coenzyme Q10 or CoQ9, in free form or its salts or bound to
peptide(s) or protein(s). Lipids, oils, waxes, fats or lipid
mixture shall mean any, lipid, oil, wax and/or fat containing any
bound or free Coenzyme Q10 and/or Coenzyme Q9.
[8031] In one embodiment, the term "the fine chemical" and the term
"the respective fine chemical" mean at least one chemical compound
with an activity of the above mentioned fine chemical.
[8032] [0016.0.18.18] Accordingly, the present invention relates to
a process comprising [8033] (a) increasing or generating the
activity of one or more of YPR138C, YBR184W, b2699 and/or b1829
protein(s) in a non-human organism in one or more parts thereof and
[8034] (b) growing the organism under conditions which permit the
production of the respective fine chemical, thus, CoQ10 or the
respective fine chemical-comprising compositions, in said
organism.
[8035] Accordingly, the present invention relates to a process
comprising [8036] (a) increasing or generating the activity of one
or more of YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729,
b3644, b3605, b2699, b0730 and/or b0175 protein(s) in a non-human
organism in one or more parts thereof and [8037] (b) growing the
organism under conditions which permit the production of the
respective fine chemical, thus, CoQ9 or the respective fine
chemical-comprising compositions, in said organism.
[8038] Accordingly, the present invention relates to a process
comprising [8039] (a) increasing or generating the activity of one
or more proteins having the activity of a protein indicated in
Table II, column 3, lines 231 to 234 or having the sequence of a
polypeptide encoded by a nucleic acid molecule indicated in Table
I, column 5 or 7, lines 231 to 234, in a non-human organism in one
or more parts thereof; and [8040] (b) growing the organism under
conditions which permit the production of the respective fine
chemical, thus, CoQ10 in said organism.
[8041] Accordingly, the present invention relates to a process
comprising [8042] (a) increasing or generating the activity of one
or more proteins having the activity of a protein indicated in
Table II, column 3, lines 235 to 242 and/or 599 to 602 or having
the sequence of a polypeptide encoded by a nucleic acid molecule
indicated in Table I, column 5 or 7, lines 235 to 242 and/or 599 to
602, in a non-human organism in one or more parts thereof; and
[8043] (b) growing the organism under conditions which permit the
production of the respective fine chemical, thus, CoQ9 in said
organism.
[8044] [0016.1.0.18] In one embodiment, the method of the present
invention confers the increase of the content of more than one of
the respective fine chemicals, i.e. of Coenzyme Q9 and/or Coenzyme
Q10.
[8045] Accordingly, the term "the fine chemical" can mean "Coenzyme
Q9", and/or "Coenzyme Q10", owing to circumstances and the context.
In order to illustrate that the meaning of the term "the fine
chemical" means "Coenzyme Q9" and/or "Coenzyme Q10" the term "the
respective fine chemical" is also used.
[8046] [0017.0.0.18] to [0018.0.0.18] see [0017.0.0.0] to
[0018.0.0.0]
[8047] [0019.0.18.18] Advantageously the process of the invention
leads to an enhanced production of the respective fine chemical.
The terms "enhanced" or "increase" mean at least a 10%, 20%, 30%,
40% or 50%, preferably at least 60%, 70%, 80%, 90% or 100%, more
preferably 150%, 200%, 300%, 400% or 500% higher production of the
respective fine chemical in comparison to the reference as defined
below, e.g. that means in comparison to an organism without the
aforementioned modification of the activity of an YPR138C, YBR184W,
b2699, b1829, and/or YPR172W, YER174C, YER156C, YDR513W, b2426,
b2703, b2729, b3644, b3605, b2699, b0730, b0175 protein, resp.
[8048] [0020.0.18.18] Surprisingly it was found, that the
transgenic expression of at least one of the Saccharomyces
cerevisiae protein(s) YPR138C or YBR184W, or the E. coli K12
protein(s) b2699 or b1829 in Arabidopsis thaliana conferred an
increase in the CoQ10 content of the transformed plants.
[8049] Surprisingly it was found, that the transgenic expression of
at least one of the
[8050] Saccharomyces cerevisiae protein(s) YPR172W, YER174C,
YER156C or YDR513W or the E. coli K12 protein(s) b2426, b2703,
b2729, b3644, b3605, b2699, b0730 or b0175 in Arabidopsis thaliana
conferred an increase in the CoQ9 content of the transformed
plants. Surprisingly it was found, that the transgenic expression
of the E. coli K12 protein b2699 in Arabidopsis thaliana conferred
an increase in the CoQ9 and CoQ10 content of the transformed
plants.
[8051] [0021.0.0.18] see [0021.0.0.0]
[8052] [0022.0.18.18] The sequence of b2426 (Accession number
NP.sub.--416921) from Escherichia coli K12 has been published in
Blattner et al., Science 277 (5331), 1453-1474,1997, and its
activity is being defined as a putative oxidoreductase with
NAD(P)-binding domain. Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of the "ribitol dehydrogenase, short-chain alcohol
dehydrogenase homology" superfamily, preferably being involved in
C-compound and carbohydrate utilization, metabolism of vitamins,
cofactors and prosthetic groups, biosynthesis of secondary products
derived from primary amino acids, aerobic aromate catabolism,
anaerobic aromate catabolism, and/or catabolism of secondary
metabolites, biosynthesis of the cysteine-aromatic group and/or
degradation of amino acids of the cysteine-aromatic group, aromate
anabolism, more preferred a protein with the activity of a putative
oxidoreductase with NAD(P)-binding domain from E. coli or its
homolog, e.g. as shown herein in Table II, column 5 or 7, line 599
or as encoded by the nucleic acid molecule shown in Table I, column
5 or 7, line 599, for the production of the respective fine
chemical, meaning of Coenzyme Q9, in particular for increasing the
amount of Coenzyme Q9, preferably Coenzyme Q9 in free or bound form
in an organism or a part thereof, as mentioned.
[8053] The sequence of b2729 (Accession number YP 026181) from
Escherichia coli K12 has been published in Blattner et al., Science
277 (5331), 1453-1474, 1997, and its activity is being defined as
hydrogenase expression/formation protein. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the "hydrogenase
expression/formation protein hypD"-superfamily, preferably a
protein with the activity of a hydrogenase expression/formation
protein from E. coli or its homolog, e.g. as shown herein in Table
II, column 5 or 7, line 601 or as encoded by the nucleic acid
molecule shown in Table I, column 5 or 7, line 601, for the
production of the respective fine chemical, meaning of Coenzyme Q9,
in particular for increasing the amount of Coenzyme Q9, preferably
Coenzyme Q9 in free or bound form in an organism or a part thereof,
as mentioned.
[8054] The sequence of b2703 (Accession number NP.sub.--417209)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as phosphotransferase system enzyme II;
glucitol/sorbitol-specific. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of the "phosphotransferase system
sorbitol-specific enzyme II, factor II"-superfamily, preferably
being involved in plasma membrane, cellular import, modification by
phosphorylation, dephosphorylation, C-compound, carbohydrate
transport, sugar binding, and/or phosphotransferase system, more
preferred a protein with the activity of a phosphotransferase
system enzyme II; glucitol/sorbitol-specific from E. coli or its
homolog, e.g. as shown herein in Table II, column 5 or 7, line 600
or as encoded by the nucleic acid molecule shown in Table I, column
5 or 7, line 600, for the production of the respective fine
chemical, meaning of Coenzyme Q9, in particular for increasing the
amount of Coenzyme Q9, preferably Coenzyme Q9 in free or bound form
in an organism or a part thereof, as mentioned. The sequence of
b3644 (Accession number NP.sub.--418101) from Escherichia coli K12
has been published in Blattner et al., Science 277 (5331),
1453-1474, 1997, and its activity is being defined as an
uncharacterized stress-induced protein. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein of the superfamily of hypothetical protein H10467,
preferably of a stress-induced protein from E. coli or its homolog,
e.g. as shown herein in Table II, column 5 or 7, line 602 or as
encoded by the nucleic acid molecule shown in Table I, column 5 or
7, line 602, for the production of the respective fine chemical,
meaning of Coenzyme Q9, preferably in free or bound form, in
particular for increasing the amount of Coenzyme Q9, preferably in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of an uncharacterized stress-induced protein is increased
or generated, e.g. from E. coli or a homolog thereof.
[8055] The sequence of b2699 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a DNA strand exchange and
recombination protein with protease and nuclease activity.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein of the superfamily of the
recombination protein recA, in particular of a protein with DNA
recombination and DNA repair, pheromone response, mating-type
determination, sex-specific protein, nucleotide binding and/or a
protease and nuclease activity, in particular a DNA strand exchange
and recombination protein with protease and nuclease activity or
its homologs, e.g. as shown herein in Table II, column 5 or 7,
lines 233, or as encoded by the nucleic acid molecule shown in
Table I, column 5 or 7, lines 233, for the production of the
respective fine chemical, meaning of reduced or oxidized Coenzyme
Q10, preferably in free or bound or derivative form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a protein with protease
and nuclease activity, in particular of a DNA strand exchange and
recombination protein with protease and nuclease activity of the
superfamily of recombination protein rec A or its homolog is
increased or generated, e.g. from E. coli or a homolog thereof.
[8056] The sequence of b1829 (ACCESSION NP.sub.--416343) from
Escherichia coli K12 has been published in Blattner et al., Science
277(5331), 1453-1474, 1997, and its activity is being defined as a
heat shock protein with protease activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein of the stress response, the pheromone response, the
mating type determination, protein modification or having a
proteolytic degradation activity or being a sex specific protein,
in particular the use of a protease, in particular of a heat shock
protein with protease activity, preferably of the superfamily of
the heat shock protein htpX, or its homologs, e.g. as shown herein
in Table II, column 5 or 7, lines 234, or as encoded by the nucleic
acid molecule shown in Table I, column 5 or 7, lines 234, for the
production of the respective fine chemical, meaning of reduced or
oxidized Coenzyme Q10, e.g. in free or bound form in an organism or
a part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a protease, in particular of
a heat shock protein with protease activity, preferably of the
superfamily of the heat shock protein htpX, is increased or
generated, e.g. from E. coli or a homolog thereof. The sequence of
b3605 (ACCESSION NP.sub.--418062) from Escherichia coli K12 has
been published in Blattner et al., Science 277(5331), 1453-1474,
1997, and its activity is being defined as a L-lactate
dehydrogenase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein of the superfamily
(S)-2-hydroxy-acid oxidase homology, in particular of a protein
with an activity in the respiration, lipid, fatty-acid and
isoprenoid metabolism, as electron/hydrogen carrier, and/or in the
breakdown of lipids, fatty acids and isoprenoids activity, e.g. a
L-lactate dehydrogenase, or its homologs, e.g. as shown herein in
Table II, column 5 or 7, lines 239, or as encoded by the nucleic
acid molecule shown in Table I, column 5 or 7, lines 239, for the
production of the respective fine chemical, meaning of reduced or
oxidized Coenzyme Q9 preferably in free or bound or derivative form
in an organism or a part thereof, as mentioned. In one embodiment,
in the process of the present invention the activity of a protein
of the superfamily (S)-2-hydroxy-acid oxidase homology, in
particular of a protein with L-lactate dehydrogenase or its homolog
is increased or generated, e.g. from E. coli or a homolog
thereof.
[8057] The sequence of b2699 (ACCESSION NP.sub.--417179) from
Escherichia coli K12 has been published in Blattner et al., Science
277(5331), 1453-1474, 1997, and its activity is being defined as a
DNA strand exchange and recombination protein with protease and
nuclease activity. Accordingly, in one embodiment, the process of
the present invention comprises the use of a protein of the
superfamily of the recombination protein recA, in particular of a
protein with DNA recombination and DNA repair, pheromone response,
mating-type determination, sex-specific protein, nucleotide binding
and/or a protease and nuclease activity, in particular a DNA strand
exchange and recombination protein with protease and nuclease
activity or its homologs, e.g. as shown herein in Table II, column
5 or 7, lines 240, or as encoded by the nucleic acid molecule shown
in Table I, column 5 or 7, lines 240, for the production of the
respective fine chemical, meaning of reduced or oxidized Coenzyme
Q9, preferably in free or bound or derivative form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a protein with protease
and nuclease activity, in particular of a DNA strand exchange and
recombination protein with protease and nuclease activity of the
superfamily of recombination protein rec A or its homolog is
increased or generated, e.g. from E. coli or a homolog thereof. The
sequence of b0730 (ACCESSION NP.sub.--415258) from Escherichia coli
K12 has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as
transcriptional regulator of succinylCoA synthetase operon and
fatty acyl response regulator. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein of
the transcription regulator GntR superfamily, in particular of a
protein having a regulation of C-compound and carbohydrate
utilization, transcriptional control, prokaryotic nucleotide,
transcriptional repressor, DNA binding transcriptional regulator of
succinylCoA synthetase operon or a fatty acid response regulator
activity, e.g. a transcriptional regulator of succinylCoA
synthetase operon and fatty acyl response regulator or its
homologs, e.g. as shown herein in Table II, column 5 or 7, lines
241, or as encoded by the nucleic acid molecule shown in Table I,
column 5 or 7, lines 241, for the production of the respective fine
chemical, meaning of reduced or oxidized Coenzyme Q9, preferably in
free or bound or derivative form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a protein transcriptional regulator of
succinylCoA synthetase operon and fatty acyl response regulator or
its homolog is increased or generated, e.g. from E. coli or a
homolog thereof.
[8058] The sequence of b0175 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a CDP-diglyceride synthase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein having a activity in the
nucleotide-metabolism, phospholipid biosynthesis, lipid, fatty-acid
or isoprenoids metabolism, cellular communication, signal
transduction, or cellular sensing and response, preferably of a
protein of the phophatidate cytidylyltransferase superfamily,
preferably of a CDP-diglyceride synthase, e.g. from E. coli or its
homolog, e.g. as shown herein, in Table II, column 5 or 7, lines
242, or as encoded by the nucleic acid molecule shown in Table I,
column 5 or 7, lines 242, for the production of the respective fine
chemical, meaning reduced or oxidized Coenzyme Q9, preferably in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention said
activity, e.g. the activity of a phosphatidate cytidylytransferase,
in particular of a CDP-diglyceride synthase, is increased or
generated, e.g. from E. coli or a homolog thereof.
[8059] The sequence of YPR138C (ACCESSION NP.sub.--015464) from
Saccharomyces cerevisiae has been published in Bussey, H et al.,
Nature 387 (6632 Suppl), 103-105 (1997) and in Goffeau et al.,
Science 274 (5287), 546-547, 1996 and its cellular activity has
characterized as NH4+ transporter. Accordingly, in one embodiment,
the process of the present invention comprises the use of a NH4+
transporter or an ammonium transport protein; or a protein of the
ammonium transporter nrgA superfamily, in particular the use of a
protein having a anion transporters (Cl, SO4, PO4, etc.), other
cation transporters (Na, K, Ca, NH4, etc.), nitrogen and sulfur
transport, cellular import, and/or plasma membrane activity for the
production of Coenzyme Q10. Accordingly, in one embodiment, the
process of the present invention comprises the use of YPR138C from
Saccharomyces cerevisiae, e.g. as indicated herein in Table II,
line 231, columns 3 or 5, or its homologue, e.g. as shown herein in
Table II, line 231, column 7, for the production of the respective
fine chemical, meaning of Coenzyme Q10, in particular for
increasing the amount of reduced and/or oxidized Coenzyme Q10,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of a NH4+ transporter is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[8060] The sequence of YBR184W (Accession NP.sub.--009743) from
Saccharomyces cerevisiae has been published in Feldmann, H. et al.,
EMBO J. 13 (24), 5795-5809 (1994) and in Goffeau et al., Science
274 (5287), 546-547, 1996 and its cellular activity has not been
identified characterized, yet. Accordingly, in one embodiment, the
process of the present invention comprises the use of YBR184W from
Saccharomyces cerevisiae, e.g. as indicated herein in Table II,
line 232, columns 3 or 5, or its homologue, e.g. as shown herein in
Table II, line 232, column 7, for the production of the respective
fine chemical, meaning of Coenzyme Q10, in particular for
increasing the amount of reduced and/or oxidized Coenzyme Q10,
preferably of Coenzyme Q10 in free or bound form in an organism or
a part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of YBR 184W from Saccharomyces
cerevisiae or a homolog thereof is increased or generated.
[8061] The sequence of YPR172W (ACCESSION NP.sub.--015498) from
Saccharomyces cerevisiae has been published in Bussey, H. et al.,
Nature 387 (6632 Suppl), 103-105 (1997) and in Goffeau et al.,
Science 274 (5287), 546-547, 1996 and its cellular activity has not
been identified characterized, yet. Accordingly, in one embodiment,
the process of the present invention comprises the use of YPR172W
from Saccharomyces cerevisiae, e.g. as indicated herein in Table
II, line 235, columns 3 or 5, or its homologue, e.g. as shown
herein in Table II, line 235, column 7, for the production of the
respective fine chemical, meaning of Coenzyme Q9, in particular for
increasing the amount of reduced and/or oxidized Coenzyme Q9,
preferably of Coenzyme Q9 in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of YPR172W from Saccharomyces
cerevisiae or a homolog thereof is increased or generated.
[8062] The sequence of YER174C (ACCESSION NP.sub.--011101) from
Saccharomyces cerevisiae has been published in Dietrich, F. S et
al., Nature 387 (6632 Suppl), 78-81 (1997) and in Goffeau et al.,
Science 274 (5287), 546-547, 1996 and its cellular activity has
characterized as hydroperoxide and superoxide-radical responsive
glutathione-dependent oxidoreductase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of hydroperoxide and superoxide-radical responsive
glutathione-dependent oxidoreductase or a protein of the
thioredoxin homology superfamily, e.g. of a protein having a
protein modification, stress response activity for the production
of Coenzyme Q9. Accordingly, in one embodiment, the process of the
present invention comprises the use of YER174C from Saccharomyces
cerevisiae, e.g. as indicated herein in Table II, line 236, columns
3 or 5, or its homologue, e.g. as shown herein in Table II, line
236, column 7, for the production of the respective fine chemical,
meaning of Coenzyme Q9, in particular for increasing the amount of
reduced and/or oxidized Coenzyme Q9 and/or Coenzyme Q9 in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a hydroperoxide and superoxide-radical responsive
glutathione-dependent oxidoreductase is increased or generated,
e.g. from Saccharomyces cerevisiae or a homolog thereof.
[8063] The sequence of YER156C (ACCESSION NP.sub.--011083) from
Saccharomyces cerevisiae has been published in Dietrich et al.,
Nature 387 (6632 Suppl), 78-81 (1997) and in Goffeau et al.,
Science 274 (5287), 546-547, 1996 and its cellular activity has not
been identified characterized, yet. Accordingly, in one embodiment,
the process of the present invention comprises the use of YER156C
or a protein of the Arabidopsis thaliana hypothetical protein
F2K15.180 superfamily for the production of Coenzyme Q9.
Accordingly, in one embodiment, the process of the present
invention comprises the use of YER156C from Saccharomyces
cerevisiae, e.g. as indicated herein in Table II, line 237, columns
3 or 5, or its homologue, e.g. as shown herein in Table II, line
237, column 7, for the production of the respective fine chemical,
meaning of Coenzyme Q9, in particular for increasing the amount of
reduced and/or oxidized Coenzyme Q9, preferably of Coenzyme Q9 in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of YER156C from Saccharomyces cerevisiae or a homolog
thereof is increased or generated.
[8064] The sequence of YDR513W (ACCESSION NP.sub.--010801) from
Saccharomyces cerevisiae has been published in Jacq et al., Nature
387 (6632 Suppl), 75-78 (1997) and in Goffeau et al., Science 274
(5287), 546-547, 1996 and its cellular activity has characterized
as glutathione reductase. Accordingly, in one embodiment, the
process of the present invention comprises the use of glutathione
reductase or a protein of the glutaredoxin superfamily, in
particular of a protein having a deoxyribonucleotide metabolism,
cytoplasm, stress response, detoxification, and/or electron
transport and membrane-associated energy conservation activity for
the production of Coenzyme Q9. Accordingly, in one embodiment, the
process of the present invention comprises the use of YDR513W from
Saccharomyces cerevisiae, e.g. as indicated herein in Table II,
line 238, columns 3 or 5, or its homologue, e.g. as shown herein in
Table II, line 238, column 7, for the production of the respective
fine chemical, meaning of Coenzyme Q9, in particular for increasing
the amount of reduced and/or oxidized Coenzyme Q9 and/or Coenzyme
Q9 in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a glutathione reductase is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[8065] [0023.0.18.18] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
Further, in the present invention, the term "homologue" relates to
the sequence of an organism having the highest sequence homology to
the herein mentioned or listed sequences of all expressed sequences
of said organism. However, the person skilled in the art knows,
that, preferably, the homologue has said
the--fine-chemical-increasing activity and, if known, the same
biological function or activity in the organism as at least one of
the protein(s) indicated in Table II, Column 3, e.g. having the
sequence of a polypeptide encoded by a nucleic acid molecule
comprising the sequence indicated in Table I, Column 5 or 7.
[8066] In one embodiment, a homolog of any one of the polypeptides
indicated in any one of lines 231 or 232 or 235 to 238 of Table II,
column 3 is a homolog having the same or a similar activity as
described herein or annotated. In particular an increase of
activity confers an increase in the content of the respective fine
chemical in the organisms. In one embodiment, the homologue is a
homolog with a sequence as indicated in any one of lines 231 or 232
or 235 to 238 of Table I or II, column 7, resp. In one embodiment,
the homologue of one of the polypeptides indicated in any one of
lines 231 or 232 or 235 to 238 of Table II, column 3, is derived
from an eukaryotic organism. In one embodiment, the homologue is
derived from Fungi. In one embodiment, the homologue of a
polypeptide indicated in any one of lines 231 or 232 or 235 to 238
of Table II, column 3, is derived from Ascomyceta. In one
embodiment, the homologue of a polypeptide indicated in any one of
lines 231 or 232 or 235 to 238 of Table II, column 3, is derived
from Saccharomycotina. In one embodiment, the homologue of a
polypeptide indicated in any one of lines 231 or 232 or 235 to 238
of Table II, column 3, is derived from Saccharomycetes. In one
embodiment, the homolog of a polypeptide indicated in any one of
lines 231 or 232 or 235 to 238 of Table II, column 3, is a
homologue being derived from Saccharomycetales. In one embodiment,
the homologue of a polypeptide indicated in any one of lines 231 or
232 or 235 to 238 of Table II, column 3, is a homologue having the
same or a similar activity being derived from Saccharomycetaceae.
In one embodiment, the homologue of a polypeptide indicated in any
one of lines 231 or 232 or 235 to 238 of Table II, column 3 is a
homologue having the same or a similar activity, in particular an
increase of activity confers an increase in the content of Coenzyme
Q10 and/or Coenzyme Q9 in a organisms or part thereof, being
derived from Saccharomycetes.
[8067] In one embodiment, the homolog of the any one of the
polypeptides indicated in any one of lines 233 or 234 or 239 to 242
and/or 599 to 602 of Table II, column 3, is a homolog having the
same or a similar activity as described herein or annotated. In
particular an increase of activity confers an increase in the
content of the respective fine chemical. In one embodiment, the
homolog is a homolog with a sequence as indicated in any one of
lines 233 or 234 or 239 to 242 and/or 599 to 602 of Table I or II,
column 7, resp. In one embodiment, the homolog of one of the
polypeptides indicated in any one of lines 233 or 234 or 239 to 242
and/or 599 to 602 of Table II, column 3, is derived from an
bacteria. In one embodiment, the homolog of a polypeptide indicated
in any one of lines 233 or 234 or 239 to 242 and/or 599 to 602 of
Table II, column 3, is derived from Proteobacteria. In one
embodiment, the homolog of a polypeptide indicated in any one of
lines 233 or 234 or 239 to 242 and/or 599 to 602 of Table II,
column 3, is a homolog having the same or a similar activity being
derived from Gammaproteobacteria. In one embodiment, the homolog of
a polypeptide indicated in any one of lines 233 or 234 or 239 to
242 and/or 599 to 602 of Table II, column 3, is derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in any one of lines 233 or 234 or 239 to 242 and/or 599
to 602 of Table II, column 3, is a homolog being derived from
Enterobacteriaceae. In one embodiment, the homolog of a polypeptide
indicated in any one of lines 233 or 234 or 239 to 242 and/or 599
to 602 of Table II, column 3, is a homolog having the same or a
similar activity, in particular an increase of activity confers an
increase in the content of Coenzyme Q10 and/or Coenzyme Q9 in a
organisms or part thereof being derived from Escherichia.
[8068] [0023.1.18.18] Homologs of the polypeptides indicated in
Table II, column 3, lines 231 to 234 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 231 to 234, respectively or may be the polypeptides
indicated in Table II, column 7, lines 231 to 234 having a Coenzyme
Q10 content and/or amount increasing activity.
[8069] Homologs of the polypeptides indicated in Table II, column
3, lines 235 to 242 and/or 599 to 602 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 235 to 242 and/or 599 to 602, respectively, or may be the
polypeptides indicated in Table II, column 7, lines 235 to 242
and/or 599 to 602 having a Coenzyme Q9 content and/or amount
increasing activity.
[8070] [0024.0.0.18] see [0024.0.0.0]
[8071] [0025.0.18.18] In accordance with the invention, a protein
or polypeptide has the "activity of an YPR138C, YBR184W, b2699,
and/or b1829, and/or YPR172W, YER174C, YER156C, YDR513W, b2426,
b2703, b2729, b3644, b3605, b2699, b0730, and/or b0175 protein" if
its de novo activity or its increased expression directly or
indirectly leads to an increased level of the respective fine
chemical, in particular of Coenzyme Q10 and/or Coenzyme Q9, resp.,
and/or polypeptides, proteins, peptides, enzymes, triglycerides,
lipids, oils, waxes, membranes, membrane fractions and/or fats
containing Coenzyme Q10 and/or Coenzyme Q9 in the organism or a
part thereof, preferably in a cell of said organism. In one
embodiment, the protein has the activities of a protein as
indicated in Table II, lines 231 to 234 and/or 235 to 242 and/or
599 to 602, columns 3 or 5. Throughout the specification, in a
preferred embodiment, the activity or preferably the biological
activity of such a protein or polypeptide or an nucleic acid
molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity protein as indicated in Table II, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, columns 3 or 5, or which has at least
10% of the original enzymatic activity, preferably at least 20%,
particularly preferably at least 30%, most particularly preferably
at least 40% in comparison to an, YPR138C, YBR184W, b2699, and/or
b1829, and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703,
b2729, b3644, b3605, b2699, b0730, and/or b0175 protein of
Saccharomyces cerevisiae or E. Coli, resp. or a combination
thereof.
[8072] [0025.3.18.18] In one embodiment, the polypeptide of the
invention or used in the method of the invention confers said
activity, e.g. the increase of the respective fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[8073] [0025.2.0.18] see [0025.2.0.0]
[8074] [0025.1.0.18] see [0025.1.0.0]
[8075] [0026.0.0.18] to [0033.0.0.18] see[0026.0.0.0] to
[0033.0.0.0]
[8076] [0034.0.18.18] Preferably, the reference, control or wild
type differs from the subject of the present invention only in the
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. as result of
an increase in the level of the nucleic acid molecule of the
present invention or used in the method of the invention or an
increase of the specific activity of the polypeptide of the
invention or the polypeptide used in the method of the invention,
e.g. by or in the expression level or activity of an protein having
the activity of a protein as indicated in Table II, column 3, lines
231 to 234 and/or 235 to 242 and/or 599 to 602 or being encoded by
a nucleic acid molecule indicated in Table I, column 5, lines 231
to 234 and/or 235 to 242 and/or 599 to 602 or its homologs, e.g. as
indicated in Table I, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[8077] [0035.0.0.18] to [0044.0.0.18] see [0035.0.0.0] to
[0044.0.0.0]
[8078] [0045.0.18.18] In one embodiment, in case the activity of
the Escherichia coli K12 protein b2426 or its homologs, e.g. as
indicated in Table II, columns 5 or 7, line 599, is increased, e.g.
the activity of a oxidoreductase with NAD(P)-binding domain is
increased. Preferably, an increase of the respective fine chemical,
preferably of Coenzyme Q9 between 38% and 77% or more is
conferred.
[8079] In one embodiment, the activity of the Escherichia coli K12
protein b3644 or its homologs e.g. an uncharacterized
stress-induced protein e.g. as indicated in Table II, columns 5 or
7, line 602, is increased, preferably, conferring the increase of
the fine chemical, preferably of Coenzyme Q9 between 37% and 130%
or more.
[8080] In one embodiment, the activity of the Escherichia coli K12
protein b2703 or its homologs e.g. phosphotransferase system enzyme
II; glucitol/sorbitol-specific, e.g. as indicated in Table II,
columns 5 or 7, line 600, is increased, preferably, conferring the
increase of the fine chemical, preferably of Coenzyme Q9 between
37% and 118% or more.
[8081] In one embodiment, the activity of the Escherichia coli K12
protein b2729 or its homologs, e.g. an hydrogenase
expression/formation protein, e.g. as indicated in Table II,
columns 5 or 7, line 601, is increased, preferably, conferring the
increase of the fine chemical, preferably of Coenzyme Q9 between
35% and 41% or more.
[8082] In one embodiment, the activity of the Escherichia coli K12
protein b1829 or its homologs, e.g. of a protein of the stress
response, the pheromone response, the mating type determination,
protein modification or having a proteolytic degradation activity
or being a sex specific protein, in particular the use of a
protease, in particular of a heat shock protein with protease
activity, preferably of the superfamily of the heat shock protein
htpX or its homologs, e.g. as indicated in Table II, columns 5 or
7, line 234, is increased, conferring an increase of the respective
fine chemical, preferably of Coenzyme Q10 between 48% and 433% or
more.
[8083] In one embodiment, the activity of the Escherichia coli K12
protein b2699 or its homologs, e.g. a DNA recombination and DNA
repair activity, a pheromone response activity, a mating-type
determination activity, a sex-specific protein activity, a
nucleotide binding activity and/or a protease and nuclease
activity, in particular a DNA strand exchange and recombination
protein with protease and nuclease activity, in particular of the
superfamily of the recombination protein recA, e.g. as indicated in
Table II, columns 5 or 7, line 233, is increased, conferring an
increase of the respective fine chemical, preferably of Coenzyme
Q10 between 62% and 220% or more.
[8084] In one embodiment, the activity of the Escherichia coli K12
protein b2699 or its homologs, e.g. a DNA recombination and DNA
repair activity, a pheromone response activity, a mating-type
determination activity, a sex-specific protein activity, a
nucleotide binding activity and/or a protease and nuclease
activity, in particular a DNA strand exchange and recombination
protein with protease and nuclease activity, in particular of the
superfamily of the recombination protein recA, e.g. as indicated in
Table II, columns 5 or 7, line 240, is increased, conferring an
increase of the respective fine chemical, preferably of Coenzyme Q9
between 34% and 253% or more.
[8085] In one embodiment, the activity of the Escherichia coli K12
protein b2699 or its homologs, e.g. a DNA recombination and DNA
repair activity, a pheromone response activity, a mating-type
determination activity, a sex-specific protein activity, a
nucleotide binding activity and/or a protease and nuclease
activity, in particular a DNA strand exchange and recombination
protein with protease and nuclease activity, in particular of the
superfamily of the recombination protein recA, e.g. as indicated in
Table II, columns 5 or 7, line 233 and 240, is increased,
preferably conferring an increase of the respective fine chemicals,
preferably of Coenzyme Q10 between 62% and 220% and Coenzyme Q9
between 34% and 253% or more.
[8086] In one embodiment, the activity of the Escherichia coli K12
protein b3605 or its homologs, e.g. of a protein of the superfamily
(S)-2-hydroxy-acid oxidase homology, in particular of a protein
with an activity in the respiration, lipid, fatty-acid and
isoprenoid metabolism, as electron/hydrogen carrier, and/or in the
breakdown of lipids, fatty acids and isoprenoids activity, e.g. a
L-lactate dehydrogenase, e.g. as indicated in Table II, columns 5
or 7, line 239, is increased, preferably conferring an increase of
the respective fine chemical, preferably of Coenzyme Q9 between 35%
and 41% or more.
[8087] In one embodiment, the activity of the Escherichia coli K12
protein b0730 or its homologs, e.g. of a protein transcriptional
regulator of succinylCoA synthetase operon and fatty acyl response
regulator, e.g. as indicated in Table II, columns 5 or 7, line 241,
is increased, preferably conferring an increase of the respective
fine chemical, preferably of Coenzyme Q9 between 30% and 203% or
more.
[8088] In one embodiment, the activity of the Escherichia coli K12
protein b0175 or its homologs, e.g. of a protein having a activity
in the nucleotide-metabolism, phospholipid biosynthesis, lipid,
fatty-acid or isoprenoids metabolism, cellular communication,
signal transduction, or cellular sensing and response, preferably
of a protein of the phophatidate cytidylyltransferase superfamily,
preferably of a CDP-diglyceride synthase, e.g. as indicated in
Table II, columns 5 or 7, line 242, is increased, preferably
conferring an increase of the respective fine chemical, preferably
of Coenzyme Q9 between 45% and 70% or more.
[8089] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YDR513W or its homologs, e.g. of glutathione
reductase or a protein of the glutaredoxin superfamily, in
particular of a protein having a deoxyribonucleotide metabolism,
cytoplasm, stress response, detoxification, and/or electron
transport and membrane-associated energy conservation activity,
e.g. as indicated in Table II, columns 5 or 7, line 238, is
increased, preferably, conferring an increase of the respective
fine chemical, preferably of Coenzyme Q9 between 31% and 73% or
more.
[8090] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER156C or its homologs, e.g. of a protein of
the Arabidopsis thaliana hypothetical protein F2K15.180
superfamily, e.g. as indicated in Table II, columns 5 or 7, line
237, is increased, preferably, conferring an increase of the
respective fine chemical, preferably of free Coenzyme Q9 between
32% and 58% or more.
[8091] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER174C or its homologs, e.g. of a Hydroperoxide
and superoxide-radical responsive glutathione-dependent
oxidoreductase or a protein of the thioredoxin homology
superfamily, e.g. of a protein having a protein modification,
stress response activity, e.g. as indicated in Table II, columns 5
or 7, line 236, is increased, preferably, conferring an increase of
the respective fine chemical, preferably of Coenzyme Q9 between 31%
and 72% or more.
[8092] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YPR172W or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 235, is increased, preferably,
conferring an increase of the respective fine chemical, preferably
of free Coenzyme Q9 between 34% and 66% or more.
[8093] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YBR184W or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 232, is increased, preferably,
conferring an increase of the respective fine chemical, preferably
of free Coenzyme Q10 between 70% and 95% or more.
[8094] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YPR138C or its homologs, e.g. of a NH4+
transporter or an ammonium transport protein; or a protein of the
ammonium transporter nrgA superfamily, in particular the use of a
protein having a anion transporters (Cl, SO4, PO4, etc.), other
cation transporters (Na, K, Ca, NH4, etc.), nitrogen and sulfur
transport, cellular import, and/or plasma membrane activity, e.g.
as indicated in Table II, columns 5 or 7, line 231, is increased,
preferably, conferring an increase of the respective fine chemical,
preferably of free Coenzyme Q10 between 65% and 257% or more.
[8095] [0046.0.18.18] In one embodiment, the activity of a protein
as disclosed in [0016.0.18.18] or its homologs, as indicated in
Table I, columns 5 or 7, lines 231 to 234, e.g. a protein with an
activity as defined in [0022.0.18.18], is increased conferring,
preferably, an increase of the respective fine chemical, preferably
of Coenzyme Q10 and of further vitamin- or coenzyme-activity-having
compounds or their precursors.
[8096] In one embodiment, the activity of a protein as disclosed in
[0016.0.18.18] or its homologs, as indicated in Table I, columns 5
or 7, lines 235 to 242 and/or 599 to 602, e.g. a protein with an
activity as defined in [0022.0.18.18], is increased conferring
preferably, an increase of the respective fine chemical, preferably
of Coenzyme Q10 and of further vitamin- or coenzyme-activity-having
compounds or their precursors.
[8097] In one embodiment the activity of one or more of the
Saccharomyces cerevisiae or Escherichia coli protein YPR138C,
YBR184W, b2699, and/or b1829, and/or YPR172W, YER174C, YER156C,
YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730, and/or
b0175 or of its homologs with an activity as described above in any
one of the paragraphs of section [0045.0.18.18], e.g. a homolog as
indicated in Table II, columns 5 or 7, lines 231 to 234 and/or 235
to 242 and/or 599 to 602, resp., is increased and confers an
increased level of the respective fine chemical; preferably of the
respective fine chemical and/or of waxes, membranes, membrane
fractions, organelles, triglycerides, lipids, oils and/or fats
containing an increased level of Coenzyme Q9 and/or Coenzyme
Q10.
[8098] [0047.0.0.18] see [0047.0.0.0]
[8099] [0048.0.18.18] The respective fine chemical can be contained
in the organism either in its free form and/or bound to lipids,
waxes, membranes, membrane fractions, proteins, fatty acids or
other parts of membranes, in particular to compounds contained in
membranes of mitochondria or plastids, resp., or mixtures thereof.
Accordingly, in one embodiment, the amount of the free form in a
membrane, an organelle, a cell, a tissue, more preferred in an
organism e.g. as a plant or a microorganism, or a part thereof, is
increased by 3% or more, especially preferably are 10% or more,
very especially preferably are more than 30% and most preferably
are 70% or more, such as 100%, 300% or 500%. Accordingly, in an
other embodiment, the amount of the bound the respective fine
chemical in a membrane, an organelle, a cell, a tissue, more
preferred in an organism, e.g. as a plant or a microorganism or
part thereof, is increased by 3% or more, especially preferably are
10% or more, very especially preferably are more than 30% and most
preferably are 70% or more, such as 100%, 300% or 500%.
[8100] [0049.0.18.18] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical has
the structure of the polypeptide described herein. In a particular
embodiment, the polypeptides used in the process of the present
invention or the polypeptide of the present invention comprises the
sequence of a consensus sequence as indicated in Table IV, column
7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
respectively, or of a polypeptide as indicated in Table II, columns
5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602, or of a
functional homologue thereof as described herein, e.g. as indicated
in Table II, column 7, lines 231 to 234 and/or 235 to 242 and/or
599 to 602, or of a polypeptide encoded by the nucleic acid
molecule characterized herein or the nucleic acid molecule
according to the invention, for example by a nucleic acid molecule
as indicated in Table I, columns 5 or 7, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, or its herein described functional
homologues and has the herein mentioned activity, in particular
conferring an increase in the Coenzyme Q10 and/or Coenzyme Q9 level
of an organism.
[8101] [0050.0.18.18] ./.
[8102] [0051.0.18.18] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g. hydrophobic or lipophilic
compositions. Depending on the choice of the organism used for the
process according to the present invention, for example a
microorganism or a plant, compositions or mixtures of the
respective fine chemical and various other coenzymes, vitamins
and/or antioxidants, e.g. vitamin B6 or vitamin E, can be
produced.
[8103] [0052.0.0.18] see [0052.0.0.0]
[8104] [0053.0.18.18] In one embodiment, the process of the present
invention comprises one or more of the following steps [8105] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention, or the polypeptide used in the
method of the invention e.g. of a polypeptide having an YPR138C,
YBR184W, b2699 and/or b1829, and/or YPR172W, YER174C, YER156C,
YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730, and/or
b0175 protein activity e.g. of a protein as indicated in Table II,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or
its homologs activity having herein-mentioned Coenzyme Q10 and/or
Coenzyme Q9 increasing activity, e.g. as indicated in Table II,
column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602;
[8106] b) stabilizing a mRNA conferring the increased expression of
a protein encoded by the nucleic acid molecule of the invention or
used in the method of the invention, e.g. of a polypeptide having a
YPR138C, YBR184W, b2699 and/or b1829, and/or YPR172W, YER174C,
YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730,
and/or b0175 protein activity, egg a protein as indicated in Table
II, column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602
or being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or
its homologs activity or of a mRNA encoding the polypeptide of the
present invention having herein-mentioned Coenzyme Q10 and/or
Coenzyme Q9 increasing activity, e.g. as indicated in Table II,
column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
[8107] c) increasing the specific activity of a protein conferring
the increased expression of a protein encoded by the nucleic acid
molecule of the invention or used in the method of the invention or
of the polypeptide of the present invention or used in the method
of the invention having herein-mentioned Coenzyme Q10 and/or
Coenzyme Q9 increasing activity, e.g. of a polypeptide having a
YPR138C, YBR184W, b2699 and/or b1829, and/or YPR172W, YER174C,
YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730,
and/or b0175 protein activity, e.g. of a protein as indicated in
Table II, column 5, lines 231 to 234 and/or 235 to 242 and/or 599
to 602 or being encoded by a nucleic acid molecule indicated in
Table I, column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to
602 or its homologs activity, e.g. as indicated in Table II, column
7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602, or
decreasing the inhibitory regulation of the polypeptide of the
invention or the polypeptide used in the method of the invention;
[8108] d) generating or increasing the expression of an endogenous
or artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or used in the method of
the invention or of the polypeptide of the invention or the
polypeptide used in the method of the invention having
herein-mentioned Coenzyme Q10 and/or Coenzyme Q9 increasing
activity, e.g. of a polypeptide having the YPR138C, YBR184W, b2699,
and/or b1829, and/or YPR172W, YER174C, YER156C, YDR513W, b2426,
b2703, b2729, b3644, b3605, b2699, b0730, and/or b0175 protein
activity, e.g. of a protein as indicated in Table II, column 5,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or being
encoded by a nucleic acid molecule indicated in Table I, column 5,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, or of their
homologs, e.g. as indicated in Table I or II, column 7, lines 231
to 234 and/or 235 to 242 and/or 599 to 602; [8109] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned Coenzyme Q10 and/or Coenzyme Q9 increasing
activity, e.g. of a polypeptide having the YPR138C, YBR184W, b2699,
and/or b1829, and/or YPR172W, YER174C, YER156C, YDR513W, b2426,
b2703, b2729, b3644, b3605, b2699, b0730, and/or b0175 protein
activity, e.g. of a protein as indicated in Table II, column 5,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or being
encoded by a nucleic acid molecule indicated in Table I, column 5,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, or of their
homologs, e.g. as indicated in Table I or II, column 7, lines 231
to 234 and/or 235 to 242 and/or 599 to 602, by adding one or more
exogenous inducing factors to the organism or parts thereof; [8110]
f) expressing a transgenic gene encoding a protein conferring the
increased expression of a polypeptide encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention, having herein-mentioned Coenzyme Q10 and/or Coenzyme Q9
increasing activity, e.g. of a polypeptide having the YPR138C,
YBR184W, b2699, and/or b1829, and/or YPR172W, YER174C, YER156C,
YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730, and/or
b0175 protein activity, e.g. of a protein as indicated in Table II,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602, or
of their homologs, e.g. as indicated in Table I or II, column 7,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, [8111] g)
increasing the copy number of a gene conferring the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or used in
the method of the invention or the polypeptide of the invention or
used in the method of the invention having herein-mentioned
Coenzyme Q10 and/or Coenzyme Q9 increasing activity, e.g. of a
polypeptide having the YPR138C, YBR184W, b2699, b1829, and/or
YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644,
b3605, b2699, b0730, b0175 protein activity, e.g. of a protein as
indicated in Table II, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, or of their homologs, e.g. as indicated in Table
I or II, column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to
602; [8112] h) Increasing the expression of the endogenous gene
encoding the polypeptide of the invention or used in the method of
the invention, e.g. a polypeptide having the YPR138C, YBR184W,
b2699 and/or b1829, and/or YPR172W, YER174C, YER156C, YDR513W,
b2426, b2703, b2729, b3644, b3605, b2699, b0730, and/or b0175
protein activity, e.g. of a protein as indicated in Table II,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602, or
of their homologs, e.g. as indicated in Table I or II, column 7,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, by adding
positive expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; [8113] i)
Modulating growth conditions of an organism in such a manner, that
the expression or activity of the gene encoding the protein of the
invention or the protein itself is enhanced for example
microorganisms or plants can be grown for example under a higher
temperature regime leading to an enhanced expression of heat shock
proteins, which can lead an enhanced fine chemical production;
and/or [8114] j) selecting of organisms with especially high
activity of the proteins of the invention from natural or from
mutagenized resources and breeding them into the target organisms,
e.g. the elite crops.
[8115] [0054.0.18.18] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of Coenzyme Q10 and/or
Coenzyme Q9 after increasing the expression or activity of the
encoded polypeptide or having the activity of a polypeptide having
an YPR138C, YBR184W, b2699 and/or b1829, and/or YPR172W, YER174C,
YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730,
and/or b0175 protein, e.g. of a protein as indicated in Table II,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602, or
of their homologs, e.g. as indicated in Table I or II, column 7,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602.
[8116] [0055.0.0.18] to [0067.0.0.18] see [0055.0.0.0] to
[0067.0.0.0]
[8117] [0068.0.9.9] The mutation is introduced in such a way that
the production of the Coenzyme Q9 and/or Coenzyme Q10 is not
adversely affected.
[8118] [0069.0.0.18] see [0069.0.0.0]
[8119] [0070.0.18.18] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or used in the method of the invention or
the polypeptide of the invention or used in the method of the
invention, for example the nucleic acid construct mentioned below,
or encoding the
[8120] YPR138C, YBR184W, b2699 and/or b1829, and/or YPR172W,
YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605,
b2699, b0730, and/or b0175 protein, e.g. of a protein as indicated
in Table II, column 5, lines 231 to 234 and/or 235 to 242 and/or
599 to 602 or being encoded by a nucleic acid molecule indicated in
Table I, column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to
602 or of its homologs, e.g. as indicated in Table II, column 7,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, into an
organism alone or in combination with other genes, it is possible
not only to increase the biosynthetic flux towards the end product,
but also to increase, modify or create de novo an advantageous,
preferably novel metabolites composition in the organism, e.g. an
advantageous hydrophopic composition comprising a higher content of
(from a viewpoint of nutritional physiology limited) coenzymes,
vitamins and/or antioxidants, e.g. vitamin B6 and/or vitamin E. In
one embodiment, the expression of a protein as indicated in Table
II, column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602
or being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or
of its homologs, e.g. as indicated in Table II, column 7, lines 231
to 234 and/or 235 to 242 and/or 599 to 602 conferring an increase
of coenzymes, in particular of Coenzyme Q9 and/or Coenzyme Q10, and
preferably of further coenzymes, vitamins, and/or antioxidants.
[8121] [0071.0.18.18] Preferably the composition further comprises
higher amounts of metabolites positively affecting or lower amounts
of metabolites negatively affecting the nutrition or health of
animals or humans provided with said compositions or organisms of
the invention or parts thereof. Likewise, the number or activity of
further genes which are required for the import or export of
nutrients or metabolites, including amino acids, fatty acids,
vitamins, coenzymes, antioxidants etc. or any one of their
precursors, required for the cell's biosynthesis of the respective
fine chemical may be increased so that the concentration of
necessary or relevant precursors, e.g. of isoprenoids, acetyl CoA,
HMG-CoA, mevalonate, Isopentenyl pyrophosphate, Geranyl
pyrophosphate, Farnesyl Pyrophosphate, or other cofactors or
intermediates within the organelle, e.g. in mitochondria or
plastids, resp., within (a) cell(s) or within the corresponding
storage compartments is increased. Owing to the increased or novel
generated activity of the polypeptide of the invention or used in
the method of the invention or owing to the increased number of
nucleic acid sequences of the invention or used in the method of
the invention and/or to the modulation of further genes which are
involved in the biosynthesis of the respective fine chemical, e.g.
by increasing the activity of enzymes synthesizing precursors, e.g.
Lovastatin, HMG-CoA Reductase, Mevalonate Kinase, or by destroying
the activity of one or more genes which are involved in the
breakdown of the respective fine chemical, it is possible to
increase the yield, production and/or production efficiency of the
respective fine chemical in the host organism, such as plants or
the microorganisms.
[8122] [0072.0.18.18] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are
antioxidants, further coenzymes, vitamins, e.g. vitamin B6 or
vitamin E, or triglycerides, fatty acids, lipids, oils and/or fats
containing Coenzyme Q10 and/or Coenzyme Q9.
[8123] [0073.0.18.18] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[8124] w) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, an
organelle, a plant or animal tissue or a plant; [8125] x)
increasing the YPR138C, YBR184W, b2699 and/or b1829, and/or
YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644,
b3605, b2699, b0730, and/or b0175 protein activity or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. of a protein as
indicated in Table II, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 or of its homologs, e.g. as indicated in Table
II, column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
and conferring an increase of the respective fine chemical in the
organism, preferably in the microorganism, the non-human animal,
the plant or animal cell, the plant or animal tissue, the organelle
or the plant, [8126] y) growing the organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant under conditions which permit the
production of the respective fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and [8127] z) if desired, recovering, optionally
isolating, the free and/or bound the respective fine chemical and,
optionally further free and/or bound vitamins, coenzymes or
antioxidants synthesized by the organism, the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[8128] [0074.0.18.18] The organism, in particular the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue, the organelle or the plant is
advantageously grown in such a way that it is not only possible to
recover, if desired isolate the free or bound the respective fine
chemical or the free and bound the respective fine chemical but as
option it is also possible to produce, recover and, if desired
isolate, other free or/and bound antioxidants, vitamins or
coenzymes and mixtures thereof.
[8129] [0075.0.0.18] to [0077.0.0.18] see [0075.0.0.0] to
[0077.0.0.0]
[8130] [0078.0.18.18] The organism such as microorganisms or plants
or the recovered, and if desired isolated, fine chemical can then
be processed further directly into foodstuffs or animal feeds or
for other applications, for example according to the disclosures
made in the following US patent publications:
[8131] U.S. Pat. No. 6,380,252: Use of L-acetylcarnitine,
L-isovalerylcarnitine, L-propionylcarnitine for increasing the
levels of IGF-1, U.S. Pat. No. 6,372,198: Dentifrice for the
mineralization and remineralization of teeth, U.S. Pat. No.
6,368,617: Dietary supplement, U.S. Pat. No. 6,350,473: Method for
treating hypercholesterolemia, hyperlipidemia, and atherosclerosis,
U.S. Pat. No. 6,335,361: Method of treating benign forgetfulness,
U.S. Pat. No. 6,329,432: Mesozeaxanthin formulations for treatment
of retinal disorders, U.S. Pat. No. 6,328,987: Cosmetic skin care
compositions containing alpha interferon, U.S. Pat. No. 6,312,703:
Compressed lecithin preparations, U.S. Pat. No. 6,306,392:
Composition comprising a carnitine and glutathione, useful to
increase the absorption of glutathione and synergize its effects,
U.S. Pat. No. 6,303,586: Supportive therapy for diabetes,
hyperglycemia and hypoglycemia, U.S. Pat. No. 6,297,281:
Association of no syntase inhibitors with trappers of oxygen
reactive forms, U.S. Pat. No. 6,294,697: Discrete-length
polyethylene glycols, U.S. Pat. No. 6,277,842: Dietary supplemental
method for fat and weight reduction, U.S. Pat. No. 6,261,250:
Method and apparatus for enhancing cardiovascular activity and
health through rhythmic limb elevation, U.S. Pat. No. 6,258,855:
Method of retarding and ameliorating carpal tunnel syndrome, U.S.
Pat. No. 6,258,848: Methods and compositions for increasing insulin
sensitivity, U.S. Pat. No. 6,258,847: Use of 2-mercaptoethanolamine
(2-MEA) and related aminothiol compounds and copper(II)-3,5
di-isopropyl salicylates and related compounds in the prevention
and treatment of various diseases, U.S. Pat. No. 6,255,354:
Preparation of a pulmonary surfactant for instillation and oral
application, U.S. Pat. No. 6,254,547: Breath methylated alkane
contour: a new marker of oxidative stress and disease, U.S. Pat.
No. 6,248,552: Enzyme-based assay for determining effects of
exogenous and endogenous factors on cellular energy production,
U.S. Pat. No. 6,248,363: Solid carriers for improved delivery of
active ingredients in pharmaceutical compositions, U.S. Pat. No.
6,245,800: Method of preventing or treating statin-induced toxic
effects using L-carnitine or an alkanoyl L-carnitine, U.S. Pat. No.
6,245,378: Nutritional supplement for facilitating skeletal muscle
adaptation to strenuous exercise and counteracting defatigation in
asthenic individuals, U.S. Pat. No. 6,242,491: Use of creatine or
creatine compounds for skin preservation, U.S. Pat. No. 6,232,346:
Composition for improvement of cellular nutrition and mitochondrial
energetics, U.S. Pat. No. 6,231,836: Folic acid dentifrice, U.S.
Pat. No. 6,228,891: Use of
2,3-dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone, U.S. Pat. No.
6,228,402: Xylitol-containing non-human foodstuff and method, U.S.
Pat. No. 6,228,347: Antioxidant gel for gingival conditions, U.S.
Pat. No. 6,218,436: Pharmaceutically active carotenoids, U.S. Pat.
No. 6,203,818: Nutritional supplement for cardiovascular health,
U.S. Pat. No. 6,200,550: Oral care compositions comprising coenzyme
Q10, U.S. Pat. No. 6,191,172: Water-soluble compositions of
bioactive lipophilic compounds, U.S. Pat. No. 6,184,255:
Pharmaceutical composition comprising coenzyme Q10, U.S. Pat. No.
6,166,077: Use of L-acetylcarnitine, L-isovalerylcarnitine,
L-propionylcarnitine for increasing the levels of IGF-1, U.S. Pat.
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6,159,508: Xylitol-containing non-human foodstuff and method, U.S.
Pat. No. 6,159,476: Herbal supplement for increased muscle strength
and endurance for athletes, U.S. Pat. No. 6,153,582: Defined
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U.S. Pat. No. 6,107,281: Compounds and their combinations for the
treatment of influenza infection, U.S. Pat. No. 6,106,286: Method
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food supplement, U.S. Pat. No. 6,086,910: Food supplements, U.S.
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[8132] The fermentation broth, fermentation products, plants or
plant products can be purified in the customary manner by
hydrolysis with strong bases, extraction and crystallization or via
thin layer chromatography and other methods known to the person
skilled in the art and described herein below. Products of these
different work-up procedures are fatty acids or fatty acid
compositions which still comprise fermentation broth, plant
particles and cell components in different amounts, advantageously
in the range of from 0 to 99% by weight, preferably below 80% by
weight, especially preferably between below 50% by weight.
[8133] [0079.0.0.18] to [0084.0.0.18] see [0079.0.0.0] to
[0084.0.0.0]
[8134] [0084.2.18.18] Coenzyme Q10 production was reported in
Agrobacterium sp., Protaminobacter rubber and Paracoccus
denitrificans. Coenzyme Q9 production was reported in Candida
tropicalis. Production of ubiquiones with side chain length of 6-10
units, e.g. including Coenzyme Q10 and Coenzyme Q9 was reported for
controlled continuous culture of phototrophic bacteria (wild-type
strains of Rhodobacter capsulatus, Rhodobacter sphaeroides,
Thiocapsa roseopersicina and Ectothiorhodospira shaposhnikovii.
Cells mostly contained one main ubiquinone, whereby the content and
composition dependent on growth conditions, substrates and other
factors. Preferred is a production of more than 0.1, preferably
more than 1 to 6 mg/g dry cells in one of said organisms or in any
other microorganism, even more preferred are more than 10 mg/g dry
cells, 20 mg/g dry cells, 50 mg/g dry cells, 100 mg/g dry cells,
200 mg/g dry cells, 300 mg/g dry cells, 500 mg/g dry cells or
more.
[8135] [0084.0.0.18] see [0084.0.0.0]
[8136] [0085.0.18.18] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [8137] a) a nucleic acid sequence as
shown in table I, lines 231 to 234 and/or 235 to 242 and/or 599 to
602, columns 5 or 7 or a derivative thereof, or [8138] b) a genetic
regulatory element, for example a promoter, which is functionally
linked to the nucleic acid sequence as shown table I, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 or a
derivative thereof, or [8139] c) (a) and (b) is/are not present in
its/their natural genetic environment or has/have been modified by
means of genetic manipulation methods, it being possible for the
modification to be, by way of example, a substitution, addition,
deletion, inversion or insertion of one or more nucleotide.
"Natural genetic environment" means the natural chromosomal locus
in the organism of origin or the presence in a genomic library. In
the case of a genomic library, the natural, genetic environment of
the nucleic acid sequence is preferably at least partially still
preserved. The environment flanks the nucleic acid sequence at
least on one side and has a sequence length of at least 50 bp,
preferably at least 500 bp, particularly preferably at least 1000
bp, very particularly preferably at least 5000 bp.
[8140] [0086.0.0.18] to [0087.0.0.18] see [0086.0.0.0] to
[0087.0.0.0]
[8141] [0088.0.18.18] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose Coenzyme
content, in particular its Coenzyme Q9 and/or Coenzyme Q10 content
is modified advantageously owing to the nucleic acid molecule of
the present invention expressed. This is important for plant
breeders since, for example, the nutritional value of plants for
feed or nutrition is dependent on the abovementioned coenzymes, in
particular on the essential coenzyme Q10, and the general amount of
coenzymes as source in feed or food. After the YPR138C, YBR184W,
b2699 and/or b1829, and/or YPR172W, YER174C, YER156C, YDR513W,
b2426, b2703, b2729, b3644, b3605, b2699, b0730, and/or b0175
protein activity, e.g. of a protein as indicated in Table II,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or
of its homologs, e.g. as indicated in Table II, column 7, lines 231
to 234 and/or 235 to 242 and/or 599 to 602, has been increased or
generated, or after the expression of nucleic acid molecule or
polypeptide according to the invention has been generated or
increased, the transgenic plant generated thus is grown on or in a
nutrient medium or else in the soil and subsequently harvested.
[8142] [0088.1.0.18] see [0088.1.0.0]
[8143] [0089.0.0.18] to [0094.0.0.18] see [0089.0.0.0] to
[0094.0.0.0]
[8144] [0095.0.18.18] It may be advantageous to increase the pool
of the respective fine chemical in the transgenic organisms by the
process according to the invention in order to isolate high amounts
of the essentially pure fine chemical.
[8145] [0096.0.18.18] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid for example a coenzyme
precursor (e.g. isoprenoids, acetyl CoA, HMG-CoA, mevalonate,
isopentyl pyrophosphate, geranyl pyrophosphate, farnesyl
pyrophosphate, etc.) transporter protein or a compound, which
increases the production of Coenzyme precursors is useful to
increase the production of the respective fine chemical (see Bao
and Ohlrogge, Plant Physiol. 1999 August; 120 (4): 1057-1062).
[8146] [0097.0.18.18] In may also be advantageous to increase the
content of the lipid bound respective fine chemical.
[8147] [0098.0.18.18] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated. The production of further antioxidants,
vitamins or coenzymes such as coenzymes Q0, Q1, Q2, Q3, Q4, Q5, Q6,
Q7, and/or Q8 or retinal, vitamin E, vitamin B6, or other fat
soluble vitamins or mixtures thereof by the process according to
the invention is advantageous, e.g. for the production of
compositions used in food and/or feed or cosmetic production.
[8148] [0099.0.18.18] In the case of the fermentation of
microorganisms, the abovementioned coenzymes may accumulate in
membrane fragments and/or in the lipophilic or hydrophobic
fraction. If microorganisms are used in the process according to
the invention, the fermentation broth can be processed after the
cultivation. Depending on the requirement, all or some of the
biomass can be removed from the fermentation broth by separation
methods such as, for example, centrifugation, filtration, decanting
or a combination of these methods, or else the biomass can be left
in the fermentation broth. The fermentation broth can subsequently
be reduced, or concentrated, with the aid of known methods such as,
for example, rotary evaporator, thin-layer evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth advantageously together with
compounds for the formulation can subsequently be processed by
lyophilization, spray drying, spray granulation or by other
methods. Preferably the Coenzymes or the lipophilic or hydrophobic
compositions are isolated from the organisms, such as the
microorganisms or plants or the culture medium in or on which the
organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[8149] [0100.0.18.18] Transgenic plants which comprise Coenzyme Q9
and Coenzyme Q10 synthesized in the process according to the
invention can advantageously be marketed directly without there
being any need for the membrane fragments, cells, cell fragments,
organelles, plastids, waxes, oils, lipids or fats comprising the
respective fine chemical to be isolated. For example, as coenzymes
may be isolated from membranes, e.g. mitochondrial or plastid
membranes, it can be sufficient to isolate only cell fractions
comprising said membrane or fragments of said membranes. Plants for
the process according to the invention are listed as meaning intact
plants and all plant parts, plant organs or plant parts such as
leaf, stem, seeds, root, tubers, anthers, fibers, root hairs,
stalks, embryos, calli, cotelydons, petioles, harvested material,
plant tissue, reproductive tissue and cell cultures which are
derived from the actual transgenic plant and/or can be used for
bringing about the transgenic plant. In this context, the seed
comprises all parts of the seed such as the seed coats, epidermal
cells, seed cells, endosperm or embryonic tissue. However, the
respective fine chemical produced in the process according to the
invention can also be isolated from the organisms, advantageously
plants, in the form of their cells, cell fragments, plastids,
organelles, membranes, membrane fragments, waxes oils, fats, lipids
and/or fatty acids. Coenzyme Q9 or Coenzyme Q10 produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts, preferably the plant
seeds. To increase the efficiency of oil extraction it is
beneficial to clean, to temper and if necessary to hull and to
flake the plant material especially the seeds. In this context,
oils, fats, lipids, waxes and/or fatty acids fractions can be
obtained by what is known as cold beating or cold pressing without
applying heat. In the case of microorganisms, the latter are, after
harvesting, for example extracted directly without further
processing steps or else, after disruption, extracted via various
methods with which the skilled worker is familiar. Chemically pure
the fine chemical comprising compositions are advantageous for
applications in the food or feed industry sector, the cosmetic
sector and especially the pharmacological industry sector.
[8150] [0101.0.18.18] see [0101.0.0.0]
[8151] [0102.0.18.18] Coenzymes can for example be detected
advantageously via LC separation methods. The unambiguous detection
for the presence of Coenzymes products can be obtained by analyzing
recombinant organisms using analytical standard methods like LC-MS,
LC-MSMS, or TLC. The material to be analyzed can be disrupted by
sonication, grinding in a glass mill, liquid nitrogen and grinding,
cooking, or via other applicable methods; see also Biotechnology of
Vitamins, Pigments and Growth Factors, Edited by Erik J. Vandamme,
London, 1989, p. 96 to 103.
[8152] [0103.0.18.18] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [8153] a) nucleic acid molecule encoding, preferably
at least the mature form, of the polypeptide shown in table II,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or
7 or a fragment thereof, which confers an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[8154] b) nucleic acid molecule comprising, preferably at least the
mature form, of the nucleic acid molecule shown in table I, lines
231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7;
[8155] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [8156] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[8157] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [8158]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [8159] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[8160] h) nucleic acid molecule comprising a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers shown in table III,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, column 7 and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [8161] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from an
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [8162] j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence shown in table IV,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, column 7 and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [8163] k) nucleic acid
molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of the polypeptide shown in table II, lines 231 to 234 and/or 235
to 242 and/or 599 to 602, columns 5 or 7 and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; and [8164] l) nucleic acid molecule which is
obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably to (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; or which comprises a sequence which is complementary
thereto.
[8165] [0103.1.18.18] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I A, columns 5 or 7, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, resp., by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of the sequence
indicated in Table I A, columns 5 or 7, lines 231 to 234 and/or 235
to 242 and/or 599 to 602, resp.: In one embodiment, the nucleic
acid molecule used in the process of the invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table I A, columns 5 or 7, lines 231 to 234 and/or 235
to 242 and/or 599 to 602, resp. In another embodiment, the nucleic
acid molecule does not encode a polypeptide of a sequence indicated
in Table II A, columns 5 or 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, resp.
[8166] [0103.2.18.18] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over a or more
sequence(s) indicated in Table I B, column 7, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, resp., by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of a or more
sequence(s) indicated in Table I B, column 7, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, resp. In one embodiment, the
nucleic acid molecule used in the process of the invention is less
than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table I B, column 7, lines 231 to 234 and/or 235 to
242 and/or 599 to 602, resp. In another embodiment, the nucleic
acid molecule does not encode a polypeptide of a sequence indicated
in Table II B, column 7, lines 231 to 234 and/or 235 to 242 and/or
599 to 602, resp.
[8167] [0104.0.18.18] In one embodiment, the nucleic acid molecule
or the invention or used in the process of the invention
distinguishes over the sequence shown in table I, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7 by one or more
nucleotides or does not consist of the sequence shown in table I,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or
7. In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence shown in table I, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, columns 5 or 7. In another
embodiment, the nucleic acid molecule does not encode a polypeptide
of the sequence shown in table II, lines 231 to 234 and/or 235 to
242 and/or 599 to 602, columns 5 or 7.
[8168] [0105.0.0.18] to [0107.0.0.18] see [0105.0.0.0] to
[0107.0.0.0]
[8169] [0108.0.18.18] Nucleic acid molecules with the sequence
shown in table I, lines 231 to 234 and/or 235 to 242 and/or 599 to
602, columns 5 or 7, nucleic acid molecules which are derived from
the amino acid sequences shown in table II, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, columns 5 or 7 or from polypeptides
comprising the consensus sequence shown in table IV, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, column 7, or their
derivatives or homologues encoding polypeptides with the enzymatic
or biological activity of an YPR138C, YBR184W, b2699 and/or b1829,
and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729,
b3644, b3605, b2699, b0730, and/or b0175 protein or conferring a
Coenzyme Q10 and/or Coenzyme Q9 increase dependent on its
expression or its activity, are advantageously increased in the
process according to the invention.
[8170] [0109.0.0.18] see [0109.0.0.0]
[8171] [0110.0.18.18] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
used in the process of the invention, e.g. with YPR138C, YBR184W,
b2699 and/or b1829, and/or YPR172W, YER174C, YER156C, YDR513W,
b2426, b2703, b2729, b3644, b3605, b2699, b0730, and/or b0175
protein activity, e.g. of a protein as indicated in Table II,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or
being encoded by a nucleic acid molecule indicated in Table I,
column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or
of its homologs, e.g. as indicated in Table
[8172] II, column 7, lines 231 to 234 and/or 235 to 242 and/or 599
to 602, can be determined from generally accessible databases.
[8173] [0111.0.0.18] see [0111.0.0.0]
[8174] [0112.0.18.18] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with YPR138C,
YBR184W, b2699 and/or b1829, and/or YPR172W, YER174C, YER156C,
YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730, and/or
b0175 protein activity and conferring Coenzyme Q10 and/or Coenzyme
Q9 increase, e.g. a protein as indicated in Table II, column 5,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or being
encoded by a nucleic acid molecule indicated in Table I, column 5,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, or of their
homologs, e.g. as indicated in Table I or II, column 7, lines 231
to 234 and/or 235 to 242 and/or 599 to 602.
[8175] [0113.0.0.18] to [0117.0.0.18] see [0113.0.0.0] to
[0117.0.0.0]
[8176] [0118.1.0.18] In one embodiment, the nucleic acid molecule
according to the invention or used in the process of the invention
originates from a plant with a high Coenzyme Q10 and/or Coenzyme Q9
content. In one embodiment, the nucleic acid molecule according to
the invention or used in the process of the invention originates
from and/or is transformed into a plant with a high Coenzyme Q10
and/or Coenzyme Q9 content.
[8177] [0118.0.0.18] to [0120.0.0.18] see [0118.0.0.0] to
[0120.0.0.0]
[8178] [0121.0.18.18] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences shown in table II, lines
231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 or
the functional homologues thereof as described herein, preferably
conferring above-mentioned activity, i.e. conferring a respective
fine chemical increase after increasing its activity, e.g. having
the activity of an YPR138C, YBR184W, b2699 and/or b1829, and/or
YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644,
b3605, b2699, b0730, and/or b0175 protein, e.g. of a protein as
indicated in Table II, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 or of its homologs, e.g. as indicated in Table
II, column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to
602.
[8179] [0122.0.0.18] to [0127.0.0.18] see [0122.0.0.0] to
[0127.0.0.0]
[8180] [0128.0.18.18] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs shown in table III, column 7,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, by means of
polymerase chain reaction can be generated on the basis of a
sequence shown herein, for example the sequence shown in table I,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or
7 or the sequences derived from table II, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, columns 5 or 7.
[8181] [0129.0.18.18] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention or used in the method of the invention are indicated in
the alignments shown in the figures. Conserved regions are those,
which show a very little variation in the amino acid in one
particular position of several homologs from different origin. The
consensus sequence shown in table IV, lines 231 to 234 and/or 235
to 242 and/or 599 to 602, column 7 is derived from said
alignments.
[8182] [0130.0.18.18] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of the
respective fine chemical, in particular of Coenzyme Q10 and/or
Coenzyme Q9 after increasing the expression or activity or having
an YPR138C, YBR184W, b2699 and/or b1829, and/or YPR172W, YER174C,
YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730,
and/or b0175 activity or further functional homologs of the
polypeptide of the invention, e.g. homologs from a protein as
indicated in Table II, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 or of its homologs, e.g. as indicated in Table
II, column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
from other organisms.
[8183] [0131.0.0.18] to [0138.0.0.18] see [0131.0.0.0] to
[0138.0.0.0]
[8184] [0139.0.18.18] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical increase, derived from
other organisms, can be encoded by other DNA sequences which
hybridize to the sequences shown in table I, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7 under relaxed
hybridization conditions and which code on expression for peptides
having the respective fine chemical-increasing activity.
[8185] [0140.0.0.18] to [0146.0.0.18] see [0140.0.0.0] to
[0146.0.0.0]
[8186] [0147.0.18.18] Further, the nucleic acid molecule of the
invention or used in the method of the invention comprises a
nucleic acid molecule, which is a complement of one of the
nucleotide sequences of above mentioned nucleic acid molecules or a
portion thereof. A nucleic acid molecule which is complementary to
one of the nucleotide sequences shown in table I, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7 is one which is
sufficiently complementary to one of the nucleotide sequences shown
in table I, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7 such that it can hybridize to one of the nucleotide
sequences shown in table I, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7, thereby forming a stable duplex.
Preferably, the hybridisation is performed under stringent
hybridization conditions. However, a complement of one of the
herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[8187] [0148.0.18.18] The nucleic acid molecule of the invention or
used in the method of the invention comprises a nucleotide sequence
which is at least about 30%, 35%, 40% or 45%, preferably at least
about 50%, 55%, 60% or 65%, more preferably at least about 70%,
80%, or 90%, and even more preferably at least about 95%, 97%, 98%,
99% or more homologous to a nucleotide sequence shown in table I,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or
7, or a functional portion thereof and preferably has above
mentioned activity, in particular having a the respective fine
chemical, in particular Coenzyme Q10 and/or Coenzyme Q9-increasing
activity after increasing the activity or an activity of an
YPR138C, YBR184W, b2699 and/or b1829, and/or YPR172W, YER174C,
YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730,
and/or b0175 gene product, e.g. a gene encoding a protein as
indicated in Table II, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 or comprising or expressing a nucleic acid
molecule indicated in Table I, column 5, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, or of their homologs, e.g. as
indicated in Table I or II, column 7, lines 231 to 234 and/or 235
to 242 and/or 599 to 602.
[8188] [0149.0.18.18] The nucleic acid molecule of the invention or
used in the method of the invention comprises a nucleotide sequence
which hybridizes, preferably hybridizes under stringent conditions
as defined herein, to one of the nucleotide sequences shown in
table I, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity, e.g. conferring an increase of the fine
chemical, and optionally, the activity of YPR138C, YBR184W, b2699
and/or b1829, and/or YPR172W, YER174C, YER156C, YDR513W, b2426,
b2703, b2729, b3644, b3605, b2699, b0730, and/or b0175, e.g. of a
protein as indicated in Table II, column 5, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, or being encoded by a nucleic acid
molecule indicated in Table I, column 5, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, or of their homologs, e.g. as
indicated in Table I or II, column 7, lines 231 to 234 and/or 235
to 242 and/or 599 to 602.
[8189] [00149.1.18.18] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 231 to 234 and/or 235 to 242 and/or 599 to
602, resp., preferably Table I B, column 7, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, resp., has further one or more of the
activities annotated or known for the a protein as indicated in
Table II, column 3, lines 231 to 234 and/or 235 to 242 and/or 599
to 602.
[8190] [0150.0.18.18] Moreover, the nucleic acid molecule of the
invention or used in the method of the invention can comprise only
a portion of the coding region of one of the sequences shown in
table I, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7, resp., preferably Table I B, column 7, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, resp., for example a
fragment which can be used as a probe or primer or a fragment
encoding a biologically active portion of the polypeptide of the
present invention or of a polypeptide used in the process of the
present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of Coenzyme Q10 and/or Coenzyme Q9 if its
activity is increased. The nucleotide sequences determined from the
cloning of the present protein-according-to-the-invention-encoding
gene allows for the generation of probes and primers designed for
use in identifying and/or cloning its homologues in other cell
types and organisms. The probe/primer typically comprises
substantially purified oligonucleotide. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 15 preferably
about 20 or 25, more preferably about 40, 50 or 75 consecutive
nucleotides of a sense strand of one of the sequences set forth,
e.g., in table I, lines 231 to 234 and/or 235 to 242 and/or 599 to
602, columns 5 or 7, resp., preferably Table I B, column 7, lines
231 to 234 and/or 235 to 242 and/or 599 to 602, resp., an
anti-sense sequence of one of the sequences, e.g., set forth in
table I, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primers pairs shown in table
III, lines 231 to 234 and/or 235 to 242 and/or 599 to 602, column 7
will result in a fragment of YPR138C, YBR184W, b2699 and/or b1829,
and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729,
b3644, b3605, b2699, b0730, and/or b0175 gene product, e.g. of a
gene encoding of a protein as indicated in Table II, column 5,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, resp.,
preferably Table II B, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, resp., or expressing a nucleic acid molecule
indicated in Table I, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 or of its homologs, e.g. as indicated in Table
II, column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to
602.
[8191] [0151.0.0.18] see [0151.0.0.0]
[8192] [0152.0.18.18] The nucleic acid molecule of the invention or
used in the method of the invention encodes a polypeptide or
portion thereof which includes an amino acid sequence which is
sufficiently homologous to the amino acid sequence shown in table
II, lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5
or 7, resp., preferably Table I B, column 7, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, resp., such that the protein
or portion thereof maintains the ability to participate in the
respective fine chemical production, in particular a Coenzyme Q10
and/or Coenzyme Q9 increasing the activity as mentioned above or as
described in the examples in plants or microorganisms is
comprised.
[8193] [0153.0.18.18] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence shown in table II, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7 such that the protein or portion
thereof is able to participate in the increase of the respective
fine chemical production. In one embodiment, a protein or portion
thereof as indicated in Table II, columns 5 or 7, lines 231 to 234
and/or 235 to 242 and/or 599 to 602 has for example an activity of
a polypeptide indicated in Table II, column 3, lines 231 to 234
and/or 235 to 242 and/or 599 to 602.
[8194] [0154.0.18.18] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence of table II, lines 231 to 234 and/or 235 to 242 and/or 599
to 602, columns 5 or 7 and having above-mentioned activity, e.g.
conferring preferably the increase of the respective fine
chemical.
[8195] [0155.0.0.18] to [0156.0.0.18] see [0155.0.0.0] to
[0156.0.0.0]
[8196] [0157.0.18.18] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences shown in
table I, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7 (and portions thereof) due to degeneracy of the
genetic code and thus encode a polypeptide of the present
invention, in particular a polypeptide having above mentioned
activity, e.g. conferring an increase in the respective fine
chemical in a organism, e.g. as that polypeptide comprising a
consensus sequence as indicated in Table IV, column 7, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, resp., or of the
polypeptides encoded by the sequence shown in table II, lines 231
to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 or the
functional homologues. Advantageously, the nucleic acid molecule of
the invention or used in the method of the invention comprises, or
in an other embodiment has, a nucleotide sequence encoding a
protein comprising, or in an other embodiment having, an amino acid
sequence as indicated in Table IV, column 7, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, resp., or as shown in table
II, lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5
or 7 or the functional homologues. In a still further embodiment,
the nucleic acid molecule of the invention or used in the method of
the invention encodes a full length protein which is substantially
homologous to an amino acid sequence shown in table II, lines 231
to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 or the
functional homologues. However, in a preferred embodiment, the
nucleic acid molecule of the present invention does not consist of
the sequence shown in table I, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7, preferably of Table I A, columns
5 or 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602.
Preferably, the nucleic acid molecule of the invention is a
functional homologue or identical to a nucleic acid molecule
indicated in Table I B, column 7, lines 231 to 234 and/or 235 to
242 and/or 599 to 602.
[8197] [0158.0.0.18] to [0160.0.0.18] see [0158.0.0.0] to
[0160.0.0.0]
[8198] [0161.0.18.18] Accordingly, in another embodiment, a nucleic
acid molecule of the invention or used in the method of the
invention is at least 15, 20, 25 or 30 nucleotides in length.
Preferably, it hybridizes under stringent conditions to a nucleic
acid molecule comprising a nucleotide sequence of the nucleic acid
molecule of the present invention or used in the process of the
present invention, e.g. comprising a sequence shown in table I,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or
7. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[8199] [0162.0.0.18] see [0162.0.0.0]
[8200] [0163.0.18.18] Preferably, a nucleic acid molecule of the
invention or used in the method of the invention that hybridizes
under stringent conditions to a sequence shown in table I, lines
231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7
corresponds to a naturally-occurring nucleic acid molecule of the
invention or used in the method of the invention. As used herein, a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA
molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein). Preferably, the nucleic acid molecule
encodes a natural protein having above-mentioned activity, e.g.
conferring the respective fine chemical increase after increasing
the expression or activity thereof or the activity of a protein of
the invention or used in the process of the invention.
[8201] [0164.0.0.18] see [0164.0.0.0]
[8202] [0165.0.18.18] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. shown in
table I, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7, resp., preferably Table I B, column 7, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, resp.,
[8203] [0166.0.0.18] to [0167.0.0.18] see [0166.0.0.0] to
[0167.0.0.0]
[8204] [0168.0.18.18] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in the sequences shown in table II, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7, resp.,
preferably Table II B, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, resp., yet retain said activity described
herein. The nucleic acid molecule can comprise a nucleotide
sequence encoding a polypeptide, wherein the polypeptide comprises
an amino acid sequence at least about 50% identical to an amino
acid sequence shown in table II, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7, resp., preferably Table II B,
column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
resp., and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to the
sequence shown in table II, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7, resp., preferably Table II B,
column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
resp., more preferably at least about 70% identical to one of the
sequences shown in table II, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7, resp., preferably Table II B,
column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
resp., even more preferably at least about 80%, 90%, 95% homologous
to the sequence shown in table II, lines 231 to 234 and/or 235 to
242 and/or 599 to 602, columns 5 or 7, resp., preferably Table II
B, column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
resp., and most preferably at least about 96%, 97%, 98%, or 99%
identical to the sequence shown in table II, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7, resp.,
preferably Table II B, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, resp.
[8205] [0169.0.0.18] to [0172.0.0.18] see [0169.0.0.0] to
[0172.0.0.0]
[8206] [0173.0.18.18] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 22609 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 22609 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[8207] [0174.0.0.18] see [0174.0.0.0]
[8208] [0175.0.18.18] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 22610 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 22610 by the above program algorithm with the
above parameter set, has a 80% homology.
[8209] [0176.0.18.18] Functional equivalents derived from one of
the polypeptides as shown in table II, lines 231 to 234 and/or 235
to 242 and/or 599 to 602, columns 5 or 7 according to the invention
by substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as shown in table II,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or
7 according to the invention and are distinguished by essentially
the same properties as a polypeptide as shown in table II, lines
231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7.
[8210] [0177.0.18.18] Functional equivalents derived from a nucleic
acid sequence as shown in table I, lines 231 to 234 and/or 235 to
242 and/or 599 to 602, columns 5 or 7 according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of a polypeptides as shown in table II, lines
231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7
according to the invention and encode polypeptides having
essentially the same properties as a polypeptide as shown in table
II, lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5
or 7.
[8211] [0178.0.0.18] see [0178.0.0.0]
[8212] [0179.0.18.18] A nucleic acid molecule encoding an
homologous to a protein sequence of table II, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7, resp.,
preferably Table II B, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, resp., can be created by introducing one or more
nucleotide substitutions, additions or deletions into a nucleotide
sequence of the nucleic acid molecule of the present invention, in
particular of table I, lines 231 to 234 and/or 235 to 242 and/or
599 to 602, columns 5 or 7, resp., preferably Table I B, column 7,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, resp., such
that one or more amino acid substitutions, additions or deletions
are introduced into the encoded protein. Mutations can be
introduced into the encoding sequences of table I, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7, resp.,
preferably Table I B, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, resp., by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
[8213] [0180.0.0.18] to [0183.0.0.18] see [0180.0.0.0] to
[0183.0.0.0]
[8214] [0184.0.18.18] Homologues of the nucleic acid sequences
used, with a sequence shown in table I, lines 231 to 234 and/or 235
to 242 and/or 599 to 602, columns 5 or 7, comprise also allelic
variants with at least approximately 30%, 35%, 40% or 45% homology,
by preference at least approximately 50%, 60% or 70%, more
preferably at least approximately 90%, 91%, 92%, 93%, 94% or 95%
and even more preferably at least approximately 96%, 97%, 98%, 99%
or more homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from a
sequences shown, preferably from table I, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, columns 5 or 7, resp., preferably
Table I B, column 7, lines 231 to 234 and/or 235 to 242 and/or 599
to 602, resp., or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[8215] [0185.0.18.18] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more of the sequences shown in
any of the table I, lines 231 to 234 and/or 235 to 242 and/or 599
to 602, columns 5 or 7, resp., preferably Table I B, column 7,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, resp., In one
embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotides not shown in any
one of table I, lines 231 to 234 and/or 235 to 242 and/or 599 to
602, columns 5 or 7, resp., preferably Table I B, column 7, lines
231 to 234 and/or 235 to 242 and/or 599 to 602, resp., In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, the nucleic
acid molecule use in the process of the invention is identical to a
sequences shown in table I, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7, resp., preferably Table I B,
column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
resp.,
[8216] [0186.0.18.18] Also preferred is that one or more nucleic
acid molecule used in the process of the invention encodes a
polypeptide comprising a sequence shown in table II, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7, resp.,
preferably Table II B, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, resp., In one embodiment, the nucleic acid
molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment used in the inventive process, the
encoded polypeptide is identical to a sequence shown in table II,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or
7, resp., preferably Table II B, column 7, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, resp.,
[8217] [0187.0.18.18] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising a sequence shown in table II, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, columns 5 or 7, resp., preferably
Table II B, column 7, lines 231 to 234 and/or 235 to 242 and/or 599
to 602, resp., and comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence of a
sequence shown in table I, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7, resp., preferably Table I B,
column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
resp.,
[8218] [0188.0.18.18] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide shown in table II,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or
7, expressed under identical conditions.
[8219] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in,
preferably Table II B, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, resp.,
[8220] [0189.0.18.18] Homologues of nucleic acid sequences shown in
table I, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7 or of the derived sequences shown in table II, lines
231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 also
mean truncated sequences, cDNA, single-stranded DNA or RNA of the
coding and noncoding DNA sequence. Homologues of said sequences are
also understood as meaning derivatives, which comprise noncoding
regions such as, for example, UTRs, terminators, enhancers or
promoter variants. The promoters upstream of the nucleotide
sequences stated can be modified by one or more nucleotide
substitution(s), insertion(s) and/or deletion(s) without, however,
interfering with the functionality or activity either of the
promoters, the open reading frame (=ORF) or with the 3'-regulatory
region such as terminators or other 3' regulatory regions, which
are far away from the ORF. It is furthermore possible that the
activity of the promoters is increased by modification of their
sequence, or that they are replaced completely by more active
promoters, even promoters from heterologous organisms. Appropriate
promoters are known to the person skilled in the art and are
mentioned herein below.
[8221] [0190.0.0.18] to [0191.0.0.18] see [0190.0.0.0] to
[0191.0.0.0]
[8222] [0191.1.18.18] In one embodiment, the organisms or a part
thereof provides according to the herein mentioned process of the
invention an increased level of the fine chemical bound to any wax,
triglycerides, lipid, oil and/or fat or any composition thereof
containing any bound or free Coenzyme Q10 and/or Coenzyme Q9, for
example bound to lipids, lipoproteins, membrane fractions,
sphingolipids, phosphoglycerides, lipids, glycolipids such as
glycosphingolipids, phospholipids such as phosphatidylethanolamine,
phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol or diphosphatidylglycerol, or as
monoacylglyceride, diacylglyceride or triacylglyceride, compared to
said control or selected organisms or parts thereof.
[8223] [0192.0.0.18] to [0203.0.0.18] see [0192.0.0.0] to
[0203.0.0.0]
[8224] [0204.0.18.18] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [8225] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide shown in table II, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7; resp., preferably Table II B,
column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
resp., or a fragment thereof conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof
[8226] b) nucleic acid molecule comprising, preferably at least the
mature form, of a nucleic acid molecule shown in table I, lines 231
to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7, resp.,
preferably Table I B, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, resp., or a fragment thereof conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [8227] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [8228]
d) nucleic acid molecule encoding a polypeptide whose sequence has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [8229] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under stringent hybridisation conditions and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [8230] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [8231] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [8232]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
the primers or primer pairs indicated in table III, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, column 7 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [8233] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from a expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [8234] j) nucleic acid molecule which encodes a
polypeptide comprising a consensus sequence shown in table IV,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, column 7 and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [8235] k) nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domain of the polypeptide shown in table II, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7, resp.,
preferably Table II B, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, resp., and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
and [8236] l) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of the nucleic acid molecule shown in table I, lines 231
to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 or a
nucleic acid molecule encoding, preferably at least the mature form
of, a polypeptide shown in table II, lines 231 to 234 and/or 235 to
242 and/or 599 to 602, columns 5 or 7, and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; or which encompasses a sequence which is
complementary thereto; whereby, preferably, the nucleic acid
molecule according to (a) to (l) distinguishes over a sequence
depicted in table I A, lines 231 to 234 and/or 235 to 242 and/or
599 to 602, columns 5 or 7 by one or more nucleotides. In one
embodiment, the nucleic acid molecule of the invention or used in
the method of the invention does not consist of a sequence shown in
table I A, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7. In an other embodiment, the nucleic acid molecule
of the present invention is at least 30% identical and less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence shown
in table I A, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7. In a further embodiment the nucleic acid molecule
does not encode a polypeptide sequence shown in table II A, lines
231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7.
Accordingly, in one embodiment, the nucleic acid molecule of the
present invention encodes in one embodiment a polypeptide which
differs at least in one or more amino acids from a polypeptide
shown in table II A, lines 231 to 234 and/or 235 to 242 and/or 599
to 602, columns 5 or 7 but does not encode a protein of a sequence
shown in table II A, lines 231 to 234 and/or 235 to 242 and/or 599
to 602, columns 5 or 7. Accordingly, in one embodiment, the protein
encoded by a sequence of a nucleic acid according to (a) to (l)
does not consist of a sequence shown in table II A, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7. In a
further embodiment, the protein of the present invention is at
least 30% identical to protein sequence depicted in table II A,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or
7 and less than 100%, preferably less than 99.999%, 99.99% or
99.9%, more preferably less than 99%, 985, 97%, 96% or 95%
identical to a sequence shown in table II A, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7. whereby, in a
further embodimen, the nucleic acid molecule according to (a) to
(l) distinguishes over a sequence depicted in table I B, lines 231
to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 by one
or more nucleotides. In one embodiment, the nucleic acid molecule
of the invention or used in the method of the invention does not
consist of a sequence shown in table I B, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, columns 5 or 7. In an other
embodiment, the nucleic acid molecule of the present invention is
at least 30% identical and less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence shown in table I b, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7. In a further
embodiment the nucleic acid molecule does not encode a polypeptide
sequence shown in table II B, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7. Accordingly, in one embodiment,
the nucleic acid molecule of the present invention encodes in one
embodiment a polypeptide which differs at least in one or more
amino acids from a polypeptide shown in table II A, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 but does
not encode a protein of a sequence shown in table II B, lines 231
to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7.
Accordingly, in one embodiment, the protein encoded by a sequence
of a nucleic acid according to (a) to (l) does not consist of a
sequence shown in table II B, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7. In a further embodiment, the
protein of the present invention is at least 30% identical to
protein sequence depicted in table II B, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, columns 5 or 7 and less than 100%,
preferably less than 99.999%, 99.99% or 99.9%, more preferably less
than 99%, 985, 97%, 96% or 95% identical to a sequence shown in
table II B, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7.
[8237] [0205.0.0.18] to [0226.0.0.18] see [0205.0.0.0] to
[0226.0.0.0]
[8238] [0227.0.18.18] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[8239] In addition to a sequence mentioned in table I, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, resp., preferably Table I
B, column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
resp., columns 5 or 7 or its derivatives, it is advantageous
additionally to express and/or mutate further genes in the
organisms. Especially advantageously, additionally at least one
further gene of the isoprenoid biosynthetic pathway such as for
acetyl CoA, HMG-CoA, Mevalonate, Isopentyl pyrophosphate, Geranyl
pyrophosphate, Farnesyl pyrophosphate e.g. HMG-CoA Reductase,
Mevalonate, Kinase, etc., is expressed in the organisms such as
plants or microorganisms. It is also possible that the regulation
of the natural genes has been modified advantageously so that the
gene and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the isoprenoids, coenzyme precursor or coenzymes,
preferably Q9 and/or Q10, as desired since, for example, feedback
regulations no longer exist to the same extent or not at all. In
addition it might be advantageously to combine one or more
sequences shown in table I, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7 with genes which generally
support or enhances to growth or yield of the target organism, for
example genes which lead to faster growth rate of microorganisms or
genes which produces stress-, pathogen, or herbicide resistant
plants.
[8240] [0227.1.18.18] In addition to the sequence mentioned in
table I, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7 or its derivatives, it is advantageous additionally
to knock out and/or mutate further genes in the organisms. E.g. it
may be advantageous, if one or more genes of the catabolic pathway
for isoprenoids or quinones are reduced, deleted or in another way
knocked out in the organisms such as plants or microorganisms.
[8241] [0228.0.18.18] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the isoprenoid
metabolism, in particular in Coenzyme Q9 or Coenzyme Q10
synthesis.
[8242] [0229.0.18.18] ./.
[8243] [0230.1.0.18] ./.
[8244] [0230.2.0.18] ./.
[8245] [0230.0.0.18] see [0230.0.0.0]
[8246] [0231.0.18.18] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a Coenzyme Q10 and/or Coenzyme Q9
degrading protein is attenuated, in particular by reducing the rate
of expression of the corresponding gene.
[8247] [0232.0.0.18] to [0276.0.0.18] see [0232.0.0.0] to
[0276.0.0.0]
[8248] [0277.0.18.18] The coenzymes produced can be isolated from
the organism by methods with which the skilled worker is familiar,
for example, via extraction, salt precipitation and/or different
chromatography methods, e.g. as mentioned above. The process
according to the invention can be conducted batchwise,
semibatchwise or continuously. The respective fine chemical
produced in the process according to the invention can be isolated
as mentioned above from the organisms, advantageously plants, in
the form of their waxes, membrane fractions, oils, fats, lipids
and/or fatty acids. Fractions comprising the coenzymes produced by
this process can be obtained by harvesting the organisms, either
from the crop in which they grow, or from the field. This can be
done via pressing or extraction of the plant parts, preferably the
plant seeds.
[8249] [0278.0.0.18] to [0283.0.0.18] see [0278.0.0.0] to
[0283.0.0.0]
[8250] [0283.0.18.18] Moreover, a native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an anti-YPR138C, YBR184W, b2699 and/or b1829,
and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729,
b3644, b3605, b2699, b0730, and/or b0175 protein antibody or an
antibody against a polypeptide as shown in table II, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 or a
antigenic part thereof, which can be produced by standard
techniques utilizing the polypeptid of the present invention or
characterized in the process of the present invention or fragment
thereof, i.e., the polypeptide of this invention. Preferred are
monoclonal antibodies specifically binding to a polypeptide as
indicated in Table II, columns 5 or 7, lines 231 to 234 and/or 235
to 242 and/or 599 to 602.
[8251] [0284.0.0.18] see [0284.0.0.0]
[8252] [0285.0.18.18] In one embodiment, the present invention
relates to a polypeptide having a sequence shown in table II, lines
231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 or
as coded by a nucleic acid molecule shown in table I, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 or
functional homologues thereof.
[8253] [0286.0.18.18] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
comprising or consisting of a consensus sequence shown in table IV,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, column 7 and
in one another embodiment, the present invention relates to a
polypeptide comprising or consisting of a consensus sequence shown
in table IV, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
column 7 whereby 20 or less, preferably 15 or 10, preferably 9, 8,
7, or 6, more preferred 5 or 4, even more preferred 3, even more
preferred 2, even more preferred 1, most preferred 0 of the amino
acids positions indicated can be replaced by any amino acid or, in
an further embodiment, can be replaced and/or absent. In one
embodiment, the present invention relates to the method of the
present invention comprising a polypeptide or to a polypeptide
comprising more than one consensus sequences (of an individual
line) as indicated in Table IV, column 7, lines 231 to 234 and/or
235 to 242 and/or 599 to 602, resp.
[8254] [0287.0.0.18] to [0289.0.0.18] see [0287.0.0.0] to
[0289.0.0.0]
[8255] [0290.0.0.18] see [0290.0.0.0]
[8256] [0291.0.18.18] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[8257] In one embodiment, said polypeptide of the invention or used
in the method of the invention distinguishes over a sequence shown
in table II A or II B, lines 231 to 234 and/or 235 to 242 and/or
599 to 602, columns 5 or 7 by one or more amino acids. In one
embodiment, polypeptide distinguishes form a sequence shown in
table II A or II B, lines 231 to 234 and/or 235 to 242 and/or 599
to 602, columns 5 or 7 by more than 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, even more
preferred are more than 40, 50, or 60 amino acids and, preferably,
the sequence of the polypeptide of the invention distinguishes from
a sequence shown in table II A or II B, lines 231 to 234 and/or 235
to 242 and/or 599 to 602, columns 5 or 7 by not more than 80% or
70% of the amino acids, preferably not more than 60% or 50%, more
preferred not more than 40% or 30%, even more preferred not more
than 20% or 10%. In an other embodiment, said polypeptide of the
invention does not consist of a sequence shown in table II A or II
B, lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5
or 7.
[8258] [0292.0.0.18] see [0292.0.0.0]
[8259] [0293.0.18.18] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or used in the process of the invention and having
a sequence which distinguishes from the sequence as shown in table
II A or II B, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7 by one or more amino acids. In another embodiment,
said polypeptide of the invention does not consist of the sequence
shown in table II A or II B, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7. In a further embodiment, said
polypeptide of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical. In one embodiment, said polypeptide
does not consist of the sequence encoded by the nucleic acid
molecules shown in table I, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7.
[8260] [0294.0.18.18] In one embodiment, the present invention
relates to a polypeptide having YPR138C, YBR184W, b2699 and/or
b1829, and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703,
b2729, b3644, b3605, b2699, b0730, and/or b0175 protein activity,
which distinguishes over the sequence depicted in table II A or II
B, lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5
or 7 by one or more amino acids, preferably by more than 5, 6, 7, 8
or 9 amino acids, preferably by more than 10, 15, 20, 25 or 30
amino acids, even more preferred are more than 40, 50, or 60 amino
acids but even more preferred by less than 70% of the amino acids,
more preferred by less than 50%, even more preferred my less than
30% or 25%, more preferred are 20% or 15%, even more preferred are
less than 10%.
[8261] [0295.0.0.18] to [0296.0.0.18] see [0295.0.0.0] to
[0296.0.0.0]
[8262] [0297.0.18.18] The language "substantially free of cellular
material" includes preparations of the polypeptide of the invention
or used in the method of the invention in which the protein is
separated from cellular components of the cells in which it is
naturally or recombinantly produced. In one embodiment, the
language "substantially free of cellular material" includes
preparations having less than about 30% (by dry weight) of
"contaminating protein", more preferably less than about 20% of
"contaminating protein", still more preferably less than about 10%
of "contaminating protein", and most preferably less than about 5%
"contaminating protein". The term "Contaminating protein" relates
to polypeptides, which are not polypeptides of the present
invention. When the polypeptide of the present invention or
biologically active portion thereof is recombinantly produced, it
is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less
than about 10%, and most preferably less than about 5% of the
volume of the protein preparation. The language "substantially free
of chemical precursors or other chemicals" includes preparations in
which the polypeptide of the present invention is separated from
chemical precursors or other chemicals, which are involved in the
synthesis of the protein. The language "substantially free of
chemical precursors or other chemicals" includes preparations
having less than about 30% (by dry weight) of chemical precursors
of a protein as indicated in Table II, column 5, lines 231 to 234
and/or 235 to 242 and/or 599 to 602 or being encoded by a nucleic
acid molecule indicated in Table I, column 5, lines 231 to 234
and/or 235 to 242 and/or 599 to 602 or of its homologs, e.g. as
indicated in Table II, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, or of non-YPR138C, YBR184W, b2699 and/or b1829,
and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729,
b3644, b3605, b2699, b0730, and/or b0175 chemicals, more preferably
less than about 20% chemical precursors of a protein as indicated
in Table II, column 5, lines 231 to 234 and/or 235 to 242 and/or
599 to 602 or being encoded by a nucleic acid molecule indicated in
Table I, column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to
602 or of its homologs, e.g. as indicated in Table II, column 7,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, or of
non-YPR138C, YBR184W, b2699 and/or b1829, and/or YPR172W, YER174C,
YER156C, YDR513W, b3605, b2699 b0730 and/or b0175 chemicals, still
more preferably less than about 10% chemical precursors of a
protein as indicated in Table II, column 5, lines 231 to 234 and/or
235 to 242 and/or 599 to 602 or being encoded by a nucleic acid
molecule indicated in Table I, column 5, lines 231 to 234 and/or
235 to 242 and/or 599 to 602 or of its homologs, e.g. as indicated
in Table II, column 7, lines 231 to 234 and/or 235 to 242 and/or
599 to 602, or of non-YPR138C, YBR184W, b2699 and/or b1829, and/or
YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644,
b3605, b2699, b0730, and/or b0175 chemicals, and most preferably
less than about 5% chemical precursors of a protein as indicated in
Table II, column 5, lines 231 to 234 and/or 235 to 242 and/or 599
to 602 or being encoded by a nucleic acid molecule indicated in
Table I, column 5, lines 231 to 234 and/or 235 to 242 and/or 599 to
602 or of its homologs, e.g. as indicated in Table II, column 7,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, or of
non-YPR138C, YBR184W, b2699, b1829, and/or YPR172W, YER174C,
YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730,
b0175 chemicals. In preferred embodiments, isolated proteins or
biologically active portions thereof lack contaminating proteins
from the same organism from which the polypeptide of the present
invention is derived. Typically, such proteins are produced by
recombinant techniques.
[8263] [0298.0.18.18] A polypeptide of the invention or used in the
method of the invention can participate in the process of the
present invention. The polypeptide or a portion thereof comprises
preferably an amino acid sequence which is sufficiently homologous
to an amino acid sequence shown in table II, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7.
[8264] [0299.0.18.18] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences as shown in table II A or II B, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7. The
preferred polypeptide of the present invention preferably possesses
at least one of the activities according to the invention and
described herein. A preferred polypeptide of the present invention
includes an amino acid sequence encoded by a nucleotide sequence
which hybridizes, preferably hybridizes under stringent conditions,
to a nucleotide sequence of table I, lines 231 to 234 and/or 235 to
242 and/or 599 to 602, columns 5 or 7 or which is homologous
thereto, as defined above.
[8265] [0300.0.18.18] Accordingly the polypeptide of the present
invention can vary from the sequences shown in table II, lines 231
to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or 7 in amino
acid sequence due to natural variation or mutagenesis, as described
in detail herein. Accordingly, the polypeptide comprise an amino
acid sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%,
65% or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence shown in table II, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7.
[8266] [0301.0.0.18] see [0301.0.0.0]
[8267] [0302.0.18.18] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., the amino acid sequence shown in table II,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602, columns 5 or
7 or the amino acid sequence of a protein homologous thereto, which
include fewer amino acids than a full length polypeptide of the
present invention or used in the process of the present invention
or the full length protein which is homologous to an polypeptide of
the present invention or used in the process of the present
invention depicted herein, and exhibit at least one activity of
polypeptide of the present invention or used in the process of the
present invention.
[8268] [0303.0.0.18] see [0303.0.0.0]
[8269] [0304.0.18.18] Manipulation of the nucleic acid molecule of
the invention or used in the method of the invention may result in
the production of protein indicated in Table II, column 5, lines
231 to 234 and/or 235 to 242 and/or 599 to 602 or being encoded by
a nucleic acid molecule indicated in Table I, column 5, lines 231
to 234 and/or 235 to 242 and/or 599 to 602 or of its homologs, e.g.
as indicated in Table II, column 7, lines 231 to 234 and/or 235 to
242 and/or 599 to 602, having differences from the wild-type
protein. These proteins may be improved in efficiency or activity,
may be present in greater numbers in the cell than is usual, or may
be decreased in efficiency or activity in relation to the wild type
protein.
[8270] [0305.0.18.18] Any mutagenesis strategies for the
polypeptide of the present invention or the polypeptide used in the
process of the present invention to result in increasing said
activity are not meant to be limiting; variations on these
strategies will be readily apparent to one skilled in the art.
Using such strategies, and incorporating the mechanisms disclosed
herein, the nucleic acid molecule and polypeptide of the invention
or used in the method of the invention may be utilized to generate
plants or parts thereof, expressing wildtype YPR138C, YBR184W,
b2699 and/or b1829, and/or YPR172W, YER174C, YER156C, YDR513W,
b2426, b2703, b2729, b3644, b3605, b2699, b0730, and/or b0175
proteins, e.g. of a protein as indicated in Table II, column 5,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or being
encoded by a nucleic acid molecule indicated in Table I, column 5,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or of its
homologs, e.g. as indicated in Table II, column 7, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, or a mutant of a protein as
indicated in Table II, column 5 or 7, lines 231 to 234 and/or 235
to 242 and/or 599 to 602 or being encoded by a nucleic acid
molecule indicated in Table I, column 5, lines 231 to 234 and/or
235 to 242 and/or 599 to 602 or of its homologs, e.g. as indicated
in Table II, column 7, lines 231 to 234 and/or 235 to 242 and/or
599 to 602, e.g. a mutated YPR138C, YBR184W, b2699 and/or b1829,
and/or YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729,
b3644, b3605, b2699, b0730, and/or b0175 protein, and encoding
nucleic acid molecules and polypeptide molecules of the invention
or used in the method of the invention such that the yield,
production, and/or efficiency of production of a desired compound
is improved.
[8271] [0306.0.0.18] to [0308.0.0.18] see [0306.0.0.0] to
[0308.0.0.0]
[8272] [0309.0.18.18] In one embodiment, an "YPR138C, YBR184W,
b2699 and/or b1829, and/or YPR172W, YER174C, YER156C, YDR513W,
b2426, b2703, b2729, b3644, b3605, b2699, b0730, and/or b0175
protein (=polypeptide)" refers to a polypeptide having an amino
acid sequence corresponding to the polypeptide of the invention or
used in the process of the invention, whereas a "non-YPR138C,
YBR184W, b2699 and/or b1829, and/or YPR172W, YER174C, YER156C,
YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730, and/or
b0175 polypeptide" or "other polypeptide" refers to a polypeptide
having an amino acid sequence corresponding to a protein which is
not substantially homologous a polypeptide of the invention,
preferably which is not substantially homologous to a polypeptide
having an YPR138C, YBR184W, b2699 and/or b1829, and/or YPR172W,
YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605,
b2699, b0730, and/or b0175 protein activity, e.g., a protein which
does not confer the activity described herein and which is derived
from the same or a different organism. In one embodiment, a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table II, columns 5 or 7, lines 231 to 234 and/or 235
to 242 and/or 599 to 602, resp., does not confer an increase of the
respective fine chemical in an organism or part thereof.
[8273] [0310.0.0.18] to [0317.0.0.18] see [0310.0.0.0] to
[0317.0.0.0]
[8274] [0318.0.18.18] In an especially preferred embodiment, the
polypeptide according to the invention furthermore also does not
have the sequences of those proteins which are encoded by a
sequences shown in table II, lines 231 to 234 and/or 235 to 242
and/or 599 to 602, columns 5 or 7.
[8275] [0319.0.0.18] to [0334.0.0.18] see [0319.0.0.0] to
[0334.0.0.0]
[8276] [0335.0.18.18] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of the nucleic acid sequences of the table I, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7 and/or homologs
thereof. As described inter alia in WO 99/32619, dsRNAi approaches
are clearly superior to traditional antisense approaches. The
invention therefore furthermore relates to double-stranded RNA
molecules (dsRNA molecules) which, when introduced into an
organism, advantageously into a plant (or a cell, tissue, organ or
seed derived there from), bring about altered metabolic activity by
the reduction in the expression of a nucleic acid sequences of the
table I, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
columns 5 or 7 and/or homologs thereof. In a double-stranded RNA
molecule for reducing the expression of an protein encoded by a
nucleic acid sequence of one of the table I, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7 and/or homologs
thereof, one of the two RNA strands is essentially identical to at
least part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[8277] [0336.0.0.18] to [0342.0.0.18] see [0336.0.0.0] to
[0342.0.0.0]
[8278] [0343.0.18.18] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence of one of the sequences shown in table I, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, columns 5 or 7 or its homolog
is not necessarily required in order to bring about effective
reduction in the expression. The advantage is, accordingly, that
the method is tolerant with regard to sequence deviations as may be
present as a consequence of genetic mutations, polymorphisms or
evolutionary divergences. Thus, for example, using the dsRNA, which
has been generated starting from a sequence of one of sequences
shown in table I, lines 231 to 234 and/or 235 to 242 and/or 599 to
602, columns 5 or 7 or homologs thereof of the one organism, may be
used to suppress the corresponding expression in another
organism.
[8279] [0344.0.0.18] to [0361.0.0.18] see [0344.0.0.0] to
[0361.0.0.0]
[8280] [0362.0.18.18] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or used in the method of the
invention, the nucleic acid construct of the invention, the
antisense molecule of the invention, the vector of the invention or
a nucleic acid molecule encoding the polypeptide of the invention,
e.g. encoding a polypeptide having an YPR138C, YBR184W, b2699
and/or b1829, and/or YPR172W, YER174C, YER156C, YDR513W, b2426,
b2703, b2729, b3644, b3605, b2699, b0730, and/or b0175 protein
activity or of a protein as indicated in Table II, column 5, lines
231 to 234 and/or 235 to 242 and/or 599 to 602 or being encoded by
a nucleic acid molecule indicated in Table I, column 5, lines 231
to 234 and/or 235 to 242 and/or 599 to 602 or of its homologs, e.g.
as indicated in Table II, column 7, lines 231 to 234 and/or 235 to
242 and/or 599 to 602. Due to the above mentioned activity the
respective fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity is increased, e.g. due to an increased expression
or specific activity of the subject matters of the invention in a
cell or an organism or a part thereof. Transgenic for a polypeptide
having an YPR138C, YBR184W, b2699 and/or b1829, and/or YPR172W,
YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605,
b2699, b0730, and/or b0175 protein activity e.g. for a protein as
indicated in Table II, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, lines 231 to 234 and/or 235 to 242
and/or 599 to 602 or of its homologs, e.g. as indicated in Table
II, column 7, lines 231 to 234 and/or 235 to 242 and/or 599 to 602,
means herein that due to modulation or manipulation of the genome,
the activity of YPR138C, YBR184W, b2699 and/or b1829, and/or
YPR172W, YER174C, YER156C, YDR513W, b2426, b2703, b2729, b3644,
b3605, b2699, b0730, and/or b0175 or a YPR138C, YBR184W, b2699
and/or b1829, and/or YPR172W, YER174C, YER156C, YDR513W, b2426,
b2703, b2729, b3644, b3605, b2699, b0730, and/or b0175-like
activity is increased in the cell or organism or part thereof, e.g.
of a protein as indicated in Table II, column 5, lines 231 to 234
and/or 235 to 242 and/or 599 to 602 or being encoded by a nucleic
acid molecule indicated in Table I, column 5, lines 231 to 234
and/or 235 to 242 and/or 599 to 602 or of its homologs, e.g. as
indicated in Table II, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602. Examples are described above in context with the
process of the invention.
[8281] [0363.0.0.18] see [0363.0.0.0]
[8282] [0364.0.18.18] A naturally occurring expression
cassette--for example the naturally occurring combination of the
YPR138C, YBR184W, b2699 and/or B1829, and/or YPR172W, YER174C,
YER156C, YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730,
and/or b0175 protein promoter with the corresponding YPR138C,
YBR184W, b2699 and or b1829, and/or YPR172W, YER174C, YER156C,
YDR513W, b2426, b2703, b2729, b3644, b3605, b2699, b0730, and/or
b0175 protein gene. e.g. the combination of the promoter of a
protein as indicated in Table II, column 5, lines 231 to 234 and/or
235 to 242 and/or 599 to 602 or being encoded by a nucleic acid
molecule indicated in Table I, column 5, lines 231 to 234 and/or
235 to 242 and/or 599 to 602 or of its homologs, e.g. as indicated
in Table II, column 7, lines 231 to 234 and/or 235 to 242 and/or
599 to 602, with the corresponding gene--becomes a transgenic
expression cassette when it is modified by non-natural, synthetic
"artificial" methods such as, for example, mutagenization. Such
methods have been described (U.S. Pat. No. 5,565,350; WO 00/15815;
also see above).
[8283] [0365.0.0.18] to [0376.0.0.18] see [0365.0.0.0] to
[0376.0.0.0]
[8284] [0377.0.18.18] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced coenzymes are isolated to high purity, preferably, to
purity of more than 70% w/w or more, more preferred are 80% or
more, in particular preferred are 90% w/w or more, even more
preferred are 95% w/w or more, e.g. 97% w/w or more, e.g. 98% w/w,
99% w/w, or 99.9% w/w.
[8285] [0378.0.18.18] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the Coenzyme Q9
or Coenzyme Q10 produced in the process can be recovered, purified
or isolated. The resulting coenzymes, e.g. compositions comprising
the former, can, if appropriate, subsequently be further purified,
if desired mixed with other active ingredients such as vitamins,
amino acids, carbohydrates, antibiotics and the like, and, if
appropriate, formulated.
[8286] [0379.0.18.18] In one embodiment, said coenzyme is the
respective fine chemical, preferably reduced Coenzyme Q9 and/or
Coenzyme Q10.
[8287] [0380.0.18.18] The Coenzyme Q9 or Coenzyme Q10 comprising
fraction or the respective fine chemical obtained in the process
are suitable as starting material for the synthesis of further
products of value. For example, they can be used in combination
with each other or alone for the production of pharmaceuticals,
foodstuffs, animal feeds or cosmetics. Accordingly, the present
invention relates a method for the production of a pharmaceuticals,
food stuff, animal feeds, nutrients or cosmetics comprising the
steps of the process according to the invention, including the
isolation of Coenzyme Q9 or Coenzyme Q10 composition produced or
the respective fine chemical produced if desired and formulating
the product with a pharmaceutical acceptable carrier or formulating
the product in a form acceptable for an application in agriculture.
A further embodiment according to the invention is the use of the
respective fine chemical produced in the process or of the
transgenic organisms in animal feeds, foodstuffs, medicines, food
supplements, cosmetics or pharmaceuticals.
[8288] [0381.0.0.18] to [0382.0.0.18] see [0381.0.0.0] to
[0382.0.0.0]
[8289] [0383.0.5.18] For preparing antioxidant, coenzyme and/or
vitamin-containing fine chemicals, in particular the respective
fine chemical-comprising fine chemicals, it is possible to use as
source organic compounds such as, for example, waxes, oils, fats
and/or lipids or a membrane comprising fraction, in particular
compositions comprising membranes of mitochondria or of
plastids.
[8290] [0384.0.0.18] see [0384.0.0.0]
[8291] [0385.0.18.18] The fermentation broths obtained in this way,
containing in particular Coenzyme Q10 and/or Coenzyme Q9 in
mixtures with other lipids, fats and/or oils and/or with other
vitamins or coenzymes or antioxidants normally have a dry matter
content of from, 1% to 50% by weight. Depending on requirements,
the biomass can be removed entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation or a combination of these methods, from the
fermentation broth or left completely in it. The fermentation broth
can then be thickened or concentrated by known methods, such as,
for example, with the aid of a rotary evaporator, thin-film
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. This concentrated fermentation broth can then be
worked up by freeze-drying, spray drying, spray granulation or by
other processes.
[8292] [0386.0.18.18] However, it is also possible to further
purify the fraction or composition comprising the respective fine
chemical, e.g. Coenzyme Q9 and/or Coenzyme Q10, as produced. For
this purpose, the product-containing composition is subjected for
example to a thin layer chromatography on silica gel plates or to a
chromatography such as a Florisil column (Bouhours J. F., J.
Chromatrogr. 1979, 169, 462), in which case the desired product or
the impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use. An alternative method to
purify the fatty acids is for example crystallization in the
presence of urea. These methods can be combined with each
other.
[8293] [0387.0.0.18] to [0392.0.0.18] see [0387.0.0.0] to
[0392.0.0.0]
[8294] [0393.0.18.18] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps:
(i) contacting e.g. hybridising, the nucleic acid molecules of a
sample, e.g. cells, tissues, plants or microorganisms or a nucleic
acid library, which can contain a candidate gene encoding a gene
product conferring an increase in the respective fine chemical
after expression, with the nucleic acid molecule of the present
invention; (j) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence shown in table I, lines 231 to 234 and/or 235 to
242 and/or 599 to 602, columns 5 or 7 and, optionally, isolating
the full length cDNA clone or complete genomic clone; (k)
introducing the candidate nucleic acid molecules in host cells,
preferably in a plant cell or a microorganism, appropriate for
producing the respective fine chemical; (l) expressing the
identified nucleic acid molecules in the host cells; (m) assaying
the respective fine chemical level in the host cells; and (n)
identifying the nucleic acid molecule and its gene product which
expression confers an increase in the respective fine chemical
level in the host cell after expression compared to the wild
type.
[8295] [0394.0.0.18] to [0399.0.0.18] see [0394.0.0.0] to
[0399.0.0.0]
[8296] [0399.1.18.18] One can think to screen for increased fine
chemical production by for example resistance to drugs blocking
Coenzyme Q10 and/or Coenzyme Q9 synthesis and looking whether this
effect is dependent on a protein as indicated in Table II, column
5, lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or being
encoded by a nucleic acid molecule indicated in Table I, column 5,
lines 231 to 234 and/or 235 to 242 and/or 599 to 602 or of its
homologs, e.g. as indicated in Table II, column 7, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, e.g. by comparing the
phenotype of nearly identical organisms with low and high activity
of a protein as indicated in Table II, column 5, lines 231 to 234
and/or 235 to 242 and/or 599 to 602 or being encoded by a nucleic
acid molecule indicated in Table I, column 5, lines 231 to 234
and/or 235 to 242 and/or 599 to 602 or of its homologs, e.g. as
indicated in Table II, column 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602.
[8297] [0400.0.0.18] to [0416.0.0.18] see [0400.0.0.0] to
[0416.0.0.0]
[8298] [0417.0.18.18] The nucleic acid molecule of the invention or
used in the method of the invention, the vector of the invention or
the nucleic acid construct of the invention may also be useful for
the production of organisms resistant to inhibitors of the
isoprenoid, in particular the Coenzyme Q9 and/or Coenzyme Q10
production biosynthesis pathways. In particular, the overexpression
of the polypeptide of the present invention may protect an organism
such as a microorganism or a plant against inhibitors, which block
the respective fine chemical synthesis in said organism.
[8299] As reduced Coenzyme Q9 and/or Coenzyme Q10 can protect
organisms against damages of oxidative stress, a increased level of
the respective fine chemical may protect plants against herbicides
which cause the toxic build-up of oxidative compounds, e.g. singlet
oxygen. Further it is known, that Coenzyme Q9 and/or Coenzyme Q10
stabilize membranes.
[8300] Accordingly, in one embodiment, the increase of the level of
the respective fine chemical is used to protect plants against
herbicides destroying membranes or cells due to the creation of
free oxygen radicals.
[8301] Examples of inhibitors or herbicides building up oxidative
stress are aryl triazion, e.g. sulfentrazone, carfentrazone, or
diphenylethers, e.g. acifluorfen, lactofen, or oxyfluorfen, or
N-Phenylphthalimide, e.g. flumiclorac or flumioxazin, substituted
ureas, e.g. fluometuron, tebuthiuron, or diuron, linuron, or
triazines, e.g. atrazine, prometryn, ametryn, metributzin,
prometon, simazine, or hexazinone, or uracils, e.g. bromacil or
terbacil.
[8302] Inhibitors of the isoprenoids pathway may also lead to an
toxic decrease of Coenzyme Q9 and/or Coenzyme Q10 in cells or
membranes, e.g. as Clomazone. Thus, in one embodiment, the present
invention relates to the use of an increase of the respective fine
chemical according to the present invention for the protection of
plants against carotenoids inhibitors as pyridines and
pyridazinones.
[8303] [0418.0.0.18] to [0423.0.0.18] see [0418.0.0.0] to
[0423.0.0.0]
[8304] [0424.0.18.18] Accordingly, the nucleic acid of the
invention or used in the method of the invention, the polypeptide
of the invention or used in the method of the invention, the
nucleic acid construct of the invention, the organisms, the host
cell, the microorganisms, the plant, plant tissue, plant cell, or
the part thereof of the invention, the vector of the invention, the
agonist identified with the method of the invention, the nucleic
acid molecule identified with the method of the present invention,
can be used for the production of the respective fine chemical or
of the respective fine chemical and one or more other antioxidants,
coenzymes or vitamins, in particular vitamin E, or vitamin B6.
Accordingly, the nucleic acid of the invention or used in the
method of the invention, or the nucleic acid molecule identified
with the method of the present invention or the complement
sequences thereof, the polypeptide of the invention, the nucleic
acid construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the
antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the respective
fine chemical in a organism or part thereof, e.g. in a cell.
[8305] [0425.0.0.18] see [0425.0.0.0]
[8306] [0425.1.18.18] There are many studies, spanning more than
two decades, reporting positive results from the use of Coenzyme
Q10 as adjunctive therapy in the treatment of congestive heart
failure, e.g. supporting ATP in the mitochondria having Antioxidant
action, stabilizing cell membranes or reducing platelet size and
limitation of platelet activity. Coenzyme Q10 has been an approved
drug in Japan for use in congestive heart failure since 1974. Early
studies of congestive heart failure focused on idiopathic dilated
cardiomyopathy, testing Coenzyme Q10 against placebo using
echocardiography to assess heart function. Echocardiographic
improvement seen in these studies was generally slow but sustained
and was accompanied by diminished fatigue, chest pain, dyspnea and
palpitations. Normal heart size and function were restored in some
patients using only Coenzyme Q10; this occurred primarily in
patients with recent onset of congestive heart failure.
[8307] It is now known that the HMG-CoA reductase inhibitors, while
very effective in lowering cholesterol levels, also significantly
lower levels of Coenzyme Q10. This may be particularly hazardous
for patients with heart failure, suggesting a possible indication
for Coenzyme Q10 in many, if not all, individuals using these
cholesterol-lowering drugs. Significant Coenzyme Q10 deficiencies
have been noted in diseased gingiva. It is not unreasonable to
hypothesize that Coenzyme Q10 might be helpful in muscular
dystrophy, cancer (e.g. reduce tumor size, remission in metastatic
breast cancer), AIDS, neurodegenerative diseases (e.g. Huntington's
disease, Familial Alzheimer's disease, Parkinson's disease), Male
infertility (Coenzyme Q10 improves sperm motility and protects
seminal fluid from free radical injury), chronic stable angina,
Significant reduction of plasma levels of lipid peroxidation, skin
photoaging, other immune dysfunctions. Muscular dystrophy is
usually associated with cardiac disease. There is also some
evidence that Coenzyme Q10 might boost energy and speed recovery of
exercise-related muscle exhaustion and damage.
[8308] Accordingly, the nucleic acid of the invention or used in
the method of the invention, the polypeptide of the invention or
used in the method of the invention, the nucleic acid construct of
the invention, the organisms, the host cell, the microorganisms,
the plant, plant tissue, plant cell, or the part thereof of the
invention, the vector of the invention, the antagonist or the
agonist identified with the method of the invention, the antibody
of the present invention, the antisense molecule of the present
invention or the nucleic acid molecule identified with the method
of the present invention, can be used for the preparation of
pharmaceutical, e.g. comprising isolated or recovered the fine
chemical, vegetable, lipids, fats or oils, in particular e.g. in
combination with other antioxidants, vitamins or coenzymes for the
treatment of congestive heart failure, heart muscle dysfunction,
(e.g. for stabilizing cell membranes), reduced platelet size,
limited of platelet activity, idiopathic dilated cardiomyopathy,
atigue, chest pain, dyspnea and palpitations, hypertension and
other manifestations of cardiovascular disease, lowering levels of
Coenzyme Q10 due to treatments with HMG-CoA reductase inhibitors,
diseased gingival, gingival inflammation, cancer, AIDS, other
immune dysfunctions, Muscular dystrophy associated with cardiac
disease, exercise-related muscle exhaustion and damage, obesity,
neurodegenerative diseases (e.g. Huntington's disease, Familial
Alzheimer's disease (e.g. in combination with vitamin B6),
Parkinson's disease), male infertility, chronic stable angina,
significant reduction of plasma levels of lipid peroxidation, skin
photoaging, lipid peroxidation or for reducing the size of tumors,
for remission in metastatic breast cancer, for improving sperm
motility, for protecting against oxidative stress or protecting
seminal fluid. The pharmaceutical composition can comprise other
coenzymes, vitamins, antioxidants or nutrients. As antioxidant the
respective fine chemical is used in one advantageous embodiment in
the reduced form.
[8309] [0425.2.18.18] Further, the nucleic acid of the invention or
used in the method of the invention, the polypeptide of the
invention or used in the method of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the
antagonist or the agonist identified with the method of the
invention, the antibody of the present invention, the antisense
molecule of the present invention or the nucleic acid molecule
identified with the method of the present invention, can be used
for the preparation of an pharmaceutical composition, e.g. in
combination with a pharmaceutical acceptable carrier, a cosmetical
composition, e.g. in combination with a emulsifier, detergent, dye
additive, softener, formulation agent, wetting agent, lubricating
agent etc, or as nutrition supplement, e.g. as oil-based capsules,
powder-filled capsules, or tablets or solubilized softgels
(microemulsions and others).
[8310] Daily doses of Coenzyme Q10 range from 5 to 300 milligrams.
Those who use Coenzyme Q10 for periodontal health take 50 to 200
milligrams daily, e.g. 100 to 150 milligrams daily. The same dose
range applies to those who take statin drugs for treatment of
hypercholesterolemia. Coenzyme Q10 is best taken with food, in
particular together with fats or oils. It is reported, that about
three weeks of daily dosing are necessary to reach maximal serum
concentrations of Coenzyme Q10. Coenzyme Q10 is also available
topically in some toothpastes and skin creams.
[8311] In one further embodiment, the nucleic acid of the invention
or used in the method of the invention, the polypeptide of the
invention or used in the method of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof, of the invention, the vector of the invention, the
antagonist or the agonist identified with the method of the
invention, the antibody of the present invention, the antisense
molecule of the present invention or the nucleic acid molecule
identified with the method of the present invention used for the
preparation of an pharmaceutical composition comprises the isolated
or recovered the respective fine chemical, or are comprised in
lipids, fats, oils, or membrane fractions of said organism, host
cell, microorganism, plant, plant tissue, plant cell, or the part
thereof, of the invention, e.g. comprising further other
antioxidants, vitamins, nutritional supplements or coenzymes, e.g.
selen, vitamin E, vitamin B6, retinol.
[8312] It is assumed that Coenzyme Q9 can be used for the above
mentioned usages as well. Coenzyme Q9 distincts from Coenzyme Q10
only by "tail" which is one isoprenoid unit shorter. Thus, it is
assumed that Coenzyme Q9 is less lipophilic and more hydrophilic
than Coenzyme Q10. Therefore, in one embodiment, it is advantageous
to use products of the process of the present invention or the
products of the present invention for production of a particular
hydrophilic cosmetic composition, e.g. in combination with a
emulsifier, detergent, dye additive, softener, formulation agent,
wetting agent, lubricating agent etc, or a particular hydrophilic
nutrition supplement, e.g. as oil-based capsules, powder-filled
capsules, or tablets or solubilized softgels (microemulsions and
others).
[8313] [0425.3.18.18] In a further embodiment the present invention
relates to the use of the agonist of the present invention, the
plant of the present invention or a part thereof, the microorganism
or the host cell of the present invention or the polypeptide of the
present invention, or a part thereof, for the production a cosmetic
composition or a pharmaceutical composition. Such a composition has
antioxidative activity, photoprotective activity, and/or tanning
activity, can be used for the treating of high levels of
cholesterol and/or lipids, to protect, treat or heal the above
mentioned diseases, or can be used for the cleaning, conditioning,
and/or treating of the skin, e.g. if combined with a
pharmaceutically or cosmetically acceptable carrier.
[8314] The Coenzyme Q9 and/or Coenzyme Q10 can be also used as
stabilizer of other colours or oxygen sensitive compounds.
[8315] [0426.0.0.18] see [0426.0.0.0]
[8316] [0426.1.0.18] ./.
[8317] [0427.0.0.18] to [0430.0.0.0] see [0427.0.0.0] to
[0430.0.0.0]
[0431.0.18.18] Example 1
Cloning SEQ ID NO: 22609 into in Escherichia coli
[8318] [0432.0.18.18] A DNA polynucleotide with a sequence as
indicated in Table I, column 5 and encoding a polypeptide as listed
in Table 1 below, e.g. SEQ ID NO: 22609 was cloned into the
plasmids pBR322 (Sutcliffe, J. G. (1979) Proc. Natl Acad. Sci. USA,
75: 3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol.
134:
[8319] 1141-1156); plasmids of the pBS series (pBSSK+, pBSSK- and
others; Stratagene, LaJolla, USA) or cosmids such as SuperCosi
(Stratagene, LaJolla, USA) or Lorist6 (Gibson, T. J. Rosenthal, A.,
and Waterson, R. H. (1987) Gene 53: 283-286) for expression in E.
coli using known, well-established procedures (see, for example,
Sambrook, J. et al. (1989) "Molecular Cloning: A Laboratory
Manual". Cold Spring Harbor Laboratory Press or Ausubel, F. M. et
al. (1994) "Current Protocols in Molecular Biology", John Wiley
& Sons).
[8320] [0433.0.0.18] to [0435.0.0.18] see [0433.0.0.0] to
[0435.0.0.0]
[8321] [0436.0.0.18] see [0436.0.0.0]
[8322] [0436.1.0.18] see [0436.1.0.0]
[0437.0.5.18] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpina (see [0437.0.5.5])
[8323] [0438.0.5.18] see [0438.0.5.5]
[8324] [0438.1.5.18] see [0438.0.5.5])
[8325] [0439.0.0.18] see [0439.0.0.18]
[8326] [0439.1.5.18] see [0439.0.5.5]
[8327] [0440.0.0.18] see [0440.0.0.0]
[8328] [0440.1.5.18] see [0440.0.5.5]
[8329] [0441.0.0.18] see [0441.0.0.0]
[8330] [0442.0.0.18] see [0442.0.0.0]
[8331] [0442.1.5.18] see [0442.0.5.5]
[8332] [0443.0.0.18] to [0445.0.0.18] see [0443.0.0.0] to
[0445.0.0.0]
[8333] [0445.1.5.18] see [0445.0.5.5]
[8334] [0445.1.9.18] to [0445.3.9.18] see [0445.1.9.9] to
[0445.3.9.9]
[8335] [0446.0.0.18] to [0451.0.0.18] see [0446.0.0.0] to
[0451.0.0.0]
[8336] [0451.1.5.18] see [0451.0.5.5])
[8337] [00451.2.0.18] see [00451.1.0.0]
[8338] [0452.0.0.18] to [0455.0.0.18] see [0452.0.0.0] to
[0455.0.0.0]
[8339] [0455.0.5.18] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a fatty acid) can be determined by growing the
modified microorganisms or the modified plant under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the elevated production of
desired product (i.e. of the lipids or a fatty acid). These
analytical techniques are known to the skilled worker and comprise
spectroscopy, thin-layer chromatography, various types of staining
methods, enzymatic and microbiological methods and analytical
chromatography such as high-performance liquid chromatography (see,
for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2,
p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.,
(1987) "Applications of HPLC in Biochemistry" in: Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et
al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery
and purification", p. 469-714, VCH: Weinheim; Better, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications). In addition to the abovementioned processes, plant
lipids are extracted from plant material as described by Cahoon et
al. (1999) Proc. Natl. Acad. Sci. USA 96 (22): 12935-12940 and
Browse et al. (1986) Analytic Biochemistry 152:141-145. The
qualitative and quantitative analysis of lipids or fatty acids is
described by Christie, William W., Advances in Lipid Methodology,
Ayr/Scotland: Oily Press (Oily Press Lipid Library; 2); Christie,
William W., Gas Chromatography and Lipids. A Practical Guide--Ayr,
Scotland: Oily Press, 1989, Repr. 1992, IX, 307 pp. (Oily Press
Lipid Library; 1); "Progress in Lipid Research, Oxford: Pergamon
Press, 1 (1952)--16 (1977) under the title: Progress in the
Chemistry of Fats and Other Lipids CODEN. (see [0455.0.5.5])
[8340] [0456.0.0.18] see [0456.0.0.0]
[0457.0.18.18] Example 9
Purification of Coenzyme Q9 or Coenzyme Q10
[8341] [0458.0.18.18] One example is the analysis of the coenzymes.
The unambiguous detection for the presence of the coenzymes
products can be obtained by analyzing recombinant organisms using
analytical standard methods, especially HPLC with UV or
electrochemical detection as for example described in The Journal
of Lipid Research, Vol. 39, 2099-2105, 1998.
[8342] Possible methods for the production and preparation of
coenzymes like Coenzyme Q10 has also been described for example in
WO2003056024, J57129695, J57202294, DE3416853 and DD-229152.
Further methods for the isolation of the respective fine chemical
can also been found in WO 9500634, Fat-Sci. Technol.; (1992) 94, 4,
153-57, DD-294280, DD-293048, JP-145413, DD-273002, DD-271128,
SU1406163, JP-166837, JP-176705, Acta-Biotechnol.; (1986) 6, 3,
277-79, DD-229152, DE3416854, DE3416853, JP-202840, JP-048433,
JP-125306, JP-087137, JP-014026, WO2003056024 and W0200240682
[8343] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[8344] [0459.0.18.18] Usually acetone or hexane is used for the
extraction of the coenzymes and further purification is achieved
either by column chromatography with a suitable resin.
[8345] If necessary, these chromatography steps may be repeated,
using identical or other chromatography resins. The skilled worker
is familiar with the selection of suitable chromatography resin and
the most effective use for a particular molecule to be
purified.
[8346] [0460.0.18.18] In addition depending on the produced fine
chemical purification is also possible with crystallization or
distillation. Both methods are well known to a person skilled in
the art.
[0461.0.18.18] Example 10
Cloning SEQ ID NO: 22609 for the Expression in Plants
[8347] [0462.0.0.18] see [0462.0.0.0]
[8348] [0463.0.18.18] SEQ ID NO: 22609 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[8349] [0464.0.0.18] to [0466.0.0.18] see [0464.0.0.0] to
[0466.0.0.0]
[8350] [0467.0.18.18] The following primer sequences were selected
for the gene SEQ ID NO: 22609:
TABLE-US-00085 i) forward primer (SEQ ID NO: 22701) atgggacaca
agcccttata ccg ii) reverse primer (SEQ ID NO: 22702) ttatcgcgat
gattttcgct gcg
[8351] [0468.0.0.18] to [0479.0.0.18] see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.18.18] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 22609
[8352] [0481.0.0.18] to [0513.0.0.18] see [0481.0.0.0] to
[0513.0.0.0]
[8353] [0514.0.18.18] The results of the different plant analyses
can be seen from the table which follows:
TABLE-US-00086 TABLE 1 Metabolite Profiling Info: ORF Metabolite
Method Min Max YBR184W Coenzyme Q10 LC 1.70 1.95 YDR513W Coenzyme
Q9 LC 1.31 1.73 YER156C Coenzyme Q9 LC 1.32 1.58 YER174C Coenzyme
Q9 LC 1.31 1.72 YPR138C Coenzyme Q10 LC 1.65 3.57 YPR172W Coenzyme
Q9 LC 1.34 1.66 b3644 Coenzyme Q9 LC 1.37 2.30 b2426 Coenzyme Q9 LC
1.38 1.77 b2703 Coenzyme Q9 LC 1.37 2.18 b2729 Coenzyme Q9 LC 1.35
1.41 b0175 Coenzyme Q9 LC 1.45 1.70 b0730 Coenzyme Q9 LC 1.30 3.03
b1829 Coenzyme Q10 LC 1.48 5.33 b2699 Coenzyme Q10 LC 1.62 3.20
b2699 Coenzyme Q9 LC 1.34 3.53 b3605 Coenzyme Q9 LC 1.35 1.41
[8354] [0515.0.0.18] to [0552.0.0.18] see [0515.0.0.0] to
[0552.0.0.0] including [0530.1.0.0] to [0530.6.0.0] as well as
[0552.2.0.0]
[0552.1.18.18] Example 15
Metabolite Profiling Info from Zea mays
[8355] Zea mays plants were engineered, grown and analysed as
described in Example 14c.
[8356] The results of the different Zea mays plants analysed can be
seen from Table 2 which follows:
TABLE-US-00087 TABLE 2 ORF_NAME Metabolite Min Max b1829 Coenzyme
Q10 1.28 1.65
[8357] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in Coenzyme Q10 in
genetically modified corn plants expressing the E. coli nucleic
acid sequence b1829.
[8358] In one embodiment, in case the activity of the E. coli
protein b1829 or its homologs, an activity being defined as a heat
shock protein or its homolog, is increased in corn plants,
preferably, an increase of the fine chemical Coenzyme Q10 between
28% and 65% is conferred.
[8359] [0552.2.0.18] see [0552.2.0.0]
[8360] [0553.0.18.18] [8361] 1. A process for the production of
Coenzyme Q10 and/or Coenzyme Q9, which comprises [8362] (a)
increasing or generating the activity of a protein as indicated in
Table II, columns 5 or 7, lines 231 to 234 and/or 235 to 242 and/or
599 to 602 or a functional equivalent thereof in a non-human
organism, or in one or more parts thereof; and [8363] (b) growing
the organism under conditions which permit the production of
Coenzyme Q10 and/or Coenzyme Q9 in said organism. [8364] 2. A
process for the production of Coenzyme Q10 and/or Coenzyme Q9,
comprising the increasing or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [8365] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 231 to
234 and/or 235 to 242 and/or 599 to 602 or a fragment thereof,
which confers an increase in the amount of Coenzyme Q10 and/or
Coenzyme Q9 in an organism or a part thereof; [8366] b) nucleic
acid molecule comprising of a nucleic acid molecule as indicated in
[8367] Table I, columns 5 or 7, lines 231 to 234 and/or 235 to 242
and/or 599 to 602; [8368] c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as a result of the degeneracy of the
genetic code and conferring an increase in the amount of Coenzyme
Q10 and/or Coenzyme Q9 in an organism or a part thereof; [8369] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of Coenzyme Q10 and/or Coenzyme Q9 in an
organism or a part thereof; [8370] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of Coenzyme Q10 and/or Coenzyme Q9 in an organism or a
part thereof; [8371] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, column 7, lines
231 to 234 and/or 235 to 242 and/or 599 to 602 and conferring an
increase in the amount of Coenzyme Q10 and/or Coenzyme Q9 in an
organism or a part thereof; [8372] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
Coenzyme Q10 and/or Coenzyme Q9 in an organism or a part thereof;
[8373] h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table IV, column 7, lines 231 to 234
and/or 235 to 242 and/or 599 to 602 and conferring an increase in
the amount of Coenzyme Q10 and/or Coenzyme Q9 in an organism or a
part thereof; and [8374] i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of Coenzyme Q10 and/or Coenzyme Q9 in an organism or a part
thereof [8375] or comprising a sequence which is complementary
thereto. [8376] 3. The process of claim 1 or 2, comprising
recovering of the free or bound Coenzyme Q10 and/or Coenzyme Q9.
[8377] 4. The process of any one of claims 1 to 3, comprising the
following steps: [8378] (a) selecting an organism or a part thereof
expressing a polypeptide encoded by the nucleic acid molecule
characterized in claim 2; [8379] (b) mutagenizing the selected
organism or the part thereof; [8380] (c) comparing the activity or
the expression level of said polypeptide in the mutagenized
organism or the part thereof with the activity or the expression of
said polypeptide of the selected organisms or the part thereof;
[8381] (d) selecting the mutated organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
[8382] (e) optionally, growing and cultivating the organisms or the
parts thereof; and [8383] (f) recovering, and optionally isolating,
the free or bound Coenzyme Q10 and/or Coenzyme Q9 produced by the
selected mutated organisms or parts thereof. [8384] 5. The process
of any one of claims 1 to 4, wherein the activity of said protein
or the expression of said nucleic acid molecule is increased or
generated transiently or stably. [8385] 6. An isolated nucleic acid
molecule comprising a nucleic acid molecule selected from the group
consisting of: [8386] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, preferably Table II B,
columns 5 or 7, lines 231 to 234 and/or 235 to 242 and/or 599 to
602, or a fragment thereof, which confers an increase in the amount
of Coenzyme Q10 and/or Coenzyme Q9 in an organism or a part
thereof; [8387] b) nucleic acid molecule comprising of a nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines 231 to
234 and/or 235 to 242 and/or 599 to 602, preferably Table I B,
columns 5 or 7, lines 231 to 234 and/or 235 to 242 and/or 599 to
602; [8388] c) nucleic acid molecule whose sequence can be deduced
from a polypeptide sequence encoded by a nucleic acid molecule of
(a) or (b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of Coenzyme Q10 and/or
Coenzyme Q9 in an organism or a part thereof; [8389] d) nucleic
acid molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of Coenzyme Q10 and/or Coenzyme Q9 in an organism or
a part thereof; [8390] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under stringent
hybridisation conditions and conferring an increase in the amount
of Coenzyme Q10 and/or Coenzyme Q9 in an organism or a part
thereof; [8391] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, column 7, lines
231 to 234 and/or 235 to 242 and/or 599 to 602 and conferring an
increase in the amount of Coenzyme Q10 and/or Coenzyme Q9 in an
organism or a part thereof; [8392] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
Coenzyme Q10 and/or Coenzyme Q9 in an organism or a part thereof;
[8393] h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table IV, column 7, lines 231 to 234
and/or 235 to 242 and/or 599 to 602 and conferring an increase in
the amount of Coenzyme Q10 and/or Coenzyme Q9 in an organism or a
part thereof; and [8394] i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of Coenzyme Q10 and/or Coenzyme Q9 in an organism or a part
thereof [8395] whereby the nucleic acid molecule distinguishes over
the sequence as indicated in Table I A, columns 5 or 7, lines 231
to 234 and/or 235 to 242 and/or 599 to 602 by one or more
nucleotides. [8396] 7. A nucleic acid construct which confers the
expression of the nucleic acid molecule of claim 6, comprising one
or more regulatory elements. [8397] 8. A vector comprising the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7. [8398] 9. The vector as claimed in claim 8,
wherein the nucleic acid molecule is in operable linkage with
regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic, host. [8399] 10. A
host cell, which has been transformed stably or transiently with
the vector as claimed in claim 8 or 9 or the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 2 to 5. [8400] 11.
The host cell of claim 10, which is a transgenic host cell. [8401]
12. The host cell of claim 10 or 11, which is a plant cell, an
animal cell, a microorganism, or a yeast cell, a fungus cell, a
prokaryotic cell, an eukaryotic cell or an archaebacterium. [8402]
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. [8403] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table II A, columns 5 or 7, lines 231 to 234 and/or
235 to 242 and/or 599 to 602 by one or more amino acids [8404] 15.
An antibody, which binds specifically to the polypeptide as claimed
in claim 14. [8405] 16. A plant tissue, propagation material,
harvested material or a plant comprising the host cell as claimed
in claim 12 which is plant cell or an Agrobacterium. [8406] 17. A
method for screening for agonists and antagonists of the activity
of a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of Coenzyme Q10 and/or
Coenzyme Q9 in an organism or a part thereof comprising: [8407] (a)
contacting cells, tissues, plants or microorganisms which express
the a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of Coenzyme Q10 and/or
Coenzyme Q9 in an organism or a part thereof with a candidate
compound or a sample comprising a plurality of compounds under
conditions which permit the expression the polypeptide; [8408] (b)
assaying the Coenzyme Q10 and/or Coenzyme Q9 level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [8409] (c)
identifying a agonist or antagonist by comparing the measured
Coenzyme Q10 and/or Coenzyme Q9 level or polypeptide expression
level with a standard Coenzyme Q10 and/or Coenzyme Q9 or
polypeptide expression level measured in the absence of said
candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. [8410] 18. A process for the
identification of a compound conferring increased Coenzyme Q10
and/or Coenzyme Q9 production in a plant or microorganism,
comprising the steps: [8411] (a) culturing a plant cell or tissue
or microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of Coenzyme Q10 and/or Coenzyme Q9 CoQ 9 in
an organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of Coenzyme Q10 and/or
Coenzyme Q9 in an organism or a part thereof; [8412] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [8413] 19. A method for the identification of a
gene product conferring an increase in Coenzyme Q10 and/or Coenzyme
Q9 production in a cell, comprising the following steps: [8414] (a)
contacting the nucleic acid molecules of a sample, which can
contain a candidate gene encoding a gene product conferring an
increase in Coenzyme Q10 and/or Coenzyme Q9 after expression with
the nucleic acid molecule of claim 6; [8415] (b) identifying the
nucleic acid molecules, which hybridise under relaxed stringent
conditions with the nucleic acid molecule of claim 6; [8416] (c)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing Coenzyme Q10 and/or Coenzyme Q9; [8417]
(d) expressing the identified nucleic acid molecules in the host
cells; [8418] (e) assaying the Coenzyme Q10 and/or Coenzyme Q9
level in the host cells; and [8419] (f) identifying nucleic acid
molecule and its gene product which expression confers an increase
in the Coenzyme Q10 and/or Coenzyme Q9 level in the host cell in
the host cell after expression compared to the wild type. [8420]
20. A method for the identification of a gene product conferring an
increase in
[8421] Coenzyme Q10 and/or Coenzyme Q9 production in a cell,
comprising the following steps: [8422] (a) identifying in a data
bank nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the Coenzyme Q10 and/or Coenzyme Q9 amount or level in an organism
or a part thereof after expression, and which are at least 20%
homolog to the nucleic acid molecule of claim 6; [8423] (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing Coenzyme Q10 and/or Coenzyme Q9; [8424]
(c) expressing the identified nucleic acid molecules in the host
cells; [8425] (d) assaying the Coenzyme Q10 and/or Coenzyme Q9
level in the host cells; and [8426] (e) identifying nucleic acid
molecule and its gene product which expression confers an increase
in the Coenzyme Q10 and/or Coenzyme Q9 level in the host cell after
expression compared to the wild type. [8427] 21. A method for the
production of an agricultural composition comprising the steps of
the method of any one of claims 17 to 20 and formulating the
compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. [8428] 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [8429] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
Coenzyme Q10 and/or Coenzyme Q9 after expression. [8430] 24. Use of
the polypeptide of claim 14 or the nucleic acid construct claim 7
or the gene product identified according to the method of claim 19
or 20 for identifying compounds capable of conferring a modulation
of Coenzyme Q10 and/or Coenzyme Q9 levels in an organism. [8431]
25. Cosmetic, pharmaceutical, nutrition composition, food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. [8432] 26. Use of the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the harvested material of
claim 16, the host cell of claim 10 to 12 or the gene product
identified according to the method of claim 19 or 20 for the
protection of vegetable fats, oils, lipids or waxes comprising the
respective fine chemical. [8433] 27. Use of the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the harvested material of
claim 16, the host cell of claim 10 to 12 or the gene product
identified according to the method of claim 19 or 20 for the
production of oils, lipids, fats or waxes comprising the respective
fine chemical derived from microorganisms. [8434] 28. Use of the
nucleic acid molecule of claim 6, the polypeptide of claim 14, the
nucleic acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20 for the production of of a pharmaceutical agent for the
treatment of congestive heart failure, heart muscle dysfunction,
reduced platelet size, limited of platelet activity, idiopathic
dilated cardiomyopathy, atigue, chest pain, dyspnea and
palpitations, hypertension and other manifestations of
cardiovascular disease, lowering levels of Coenzyme Q10 due to
treatments with HMG-CoA reductase inhibitors, diseased gingival,
gingival inflammation, cancer, AIDS, other immune dysfunctions,
Muscular dystrophy associated with cardiac disease,
exercise-related muscle exhaustion and damage, obesity,
neurodegenerative diseases, male infertility, chronic stable
angina, significant reduction of plasma levels of lipid
peroxidation, skin photoaging, lipid peroxidation or for reducing
the size of tumors, for remission in metastatic breast cancer, for
improving sperm motility, for protecting against oxidative stress,
for stabilizing cell membranes or protecting seminal fluid or a
cosmetic agent or a nutrition supplement for food or feed.
[8435] [0554.0.0.18] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[8436] [0000.0.0.19] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[8437] [0001.0.0.19] for the disclosure of this paragraph see
[0001.0.0.0].
[8438] [0002.0.19.19] Plants produce several important secondary
metabolites from phenylalanine through the phenylpropanoid pathway.
Such substances include flavonoids, lignins, tannins, salicylic
acid and hydroxycinnamic acid esters. Recent work on the
phenylpropanoid pathway has shown that the traditional view of
lignin biosynthesis is incorrect. Although the hydroxylation and
methylation reactions of the pathway were long thought to occur at
the level of the free hydroxycinnamic acids, it turns now out, that
the enzymes catalyzing phenylpropanoid 3-hydroxylation and
3-O-methylation reactions uses shikimate and CoA conjugates as
substrates. The recent cloning of a aldehyde dehydrogenase involved
in ferulic acid and sinapic acid biosynthesis suggest that both
substances are derived at least in part through oxidation of
coniferaldehyde and sinapaldehyde (see Nair et al., 2004, Plant
Cell, 16, 544-554 and citations therein).
[8439] [0003.0.0.19] Ferulic acid is a substance found in the seeds
and leaves of most plants, especially in the brans of grasses such
as wheat, rice, and oats. Its chemical structure strongly resembles
that of curcumin, the substance responsible for the yellow color of
the spice turmeric.
[8440] [0004.0.0.19] The amount of ferulic acid in plant materials
varies widely depending on the species and growing conditions;
supplements are therefore a more reliable source of this substance
than food or unprocessed herbal materials.
[8441] [0005.0.0.19] Ferulic acid has antioxidant properties that
make it an important anti-aging supplement, and they also
contribute to ferulic acid's other potential uses. These include
applications in diabetes, cardiovascular disease, cancer,
neuroprotection, bone degeneration, menopause, immunity, and
(perhaps) athletic performance.
[8442] [0006.0.0.19] In male rats fed a high cholesterol diet,
ferulic acid supplementation significantly lowered total
cholesterol and triglyceride concentrations in the blood, as
compared to a control group. Moreover, HDL (good cholesterol') is
increased with ferulic acid supplementation.
[8443] Like many other dietary substances, ferulic acid is an
antioxidant--but it is an unusually good one. It is especially good
at neutralizing the free radicals known as `superoxide`, `hydroxyl
radical`, and `nitric oxide`. It acts synergistically with other
antioxidants, giving them extra potency. In addition, ferulic acid
can be activated to even higher antioxidant activity by exposure to
UV light, suggesting that it might help to protect skin from sun
damage.
[8444] In microbiological applications ferulic acid is useful as a
substrate for vanillin production, as for example described in WO
9735999 or DE19960106 or for melanin production (WO 9720944).
[8445] [0007.0.0.19] Cinnamic acids, which include caffeic and
ferulic acids, are also powerful antioxidants. Experiments have
found that these compounds can stop the growth of cancer cells.
[8446] In addition sinapic acid is an intermediate in syringyl
lignin biosynthesis in angiosperms, and in some taxa serves as a
precursor for soluble secondary metabolites. The biosynthesis and
accumulation of the sinapate esters sinapoylglucose,
sinapoylmalate, and sinapoylcholine are developmentally regulated
in at least Arabidopsis and other members of the Brassicaceae
(Ruegger et al., 1999, 119(1): 101-10, 1999).
[8447] Due to these interesting physiological roles and
agrobiotechnological potential of ferulic acid or sinapic acid
there is a need to identify the genes of enzymes and other proteins
involved in ferulic acid or sinapic acid metabolism, and to
generate mutants or transgenic plant lines with which to modify the
ferulic acid or sinapic acid content in plants.
[8448] [0008.0.19.19] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce ferulic acid or sinapic acid in
plants. That type of production permits control over quality,
quantity and selection of the most suitable and efficient producer
organisms. The latter is especially important for commercial
production economics and therefore availability to consumers. In
addition it is desirable to produce ferulic acid or sinapic acid in
plants in order to increase plant productivity and resistance
against biotic and abiotic stress as discussed before.
[8449] Methods of recombinant DNA technology have been used for
some years to improve the production of fine chemicals in
microorganisms and plants by amplifying individual biosynthesis
genes and investigating the effect on production of fine chemicals.
It is for example reported, that the xanthophyll astaxanthin could
be produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. Those results indicated
that about 75 percent of the carotenoids found in the flower of the
transformed plant contained a keto group.
[8450] [0009.0.19.19] Thus, it would be advantageous if an algae,
plant or other microorganism were available which produce large
amounts ferulic acid or sinapic acid. The invention discussed
hereinafter relates in some embodiments to such transformed
prokaryotic or eukaryotic microorganisms.
[8451] It would also be advantageous if plants were available whose
roots, leaves, stem, fruits or flowers produced large amounts of
ferulic acid or sinapic acid. The invention discussed hereinafter
relates in some embodiments to such transformed plants.
[8452] [0010.0.19.19] Therefore improving the quality of foodstuffs
and animal feeds is an important task of the food-and-feed
industry. This is necessary since, for example ferulic acid or
sinapic acid, as mentioned above, which occur in plants and some
microorganisms are limited with regard to the supply of mammals.
Especially advantageous for the quality of foodstuffs and animal
feeds is as balanced as possible a specific ferulic acid or sinapic
acid profile in the diet since an excess of ferulic acid or sinapic
acid above a specific concentration in the food has a positive
effect. A further increase in quality is only possible via addition
of further ferulic acid or sinapic acid, which are limiting.
[8453] [0011.0.19.19] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add ferulic acid or sinapic
acid in a balanced manner to suit the organism.
[8454] [0012.0.19.19] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
proteins which participate in the biosynthesis of ferulic acid or
sinapic acid and make it possible to produce them specifically on
an industrial scale without unwanted byproducts forming. In the
selection of genes for biosynthesis two characteristics above all
are particularly important. On the one hand, there is as ever a
need for improved processes for obtaining the highest possible
contents of ferulic acid or sinapic acid; on the other hand as less
as possible byproducts should be produced in the production
process.
[8455] [0013.0.0.19] for the disclosure of this paragraph see
[0013.0.0.0].
[8456] [0014.0.19.19] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a ferulic acid or sinapic
acid. Accordingly, in the present invention, the term "the fine
chemical" as used herein relates to a ferulic acid or sinapic acid.
Further, the term "the fine chemicals" as used herein also relates
to fine chemicals comprising ferulic acid or sinapic acid.
[8457] [0015.0.19.19] In one embodiment, the term "the fine
chemical" or "the respective fine chemical" means at least one
chemical compound with ferulic acid or sinapic acid activity.
[8458] In one embodiment, the term "the fine chemical" means
ferulic acid. In one embodiment, the term "the fine chemical" means
sinapic acid depending on the context in which the term is used.
Throughout the specification the term "the fine chemical" means
ferulic acid or sinapic acid, its salts, ester, thioester or in
free form or bound to other compounds such sugars or sugarpolymers,
like glucoside, e.g. diglucoside.
[8459] [0016.0.19.19] Accordingly, the present invention relates to
a process comprising [8460] (a) increasing or generating the
activity of one or more b0196, b0730, b1896, b2414, b3074, b3172,
YBR184W, YDR513W or b2818 protein(s) in a non-human organism in one
or more parts thereof; and [8461] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
ferulic acid or sinapic acid in said organism.
[8462] Accordingly, the present invention relates to a process
comprising.
[8463] (a) increasing or generating the activity of one or more
proteins having the activity of a protein indicated in Table II,
column 3, lines 243 to 250 and 603, resp. or having the sequence of
a polypeptide encoded by a nucleic acid molecule indicated in Table
I, column 5 or 7, lines 243 to 250 and 603, resp. in a non-human
organism in one or more parts thereof; and
growing the organism under conditions which permit the production
of the fine chemical, thus, ferulic acid or sinapic acid, in said
organism.
[8464] [0016.1.19.19] Accordingly, the term "the fine chemical"
means "ferulic acid" in relation to all sequences listed in Table
I, lines 243, 244, 246, 247, 249 or homologs thereof and means
"sinapic acid" in relation to the sequence listed in Table I, lines
245, 248, 250, 603 or homologs thereof. Accordingly, the term "the
fine chemical" can mean "ferulic acid" or "sinapic acid", owing to
circumstances and the context. In order to illustrate that the
meaning of the term "the respective fine chemical" means "ferulic
acid" or "sinapic acid" owing to the sequences listed in the
context the term "the respective fine chemical" is also used.
[8465] [0017.0.0.19] and [0018.0.0.19] for the disclosure of the
paragraphs [0017.0.0.19] and [0018.0.0.19] see paragraphs
[0017.0.0.0] and [0018.0.0.0] above.
[8466] [0019.0.19.19] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the respective fine chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table II, column 3, lines 243 to 250 and 603 or
encoded by nucleic acid molecule indicated in Table I, columns 5 or
7, lines 243 to 250 and 603.
[8467] [0020.0.19.19] Surprisingly it was found, that the
transgenic expression of the Escherichia coli K12 protein b0196,
b0730, b1896, b2414, b3074, b3172, b2818 or
[8468] Saccharomyces cerevisiae protein YBR184W or YDR513W in
Arabidopsis thaliana conferred an increase in ferulic acid or
sinapic acid ("the fine chemical" or "the fine respective
chemical") in respect to said proteins and their homologs as wells
as the encoding nucleic acid molecules, in particular as indicated
in Table II, column 3, lines 243 to 250 and 603 content of the
transformed plants.
[8469] [0021.0.0.19] for the disclosure of this paragraph see
[0021.0.0.0] above.
[8470] [0022.0.19.19] The sequence of b0196 from Escherichia coli
K12 has been published in Blattner, F. R. et al., Science 277
(5331), 1453-1474 (1997) and its activity is being defined as
regulator in colanic acid synthesis. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein b0196 from
[8471] Escherichia coli K12 or its homolog, e.g. as shown herein,
for the production of the respective fine chemical, in particular
for increasing the amount offerulic acid, preferably in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the protein b0196 is increased.
[8472] The sequence of b0730 from Escherichia coli K12 has been
published in Blattner F. R. et al., Science 277:1453-1474(1997) and
its activity is being defined as a transcriptional regulator of
succinyl Co Asynthase operon and fatty acyl responsive regulator.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein b0730 from Escherichia
coli K12 or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of ferulic acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein b0730
is increased.
[8473] The sequence of b1896 from Escherichia coli K12 has been
published in Blattner F. R. et al., Science 277:1453-1474(1997) and
its activity is being defined as a protein having
trehalose-6-phosphate synthase activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein b1896 from Escherichia coli K12 or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of sinapic acid,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein b1896 is increased.
[8474] The sequence of b2414 from Escherichia coli K12 has been
published in Blattner F. R. et al., Science 277:1453-1474(1997) and
its function is being defined as a subunit of cysteine synthase A
and O-acetylserine sulfhydrolase A, a PLP-dependent enzyme.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein b2414 from Escherichia
coli K12 or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of ferulic acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein b2414
is increased.
[8475] The sequence of b3074 from Escherichia coli K12 has been
published in Blattner F. R. et al., Science 277:1453-1474(1997) and
its activity is being defined as a putative tRNA synthetase
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein b3074 from Escherichia
coli K12 or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of ferulic acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein b3074
is increased.
[8476] The sequence of b3172 from Escherichia coli K12 has been
published in Blattner F. R. et al., Science 277:1453-1474(1997) and
its activity is being defined as a protein having argininosuccinate
synthetase activity. Accordingly, in one embodiment, the process of
the present invention comprises the use of a protein b3172 from
Escherichia coli K12 or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, in particular for
increasing the amount of sinapic acid, preferably in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the protein b3172 is increased.
[8477] The sequence of YBR184W from Saccharomyces cerevisiae has
been published in Goffeau, A. et al., Science 274 (5287), 546-547
(1996) and its activity is being defined as an unclassified
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein YBR184W from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of ferulic acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the unspecified
protein YBR184W is increased.
[8478] The sequence of YDR513W from Saccharomyces cerevisiae has
been published in Jacq, C. et al., Nature 387 (6632 Suppl), 75-78
(1997) and its activity is being defined as a protein having
glutaredoxin (thioltransferase) (glutathione reductase) activity.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein YDR513W having said
activity, for the production of the respective fine chemical, in
particular for increasing the amount of sinapic acid, preferably in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of the protein YDR513W is increased. The sequence of b2818
(Accession number NP.sub.--417295) from Escherichia coli K12 has
been published in Blattner et al., Science 277 (5331), 1453-1474,
1997, and its activity is being defined as a N-acetylglutamate
synthase (amino acid N-acetyltransferase). Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of amino-acid acetyltransferase,
acetylglutamate kinase superfamily, preferably a protein with the
activity of a a N-acetylglutamate synthase (amino acid
N-acetyltransferase) from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of sinapic
acid, in particular for increasing the amount of sinapic acid,
preferably sinapic acid in free or bound form in an organism or a
part thereof, as mentioned.
[8479] [0023.0.19.19] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[8480] In one embodiment, the homolog of any one of the
polypeptides indicated in Table II, column 3, lines 243, 244, 246,
247 or 249 is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms preferably
ferulic acid.
[8481] In one embodiment, the homolog of the polypeptides indicated
in Table II, column 3, line 245, 248, 250 or 603 is a homolog
having the same or a similar activity. In particular an increase of
activity confers an increase in the content of the respective fine
chemical in the organisms preferably sinapic acid.
[8482] [0023.0.19.19] Homologs of the polypeptides indicated in
Table II, column 3, lines 243 to 248, 250 and 603 may be the
polypeptides encoded by the nucleic acid molecules indicated in
Table I, column 7, lines 243 to 248, 250 and 603 or may be the
polypeptides indicated in Table II, column 7, lines 243 to 248, 250
and 603.
[8483] Homologs of the polypeptides indicated in Table II, column
3, lines 243, 244, 246, 247 may be the polypeptides encoded by the
nucleic acid molecules indicated in Table I, column 7, lines 243,
244, 246, 247, respectively or may be the polypeptides indicated in
Table II, column 7, lines 243, 244, 246, 247, having a ferulic acid
content and/or amount increasing activity.
[8484] Homologs of the polypeptide indicated in Table II, column 3,
lines 245, 248, 250, 603 may be the polypeptides encoded by the
nucleic acid molecules indicated in Table I, column 7, lines 245,
248, 250, 603 respectively or may be the polypeptides indicated in
Table II, column 7, lines 245, 248, 250, 603 having a sinapic acid
content and/or amount increasing activity.
[8485] [0023.1.0.19] Homologs of the polypeptides polypeptide
indicated in Table II, column 3, lines 243 to 250 and 603 may be
the polypeptides encoded by the nucleic acid molecules polypeptide
indicated in Table I, column 7, lines 243 to 250 and 603 or may be
the polypeptides indicated in Table II, column 7, lines 243 to 250
and 603.
[8486] [0024.0.0.19] for the disclosure of this paragraph see
[0024.0.0.0] above.
[8487] [0025.0.19.19] In accordance with the invention, a protein
or polypeptide has the "activity of a protein of the invention",
e.g. the activity of a protein indicated in Table II, column 3,
lines 243 to 250 and 603 if its de novo activity, or its increased
expression directly or indirectly leads to an increased ferulic
acid or sinapic acid level, resp., in the organism or a part
thereof, preferably in a cell of said organism. In a preferred
embodiment, the protein or polypeptide has the above-mentioned
additional activities of a protein indicated in Table II, column 3,
lines 243 to 250 and 603. Throughout the specification the activity
or preferably the biological activity of such a protein or
polypeptide or an nucleic acid molecule or sequence encoding such
protein or polypeptide is identical or similar if it still has the
biological or enzymatic activity of any one of the proteins
indicated in Table II, column 3, lines 243 to 250 and 603, or which
has at least 10% of the original enzymatic activity, preferably
20%, particularly preferably 30%, most particularly preferably 40%
in comparison to any one of the proteins indicated in Table II,
column 3, lines 243 to 250 and 603 of Escherichia coli K12 or
Saccharomyces cerevisiae respectively.
[8488] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[8489] [0025.1.0.19] and [0025.2.0.19] for the disclosure of the
paragraphs [0025.1.0.19] and [0025.2.0.19] see [0025.1.0.0] and
[0025.2.0.0] above.
[8490] [0026.0.0.19] to [0033.0.0.19] for the disclosure of the
paragraphs [0026.0.0.19] to [0033.0.0.19] see [0026.0.0.0] to
[0033.0.0.0] above.
[8491] [0034.0.19.19] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of an protein having the activity of a
protein as indicated in Table II, column 3, lines 243 to 250 and
603 or being encoded by a nucleic acid molecule indicated in Table
I, column 5, lines 243 to 250 and 603 or its homologs, e.g. as
indicated in Table I, column 7, lines 243 to 250 and 603, its
biochemical or genetic causes. It therefore shows the increased
amount of the respective fine chemical.
[8492] [0035.0.0.19] to [0044.0.0.19] for the disclosure of the
paragraphs [0035.0.0.19] to [0044.0.0.19] see paragraphs
[0035.0.0.0] to [0044.0.0.0] above.
[8493] [0045.0.19.19] In case the activity of the Escherichia coli
K12 protein b0196 or its homologs, e.g. as indicated in Table II,
columns 5 or 7, line 243 is increased, in one embodiment the
increase of the respective fine chemical, preferably of ferulic
acid acid between 10% and 25% or more is conferred.
[8494] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0730 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 244 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of ferulic acid between 38% and 97% or more is conferred.
[8495] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 245 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of sinapic acid between 38% and 98% or more is conferred.
[8496] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2414 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 246 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of ferulic acid between 34% and 86% or more is conferred.
[8497] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3074 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 247 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of ferulic acid between 35% and 73% or more is conferred.
[8498] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3172 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 248 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of sinapic acid between 31% and 89% or more is conferred.
[8499] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YBR184W or its homologs as indicated in Table II,
columns 5 or 7, line 249, is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of ferulic acid between 30% and 37% or more is conferred.
[8500] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YDR513W or its homologs as indicated in Table
II, columns 5 or 7, line 250, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of sinapic acid between 30% and 39% or more is conferred.
[8501] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2818 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 603 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of sinapic acid between 27% and 54% or more is conferred.
[8502] [0046.0.0.19] In one embodiment, in case the activity of the
Escherichia coli K12 protein b0196 or its homologs, e.g. a
regulator in colanic acid synthesis is increased, preferably an
increase of the fine chemical ferulic acid is conferred.
[8503] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0730 or its homologs, e.g. a transcriptional
regulator of succinylCoA synthetase operon and fatty acyl response
regulator increased, preferably an increase of the fine chemical
ferulic acid is conferred.
[8504] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs, e.g. a
trehalose-6-phosphate synthase 5 is increased, preferably an
increase of the fine chemical sinapic acid is conferred.
[8505] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2414 or its homologs, e.g. a subunit of cysteine
synthase A and O-acetylserine sulfhydrolase A, PLP-dependent enzyme
is increased, preferably an increase of the fine chemical ferulic
acid is conferred.
[8506] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3074 or its homologs, e.g. a putative tRNA
synthetase is increased, preferably an increase of the fine
chemical ferulic acid is conferred.
[8507] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3172 or its homologs, e.g. a protein having
argininosuccinate synthetase activity is increased, preferably an
increase of the fine chemical sinapic acid is conferred.
[8508] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YBR184W or its homologs is increased, preferably
an increase of the fine chemical ferulic acid is conferred.
[8509] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YDR513W or its homologs is increased, preferably
an increase of the fine chemical sinapic acid is conferred.
[8510] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2818 or its homologs, e.g. a N-acetylglutamate
synthase (amino acid N-acetyltransferase) is increased, preferably,
in one embodiment the increase of the respective fine chemical,
preferably an increase of the fine chemical sinapic acid is
conferred.
[8511] [[0047.0.0.19] and [0048.0.0.19] for the disclosure of the
paragraphs [0047.0.0.19] and [0048.0.0.19] see paragraphs
[0047.0.0.0] and [0048.0.0.0] above.
[8512] [0049.0.19.19] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
ferulic acid preferably has the structure of the polypeptide
described herein. In a particular embodiment, the polypeptides used
in the process of the present invention or the polypeptide of the
present invention comprises the sequence of a consensus sequence as
indicated in Table IV, columns 7, lines 243, 244, 246, 247 or of a
polypeptide as indicated in Table II, columns 5 or 7, lines 243,
244, 246, 247 and/or 249 or of a functional homologue thereof as
described herein, or of a polypeptide encoded by the nucleic acid
molecule characterized herein or the nucleic acid molecule
according to the invention, for example by a nucleic acid molecule
as indicated in Table I, columns 5 or 7, lines 243, 244, 246, 247
and/or 249 or its herein described functional homologues and has
the herein mentioned activity conferring an increase in the ferulic
acid level.
[8513] A protein having an activity conferring an increase in the
amount or level of the sinapic preferably has the structure of the
polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence as indicated in Table IV, column 7, line 245,
248, 250 and/or 603 or of a polypeptide as indicated in Table II,
columns 5 or 7, line 245, 248, 250 and/or 603 or of a functional
homologue thereof as described herein, or of a polypeptide encoded
by the nucleic acid molecule characterized herein or the nucleic
acid molecule according to the invention, for example by a nucleic
acid molecule as indicated in Table I, columns 5 or 7, line 245,
248, 250 and/or 603 or its herein described functional homologues
and has the herein mentioned activity conferring an increase in the
sinapic level.
[8514] [0050.0.19.19] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of ferulic acid or sinapic acid, resp., or their ester, or
glucoside thereof, e.g the diglucoside thereof.
[8515] [0051.0.19.19] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g compositions comprising ferulic acid or
sinapic acid. Depending on the choice of the organism used for the
process according to the present invention, for example a
microorganism or a plant, compositions or mixtures of ferulic acid
or sinapic acid can be produced.
[8516] [0052.0.0.19] for the disclosure of this paragraph see
paragraph [0052.0.0.0] above.
[8517] [0053.0.19.19] In one embodiment, the process of the present
invention comprises one or more of the following steps [8518] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 243
to 250 and 603 or its homologs, e.g. as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603, activity having
herein-mentioned the respective fine chemical increasing activity;
[8519] b) stabilizing a mRNA conferring the increased expression of
a protein encoded by the nucleic acid molecule of the invention,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 243 to 250 and 603 or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 243
to 250 and 603, or of a mRNA encoding the polypeptide of the
present invention having herein-mentioned the respective fine
chemical increasing activity; [8520] c) increasing the specific
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the present invention having herein-mentioned
the respective fine chemical increasing activity, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 243 to 250 and 603 or its homologs activity,
e.g. as indicated in Table II, columns 5 or 7, lines 243 to 250 and
603, or decreasing the inhibitory regulation of the polypeptide of
the invention; [8521] d) generating or increasing the expression of
an endogenous or artificial transcription factor mediating the
expression of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention having herein-mentioned the
respective fine chemical increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 243 to 250 and 603 or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 243 to 250 and 603;
[8522] e) stimulating activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 243 to 250 and
603 or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603, by adding one or more
exogenous inducing factors to the organism or parts thereof; [8523]
f) expressing a transgenic gene encoding a protein conferring the
increased expression of a polypeptide encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention, having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 243 to 250 and
603 or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603, and/or [8524] g)
increasing the copy number of a gene conferring the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or the
polypeptide of the invention having herein-mentioned the respective
fine chemical increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 243
to 250 and 603 or its homologs, e.g. as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603, activity. [8525] h)
Increasing the expression of the endogenous gene encoding the
polypeptide of the invention, e.g. a polypeptide having an activity
of a protein as indicated in Table II, column 3, lines 243 to 250
and 603 or its homologs activity, e.g. as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603, by adding positive
expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; [8526]
and/or [8527] i) Modulating growth conditions of an organism in
such a manner, that the expression or activity of the gene encoding
the protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead to an enhanced respective fine
chemical production. [8528] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, e.g. the elite crops.
[8529] [0054.0.19.19] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table II, columns 3 or 5,
lines 243 to 250 and 603, resp., or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 243 to 250 and 603,
resp.
[8530] [0055.0.0.19] to [0067.0.0.19] for the disclosure of the
paragraphs [0055.0.0.19] to [0067.0.0.19] see paragraphs
[0055.0.0.0] to [0067.0.0.0] above.
[8531] [0068.0.19.19] The mutation is introduced in such a way that
the production of ferulic acid or sinapic acid is not adversely
affected.
[8532] [0069.0.0.19] for the disclosure of this paragraph see
paragraph [0069.0.0.0] above.
[8533] [0070.0.19.19] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolite composition in the organism, e.g. an advantageous
composition of ferulic acid and sinapic acid or their biochemical
derivatives, e.g. comprising a higher content of (from a viewpoint
of nutritional physiology limited) ferulic acid and/or sinapic acid
or their derivatives.
[8534] [0071.0.0.19] for the disclosure of this paragraph see
paragraph [0071.0.0.0] above.
[8535] [0072.0.0.19] %
[8536] [0073.0.19.19] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
(c) providing a non-human organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant; (d) increasing an activity of a polypeptide of the
invention or a homolog thereof, e.g. as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603, or of a polypeptide being
encoded by the nucleic acid molecule of the present invention and
described below, e.g. conferring an increase of the respective fine
chemical in an organism, preferably in a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a
plant, (e) growing an organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant under conditions which permit the production of the
respective fine chemical in the organism, preferably the
microorganism, the plant cell, the plant tissue or the plant; and
(f) if desired, recovering, optionally isolating, the free and/or
bound the respective fine chemical synthesized by the organism, the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue or the plant.
[8537] [0074.0.19.19] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound respective fine chemical.
[8538] [0075.0.0.19] to [0077.0.0.19] for the disclosure of the
paragraphs [0075.0.0.19] to [0077.0.0.19] see paragraphs
[0075.0.0.0] to [0077.0.0.0] above.
[8539] [0078.0.19.19] The organism such as microorganisms or plants
or the recovered, and if desired isolated, the respective fine
chemical can then be processed further directly into foodstuffs or
animal feeds or for other applications. The fermentation broth,
fermentation products, plants or plant products can be purified
with methods known to the person skilled in the art. Products of
these different work-up procedures are ferulic acid or sinapic acid
or comprising compositions of ferulic acid and sinapic acid still
comprising fermentation broth, plant particles and cell components
in different amounts, advantageously in the range of from 0 to 99%
by weight, preferably below 80% by weight, especially preferably
below 50% by weight.
[8540] [0079.0.0.19] to [0084.0.0.19] for the disclosure of the
paragraphs [0079.0.0.19] to [0084.0.0.19] see paragraphs
[0075.0.0.0] to [0084.0.0.0] above.
[8541] [0085.0.19.19] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [8542] a) a nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 243 to 250 and 603, or
a derivative thereof, or [8543] b) a genetic regulatory element,
for example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 243 to
250 and 603, or a derivative thereof, or [8544] c) (a) and (b)
is/are not present in its/their natural genetic environment or
has/have been modified by means of genetic manipulation methods, it
being possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[8545] [0086.0.0.19] and [0087.0.0.19] for the disclosure of the
paragraphs [0086.0.0.19] and [0087.0.0.19] see paragraphs
[0086.0.0.0] and [0087.0.0.0] above.
[8546] [0088.0.19.19] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for animals such as poultry is dependent on the
abovementioned fine chemicals or the plants are more resistant to
biotic and abiotic stress and the yield is increased.
[8547] [0088.1.0.19] for the disclosure of this paragraph see
paragraph [0088.1.0.0] above.
[8548] [0089.0.0.19] to [0094.0.0.19] for the disclosure of the
paragraphs [0089.0.0.19] to [0094.0.0.19] see paragraphs
[0089.0.0.0] to [0094.0.0.0] above.
[8549] [0095.0.19.19] It may be advantageous to increase the pool
of ferulic acid or sinapic acid in the transgenic organisms by the
process according to the invention in order to isolate high amounts
of the pure respective fine chemical and/or to obtain increased
resistance against biotic and abiotic stresses and to obtain higher
yield.
[8550] [0096.0.19.19] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example in
the organism, is useful to increase the production of the
respective fine chemical.
[8551] [0097.0.0.19] for the disclosure of this paragraph see
paragraph [0097.0.0.0] above.
[8552] [0098.0.19.19] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated.
[8553] [0099.0.19.19] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[8554] [0100.0.19.19] Transgenic plants which comprise the fine
chemicals such as ferulic acid or sinapic acid synthesized in the
process according to the invention can advantageously be marketed
directly without there being any need for the fine chemicals
synthesized to be isolated. Plants for the process according to the
invention are listed as meaning intact plants and all plant parts,
plant organs or plant parts such as leaf, stem, seeds, root,
tubers, anthers, fibers, root hairs, stalks, embryos, calli,
cotelydons, petioles, harvested material, plant tissue,
reproductive tissue and cell cultures which are derived from the
actual transgenic plant and/or can be used for bringing about the
transgenic plant. In this context, the seed comprises all parts of
the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue.
[8555] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, as extracts, e.g. ether, alcohol,
or other organic solvents or water containing extract and/or free
fine chemicals. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pretreated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[8556] Because ferulic acid or sinapic acid in microorganisms are
localized intracellular, their recovery essentially comes down to
the isolation of the biomass. Well-established approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Of the residual
hydrocarbon, adsorbed on the cells, has to be removed. Solvent
extraction or treatment with surfactants have been suggested for
this purpose.
[8557] [0101.0.19.19] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[8558] [0102.0.19.19] Ferulic acid or sinapic acid can for example
be detected advantageously via HPLC, LC or GC separation methods.
The unambiguous detection for the presence of ferulic acid or
sinapic acid containing products can be obtained by analyzing
recombinant organisms using analytical standard methods: LC, LC-MS,
MS or TLC). The material to be analyzed can be disrupted by
sonication, grinding in a glass mill, liquid nitrogen and grinding,
cooking, or via other applicable methods.
[8559] [0103.0.19.19] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [8560] a) nucleic acid molecule encoding, preferably
at least the mature form, of the polypeptide having a sequence as
indicated in Table II, columns 5 or 7, lines 243 to 250 and 603, or
a fragment thereof, which confers an increase in the amount of the
respective fine chemical in an organism or a part thereof; [8561]
b) nucleic acid molecule comprising, preferably at least the mature
form, of a nucleic acid molecule having a sequence as indicated in
Table I, columns 5 or 7, lines 243 to 250 and 603, [8562] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [8563] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[8564] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [8565]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [8566] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [8567] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers pairs
having a sequence as indicated in Table III, columns 7, lines 243
to 250 and 603, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [8568]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [8569] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequences as indicated in Table IV, column 7,
lines 243 to 250 and 603 and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[8570] k) nucleic acid molecule comprising one or more of the
nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of the polypeptide indicated in Table
II, columns 5 or 7, lines 243 to 250 and 603, and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; and [8571] l) nucleic acid molecule
which is obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably to (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; or which comprises a sequence which is complementary
thereto.
[8572] [00103.1.19.19] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 243 to 250
and 603, by one or more nucleotides. In one embodiment, the nucleic
acid molecule used in the process of the invention does not consist
of the sequence shown in indicated in Table I A, columns 5 or 7,
lines 243 to 250 and 603. In one embodiment, the nucleic acid
molecule used in the process of the invention is less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence indicated in
Table I A, columns 5 or 7, lines 243 to 250 and 603. In another
embodiment, the nucleic acid molecule does not encode a polypeptide
of a sequence indicated in Table II A, columns 5 or 7, lines 243 to
250 and 603.
[8573] [00103.2.19.19] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, lines 243 to 250
and 603, by one or more nucleotides. In one embodiment, the nucleic
acid molecule used in the process of the invention does not consist
of the sequence shown in indicated in Table I B, columns 5 or 7,
lines 243 to 250 and 603. In one embodiment, the nucleic acid
molecule used in the process of the invention is less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence indicated in
Table I B, columns 5 or 7, lines 243 to 250 and 603. In another
embodiment, the nucleic acid molecule does not encode a polypeptide
of a sequence indicated in Table II B, columns 5 or 7, lines 243 to
250 and 603.
[8574] [0104.0.19.19] In one embodiment, the nucleic acid molecule
used in the process of the present invention distinguishes over the
sequence indicated in Table I, columns 5 or 7, lines 243 to 250 and
603 by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence indicated in Table I, columns 5 or 7, lines 243 to 250
and 603 In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence indicated in Table I, columns 5 or 7,
lines 243 to 250 and 603. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table II, columns 5 or 7, lines 243 to 250 and 603.
[8575] [0105.0.0.19] to [0107.0.0.19] for the disclosure of the
paragraphs [0105.0.0.19] to [0107.0.0.19] see paragraphs
[0105.0.0.0] and [0107.0.0.0] above.
[8576] [0108.0.19.19] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 243 to 250 and 603,
nucleic acid molecules which are derived from an amino acid
sequences as indicated in Table II, columns 5 or 7, lines 243 to
250 and 603 or from polypeptides comprising the consensus sequence
as indicated in Table IV, column 7, lines 243 to 250 and 603, or
their derivatives or homologues encoding polypeptides with the
enzymatic or biological activity of an activity of a polypeptide as
indicated in Table II, column 3, 5 or 7, lines 243 to 250 and 603,
e.g. conferring the increase of the respective fine chemical,
meaning ferulic acid or sinapic acid, resp., after increasing its
expression or activity, are advantageously increased in the process
according to the invention.
[8577] [0109.0.19.19] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular ferulic
acid or sinapic acid, produced in the process according to the
invention.
[8578] [0110.0.0.19] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide used in the method
of the invention or used in the process of the invention, e.g. of a
protein as shown in Table II, columns 5 or 7, lines 243 to 250 and
603 or being encoded by a nucleic acid molecule indicated in Table
I, columns 5 or 7, lines 243 to 250 and 603 or of its homologs,
e.g. as indicated in Table II, columns 5 or 7, lines 243 to 250 and
603 can be determined from generally accessible databases.
[8579] [0111.0.0.19] for the disclosure of this paragraph see
[0111.0.0.0] above.
[8580] [0112.0.19.19] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table II, column 3, lines 243 to
250 and 603 or having the sequence of a polypeptide as indicated in
Table II, columns 5 and 7, lines 243 to 250 and 603 and conferring
an increase in the ferulic acid or sinapic acid level.
[8581] [0113.0.0.19] to [0120.0.0.19] for the disclosure of the
paragraphs [0113.0.0.19] to [0120.0.0.19] see paragraphs
[0113.0.0.0] and [0120.0.0.0] above.
[8582] [0121.0.19.19] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603 or the functional
homologues thereof as described herein, preferably conferring
above-mentioned activity, i.e. conferring a ferulic acid level
increase after increasing the activity of the polypeptide sequences
indicated in Table II, columns 5 or 7, lines 243, 244, 246, 247,
249 or conferring a sinapic acid level increase after increasing
the activity of the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 245, 248 250 and 603.
[8583] [0122.0.0.19] to [0127.0.0.19] for the disclosure of the
paragraphs [0122.0.0.19] to [0127.0.0.19] see paragraphs
[0122.0.0.0] and [0127.0.0.0] above.
[8584] [0128.0.19.19] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, column 7,
lines 243 to 250 and 603, by means of polymerase chain reaction can
be generated on the basis of a sequence shown herein, for example
the sequence as indicated in Table I, columns 5 or 7, lines 243 to
250 and 603, resp. or the sequences derived from a sequences as
indicated in Table II, columns 5 or 7, lines 243 to 250 and 603,
resp.
[8585] [0129.0.19.19] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence shown in Table IV, column
7, lines 243 to 248, 250 and 603 is derived from said
alignments.
[8586] [0130.0.19.19] for the disclosure of this paragraph see
[0130.0.0.0].
[8587] [0131.0.0.19] to [0138.0.0.19] for the disclosure of the
paragraphs [0131.0.0.19] to [0138.0.0.19] see paragraphs
[0131.0.0.0] to [0138.0.0.0] above.
[8588] [0139.0.19.19] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical level increase,
derived from other organisms, can be encoded by other DNA sequences
which hybridise to a sequence indicated in Table I, columns 5 or 7,
lines 243, 244, 246, 247, 249, preferably of Table I B, columns 5
or 7, lines 243, 244, 246, 247, 249 for ferulic acid or indicated
in Table I, columns 5 or 7, lines 245, 248, 250, 603, preferably of
Table I B, columns 5 or 7, lines 245, 248, 250, 603 for sinapic
under relaxed hybridization conditions and which code on expression
for peptides having the respective fine chemical, i.e. ferulic acid
or sinapic acid, resp., increasing-activity.
[8589] [0140.0.0.19] to [0146.0.0.19] for the disclosure of the
paragraphs [0140.0.0.19] to [0146.0.0.19] see paragraphs
[0140.0.0.0] and [0146.0.0.0] above.
[8590] [0147.0.19.19] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 243 to 250 and 603, preferably of Table I
B, columns 5 or 7, lines 243 to 250 and 603 is one which is
sufficiently complementary to one of said nucleotide sequences such
that it can hybridise to one of said nucleotide sequences, thereby
forming a stable duplex. Preferably, the hybridisation is performed
under stringent hybridization conditions. However, a complement of
one of the herein disclosed sequences is preferably a sequence
complement thereto according to the base pairing of nucleic acid
molecules well known to the skilled person. For example, the bases
A and G undergo base pairing with the bases T and U or C, resp. and
visa versa. Modifications of the bases can influence the
base-pairing partner.
[8591] [0148.0.19.19] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines
243 to 250 and 603, preferably of Table I B, columns 5 or 7, lines
243 to 250 and 603 or a portion thereof and preferably has above
mentioned activity, in particular having a ferulic acid or sinapic
acid increasing activity after increasing the activity or an
activity of a product of a gene encoding said sequences or their
homologs.
[8592] [0149.0.19.19] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 243 to 250 and 603, preferably of Table I B, columns 5 or 7,
lines 243 to 250 and 603 or a portion thereof and encodes a protein
having above-mentioned activity, e.g. conferring a of ferulic acid
or sinapic acid increase, resp., and optionally, the activity of
protein indicated in Table II, column 5, lines 243 to 250 and 603,
preferably of Table II B, columns 5 or 7, lines 243 to 250 and
603.
[8593] [00149.1.19.19] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table I, columns 5 or 7, lines 243 to 250
and 603, preferably of Table I B, columns 5 or 7, lines 243 to 250
and 603 has further one or more of the activities annotated or
known for a protein as indicated in Table II, column 3, lines 243
to 250 and 603, preferably of Table II B, columns 5 or 7, lines 243
to 250 and 603.
[8594] [0150.0.19.19] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table I, columns 5 or 7, lines 243 to
250 and 603, preferably of Table I B, columns 5 or 7, lines 243 to
250 and 603 for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of ferulic acid or sinapic
acid, resp., if its activity is increased. The nucleotide sequences
determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table I, columns
5 or 7, lines 243 to 250 and 603, an anti-sense sequence of one of
the sequences, e.g., as indicated in Table I, columns 5 or 7, lines
243 to 250 and 603, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table III, column 7, lines 243 to 250 and 603 will result in a
fragment of a polynucleotide sequence as indicated in Table I,
columns 5 or 7, lines 243 to 250 and 603 or its gene product.
Preferred is Table II B, column 7, lines 243 to 250 and 603.
[8595] [0151.0.0.19]: for the disclosure of this paragraph see
[0151.0.0.0] above.
[8596] [0152.0.19.19] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 243 to 250
and 603 such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular a ferulic acid (lines 243, 244, 246, 247, 249) or
sinapic acid (lines 245, 248, 250, 603) increasing activity as
mentioned above or as described in the examples in plants or
microorganisms is comprised.
[8597] [0153.0.19.19] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 243
to 250 and 603 such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table II, columns 5 or 7, lines 243 to 250 and 603 has
for example an activity of a polypeptide indicated in Table II,
column 3, lines 243 to 250 and 603.
[8598] [0154.0.19.19] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 243 to 250
and 603 and has above-mentioned activity, e.g. conferring
preferably the increase of the respective fine chemical.
[8599] [0155.0.0.19] and [0156.0.0.19] for the disclosure of the
paragraphs [0155.0.0.19] and [0156.0.0.19] see paragraphs
[0155.0.0.0] and [0156.0.0.0] above.
[8600] [0157.0.19.19] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences as
indicated in Table I, columns 5 or 7, lines 243 to 250 and 603 (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
polypeptides comprising the sequence as indicated in Table IV,
column 7, lines 243 to 248, 250 and 603 or as polypeptides depicted
in Table II, columns 5 or 7, lines 243 to 250 and 603 or the
functional homologues. Advantageously, the nucleic acid molecule of
the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, an amino acid sequence of a consensus sequences
as indicated in Table IV, column 7, lines 243 to 248, 250 and 603
or of the polypeptide as indicated in Table II, columns 5 or 7,
lines 243 to 250 and 603, resp., or the functional homologues. In a
still further embodiment, the nucleic acid molecule of the
invention encodes a full length protein which is substantially
homologous to an amino acid sequence comprising a consensus
sequence as indicated in Table IV, column 7, lines 243 to 248, 250
and 603 or of a polypeptide as indicated in Table II, columns 5 or
7, lines 243 to 250 and 603 or the functional homologues. However,
in a preferred embodiment, the nucleic acid molecule of the present
invention does not consist of a sequence as indicated in Table I,
columns 5 or 7, lines 243 to 250 and 603, resp., preferably as
indicated in Table I A, columns 5 or 7, lines 243 to 250 and/or
603. Preferably the nucleic acid molecule of the invention is a
functional homologue or identical to a nucleic acid molecule
indicated in Table I B, columns 5 or 7, lines 243 to 250 and/or
603.
[8601] [0158.0.0.19] to [0160.0.0.19] for the disclosure of the
paragraphs [0158.0.0.19] to [0160.0.0.19] see paragraphs
[0158.0.0.0] to [0160.0.0.0] above.
[8602] [0161.0.19.19] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 243 to 250
and 603. The nucleic acid molecule is preferably at least 20, 30,
50, 100, 250 or more nucleotides in length.
[8603] [0162.0.0.19] for the disclosure of this paragraph see
paragraph [0162.0.0.0] above.
[8604] [0163.0.19.19] Preferably, a nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 243 to 250 and 603
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the increase of
the amount of the respective fine chemical in a organism or a part
thereof, e.g. a tissue, a cell, or a compartment of a cell, after
increasing the expression or activity thereof or the activity of a
protein of the invention or used in the process of the
invention.
[8605] [0164.0.0.19] for the disclosure of this paragraph see
paragraph [0164.0.0.0] above.
[8606] [0165.0.19.19] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 243 to 250 and 603,
resp.
[8607] [0166.0.0.19] and [0167.0.0.19] for the disclosure of the
paragraphs [0166.0.0.19] and [0167.0.0.19] see paragraphs
[0166.0.0.0] and [0167.0.0.0] above.
[8608] [0168.0.19.19] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 243 to 250 and 603, resp., yet retain said activity described
herein. The nucleic acid molecule can comprise a nucleotide
sequence encoding a polypeptide, wherein the polypeptide comprises
an amino acid sequence at least about 50% identical to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 243
to 250 and 603, resp., and is capable of participation in the
increase of production of the respective fine chemical after
increasing its activity, e.g. its expression. Preferably, the
protein encoded by the nucleic acid molecule is at least about 60%
identical to a sequence as indicated in Table II, columns 5 or 7,
lines 243 to 250 and 603, resp., more preferably at least about 70%
identical to one of the sequences as indicated in Table II, columns
5 or 7, lines 243 to 250 and 603, resp., even more preferably at
least about 80%, 90%, 95% homologous to a sequence as indicated in
Table II, columns 5 or 7, lines 243 to 250 and 603, resp., and most
preferably at least about 96%, 97%, 98%, or 99% identical to the
sequence as indicated in Table II, columns 5 or 7, lines 243 to 250
and 603.
[8609] Accordingly, the invention relates to nucleic acid molecules
encoding a polypeptide having above-mentioned activity, e.g.
conferring an increase in the the respective fine chemical in an
organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 243 to 250 and 603, preferably of Table II B, column 7, lines
243 to 250 and 603 yet retain said activity described herein. The
nucleic acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table II, columns 5 or 7, lines 243 to 250 and 603,
preferably of Table II B, column 7, lines 243 to 250 and 603 and is
capable of participation in the increase of production of the
respective fine chemical after increasing its activity, e.g. its
expression. Preferably, the protein encoded by the nucleic acid
molecule is at least about 60% identical to a sequence as indicated
in Table II, columns 5 or 7, lines 243 to 250 and 603, preferably
of Table II B, column 7, lines 243 to 250 and 603, more preferably
at least about 70% identical to one of the sequences as indicated
in Table II, columns 5 or 7, lines 243 to 250 and 603, preferably
of Table II B, column 7, lines 243 to 250 and 603, even more
preferably at least about 80%, 90%, or 95% homologous to a sequence
as indicated in Table II, columns 5 or 7, lines 243 to 250 and 603,
preferably of Table II B, column 7, lines 243 to 250 and 603, and
most preferably at least about 96%, 97%, 98%, or 99% identical to
the sequence as indicated in Table II, columns 5 or 7, lines 243 to
250 and 603, preferably of Table II B, column 7, lines 243 to 250
and 603.
[8610] [0169.0.0.19] to [0172.0.0.19] for the disclosure of the
paragraphs [0169.0.0.19] to [0172.0.0.19] see paragraphs
[0169.0.0.0] to [0172.0.0.0] above.
[8611] [0173.0.19.19] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 24071 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 24071 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[8612] [0174.0.0.19]: for the disclosure of this paragraph see
paragraph [0174.0.0.0] above.
[8613] [0175.0.19.19] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 24072 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 24072 by the above program algorithm with the
above parameter set, has a 80% homology.
[8614] [0176.0.19.19] Functional equivalents derived from one of
the polypeptides as indicated in Table II, columns 5 or 7, lines
243 to 250 and 603, resp., according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603, resp., according to the
invention and are distinguished by essentially the same properties
as a polypeptide as indicated in Table II, columns 5 or 7, lines
243 to 250 and 603, resp.
[8615] [0177.0.19.19] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 243 to
250 and 603, resp., according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603, resp., according to the
invention and encode polypeptides having essentially the same
properties as a polypeptide as indicated in Table II, columns 5 or
7, lines 243 to 250 and 603, resp.
[8616] [0178.0.0.19] for the disclosure of this paragraph see
[0178.0.0.0] above.
[8617] [0179.0.19.19] A nucleic acid molecule encoding a homologous
to a protein sequence as indicated in Table II, columns 5 or 7,
lines 243 to 250 and 603, resp., can be created by introducing one
or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table I, columns 5 or 7,
lines 243 to 250 and 603, resp., such that one or more amino acid
substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced into the encoding
sequences as indicated in Table I, columns 5 or 7, lines 243 to 250
and 603, resp., by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis.
[8618] [0180.0.0.19] to [0183.0.0.19] for the disclosure of the
paragraphs [0180.0.0.19] to [0183.0.0.19] see paragraphs
[0180.0.0.0] to [0183.0.0.0] above.
[8619] [0184.0.19.19] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table I, columns 5 or 7,
lines 243 to 250 and 603, resp., or of the nucleic acid sequences
derived from a sequences as indicated in Table II, columns 5 or 7,
lines 243 to 250 and 603, preferably of Table II B, column 7, lines
243 to 250 and 603, resp., comprise also allelic variants with at
least approximately 30%, 35%, 40% or 45% homology, by preference at
least approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
I, columns 5 or 7, lines 243 to 250 and 603, resp., or from the
derived nucleic acid sequences, the intention being, however, that
the enzyme activity or the biological activity of the resulting
proteins synthesized is advantageously retained or increased.
[8620] [0185.0.19.19] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table I, columns 5 or 7, lines 243 to 250 and 603, preferably of
Table I B, column 7, lines 243 to 250 and 603, resp. In one
embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotides not shown in any
one of sequences as indicated in Table I, columns 5 or 7, lines 243
to 250 and 603, preferably of Table I B, column 7, lines 243 to 250
and 603, resp. In one embodiment, the nucleic acid molecule
comprises less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or
40 further nucleotides. In a further embodiment, the nucleic acid
molecule comprises less than 30, 20 or 10 further nucleotides. In
one embodiment, a nucleic acid molecule used in the process of the
invention is identical to a sequence as indicated in Table I,
columns 5 or 7, lines 243 to 250 and 603, preferably of Table I B,
column 7, lines 243 to 250 and 603, resp.
[8621] [0186.0.19.19] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 243 to 250 and 603, preferably of Table II B, column
7, lines 243 to 250 and 603, resp. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table II, columns 5 or 7, lines 243 to 250 and 603, preferably
of Table II B, column 7, lines 243 to 250 and 603, resp.
[8622] [0187.0.19.19] In one embodiment, a nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II, columns 5 or 7,
lines 243 to 250 and 603, preferably of Table II B, column 7, lines
243 to 250 and 603, resp., comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a sequences as indicated in Table II, columns 5
or 7, lines 243 to 250 and 603, preferably of Table II B, column 7,
lines 243 to 250 and 603, resp.
[8623] [0188.0.19.19] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 243 to 250 and 603, resp., and is
expressed under identical conditions. In one embodiment, the
polypeptide of the invention is a homolog consisting or or
comprising the sequence as indicated in Table II B, column 7, lines
243 to 250 and 603,
[8624] [0189.0.19.19] Homologues of a sequences as indicated in
Table I, columns 5 or 7, lines 243 to 250 and 603, resp., or of a
derived sequences as indicated in Table II, columns 5 or 7, lines
243 to 250 and 603, resp., also mean truncated sequences, cDNA,
single-stranded DNA or RNA of the coding and noncoding DNA
sequence. Homologues of said sequences are also understood as
meaning derivatives, which comprise noncoding regions such as, for
example, UTRs, terminators, enhancers or promoter variants. The
promoters upstream of the nucleotide sequences stated can be
modified by one or more nucleotide substitution(s), insertion(s)
and/or deletion(s) without, however, interfering with the
functionality or activity either of the promoters, the open reading
frame (=ORF) or with the 3'-regulatory region such as terminators
or other 3' regulatory regions, which are far away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[8625] [0190.0.0.19], [0191.0.0.19], [00191.1.0.19] and
[0192.0.0.19] to [0203.0.0.19] for the disclosure of the paragraphs
[0190.0.0.19], [0191.0.0.19], [0191.1.0.19] and [0192.0.0.19] to
[0203.0.0.19] see paragraphs [0190.0.0.0], [0191.0.0.0],
[0191.1.0.0] and [0192.0.0.0] to [0203.0.0.0] above.
[8626] [0204.0.19.19] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [8627] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, columns 5 or 7, lines 243 to
250 and 603, preferably of Table II B, column 7, lines 243 to 250
and 603, resp.; or a fragment thereof conferring an increase in the
amount of the respective fine chemical, i.e. ferulic acid (lines
243, 244, 246, 247, 249) or sinapic acid (lines 245, 248, 250,
603), resp., in an organism or a part thereof [8628] b) nucleic
acid molecule comprising, preferably at least the mature form, of a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 243 to 250 and 603, preferably of Table I B, column 7, lines
243 to 250 and 603, resp., or a fragment thereof conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [8629] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [8630]
d) nucleic acid molecule encoding a polypeptide whose sequence has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [8631] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [8632] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [8633] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [8634]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
primers or primer pairs as indicated in Table III, column 7, lines
243 to 250 and 603 and conferring an increase in the amount of the
respective fine chemical, i.e. ferulic acid (lines 243, 244, 246,
247, 249) or sinapic acid (lines 245, 248, 250, 603), resp., in an
organism or a part thereof; [8635] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from a expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [8636] j) nucleic acid molecule which encodes a
polypeptide comprising a consensus sequence as indicated in Table
IV, column 7, lines 243 to 248, 250 and 603 and conferring an
increase in the amount of the respective fine chemical, i.e.
ferulic acid (lines 243, 244, 246, 247, 249) or sinapic acid (lines
245, 248, 250, 603), resp., in an organism or a part thereof;
[8637] k) nucleic acid molecule encoding the amino acid sequence of
a polypeptide encoding a domaine of a polypeptide as indicated in
Table II, columns 5 or 7, lines 243 to 250 and 603, preferably of
Table II B, column 7, lines 243 to 250 and 603, resp., and
conferring an increase in the amount of the respective fine
chemical, i.e. ferulic acid (lines 243, 244, 246, 247, 249) or
sinapic acid (lines 245, 248, 250, 603), resp., in an organism or a
part thereof; and [8638] l) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 243 to 250 and 603, resp., or a nucleic acid
molecule encoding, preferably at least the mature form of, a
polypeptide as indicated in Table II, columns 5 or 7, lines 243 to
250 and 603, resp., and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
encompasses a sequence which is complementary thereto; whereby,
preferably, the nucleic acid molecule according to (a) to (l)
distinguishes over a sequence as indicated in Table IA or IB,
columns 5 or 7, lines 243 to 250 and 603, resp., by one or more
nucleotides. In one embodiment, the nucleic acid molecule of the
invention does not consist of the sequence as indicated in Table IA
or IB, columns 5 or 7, lines 243 to 250 and 603, resp. In an other
embodiment, the nucleic acid molecule of the present invention is
at least 30% identical and less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence as indicated in Table IA or IB,
columns 5 or 7, lines 243 to 250 and 603, resp. In a further
embodiment the nucleic acid molecule does not encode a polypeptide
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
243 to 250 and 603, resp. Accordingly, in one embodiment, the
nucleic acid molecule of the present invention encodes in one
embodiment a polypeptide which differs at least in one or more
amino acids from a polypeptide indicated in Table IIA or IIB,
columns 5 or 7, lines 243 to 250 and 603 does not encode a protein
of a sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 243 to 250 and 603. Accordingly, in one embodiment, the
protein encoded by a sequences of a nucleic acid according to (a)
to (l) does not consist of a sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 243 to 250 and 603. In a further
embodiment, the protein of the present invention is at least 30%
identical to a protein sequence indicated in Table IIA or IIB,
columns 5 or 7, lines 243 to 250 and 603 and less than 100%,
preferably less than 99.999%, 99.99% or 99.9%, more preferably less
than 99%, 98%, 97%, 96% or 95% identical to a sequence as indicated
in Table IIA or IIB, columns 5 or 7, lines 243 to 250 and 603.
[8639] [0205.0.0.19] and [0206.0.0.19] for the disclosure of the
paragraphs [0205.0.0.19] and [0206.0.0.19] see paragraphs
[0205.0.0.0] and [0206.0.0.0] above.
[8640] [0207.0.19.19] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the glutamic acid metabolism, the
phosphoenolpyruvate metabolism, the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[8641] [0208.0.0.19] to [0226.0.0.19] for the disclosure of the
paragraphs [0208.0.0.19] to [0226.0.0.19] see paragraphs
[0208.0.0.0] to [0226.0.0.0] above.
[8642] [0227.0.19.19] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[8643] In addition to a sequence indicated in Table I, columns 5 or
7, lines 243 to 250 and 603 or its derivatives, it is advantageous
to express and/or mutate further genes in the organisms. Especially
advantageously, additionally at least one further gene of the
glutamic acid or phosphoenolpyruvate metabolic pathway, is
expressed in the organisms such as plants or microorganisms. It is
also possible that the regulation of the natural genes has been
modified advantageously so that the gene and/or its gene product is
no longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the fine
chemicals desired since, for example, feedback regulations no
longer exist to the same extent or not at all. In addition it might
be advantageously to combine one or more of the sequences indicated
in Table I, columns 5 or 7, lines 243 to 250 and 603, resp., with
genes which generally support or enhances to growth or yield of the
target organisms, for example genes which lead to faster growth
rate of microorganisms or genes which produces stress-, pathogen,
or herbicide resistant plants.
[8644] [0228.0.19.19] %
[8645] [0229.0.19.19] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences indicated
in Table I, columns 5 or 7, lines 243 to 250 and 603 used in the
process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the aromatic amino acid
pathway, such as tryptophan, phenylalanine or tyrosine. These genes
can lead to an increased synthesis of the essential amino acids
tryptophan, phenylalanine or tyrosine.
[8646] [0230.0.0.19] for the disclosure of this paragraph see
paragraph [0230.0.0.0] above.
[8647] [0231.0.19.19] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a ferulic acid or sinapic acid
degrading protein is attenuated, in particular by reducing the rate
of expression of the corresponding gene. A person skilled in the
art knows for example, that the inhibition or repression of a
ferulic acid or sinapic acid degrading enzyme will result in an
increased ferulic acid and/or sinapic acid accumulation in the
plant.
[8648] [0232.0.0.19] to [0276.0.0.19] for the disclosure of the
paragraphs [0232.0.0.19] to [0276.0.0.19] see paragraphs
[0232.0.0.0] to [0276.0.0.0] above.
[8649] [0277.0.19.19] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously.
[8650] [0278.0.0.19] to [0282.0.0.19] for the disclosure of the
paragraphs [0278.0.0.19] to [0282.0.0.19] see paragraphs
[0278.0.0.0] to [0282.0.0.0] above.
[8651] [0283.0.19.19] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells, for example using the antibody
of the present invention as described below, e.g. an antibody
against a protein as indicated in Table II, column 3, lines 243 to
250 and 603, resp., or an antibody against a polypeptide as
indicated in Table II, columns 5 or 7, lines 243 to 250 and 603,
resp., which can be produced by standard techniques utilizing the
polypeptid of the present invention or fragment thereof. Preferred
are monoclonal antibodies.
[8652] [0284.0.0.19] for the disclosure of this paragraph see
[0284.0.0.0] above.
[8653] [0285.0.19.19] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 243 to 250 and 603, resp., or as coded by
a nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 243 to 250 and 603, resp., or functional homologues
thereof.
[8654] [0286.0.19.19] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 243 to 248, 250 and 603 and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table IV, column 7, lines 243 to 248, 250 and 603 whereby 20 or
less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred
5 or 4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid.
[8655] [0287.0.0.19] to [0290.0.0.19] for the disclosure of the
paragraphs [0287.0.0.19] to [0290.0.0.19] see paragraphs
[0287.0.0.0] to [0290.0.0.0] above.
[8656] [0291.0.19.19] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[8657] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 243 to 250 and 603, resp., by one or more
amino acids. In one embodiment, polypeptide distinguishes from a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
243 to 250 and 603, resp., by more than 5, 6, 7, 8 or 9 amino
acids, preferably by more than 10, 15, 20, 25 or 30 amino acids,
even more preferred are more than 40, 50, or 60 amino acids and,
preferably, the sequence of the polypeptide of the invention
distinguishes from a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 243 to 250 and 603, resp., by not more than
80% or 70% of the amino acids, preferably not more than 60% or 50%,
more preferred not more than 40% or 30%, even more preferred not
more than 20% or 10%. In an other embodiment, said polypeptide of
the invention does not consist of a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 243 to 250 and 603.
[8658] [0292.0.0.19] for the disclosure of this paragraph see
[0292.0.0.0] above.
[8659] [0293.0.19.19] In one embodiment, the invention relates to a
polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or by a nucleic acid molecule used in the process of the
invention. In one embodiment, the polypeptide of the invention has
a sequence which distinguishes from a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 243 to 250 and 603, resp.,
by one or more amino acids. In an other embodiment, said
polypeptide of the invention does not consist of the sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 243 to 250 and
603, resp. In a further embodiment, said polypeptide of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by a nucleic acid molecules as indicated in
Table IA or IB, columns 5 or 7, lines 243 to 250 and 603, resp.
[8660] [0294.0.19.19] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table IIA or IIB, column 3, lines 243 to 250 and 603,
resp., which distinguishes over a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 243 to 250 and 603, resp., by one
or more amino acids, preferably by more than 5, 6, 7, 8 or 9 amino
acids, preferably by more than 10, 15, 20, 25 or 30 amino acids,
even more preferred are more than 40, 50, or 60 amino acids but
even more preferred by less than 70% of the amino acids, more
preferred by less than 50%, even more preferred my less than 30% or
25%, more preferred are 20% or 15%, even more preferred are less
than 10%.
[8661] [0295.0.0.19] to [0297.0.0.19] for the disclosure of the
paragraphs [0295.0.0.19] to [0297.0.0.19] see paragraphs
[0295.0.0.0] to [0297.0.0.0] above.
[8662] [00297.1.0.19] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity and/or the amino acid
sequence of a polypeptide indicated in Table II, columns 3, 5 or 7,
lines 243 to 250 and 603.
[8663] [0298.0.19.19] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table II, columns 5 or 7, lines 243 to 250 and 603,
resp. The portion of the protein is preferably a biologically
active portion as described herein. Preferably, the polypeptide
used in the process of the invention has an amino acid sequence
identical to a sequence as indicated in Table II, columns 5 or 7,
lines 243 to 250 and 603, resp.
[8664] [0299.0.19.19] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
243 to 250 and 603, resp. The preferred polypeptide of the present
invention preferably possesses at least one of the activities
according to the invention and described herein. A preferred
polypeptide of the present invention includes an amino acid
sequence encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions, to a nucleotide
sequence as indicated in Table I, columns 5 or 7, lines 243 to 250
and 603, resp., or which is homologous thereto, as defined
above.
[8665] [0300.0.19.19] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603, resp., in amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of as indicated in Table IIA or IIB,
columns 5 or 7, lines 243 to 250 and 603, resp.
[8666] [0301.0.0.19] for the disclosure of this paragraph see
[0301.0.0.0] above.
[8667] [0302.0.19.19] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table II, columns 5 or 7, lines 243 to 250 and 603, resp., or the
amino acid sequence of a protein homologous thereto, which include
fewer amino acids than a full length polypeptide of the present
invention or used in the process of the present invention or the
full length protein which is homologous to an polypeptide of the
present invention or used in the process of the present invention
depicted herein, and exhibit at least one activity of polypeptide
of the present invention or used in the process of the present
invention.
[8668] [0303.0.0.19] for the disclosure of this paragraph see
[0303.0.0.0] above.
[8669] [0304.0.19.19] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 243 to 250 and 603 but having differences in
the sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[8670] [0305.0.0.19] to [0308.0.0.19] for the disclosure of the
paragraphs [0305.0.0.19] to [0308.0.0.19] see paragraphs
[0305.0.0.0 to [0308.0.0.0] above.
[8671] [0309.0.19.19] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603, resp., refers to a
polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas an "other polypeptide" not being indicated in
Table II, columns 5 or 7, lines 243 to 250 and 603, resp., refers
to a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous to a polypeptide of
the invention, preferably which is not substantially homologous to
a polypeptide as indicated in Table II, columns 5 or 7, lines 243
to 250 and 603, resp., e.g., a protein which does not confer the
activity described herein or annotated or known for as indicated in
Table II, column 3, lines 243 to 250 and 603, resp., and which is
derived from the same or a different organism. In one embodiment,
an "other polypeptide" not being indicated in Table II, columns 5
or 7, lines 243 to 250 and 603, resp., does not confer an increase
of the respective fine chemical in an organism or part thereof.
[8672] [0310.0.0.19] to [0334.0.0.19] for the disclosure of the
paragraphs [0310.0.0.19] to [0334.0.0.19] see paragraphs
[0310.0.0.0] to [0334.0.0.0] above.
[8673] [0335.0.19.19] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in
[8674] Table I, columns 5 or 7, lines 243 to 250 and 603, resp.,
and/or homologs thereof. As described inter alia in WO 99/32619,
dsRNAi approaches are clearly superior to traditional antisense
approaches. The invention therefore furthermore relates to
double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived there from), bring about
altered metabolic activity by the reduction in the expression of a
nucleic acid sequences as indicated in Table I, columns 5 or 7,
lines 243 to 250 and 603, resp., and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of
aprotein encoded by a nucleic acid sequence as indicated in Table
I, columns 5 or 7, lines 243 to 250 and 603, resp., and/or homologs
thereof, one of the two RNA strands is essentially identical to at
least part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[8675] [0336.0.0.19] to [0342.0.0.19] for the disclosure of the
paragraphs [0336.0.0.19] to [0342.0.0.19] see paragraphs
[0336.0.0.0] to [0342.0.0.0] above.
[8676] [0343.0.19.19] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 243 to 250
and 603, resp., or its homolog is not necessarily required in order
to bring about effective reduction in the expression. The advantage
is, accordingly, that the method is tolerant with regard to
sequence deviations as may be present as a consequence of genetic
mutations, polymorphisms or evolutionary divergences. Thus, for
example, using the dsRNA, which has been generated starting from a
sequence as indicated in Table I, columns 5 or 7, lines 243 to 250
and 603, resp., or homologs thereof of the one organism, may be
used to suppress the corresponding expression in another
organism.
[8677] [0344.0.0.19] to [0361.0.0.19] for the disclosure of the
paragraphs [0344.0.0.19] to [0361.0.0.19] see paragraphs
[0344.0.0.0] to [0361.0.0.0] above.
[8678] [0362.0.19.19] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, columns 5 or 7, lines 243 to 250 and 603, resp., e.g.
encoding a polypeptide having protein activity, as indicated in
Table II, columns 3, lines 243 to 250 and 603, resp., Due to the
abovementioned activity the respective fine chemical content in a
cell or an organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. Transgenic for a polypeptide having an activity of a
polypeptide as indicated in Table II, columns 5 or 7, lines 243 to
250 and 603, resp., means herein that due to modulation or
manipulation of the genome, an activity as annotated for a
polypeptide as indicated in Table II, column 3, lines 243 to 250
and 603, e.g. having a sequence as indicated in Table II, columns 5
or 7, lines 243 to 250 and 603, resp., is increased in a cell or an
organism or a part thereof. Examples are described above in context
with the process of the invention.
[8679] [0363.0.0.19] for the disclosure of this paragraph see
[0363.0.0.0] above.
[8680] [0364.0.19.19] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table II, column 3, lines 243 to 250 and 603, resp.
with the corresponding protein-encoding sequence as indicated in
Table I, column 5, lines 243 to 250 and 603, resp., becomes a
transgenic expression cassette when it is modified by non-natural,
synthetic "artificial" methods such as, for example,
mutagenization. Such methods have been described (U.S. Pat. No.
5,565,350; WO 00/15815; also see above).
[8681] [0365.0.0.19] to [0373.0.0.19] for the disclosure of the
paragraphs [0365.0.0.19], to [0373.0.0.19] see paragraphs
[0365.0.0.0] to [0373.0.0.0] above.
[8682] [0374.0.19.19] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. ferulic acid or sinapic acid,
in particular the respective fine chemical, produced in the process
according to the invention may, however, also be isolated from the
plant in the form of their free ferulic acid or sinapic acid, in
particular the free respective fine chemical, or bound in or to
compounds or moieties, like glucosides, e.g. diglucosides. The
respective fine chemical produced by this process can be harvested
by harvesting the organisms either from the culture in which they
grow or from the field. This can be done via expressing, grinding
and/or extraction, salt precipitation and/or ion-exchange
chromatography or other chromatographic methods of the plant parts,
preferably the plant seeds, plant fruits, plant tubers and the
like.
[8683] [0375.0.0.19] and [0376.0.0.19] for the disclosure of the
paragraphs [0375.0.0.19] and [0376.0.0.19] see paragraphs
[0375.0.0.0] and [0376.0.0.0] above.
[8684] [0377.0.19.19] Accordingly, the present invention relates
also to a process whereby the produced ferulic acid or sinapic acid
is isolated.
[8685] [0378.0.19.19] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the ferulic acid
or sinapic acid produced in the process can be isolated. The
resulting ferulic acid or sinapic acid can, if appropriate,
subsequently be further purified, if desired mixed with other
active ingredients such as vitamins, amino acids, carbohydrates,
antibiotics and the like, and, if appropriate, formulated.
[8686] [0379.0.19.19] In one embodiment, ferulic acid and sinapic
are a mixture of the respective fine chemicals.
[8687] [0380.0.19.19] The ferulic acid or sinapic acid obtained in
the process are suitable as starting material for the synthesis of
further products of value. For example, they can be used in
combination with each other or alone for the production of
pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the ferulic acid or sinapic
acid composition produced or the respective fine chemical produced
if desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the ferulic acid or sinapic acid
produced in the process or of the transgenic organisms in animal
feeds, foodstuffs, medicines, food supplements, cosmetics or
pharmaceuticals or for the production of ferulic acid or sinapic
acid e.g. after isolation of the respective fine chemical or
without, e.g. in situ, e.g in the organism used for the process for
the production of the respective fine chemical.
[8688] [0381.0.0.19] to [0384.0.0.19] for the disclosure of the
paragraphs [0381.0.0.19], to [0384.0.0.19] see paragraphs
[0381.0.0.0] to [0384.0.0.0] above.
[8689] [0385.0.19.19] The fermentation broths obtained in this way,
containing in particular ferulic acid or sinapic acid in mixtures
with other organic acids, aminoacids, polypeptides or
polysaccarides, normally have a dry matter content of from 1 to 70%
by weight, preferably 7.5 to 25% by weight. Sugar-limited
fermentation is additionally advantageous, e.g. at the end, for
example over at least 30% of the fermentation time. This means that
the concentration of utilizable sugar in the fermentation medium is
kept at, or reduced to, 0 to 10 g/l, preferably to 0 to 3 g/I
during this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed or isolated
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation, coagulation/flocculation
or a combination of these methods, from the fermentation broth or
left completely in it.
[8690] The fermentation broth can then be thickened or concentrated
by known methods, such as, for example, with the aid of a rotary
evaporator, thin-film evaporator, falling film evaporator, by
reverse osmosis or by nanofiltration. This concentrated
fermentation broth can then be worked up by freeze-drying, spray
drying, spray granulation or by other processes.
[8691] [0386.0.19.19] Accordingly, it is possible to purify the
ferulic acid or sinapic acid produced according to the invention
further. For this purpose, the product-containing composition is
subjected for example to separation via e.g. an open column
chromatography or HPLC in which case the desired product or the
impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use.
[8692] [0387.0.0.19] to [0392.0.0.19] for the disclosure of the
paragraphs [0387.0.0.19] to [0392.0.0.19] see paragraphs
[0387.0.0.0] to [0392.0.0.0] above.
[8693] [0393.0.19.19] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps:
[8694] (c) contacting e.g. hybridising, the nucleic acid molecules
of a sample, e.g. cells, tissues, plants or microorganisms or a
nucleic acid library, which can contain a candidate gene encoding a
gene product conferring an increase in the respective fine chemical
after expression, with the nucleic acid molecule of the present
invention; [8695] b. identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table I, columns 5 or 7, lines
243 to 250 and 603, preferably in Table IB, columns 5 or 7, lines
243 to 250 and 603 resp., and, optionally, isolating the full
length cDNA clone or complete genomic clone; [8696] c. introducing
the candidate nucleic acid molecules in host cells, preferably in a
plant cell or a microorganism, appropriate for producing the
respective fine chemical; [8697] d. expressing the identified
nucleic acid molecules in the host cells; [8698] e. assaying the
respective fine chemical level in the host cells; and [8699] f.
identifying the nucleic acid molecule and its gene product which
expression confers an increase in the respective fine chemical
level in the host cell after expression compared to the wild
type.
[8700] [0394.0.0.19] to [0398.0.0.19] for the disclosure of the
paragraphs [0394.0.0.19] to [0398.0.0.19] see paragraphs
[0394.0.0.0] to [0398.0.0.0] above.
[8701] [0399.0.19.19] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increase of the respective fine chemical production in a
plant or microorganism, comprising the steps:
(o) culturing a cell or tissue or microorganism or maintaining a
plant expressing the polypeptide according to the invention or a
nucleic acid molecule encoding said polypeptide and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and the polypeptide of the present invention or used
in the process of the invention; and (p) identifying if the
compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout
system.
[8702] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in respective fine chemical
production.
[8703] [00399.1.19.19] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603 or a homolog thereof, e.g.
comparing the phenotype of nearly identical organisms with low and
high activity of a protein as indicated in Table II, columns 5 or
7, lines 243 to 250 and 603 after incubation with the drug.
[8704] [0400.0.0.19] to [0416.0.0.19] for the disclosure of the
paragraphs [0400.0.0.19] to [0416.0.0.19] see paragraphs
[0400.0.0.0] to [0416.0.0.0] above.
[8705] [0417.0.19.19] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the ferulic acid or sinapic acid
biosynthesis pathways. In particular, the overexpression of the
polypeptide of the present invention may protect an organism such
as a microorganism or a plant against inhibitors, which block the
ferulic acid or sinapic acid synthesis.
[8706] [0418.0.0.19] to [0423.0.0.19] for the disclosure of the
paragraphs [0418.0.0.19] to [0423.0.0.19] see paragraphs
[0418.0.0.0] to [0423.0.0.0] above.
[8707] [0424.0.19.19] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other organic acids.
Accordingly, the nucleic acid of the invention, or the nucleic acid
molecule identified with the method of the present invention or the
complement sequences thereof, the polypeptide of the invention, the
nucleic acid construct of the invention, the organisms, the host
cell, the microorganisms, the plant, plant tissue, plant cell, or
the part thereof of the invention, the vector of the invention, the
antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the respective
fine chemical in a organism or part thereof, e.g. in a cell.
[8708] [0425.0.0.19] to [0434.0.0.19] for the disclosure of the
paragraphs [0425.0.0.19] to [0434.0.0.19] see paragraphs
[0425.0.0.0] to [0434.0.0.0] above.
[0435.0.19.19] Example 3
In-Vivo and In-Vitro Mutagenesis
[8709] [0436.0.19.19] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of ferulic acid or sinapic
acid can be carried out by passing a plasmid DNA (or another vector
DNA) containing the desired nucleic acid sequence or nucleic acid
sequences, e.g. the nucleic acid molecule of the invention or the
vector of the invention, through E. coli and other microorganisms
(for example Bacillus spp. or yeasts such as Saccharomyces
cerevisiae) which are not capable of maintaining the integrity of
its genetic information. Usual mutator strains have mutations in
the genes for the DNA repair system [for example mutHLS, mutD, mutT
and the like; for comparison, see Rupp, W. D. (1996) DNA repair
mechanisms in Escherichia coli and Salmonella, pp. 2277-2294, ASM:
Washington]. The skilled worker knows these strains. The use of
these strains is illustrated for example in Greener, A. and
Callahan, M. (1994) Strategies 7; 32-34.
[8710] In-vitro mutation methods such as increasing the spontaneous
mutation rates by chemical or physical treatment are well known to
the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[8711] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[8712] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.19.19] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces Cerevisiae and
Mortierella alpina
[8713] [0438.0.19.19] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology", VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[8714] [0439.0.19.19] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255f) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[8715] [0440.0.19.19] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002,) teaches a homolog recombination system dependent
on rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this
system for homologous recombination are vectors derived from the
Ylp series. Plasmid vectors derived for example from the
2.mu.-Vector are known by the skilled worker and used for the
expression in yeasts. Other preferred vectors are for example
pART1, pCHY21 or pEVP11 as they have been described by McLeod et
al. (EMBO J. 1987, 6:729-736) and Hoffman et al. (Genes Dev. 5,
1991: :561-571.) or Russell et al. (J. Biol. Chem. 258, 1983:
143-149.). Other beneficial yeast vectors are plasmids of the REP,
REP-X, pYZ or RIP series.
[8716] [0441.0.0.19] to [0443.0.0.19] for the disclosure of the
paragraphs [0441.0.0.19] to [0443.0.0.19] see paragraphs
[0441.0.0.0] to [0443.0.0.0] above.
[0444.0.19.19] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[8717] [0445.0.19.19] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad.
[8718] Sin. (2000) 41:41-48]. Mortierella can be grown at
20.degree. C. in a culture medium containing: 10 g/l glucose, 5 g/l
yeast extract at pH 6.5. Furthermore Jang et al. teaches a
submerged basal medium containing 20 g/l soluble starch, 5 g/l
Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l KH.sub.2PO.sub.4, and
0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[8719] [0446.0.0.19] to [0450.0.0.19] for the disclosure of the
paragraphs [0446.0.0.19] to [0450.0.0.19] see paragraphs
[0446.0.0.0] to [0450.0.0.0] above.
[8720] [0451.0.5.19], [0452.0.0.19] and [0453.0.0.19] for the
disclosure of the paragraphs [0451.0.5.19], [0452.0.0.19] and
[0453.0.0.19] see [0451.0.5.5], [0452.0.0.0] and [0453.0.0.0]
above.
[8721] [0454.0.19.19] Analysis of the effect of the nucleic acid
molecule on the production of ferulic acid or sinapic acid
[8722] [0455.0.19.19] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a ferulic acid or sinapic acid) can be determined
by growing the modified microorganisms or the modified plant under
suitable conditions (such as those described above) and analyzing
the medium and/or the cellular components for the elevated
production of desired product (i.e. of ferulic acid or sinapic
acid). These analytical techniques are known to the skilled worker
and comprise spectroscopy, thin-layer chromatography, various types
of staining methods, enzymatic and microbiological methods and
analytical chromatography such as high-performance liquid
chromatography (see, for example, Ullman, Encyclopedia of
Industrial Chemistry, Vol. A2, p. 89-90 and p. 443-613, VCH:
Weinheim (1985); Fallon, A., et al., (1987) "Applications of HPLC
in Biochemistry" in: Laboratory Techniques in Biochemistry and
Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol.
3, Chapter III: "Product recovery and purification", p. 469-714,
VCH: Weinheim; Better, P. A., et al. (1988) Bioseparations:
downstream processing for Biotechnology, John Wiley and Sons;
Kennedy, J. F., and Cabral, J. M. S. (1992) Recovery processes for
biological Materials, John Wiley and Sons; Shaeiwitz, J. A., and
Henry, J. D. (1988) Biochemical Separations, in: Ullmann's
Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, p. 1-27,
VCH: Weinheim; and Dechow, F. J. (1989) Separation and purification
techniques in biotechnology, Noyes Publications).
[8723] [0456.0.0.19] for the disclosure of this paragraph see
[0456.0.0.0] above.
[0457.0.19.19] Example 9
Purification of Ferulic Acid or Sinapic Acid
[8724] [0458.0.19.19] Abbreviations; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[8725] The unambiguous detection for the presence of ferulic acid
or sinapic acid can be obtained by analyzing recombinant organisms
using analytical standard methods: LC, LC-MSMS or TLC, as
described. The total amount produced in the organism for example in
yeasts used in the inventive process can be analysed for example
according to the following procedure:
[8726] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods.
[8727] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[8728] A typical sample pretreatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography.
[8729] For analysis, solvent delivery and aliquot removal can be
accomplished with a robotic system comprising a single injector
valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000
W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of
50% potassium hydroxide hydro-ethanolic solution (4 water--1
ethanol) can be added to each vial, followed by the addition of 3
ml of octanol. The saponification treatment can be conducted at
room temperature with vials maintained on an IKA HS 501 horizontal
shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours
at 250 movements/minute, followed by a stationary phase of
approximately one hour.
[8730] Following saponification, the supernatant can be diluted
with 0.17 ml of methanol. The addition of methanol can be conducted
under pressure to ensure sample homogeneity. Using a 0.25 ml
syringe, a 0.1 ml aliquot can be removed and transferred to HPLC
vials for analysis.
[8731] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete
with a quaternary pump, vacuum degassing system, six-way injection
valve, temperature regulated autosampler, column oven and
Photodiode Array detector can be used [Agilent Technologies
available through Ultra Scientific Inc., 250 Smith Street, North
Kingstown, R.I.]. The column can be a Waters YMC30, 5-micron,
4.6.times.250 mm with a guard column of the same material [Waters,
34 Maple Street, Milford, Mass.]. The solvents for the mobile phase
can be 81 methanol: 4 water: 15 tetrahydrofuran (THF) stabilized
with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injections were
20 l. Separation can be isocratic at 30.degree. C. with a flow rate
of 1.7 ml/minute. The peak responses can be measured by absorbance
at 447 nm.
[8732] [0459.0.19.19] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the ferulic acid or sinapic acid can be further
purified with a so-called RTHPLC. As eluent acetonitrile/water or
chloroform/acetonitrile mixtures can be used. If necessary, these
chromatography steps may be repeated, using identical or other
chromatography resins. The skilled worker is familiar with the
selection of suitable chromatography resin and the most effective
use for a particular molecule to be purified.
[8733] [0460.0.0.19] for the disclosure of this paragraph see
[0460.0.0.0] above.
[0461.0.19.19] Example 10
Cloning SEQ ID NO: 24071, 24083, 24177, 24353, 24865, 25117, 25357,
25361 or 92604 for the Expression in Plants
[8734] [0462.0.0.19] for the disclosure of this paragraph see
[0462.0.0.0] above.
[8735] [0463.0.19.19] SEQ ID NO: 24071, 24083, 24177, 24353, 24865,
25117, 25357, 25361 or 92604 is amplified by PCR as described in
the protocol of the Pfu Turbo or DNA Herculase polymerase
(Stratagene).
[8736] [0464.0.0.19] to [0466.0.0.19] for the disclosure of the
paragraphs [0464.0.0.19] to [0466.0.0.19] see paragraphs
[0464.0.0.0] to [0466.0.0.0] above.
[8737] [0466.1.0.19] In case the Herculase enzyme can be used for
the amplification, the PCR amplification cycles were as follows: 1
cycle of 2-3 minutes at 94.degree. C., followed by 25-30 cycles of
in each case 30 seconds at 94.degree. C., 30 seconds at
55-60.degree. C. and 5-10 minutes at 72.degree. C., followed by 1
cycle of 10 minutes at 72.degree. C., then 4.degree. C.
[8738] [0467.0.19.19] The following primer sequences were selected
for the gene SEQ ID NO: 24071:
TABLE-US-00088 i) forward primer (SEQ ID NO: 24081) atgcgtgctt
taccgatctg ttta ii) reverse primer (SEQ ID NO: 24082) ttatttcgcc
gtaatgttaa gcgcag
[8739] The following primer sequences were selected for the gene
SEQ ID NO: 24083:
TABLE-US-00089 i) forward primer (SEQ ID NO: 24175) atgggacaca
agcccttata ccg ii) reverse primer (SEQ ID NO: 24176) ttatcgcgat
gattttcgct gcg
[8740] The following primer sequences were selected for the gene
SEQ ID NO: 24177:
TABLE-US-00090 i) forward primer (SEQ ID NO: 24351) atgagtcgtt
tagtcgtagt atcta ii) reverse primer (SEQ ID NO: 24152) ttacgcaagc
tttggaaagg tagc
[8741] The following primer sequences were selected for the gene
SEQ ID NO: 24353:
TABLE-US-00091 i) forward primer (SEQ ID NO: 24863) atgagtaaga
tttttgaaga taac ii) reverse primer (SEQ ID NO: 24864) ttactgttgc
aattctttct cagtg
[8742] The following primer sequences were selected for the gene
SEQ ID NO: 24865:
TABLE-US-00092 i) forward primer (SEQ ID NO: 25115) atggaaaccg
tggcttacgc tg ii) reverse primer (SEQ ID NO: 24116) ttatacgacg
cgtacgcccg c
[8743] The following primer sequences were selected for the gene
SEQ ID NO: 25117:
TABLE-US-00093 i) forward primer (SEQ ID NO: 25355) atgacgacga
ttctcaagca tctc ii) reverse primer (SEQ ID NO: 25356) ttactggcct
ttgttttcca gattc
[8744] The following primer sequences were selected for the gene
SEQ ID NO: 25357:
TABLE-US-00094 i) forward primer (SEQ ID NO: 25359) atgtaccaaa
ataatgtatt gaatgct ii) reverse primer (SEQ ID NO: 25360) tcaatagtgc
attaactctc ccatt
[8745] The following primer sequences were selected for the gene
SEQ ID NO: 25361:
TABLE-US-00095 i) forward primer (SEQ ID NO: 25495) atggagacca
atttttcctt cgact ii) reverse primer (SEQ ID NO: 25496) ctattgaaat
accggcttca atattt
[8746] The following primer sequences were selected for the gene
SEQ ID NO: 92604:
TABLE-US-00096 i) forward primer (SEQ ID NO: 92658) atggtaaagg
aacgtaaaac cgagt ii) reverse primer (SEQ ID NO: 92659) ttaccctaaa
tccgccatca acac
[8747] [0468.0.0.19] to [0470.0.0.19] for the disclosure of the
paragraphs [0468.0.0.19] to [0470.0.0.19] see paragraphs
[0468.0.0.0] to [0470.0.0.0] above.
[8748] [0470.1.19.19] The PCR-products, produced by Pfu Turbo DNA
polymerase, were phosphorylated using a T4 DNA polymerase using a
standard protocol (e.g. MBI Fermentas) and cloned into the
processed binary vector.
[8749] [0471.0.0.19] for the disclosure of this paragraph see
[0471.0.0.0] above.
[8750] [0471.1.19.19] The DNA termini of the PCR-products, produced
by Herculase DNA polymerase, were blunted in a second synthesis
reaction using Pfu Turbo DNA polymerase. The composition for the
protocol of the blunting the DNA-termini was as follows: 0.2 mM
blunting dTTP and 1.25 u Pfu Turbo DNA polymerase. The reaction was
incubated at 72.degree. C. for 30 minutes. Then the PCR-products
were phosphorylated using a T4 DNA polymerase using a standard
protocol (e.g. MBI Fermentas) and cloned into the processed vector
as well.
[8751] [0472.0.0.19] to [0479.0.0.19] for the disclosure of the
paragraphs [0472.0.0.19] to [0479.0.0.19] see paragraphs
[0472.0.0.0] to [0479.0.0.0] above.
[0480.0.19.19] Example 11
Generation of Transgenic Plants which Express SEQ ID NO:24071,
24083, 24177, 24353, 24865, 25117, 25357, 25361 or 92604
[8752] [0481.0.0.19] to [0513.0.0.19] for the disclosure of the
paragraphs [0481.0.0.19] to [0513.0.0.19] see paragraphs
[0481.0.0.0] to [0513.0.0.0] above.
[8753] [0514.0.19.19] As an alternative, ferulic acid acid can be
detected as described in Mattila, P. and Kumpulainen J., J. Agric
Food Chem. 2002 Jun. 19; 50(13):3660-7.
[8754] As an alternative, sinapic acid can be detected as described
in Noda, M. and Matsumoto, M., Biochim Biophys Acta. 1971 Feb. 2;
231(1):131-3.
[8755] The results of the different plant analyses can be seen from
the table 1 which follows:
TABLE-US-00097 TABLE 1 ORF Metabolite Method Min Max b0196 Ferulic
acid LC 1.10 1.25 b0730 Ferulic acid LC 1.38 1.97 b1896 Sinapic
Acid GC 1.38 1.98 b2414 Ferulic acid LC 1.34 1.86 b3074 Ferulic
acid LC 1.35 1.73 b3172 Sinapic Acid GC 1.31 1.89 YBR184W Ferulic
acid LC 1.30 1.37 YDR513W Sinapic Acid GC 1.30 1.39 b2818 Sinapic
Acid GC 1.27 1.54
[8756] Column 2 shows the metabolite ferulic acid or sinapic acid
analyzed. Columns 4 and 5 shows the ratio of the analyzed
metabolite between the transgenic plants and the wild type;
Increase of the metabolite: Max: maximal x-fold (normalised to wild
type)-Min: minimal x-fold (normalised to wild type). Decrease of
the metabolite: Max: maximal x-fold (normalised to wild type)
(minimal decrease), Min: minimal x-fold (normalised to wild type)
(maximal decrease). Column 3 indicates the analytical method.
[8757] [0515.0.0.19] to [0530.0.0.19] for the disclosure of the
paragraphs [0515.0.0.19] to [0530.0.0.19] see paragraphs
[0515.0.0.0] to [0530.0.0.0] above.
[8758] [0530.1.0.19] to [0530.6.0.19] for the disclosure of the
paragraphs [0530.1.0.19] to [0530.6.0.19] see paragraphs
[0530.1.0.0] to [0530.6.0.0] above.
[8759] [0531.0.0.19] to [0552.0.0.19] for the disclosure of the
paragraphs [0531.0.0.19] to [0552.0.0.19] see paragraphs
[0531.0.0.0] to [0552.0.0.0] above.
[8760] [0552.1.0.19] %
[8761] [0552.2.0.19] for the disclosure of this paragraph see
[0552.2.0.0] above.
[8762] [0553.0.19.19] [8763] 1. A process for the production of
ferulic acid or sinapic acid, which comprises [8764] (a) increasing
or generating the activity of a protein as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603 or a functional equivalent
thereof in a non-human organism or in one or more parts thereof;
and [8765] (b) growing the organism under conditions which permit
the production of ferulic acid or sinapic acid in said organism.
[8766] 2. A process for the production of ferulic acid or sinapic
acid, comprising the increasing or generating in an organism or a
part thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [8767] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 243 to
250 and 603 or a fragment thereof, which confers an increase in the
amount of ferulic acid or sinapic acid in an organism or a part
thereof; [8768] b) nucleic acid molecule comprising of a nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines 243 to
250 and 603; [8769] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of ferulic acid or
sinapic acid in an organism or a part thereof; [8770] d) nucleic
acid molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of ferulic acid or sinapic acid in an organism or a
part thereof; [8771] e) nucleic acid molecule which hybridizes with
a nucleic acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of ferulic acid
or sinapic acid in an organism or a part thereof; [8772] f) nucleic
acid molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table III, column 7, lines 243 to 250 and 603 and conferring an
increase in the amount of ferulic acid or sinapic acid in an
organism or a part thereof; [8773] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
ferulic acid or sinapic acid in an organism or a part thereof;
[8774] h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table IV, column 7, lines 243 to 250 and
603 and conferring an increase in the amount of ferulic acid or
sinapic acid in an organism or a part thereof; and [8775] i)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment thereof having at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (k) and
conferring an increase in the amount of ferulic acid or sinapic
acid in an organism or a part thereof. [8776] or comprising a
sequence which is complementary thereto. [8777] 3. The process of
claim 1 or 2, comprising recovering of the free or bound ferulic
acid or sinapic acid. [8778] 4. The process of any one of claims 1
to 3, comprising the following steps: [8779] a) selecting an
organism or a part thereof expressing a polypeptide encoded by the
nucleic acid molecule characterized in claim 2; [8780] b)
mutagenizing the selected organism or the part thereof; [8781] c)
comparing the activity or the expression level of said polypeptide
in the mutagenized organism or the part thereof with the activity
or the expression of said polypeptide of the selected organisms or
the part thereof; [8782] d) selecting the mutated organisms or
parts thereof, which comprise an increased activity or expression
level of said polypeptide compared to the selected organism or the
part thereof; [8783] e) optionally, growing and cultivating the
organisms or the parts thereof; and [8784] f) recovering, and
optionally isolating, the free or bound ferulic acid or sinapic
acid produced by the selected mutated organisms or parts thereof.
[8785] 5. The process of any one of claims 1 to 4, wherein the
activity of said protein or the expression of said nucleic acid
molecule is increased or generated transiently or stably. [8786] 6.
An isolated nucleic acid molecule comprising a nucleic acid
molecule selected from the group consisting of: [8787] a) nucleic
acid molecule encoding of a polypeptide as indicated in Table II,
columns 5 or 7, lines 243 to 250 and 603 or a fragment thereof,
which confers an increase in the amount of ferulic acid or sinapic
acid in an organism or a part thereof; [8788] b) nucleic acid
molecule comprising of a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 243 to 250 and 603; [8789] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of ferulic acid or sinapic
acid in an organism or a part thereof; [8790] d) nucleic acid
molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of ferulic acid or sinapic acid in an organism or a
part thereof; [8791] e) nucleic acid molecule which hybridizes with
a nucleic acid molecule of (a) to (c) under r stringent
hybridization conditions and conferring an increase in the amount
of ferulic acid or sinapic acid in an organism or a part thereof;
[8792] f) nucleic acid molecule which encompasses a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers or
primer pairs as indicated in Table III, column 7, lines 243 to 250
and 603 and conferring an increase in the amount of ferulic acid or
sinapic acid in an organism or a part thereof; [8793] g) nucleic
acid molecule encoding a polypeptide which is isolated with the aid
of monoclonal antibodies against a polypeptide encoded by one of
the nucleic acid molecules of (a) to (f) and conferring an increase
in the amount of ferulic acid or sinapic acid in an organism or a
part thereof; [8794] h) nucleic acid molecule encoding a
polypeptide comprising a consensus as indicated in Table IV, column
7, lines 243 to 250 and 603 and conferring an increase in the
amount of in an organism or a part thereof; and [8795] i) nucleic
acid molecule which is obtainable by screening a suitable nucleic
acid library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of ferulic acid or sinapic acid in an organism or a part
thereof. [8796] whereby the nucleic acid molecule distinguishes
over the sequence as indicated in Table IA, columns 5 or 7, lines
243 to 250 and 603 by one or more nucleotides. [8797] 7. A nucleic
acid construct which confers the expression of the nucleic acid
molecule of claim 6, comprising one or more regulatory elements.
[8798] 8. A vector comprising the nucleic acid molecule as claimed
in claim 6 or the nucleic acid construct of claim 7. [8799] 9. The
vector as claimed in claim 8, wherein the nucleic acid molecule is
in operable linkage with regulatory sequences for the expression in
a prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [8800] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [8801] 11. The host cell of claim 10, which is a
transgenic host cell. [8802] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [8803] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [8804] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table IIA, columns 5
or 7, lines 243 to 250 and 603 by one or more amino acids. [8805]
15. An antibody, which binds specifically to the polypeptide as
claimed in claim 14. [8806] 16. A plant tissue, propagation
material, harvested material or a plant comprising the host cell as
claimed in claim 12 which is plant cell or an Agrobacterium. [8807]
17. A method for screening for agonists and antagonists of the
activity of a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of ferulic acid or
sinapic acid in an organism or a part thereof comprising: [8808]
(a) contacting cells, tissues, plants or microorganisms which
express the a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of ferulic acid or
sinapic acid in an organism or a part thereof with a candidate
compound or a sample comprising a plurality of compounds under
conditions which permit the expression the polypeptide; [8809] (b)
assaying the ferulic acid or sinapic acid level or the polypeptide
expression level in the cell, tissue, plant or microorganism or the
media the cell, tissue, plant or microorganisms is cultured or
maintained in; and [8810] (c) identifying a agonist or antagonist
by comparing the measured ferulic acid or sinapic acid level or
polypeptide expression level with a standard ferulic acid or
sinapic acid or polypeptide expression level measured in the
absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. [8811] 18. A process for
the identification of a compound conferring increased ferulic acid
or sinapic acid production in a plant or microorganism, comprising
the steps: [8812] a) culturing a plant cell or tissue or
microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of ferulic acid or sinapic acid in an
organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of ferulic acid or sinapic
acid in an organism or a part thereof; [8813] b) identifying if the
compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout system.
[8814] 19. A method for the identification of a gene product
conferring an increase in ferulic acid or sinapic acid production
in a cell, comprising the following steps: [8815] a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in ferulic acid
or sinapic acid after expression with the nucleic acid molecule of
claim 6; [8816] b) identifying the nucleic acid molecules, which
hybridise under relaxed stringent conditions with the nucleic acid
molecule of claim 6; [8817] c) introducing the candidate nucleic
acid molecules in host cells appropriate for producing ferulic acid
or sinapic acid; [8818] d) expressing the identified nucleic acid
molecules in the host cells; [8819] e) assaying the ferulic acid or
sinapic acid level in the host cells; and [8820] f) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the ferulic acid or sinapic acid level in the host
cell in the host cell after expression compared to the wild type.
[8821] 20. A method for the identification of a gene product
conferring an increase in ferulic acid or sinapic acid production
in a cell, comprising the following steps: [8822] a) identifying in
a data bank nucleic acid molecules of an organism; which can
contain a candidate gene encoding a gene product conferring an
increase in the ferulic acid or sinapic acid amount or level in an
organism or a part thereof after expression, and which are at least
20% homolog to the nucleic acid molecule of claim 6; [8823] b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing ferulic acid or sinapic acid; [8824] c)
expressing the identified nucleic acid molecules in the host cells;
[8825] d) assaying the ferulic acid or sinapic acid level in the
host cells; and [8826] e) identifying nucleic acid molecule and its
gene product which expression confers an increase in the ferulic
acid or sinapic acid level in the host cell after expression
compared to the wild type. [8827] 21. A method for the production
of an agricultural composition comprising the steps of the method
of any one of claims 17 to 20 and formulating the compound
identified in any one of claims 17 to 20 in a form acceptable for
an application in agriculture. [8828] 22. A composition comprising
the nucleic acid molecule of claim 6, the polypeptide of claim 14,
the nucleic acid construct of claim 7, the vector of any one of
claim 8 or 9, an antagonist or agonist identified according to
claim 17, the compound of claim 18, the gene product of claim 19 or
20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. [8829] 23. Use of the nucleic acid molecule as
claimed in claim 6 for the identification of a nucleic acid
molecule conferring an increase of ferulic acid or sinapic acid
after expression. [8830] 24. Use of the polypeptide of claim 14 or
the nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of ferulic acid or sinapic acid
levels in an organism.
[8831] 25. Agrochemical, pharmaceutical, food or feed composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20. [8832] 26. The method of any one of
claims 1 to 5, the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20, wherein the fine chemical is ferulic
acid or sinapic acid.
[8833] [0554.0.0.19] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[8834] [0000.0.0.20] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[8835] [0001.0.0.20] see [0001.0.0.0]
[8836] [0002.0.20.20]
[8837] Plants produce very long chain fatty acids such as behenic
acid (C22:0), lignoceric acid (C24:0), cerotic acid (C26:0) and/or
melissic acid (C30:0).
[8838] Very long-chain fatty acids (VLCFAs) are synthesized by a
membrane-bound fatty acid elongation complex (elongase, FAE) using
acyl-CoA substrates. The first reaction of elongation involves
condensation of malonyl-CoA with a long chain substrate producing a
.beta.-ketoacyl-CoA. Subsequent reactions are reduction of
.beta.-hydroxyacyl-CoA, dehydration to an enoyl-CoA, followed by a
second reduction to form the elongated acyl-CoA. The
.beta.-ketoacyl-CoA synthase (KCS) catalyzing the condensation
reaction plays a key role in determining the chain length of fatty
acid products found in seed oils and is the rate-limiting enzyme
for seed VLCFA production (Lassner et al., Plant Cell, 8(1996),
281-292).
[8839] The elongation process can be repeated to yield members that
are 20, 22, and 24 carbons long. Although such very long chain
fatty acids are minor components of the lipid membranes of the
body, they undoubtedly perform valuable functions, apparently
helping to stabilize membranes, especially those in peripheral
nerve cells.
[8840] Behenic acid (22:0) (docosanoic acid) is a component of
rapeseed oil (up to 2%) and peanut oil (1-5%).
[8841] Behenic acid is used to give hair conditioners and
moisturizers their smoothing properties.
[8842] Lignoceric acid (24:0) (tetracosanoic acid) is a component
of rapeseed oil (up to 1%) and peanut oil (1-3%).
[8843] Cerotic acid (26:0) (hexacosanoic acid) is a component of
beeswax.
[8844] Echinacea angustifolia extracts are sold as natural health
products comprising the very long chain fatty acid cerotic
acid.
[8845] Cerotic acid is used in cosmetics as a constituent in
hairstyling products.
[8846] Melissic acid (C30:0) (triacontanoic acid) is a component of
beeswax.
[8847] Beeswax (cera alba) is obtained from the product excreted by
certain glands of the honeybee from which the honeycomb is made. It
is freed of solid impurities by melting and centrifugation (cera
flava). Finally, it is bleached completely white (cera alba).
Beeswax consists of 10-15 percent paraffin carbohydrates, 35-37
percent esters of C16 to C36 fatty acids and about 15 percent
cerotic acid, melissic acid and their homologues. Beeswax is used
as a thickener and a humectant in the manufacture of ointments,
creams, lipsticks and other cosmetics and skincare products as an
emulsifier, emollient, moisturizer and film former.
[8848] Beeswax is also used for the production of candles.
[8849] Wax is a general term used to refer to the mixture of
long-chain apolar lipids forming a protective coating (cutin in the
cuticle) on plant leaves and fruits but also in animals (wax of
honeybee, cuticular lipids of insects, spermaceti of the sperm
whale, skin lipids, uropygial glands of birds, depot fat of
planktonic crustacea), algae, fungi and bacteria.
[8850] Many of the waxes found in nature have commercial uses in
the lubricant, food and cosmetic industry. Jojoba oil has long been
suggested as a putative resource of wax, since this desert shrub is
unusual in its capacity to produce waxes rather than
triacylglycerols (TAG) as seed storage lipids. These waxes are
esters of very-long-chain-fatty acids and fatty alcohols (Miwa,
1971, J Am Oil Chem Soc 48, 259-264). As the production cost for
jojoba wax, which is primarily used for cosmetic applications, is
high, there is a need to engineer crop plants to produce high level
of wax esters in its seed oil.
[8851] Plant aerial surfaces are covered by epicuticular waxes,
complex mixtures of very long (C.sub.20-C.sub.34) fatty acids,
alkanes, aldehydes, ketones and esters. In addition to repelling
atmospheric water they prevent dessication and are therefore an
important determinant of drought resistance (Riederer and
Schreiber, 2001, J. Exp. Bot 52, 2023-2032). Beside abiotic stress
resistance the wax layer is part of the plant defense against
biotic stressor, especially insects as for example described by
Marcell and Beattie, 2002, Mol Plant Microbe Interact. 15(12),
1236-44. Furthermore they provide stability to pollen grains, thus
influencing fertility and productivity.
[8852] Very-long-chain fatty acids (VLCFAs), consisting of more
than 18 carbon atoms like behenic acid, lignoceric acid, cerotic
acid and melissic acid, are essential components for the vitality
of higher plants. The key enzyme of VLCFA biosynthesis, the
extraplastidary fatty acid elongase, is shown for to be the primary
target site of chloroacetamide herbicides. With an analysis of the
fatty acid composition and the metabolism of 14C-labelled
precursors (sterate, malonate, acetate), the reduction of VLCFAs
was determined in vivo. The inhibition of the recombinant protein
substantiates the first and rate-limiting step of VLCFA
biosynthesis, the condensation of acyl-CoA with malonyl-CoA to
.beta.-ketoacyl-CoA, to be the primary target site of
chloroacetamides (150=10-100 nM). The concentration of VLCFAs
within the untreated cell is low, the very-long-chain compounds are
found mainly in plasma membrane lipids and epicuticular waxes. A
shift of fatty acids towards shorter chain length or even the
complete depletion of very-long-chain components is the consequence
of the inhibition of VLCFA biosynthesis. Especially the loss of
plasma membrane VLCFAs is involved in phytotoxic effects of
chloroacetamides such as the inhibition of membrane biogenesis and
mitosis (Matthes, B.,
http://www.ub.uni-konstanz.de/kops/volltexte/2001/661/).
[8853] Increased wax production in transgenic plants has for
example been reported by Broun et al., 2004, Proc Natl. Acad. Sci,
101, 4706-4711. The authors overexpressed the transcriptional
activator WIN1 in Arabidopsis, leading to increased wax load on
anal organs. As this resulted in a complex change in the wax
profile and the transgenic overexpressors had characteristic
alterations in growth and development (Broun et al., 2004, Proc
Natl. Acad. Sci, 101, 4706-4711) there is still a need for a more
controlled increased production of defined VLCFAs.
[8854] Very long chain fatty alcohols obtained from plant waxes and
beeswax have also been reported to lower plasma cholesterol in
humans and existing data support the hypothesis that VLCFA exert
regulatory roles in cholesterol metabolism in the peroxisome and
also alter LDL uptake and metabolism (discussed in Hargrove et al.,
2004, Exp Biol Med (Maywood), 229(3): 215-26).
[8855] Due to these interesting physiological roles and the
nutritional, cosmetic and agrobiotechnological potential of behenic
acid (C22:0), lignoceric acid (C24:0), cerotic acid (C26:0) and
melissic acid (C30:0) there is a need to identify the genes of
enzymes and other proteins involved in behenic acid, lignoceric
acid, cerotic acid or melissic acid metabolism, and to generate
mutants or transgenic plant lines with which to modify the behenic
acid, lignoceric acid, cerotic acid or melissic acid content in
plants.
[8856] [0003.0.0.20] %
[8857] [0004.0.0.20] %
[8858] [0005.0.0.20] %
[8859] [0006.0.0.20] %
[8860] [0007.0.0.20] %
[8861] [0008.0.20.20] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce behenic acid, lignoceric acid,
cerotic acid or melissic acid in plants. That type of production
permits control over quality, quantity and selection of the most
suitable and efficient producer organisms. The latter is especially
important for commercial production economics and therefore
availability to consumers. In addition it is desirable to produce
behenic acid, lignoceric acid, cerotic acid or melissic acid in
plants in order to increase plant productivity and resistance
against biotic and abiotic stress as discussed before.
[8862] Methods of recombinant DNA technology have been used for
some years to improve the production of fine chemicals in
microorganisms and plants by amplifying individual biosynthesis
genes and investigating the effect on production of fine chemicals.
It is for example reported, that the xanthophyll astaxanthin could
be produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. Those results indicated
that about 75 percent of the carotenoids found in the flower of the
transformed plant contained a keto group.
[8863] [0009.0.20.20] Thus, it would be advantageous if an algae,
plant or other microorganism were available which produce large
amounts behenic acid, lignoceric acid, cerotic acid or melissic
acid. The invention discussed hereinafter relates in some
embodiments to such transformed prokaryotic or eukaryotic
microorganisms.
[8864] It would also be advantageous if plants were available whose
roots, leaves, stem, fruits or flowers produced large amounts of
behenic acid, lignoceric acid, cerotic acid or melissic acid. The
invention discussed hereinafter relates in some embodiments to such
transformed plants.
[8865] [0010.0.20.20] Therefore improving the quality of foodstuffs
and animal feeds is an important task of the food-and-feed
industry. This is necessary since, for example behenic acid,
lignoceric acid, cerotic acid or melissic acid, as mentioned above,
which occur in plants and some microorganisms are limited with
regard to the supply of mammals. Especially advantageous for the
quality of foodstuffs and animal feeds is as balanced as possible a
specific behenic acid, lignoceric acid, cerotic acid or melissic
acid profile in the diet since an excess of behenic acid,
lignoceric acid, cerotic acid or melissic acid above a specific
concentration in the food has a positive effect. A further increase
in quality is only possible via addition of further behenic acid,
lignoceric acid, cerotic acid or melissic acid, which are
limiting.
[8866] [0011.0.20.20] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add behenic acid, lignoceric
acid, cerotic acid or melissic acid in a balanced manner to suit
the organism.
[8867] [0012.0.20.20] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
proteins which participate in the biosynthesis of behenic acid,
lignoceric acid, cerotic acid or melissic acid and make it possible
to produce them specifically on an industrial scale without
unwanted byproducts forming. In the selection of genes for
biosynthesis two characteristics above all are particularly
important. On the one hand, there is as ever a need for improved
processes for obtaining the highest possible contents of behenic
acid, lignoceric acid, cerotic acid or melissic acid; on the other
hand as less as possible byproducts should be produced in the
production process.
[8868] [0013.0.0.20] see [0013.0.0.0]
[8869] [0014.0.20.20] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a behenic acid, lignoceric
acid, cerotic acid or melissic acid. Accordingly, in the present
invention, the term "the fine chemical" as used herein relates to a
behenic acid, lignoceric acid, cerotic acid or melissic acid.
Further, the term "the fine chemicals" as used herein also relates
to fine chemicals comprising behenic acid, lignoceric acid, cerotic
acid or melissic acid.
[8870] In one embodiment, the term "the fine chemical" means
behenic acid. In one embodiment, the term "the fine chemical" means
lignoceric acid depending on the context in which the term is used.
In another embodiment, the term "the fine chemical" means cerotic
acid. In a further another embodiment, the term "the fine chemical"
means melissic acid. Throughout the specification the term "the
fine chemical" means behenic acid, lignoceric acid, cerotic acid or
melissic acid, its salts, ester, thioester or in free form or bound
to other compounds such sugars or sugarpolymers, like glucoside,
e.g. diglucoside.
[8871] [0016.0.20.20] Accordingly, the present invention relates to
a process comprising [8872] (a) increasing or generating the
activity of one or more b0019, b0880, b1886, b1896, b3938, YDR513W,
YER156C, YGL205W, YHR201C, YLR255C, YPR138C, b0255 protein(s) in a
non-human organism in one or more parts thereof; and [8873] (b)
growing the organism under conditions which permit the production
of the fine chemical, thus behenic acid, lignoceric acid, cerotic
acid or melissic acid in said organism.
[8874] Accordingly, the present invention relates to a process
comprising [8875] (a) increasing or generating the activity of one
or more proteins having the activity of a protein indicated in
Table IIA or IIB, column 3, lines 251 to 261, 627, resp. or having
the sequence of a polypeptide encoded by a nucleic acid molecule
indicated in Table IA or IB, column 5 or 7, lines 251 to 261, 627,
resp. in a non-human organism in one or more parts thereof; and
growing the organism under conditions which permit the production
of the fine chemical, thus, behenic acid, lignoceric acid, cerotic
acid or melissic acid, in said organism.
[8876] [0016.1.20.20] Accordingly, the term "the fine chemical"
means "behenic acid" in relation to all sequences listed in Table
IA or IB, line 258 or homologs thereof, means "lignoceric acid" in
relation to the sequence listed in Table IA or IB, lines 251, 255,
259, 261 or 627 homologs thereof, means "melissic acid" in relation
to the sequences listed in Table IA or IB, lines 256, 257 or 260.
Accordingly, the term "the fine chemical" can mean "behenic acid",
"lignoceric acid", "cerotic acid" or "melissic acid", owing to
circumstances and the context. In order to illustrate that the
meaning of the term "the respective fine chemical" means "behenic
acid", "lignoceric acid", "cerotic acid" or "melissic acid" owing
to the sequences listed in the context the term "the respective
fine chemical" is also used.
[8877] [0017.0.0.20] to [0018.0.0.20]: see [0017.0.0.0] to
[0018.0.0.0]
[8878] [0019.0.20.20] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the respective fine chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table IIA or IIB, column 3, lines 251 to 261, 627 or
encoded by nucleic acid molecule indicated in Table IA or IB,
columns 5 or 7, lines 251 to 261, 627.
[8879] [0020.0.20.20] Surprisingly it was found, that the
transgenic expression of the Escherichia coli K12 protein b0019,
b0880, b1886, b1896, b3938, b0255 or Saccharomyces cerevisiae
protein YDR513W, YER156C, YGL205W, YHR201C, YLR255C, YPR138C in
Arabidopsis thaliana conferred an increase in behenic acid,
lignoceric acid, cerotic acid or melissic acid ("the fine chemical"
or "the fine respective chemical") in respect to said proteins and
their homologs as wells as the encoding nucleic acid molecules, in
particular as indicated in Table IIA or IIB, column 3, lines 251 to
261, 627 content of the transformed plants.
[8880] [0021.0.0.20] see [0021.0.0.0]
[8881] [0022.0.20.20] The sequence of b0019 from Escherichia coli
K12 has been published in Blattner, F. R. et al., Science 277
(5331), 1453-1474 (1997) and its activity is being defined as a
protein having Na+/H+ antiporter activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein b0019 from Escherichia coli K12 or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of lignoceric
acid and/or cerotic acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein b0019
is increased.
[8882] The sequence of b0880 from Escherichia coli K12 has been
published in Blattner, F. R. et al, Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a protein having stress
induced DNA replication inhibitor activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein b0880 from Escherichia coli K12 or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of cerotic acid,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein b0880 is increased.
[8883] The sequence of b1886 from Escherichia coli K12 has been
published in Blattner, F. R. et al., Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a methyl-accepting
chemotaxis protein II, aspartate sensor receptor. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a protein b1886 from Escherichia coli K12 or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of cerotic acid,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein b1886 is increased.
[8884] The sequence of b1896 from Escherichia coli K12 has been
published in Blattner, F. R. et al., Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a protein having
trehalose-6-phosphate synthase activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein b1896 from Escherichia coli K12 or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of cerotic acid,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein b1896 is increased.
[8885] The sequence of b3938 from Escherichia coli K12 has been
published in Blattner, F. R. et al., Science 277 (5331), 1453-1474
(1997) and its activity is being defined as transcriptional
repressor for methionine biosynthesis. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein b3938 from Escherichia coli K12 or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of lignoceric
acid, preferably in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of the protein b3938 is
increased.
[8886] The sequence of YDR513W from Saccharomyces cerevisiae has
been published in Jacq, C. et al., Nature 387 (6632 Suppl), 75-78
(1997) and its activity is being defined as a protein having
glutathione reductase activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
YDR513W or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, in particular for increasing the
amount of melissic acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein
YDR513W is increased.
[8887] The sequence of YER156C from Saccharomyces cerevisiae has
been published in Dietrich, F. S. et al., Nature 387 (6632 Suppl),
78-81 (1997) and its activity has not been characterized yet.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein YER156C or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of melissic acid,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein YER156C is increased.
[8888] The sequence of YGL205W from Saccharomyces cerevisiae has
been published in Tettelin, H. et al., Nature 387 (6632 Suppl),
81-84 (1997) and its activity is being defined as a protein having
fatty-acyl coenzyme A oxidase activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein
[8889] YGL205W or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, in particular for
increasing the amount of behenic acid, preferably in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the protein YGL205W is increased.
[8890] The sequence of YHR201C from Saccharomyces cerevisiae has
been published in Goffeau, A. et al., Science 274 (5287), 546-547
(1996) and its activity is being defined as a protein having
exopolyphosphatase activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
YHR201C or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, in particular for increasing the
amount of lignoceric acid and/or cerotic acid, preferably in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention the
activity of the protein YHR201C is increased.
[8891] The sequence of YLR255C from Saccharomyces cerevisiae has
been published in the EMBL Data Library, February 1995 and its
activity has not been characterized yet. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein YLR255C or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, in particular for
increasing the amount of melissic acid, preferably in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the protein YLR255C is increased.
[8892] The sequence of YPR138C from Saccharomyces cerevisiae has
been published in Bussey, H. et al., Nature 387 (6632 Suppl),
103-105 (1997) and its activity is being defined as a protein
having NH4+ transporter activity. Accordingly, in one embodiment,
the process of the present invention comprises the use of a protein
YPR138C or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, in particular for increasing the
amount of lignoceric acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein
YPR138C is increased.
[8893] The sequence of b0255 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a CP4-6 prophage; IS911
homolog. Accordingly, in one embodiment, the process of the present
invention comprises the use of said CP4-6 prophage; IS911 homolog
protein from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of linoleic acid (ist
linoleic acid korrekt?) or lignoceric acid and/or tryglycerides,
lipids, oils and/or fats containing linoleic acid or lignoceric
acid, in particular for increasing the amount of linoleic acid or
lignoceric acid and/or tryglycerides, lipids, oils and/or fats
containing linoleic acid and/or lignoceric acid, preferably
linoleic acid or lignoceric acid in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of said CP4-6
prophage; IS911 homolog protein is increased or generated, e.g.
from E. coli or a homolog thereof.
[8894] [0023.0.20.20] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[8895] In one embodiment, the homolog of any one of the
polypeptides indicated in Table IIA or IIB, column 3, lines 251,
255, 259, 261 and 627 is a homolog having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of the respective fine chemical in the organisms
preferably lignoceric acid.
[8896] In one embodiment, the homolog of the polypeptides indicated
in Table IIA or IIB, column 3, lines 251, 252, 253, 254 and 259 is
a homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms preferably cerotic
acid.
[8897] In one embodiment, the homolog of any one of the
polypeptides indicated in Table IIA or IIB, column 3, lines 256,
257 and 260 is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms preferably
melissic acid.
[8898] In one embodiment, the homolog of any one of the
polypeptides indicated in Table IIA or IIB, column 3, line 258 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms preferably behenic
acid.
[8899] Homologs of the polypeptides indicated in Table IIA or IIB,
column 3, lines 251, 255, 259, 261 and 627 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table IA or IB,
column 7, lines 251, 255, 259, 261 and 627 or may be the
polypeptides indicated in Table IIA or IIB, column 7, lines 251,
255, 259, 261 and 627 having a lignoceric acid content and/or
amount increasing activity.
[8900] Homologs of the polypeptides indicated in Table IIA or IIB,
column 3, lines 251, 252, 253, 254 and 259 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table IA or IB,
column 7, lines 251, 252, 253, 254 and 259, respectively or may be
the polypeptides indicated in Table IIA or IIB, column 7, lines
251, 252, 253, 254 and 259 having a cerotic acid content and/or
amount increasing activity.
[8901] Homologs of the polypeptide indicated in Table IIA or IIB,
column 3, lines 256, 257 and 260 may be the polypeptides encoded by
the nucleic acid molecules indicated in Table IA or IB, column 7,
lines 256, 257 and 260, respectively or may be the polypeptides
indicated in Table IIA or IIB, column 7, lines 256, 257 and 260,
having a melissic acid content and/or amount increasing
activity.
[8902] Homologs of the polypeptide indicated in Table IIA or IIB,
column 3, line 258 may be the polypeptides encoded by the nucleic
acid molecules indicated in Table IA or IB, column 7, line 258,
respectively or may be the polypeptides indicated in Table IIA or
IIB, column 7, line 258, having a behenic content and/or amount
increasing activity.
[8903] [0024.0.0.20] see [0024.0.0.0]
[8904] [0025.0.20.20] In accordance with the invention, a protein
or polypeptide has the "activity of a protein of the invention",
e.g. the activity of a protein indicated in Table IIA or IIB,
column 3, lines 251 to 261, 627 if its de novo activity, or its
increased expression directly or indirectly leads to an increased
behenic acid, lignoceric acid, cerotic acid or melissic acid level,
resp., in the organism or a part thereof, preferably in a cell of
said organism. In a preferred embodiment, the protein or
polypeptide has the above-mentioned additional activities of a
protein indicated in Table IIA or IIB, column 3, lines 251 to 261,
627. Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of any one of the proteins indicated in Table IIA or IIB,
column 3, lines 251 to 261, 627, or which has at least 10% of the
original enzymatic activity, preferably 20%, particularly
preferably 30%, most particularly preferably 40% in comparison to
any one of the proteins indicated in Table IIA or IIB, column 3,
line 251 to 255 of Escherichia coli K12 or line 256 to 261 of
Saccharomyces cerevisiae respectively.
[8905] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table IA
or IB, column 4 and is expressed in an organism, which is
evolutionary distant to the origin organism. For example origin and
expressing organism are derived from different families, orders,
classes or phylums whereas origin and the organism indicated in
Table IA or IB, column 4 are derived from the same families,
orders, classes or phylums.
[8906] [0025.1.0.20] see [0025.1.0.0]
[8907] [0026.0.0.20] to [0033.0.0.20]: see [0026.0.0.0] to
[0033.0.0.0]
[8908] [0034.0.20.20] Preferably, the reference, control or wild
type differs from the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of a protein having the activity of a
protein as indicated in Table IIA or IIB, column 3, lines 251 to
261, 627 or being encoded by a nucleic acid molecule indicated in
Table IA or IB, column 5, lines 251 to 261, 627 or its homologs,
e.g. as indicated in Table IA or IB, column 7, lines 251 to 261,
627, its biochemical or genetic causes. It therefore shows the
increased amount of the respective fine chemical.
[8909] [0035.0.0.20] to [0038.0.0.20]: see [0035.0.0.0] to
[0038.0.0.0]
[8910] [0039.0.0.20]: see [0039.0.0.0]
[8911] [0040.0.0.20] to [0044.0.0.20]: see [0040.0.0.0] to
[0044.0.0.0]
[8912] [0045.0.20.20] In one embodiment, in case the activity of
the Escherichia coli K12 protein b0019 or its homologs, e.g. as
indicated in Table IIA or IIB, columns 5 or 7, line 251 is
increased, preferably, in one embodiment the increase of the
respective fine chemical, preferably of cerotic acid between 40%
and 150% or more is conferred.
[8913] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0019 or its homologs, e.g. as indicated in Table
IIA or IIB, columns 5 or 7, line 251 is increased, preferably, in
one embodiment the increase of the respective fine chemical,
preferably of lignoceric acid between 46% and 224% or more is
conferred.
[8914] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0880 or its homologs, e.g. as indicated in Table
IIA or IIB, columns 5 or 7, line 252 is increased, preferably, in
one embodiment the increase of the respective fine chemical,
preferably of cerotic acid between 172% and 294% or more is
conferred.
[8915] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1886 or its homologs, e.g. as indicated in Table
IIA or IIB, columns 5 or 7, line 253 is increased, preferably, in
one embodiment the increase of the respective fine chemical,
preferably of cerotic acid between 39% and 335% or more is
conferred.
[8916] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1896 or its homologs, e.g. as indicated in Table
IIA or IIB, columns 5 or 7, line 254 is increased, preferably, in
one embodiment the increase of the respective fine chemical,
preferably of cerotic acid between 39% and 75% or more is
conferred.
[8917] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3938 or its homologs, e.g. as indicated in Table
IIA or IIB, columns 5 or 7, line 255 is increased, preferably, in
one embodiment the increase of the respective fine chemical,
preferably of lignoceric acid between 37% and 89% or more is
conferred.
[8918] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YDR513W or its homologs as indicated in Table IIA
or IIB, columns 5 or 7, line 256, is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of melissic acid between 31% and 108% or more is conferred.
[8919] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YER156C or its homologs as indicated in Table
IIA or IIB, columns 5 or 7, line 257, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of melissic acid between 33% and 73% or more is
conferred.
[8920] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YGL205W or its homologs as indicated in Table
IIA or IIB, columns 5 or 7, line 258, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of behenic acid between 74% and 132% or more is
conferred.
[8921] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YHR201C or its homologs as indicated in Table
IIA or IIB, columns 5 or 7, line 259, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of cerotic acid between 46% and 227% or more is
conferred.
[8922] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YHR201C or its homologs as indicated in Table
IIA or IIB, columns 5 or 7, line 259, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of lignoceric acid between 29% and 271% or more is
conferred.
[8923] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YLR255C or its homologs as indicated in Table
IIA or IIB, columns 5 or 7, line 260, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of melissic acid between 34% and 88% or more is
conferred.
[8924] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YPR138C or its homologs as indicated in Table
IIA or IIB, columns 5 or 7, line 261, is increased, preferably, in
one embodiment an increase of the respective fine chemical,
preferably of lignoceric acid between 92% and 160% or more is
conferred.
[8925] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0255 or its homologs, e.g. as indicated in Table
IIA or IIB, columns 5 or 7, line 627 is increased, preferably, in
one embodiment the increase of the respective fine chemical,
preferably of lignoceric acid between 35% and 115% or more is
conferred.
[8926] [0046.0.0.20] %
[8927] [0047.0.0.20] to [0048.0.0.20]: see [0047.0.0.0] to
[0048.0.0.0]
[8928] [0049.0.20.20] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
lignoceric acid preferably has the structure of the polypeptide
described herein. In a particular embodiment, the polypeptides used
in the process of the present invention or the polypeptide of the
present invention comprises the sequence of a consensus sequence as
shown in SEQ ID NO: 27297, 27298 or 27309, 27310 or 27322, 27323,
27324 or 27325, 27326, 27327, 27328, 97769, 97770 or as indicated
in Table IV, columns 7, lines 251, 255, 259, 261 and 627 or of a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
251, 255, 259, 261 and 627 or of a functional homologue thereof as
described herein, or of a polypeptide encoded by the nucleic acid
molecule characterized herein or the nucleic acid molecule
according to the invention, for example by a nucleic acid molecule
as indicated in Table IA or IB, columns 5 or 7, lines 251, 255,
259, 261 or 627 or its herein described functional homologues and
has the herein mentioned activity conferring an increase in the
lignoceric acid level.
[8929] A protein having an activity conferring an increase in the
amount or level of the respective fine chemical cerotic acid
preferably has the structure of the polypeptide described herein.
In a particular embodiment, the polypeptides used in the process of
the present invention or the polypeptide of the present invention
comprises the sequence of a consensus sequence as shown in SEQ ID
NO: 27297, 27298 or 27299, 27300, 27301, 27302 or 27303, 27304 or
27305, 27306, 27307, 27308 or 27322, 27323, 27324 or as indicated
in Table IV, columns 7, lines 251, 252, 253, 254 and 259 or of a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
251, 252, 253, 254 and 259 or of a functional homologue thereof as
described herein, or of a polypeptide encoded by the nucleic acid
molecule characterized herein or the nucleic acid molecule
according to the invention, for example by a nucleic acid molecule
as indicated in Table IA or IB, columns 5 or 7, lines 251, 252,
253, 254 and 259 or its herein described functional homologues and
has the herein mentioned activity conferring an increase in the
cerotic acid level.
[8930] A protein having an activity conferring an increase in the
amount or level of the respective fine chemical melissic acid
preferably has the structure of the polypeptide described herein.
In a particular embodiment, the polypeptides used in the process of
the present invention or the polypeptide of the present invention
comprises the sequence of a consensus sequence as shown in SEQ ID
NO: 27311, 27312, 27313 or 27314, 27315, 27316, 27317, 27318, 27319
or as indicated in Table IV, columns 7, lines 256 and 257 or of a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
256, 257 and 260 or of a functional homologue thereof as described
herein, or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table IA or IB, columns 5 or 7, lines 256, 257 and 260 or its
herein described functional homologues and has the herein mentioned
activity conferring an increase in the melissic acid level.
[8931] A protein having an activity conferring an increase in the
amount or level of the respective fine chemical behenic acid
preferably has the structure of the polypeptide described herein.
In a particular embodiment, the polypeptides used in the process of
the present invention or the polypeptide of the present invention
comprises the sequence of a consensus sequence as shown in SEQ ID
NO: 27320, 27321 or as indicated in Table IV, columns 7, line 258
or of a polypeptide as indicated in Table IIA or IIB, columns 5 or
7, line 258 or of a functional homologue thereof as described
herein, or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table IA or IB, columns 5 or 7, line 258 or its herein described
functional homologues and has the herein mentioned activity
conferring an increase in the behenic acid level.
[8932] [0050.0.20.20] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of behenic acid, lignoceric acid, cerotic acid or melissic
acid, resp., or their ester, or glucoside thereof, e.g the
diglucoside thereof.
[8933] [0051.0.20.20] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g compositions comprising behenic acid,
lignoceric acid, cerotic acid or melissic acid. Depending on the
choice of the organism used for the process according to the
present invention, for example a microorganism or a plant,
compositions or mixtures of behenic acid, lignoceric acid, cerotic
acid or melissic acid can be produced.
[8934] [0052.0.0.20] see [0052.0.0.0]
[8935] [0053.0.20.20] In one embodiment, the process of the present
invention comprises one or more of the following steps [8936] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention, e.g. of a polypeptide having an
activity of a protein as indicated in Table IIA or IIB, column 3,
lines 251 to 261, 627 or its homologs, e.g. as indicated in Table
IIA or IIB, columns 5 or 7, lines 251 to 161, activity having
herein-mentioned the respective fine chemical increasing activity;
[8937] b) stabilizing a mRNA conferring the increased expression of
a protein encoded by the nucleic acid molecule of the invention,
e.g. of a polypeptide having an activity of a protein as indicated
in Table IIA or IIB, column 3, lines 251 to 261, 627 or its
homologs activity, e.g. as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627, or of a mRNA encoding the polypeptide
of the present invention having herein-mentioned the respective
fine chemical increasing activity; [8938] c) increasing the
specific activity of a protein conferring the increased expression
of a protein encoded by the nucleic acid molecule of the invention
or of the polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table IIA or IIB, column 3, lines 251 to 261, 627 or its
homologs activity, e.g. as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627, or decreasing the inhibitory
regulation of the polypeptide of the invention; [8939] d)
generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table IIA or IIB, column 3, lines 251 to
261, 627 or its homologs activity, e.g. as indicated in Table IIA
or IIB, columns 5 or 7, lines 251 to 261, 627; [8940] e)
stimulating activity of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
present invention or a polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table IIA or IIB, column 3, lines 251 to 261, 627 or its
homologs activity, e.g. as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627, by adding one or more exogenous
inducing factors to the organism or parts thereof; [8941] f)
expressing a transgenic gene encoding a protein conferring the
increased expression of a polypeptide encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention, having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table IIA or IIB, column 3, lines 251 to
261, 627 or its homologs activity, e.g. as indicated in Table IIA
or IIB, columns 5 or 7, lines 251 to 261, 627, and/or [8942] g)
increasing the copy number of a gene conferring the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or the
polypeptide of the invention having herein-mentioned the respective
fine chemical increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table IIA or IIB, column 3,
lines 251 to 261, 627 or its homologs, e.g. as indicated in Table
IIA or IIB, columns 5 or 7, lines 251 to 261, 627. [8943] h)
Increasing the expression of the endogenous gene encoding the
polypeptide of the invention, e.g. a polypeptide having an activity
of a protein as indicated in Table IIA or IIB, column 3, lines 251
to 261, 627 or its homologs activity, e.g. as indicated in Table
IIA or IIB, columns 5 or 7, 251 to 261, 627, by adding positive
expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; and/or
[8944] i) Modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead to an enhanced respective fine
chemical production. [8945] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, e.g. the elite crops.
[8946] [0054.0.20.20] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table IIA or IIB, columns 3
or 5, lines 251 to 261, 627, resp., or its homologs activity, e.g.
as indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627, resp.
[8947] [0055.0.0.20] to [0067.0.0.20]: see [0055.0.0.0] to
[0067.0.0.0]
[8948] [0068.0.20.20] The mutation is introduced in such a way that
the production of behenic acid, lignoceric acid, cerotic acid or
melissic acid is not adversely affected.
[8949] [0069.0.0.20] see [0069.0.0.0]
[8950] [0070.0.20.20] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolite composition in the organism, e.g. an advantageous
composition of behenic acid, lignoceric acid, cerotic acid and/or
melissic acid or their biochemical derivatives, e.g. comprising a
higher content of (from a viewpoint of nutritional physiology
limited) behenic acid, lignoceric acid, cerotic acid and/or
melissic acid or their derivatives.
[8951] [0071.0.0.20] see [0071.0.0.0]
[8952] [0072.0.0.20] %
[8953] [0073.0.20.20] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
[8954] providing a non-human organism, preferably a microorganism,
a non-human animal, a plant or animal cell, a plant or animal
tissue or a plant;
[8955] increasing an activity of a polypeptide of the invention or
a homolog thereof, e.g. as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627, or of a polypeptide being encoded by
the nucleic acid molecule of the present invention and described
below, e.g. conferring an increase of the respective fine chemical
in an organism, preferably in a microorganism, a non-human animal,
a plant or animal cell, a plant or animal tissue or a plant,
[8956] growing an organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a plant
under conditions which permit the production of the respective fine
chemical in the organism, preferably the microorganism, the plant
cell, the plant tissue or the plant; and
[8957] if desired, recovering, optionally isolating, the free
and/or bound respective fine chemical synthesized by the organism,
the microorganism, the non-human animal, the plant or animal cell,
the plant or animal tissue or the plant.
[8958] [0074.0.20.20] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound respective fine chemical.
[8959] [0075.0.0.20] to [0077.0.0.20]: see [0075.0.0.0] to
[0077.0.0.0]
[8960] [0078.0.20.20] The organism such as microorganisms or plants
or the recovered, and if desired isolated, respective fine chemical
can then be processed further directly into foodstuffs or animal
feeds or for other applications. The fermentation broth,
fermentation products, plants or plant products can be purified
with methods known to the person skilled in the art. Products of
these different work-up procedures are behenic acid, lignoceric
acid, cerotic acid or melissic acid or comprising compositions of
behenic acid, lignoceric acid, cerotic acid or melissic acid still
comprising fermentation broth, plant particles and cell components
in different amounts, advantageously in the range of from 0 to 99%
by weight, preferably below 80% by weight, especially preferably
below 50% by weight.
[8961] [0079.0.0.20] to [0084.0.0.20]: see [0079.0.0.0] to
[0084.0.0.0]
[8962] [0084.0.0.20] %
[8963] [0085.0.20.20] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [8964] a) a nucleic acid sequence as
indicated in Table IA or IB, columns 5 or 7, lines 251 to 261, 627,
or a derivative thereof, or [8965] b) a genetic regulatory element,
for example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table IA or IB, columns 5 or 7, lines
251 to 261, 627, or a derivative thereof, or [8966] c) (a) and (b)
is/are not present in its/their natural genetic environment or
has/have been modified by means of genetic manipulation methods, it
being possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[8967] [0086.0.0.20] to [0087.0.0.20]: see [0086.0.0.0] to
[0087.0.0.0]
[8968] [0088.0.20.20] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned fine
chemicals or the plants are more resistant to biotic and abiotic
stress and the yield is increased.
[8969] [0088.1.0.20] see [0088.1.0.0]
[8970] [0089.0.0.20] to [0090.0.0.20]: see [0089.0.0.0] to
[0090.0.0.0]
[8971] [0091.0.0.20] see [0091.0.0.0]
[8972] [0092.0.0.20] to [0094.0.0.20]: see [0092.0.0.0] to
[0094.0.0.0]
[8973] [0095.0.20.20] It may be advantageous to increase the pool
of behenic acid, lignoceric acid, cerotic acid or melissic acid in
the transgenic organisms by the process according to the invention
in order to isolate high amounts of the pure respective fine
chemical and/or to obtain increased resistance against biotic and
abiotic stresses and to obtain higher yield.
[8974] [0096.0.20.20] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example in
the organism, is useful to increase the production of the
respective fine chemical.
[8975] [0097.0.0.20] %
[8976] [0098.0.20.20] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated.
[8977] [0099.0.20.20] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[8978] [0100.0.20.20] Transgenic plants which comprise the fine
chemicals such as behenic acid, lignoceric acid, cerotic acid or
melissic acid synthesized in the process according to the invention
can advantageously be marketed directly without there being any
need for the fine chemicals synthesized to be isolated. Plants for
the process according to the invention are listed as meaning intact
plants and all plant parts, plant organs or plant parts such as
leaf, stem, seeds, root, tubers, anthers, fibers, root hairs,
stalks, embryos, calli, cotelydons, petioles, harvested material,
plant tissue, reproductive tissue and cell cultures which are
derived from the actual transgenic plant and/or can be used for
bringing about the transgenic plant. In this context, the seed
comprises all parts of the seed such as the seed coats, epidermal
cells, seed cells, endosperm or embryonic tissue.
[8979] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, as extracts, e.g. ether, alcohol,
or other organic solvents or water containing extract and/or free
fine chemicals. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pretreated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[8980] Because behenic acid, lignoceric acid, cerotic acid or
melissic acid in microorganisms are localized intracellular, their
recovery essentially comes down to the isolation of the biomass.
Well-established approaches for the harvesting of cells include
filtration, centrifugation and coagulation/flocculation as
described herein. Of the residual hydrocarbon, adsorbed on the
cells, has to be removed. Solvent extraction or treatment with
surfactants have been suggested for this purpose.
[8981] [0101.0.20.20] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michel, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[8982] [0102.0.20.20] Behenic acid, lignoceric acid, cerotic acid
or melissic acid can for example be analyzed advantageously via
HPLC, LC or GC separation and MS (masspectrometry) detection
methods. The unambiguous detection for the presence of behenic
acid, lignoceric acid, cerotic acid or melissic acid containing
products can be obtained by analyzing recombinant organisms using
analytical standard methods: LC, LC-MS, MS or TLC). The material to
be analyzed can be disrupted by sonication, grinding in a glass
mill, liquid nitrogen and grinding, cooking, or via other
applicable methods.
[8983] [0103.0.20.20] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [8984] a) nucleic acid molecule encoding, preferably
at least the mature form, of the polypeptide having a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627, or a fragment thereof, which confers an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[8985] b) nucleic acid molecule comprising, preferably at least the
mature form, of a nucleic acid molecule having a sequence as
indicated in Table IA or IB, columns 5 or 7, lines 251 to 261, 627,
[8986] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [8987] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[8988] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [8989]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [8990] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [8991] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primer pairs
having a sequence as indicated in Table III, columns 7, lines 251
to 261, 627, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [8992]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [8993] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence having
sequences as indicated in Table IV, column 7, lines 251 to 261, 627
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [8994] k) nucleic acid
molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of the polypeptide indicated in Table IIA or IIB, columns 5 or 7,
lines 251 to 261, 627, and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof; and
[8995] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[8996] [0104.0.20.20] In one embodiment, the nucleic acid molecule
used in the process of the present invention distinguishes over the
sequence indicated in Table IA or IB, columns 5 or 7, lines 251 to
261, 627 by one or more nucleotides. In one embodiment, the nucleic
acid molecule used in the process of the invention does not consist
of the sequence indicated in Table IA or IB, columns 5 or 7, lines
251 to 261, 627. In one embodiment, the nucleic acid molecule of
the present invention is less than 100%, 99.999%, 99.99%, 99.9% or
99% identical to the sequence indicated in Table IA or IB, columns
5 or 7, lines 251 to 261, 627. In another embodiment, the nucleic
acid molecule does not encode a polypeptide of a sequence indicated
in Table IIA or IIB, columns 5 or 7, lines 251 to 261, 627.
[8997] [0105.0.0.20] to [0107.0.0.20]: see [0105.0.0.0] to
[0107.0.0.0]
[8998] [0108.0.20.20] Nucleic acid molecules with the sequence as
indicated in Table IA or IB, columns 5 or 7, lines 251 to 261, 627,
nucleic acid molecules which are derived from an amino acid
sequences as indicated in Table IIA or IIB, columns 5 or 7, lines
251 to 261, 627 or from polypeptides comprising the consensus
sequence as indicated in Table IV, column 7, lines 251 to 261, 627,
or their derivatives or homologues encoding polypeptides with the
enzymatic or biological activity of an activity of a polypeptide as
indicated in Table IIA or IIB, column 3, 5 or 7, lines 251 to 261,
627, e.g. conferring the increase of the respective fine chemical,
meaning behenic acid, lignoceric acid, cerotic acid or melissic
acid, resp., after increasing its expression or activity, are
advantageously increased in the process according to the
invention.
[8999] [0109.0.20.20] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular behenic
acid, lignoceric acid, cerotic acid or melissic acid, produced in
the process according to the invention.
[9000] [0110.0.0.20] see [0110.0.0.0]
[9001] [0111.0.0.20] see [0111.0.0.0]
[9002] [0112.0.20.20] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table IIA or IIB, column 3, lines
251 to 261, 627 or having the sequence of a polypeptide as
indicated in Table IIA or IIB, columns 5 and 7, lines 251 to 261,
627 and conferring an increase in the behenic acid, lignoceric
acid, cerotic acid or melissic acid level.
[9003] [0113.0.0.20] to [0114.0.0.20]: see [0113.0.0.0] to
[0114.0.0.0]
[9004] [0115.0.0.20] see [0115.0.0.0]
[9005] [0116.0.0.20] to [0120.0.0.20] see [0116.0.0.0] to
[0120.0.0.0]
[9006] [0120.1.0.20]: %
[9007] [0121.0.20.20] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table IIA or
IIB, columns 5 or 7, lines 251 to 261, 627 or the functional
homologues thereof as described herein, preferably conferring
above-mentioned activity, i.e. conferring a lignoceric acid level
increase after increasing the activity of the polypeptide sequences
indicated in Table
[9008] IIA or IIB, columns 5 or 7, lines 251, 255, 259 or 261,
conferring a cerotic acid level increase after increasing the
activity of the polypeptide sequences indicated in Table IIA or
IIB, columns 5 or 7, lines 251, 252, 253, 254 or 259, conferring a
melissic acid level increase after increasing the activity of the
polypeptide sequences indicated in Table IIA or IIB, columns 5 or
7, lines 256, 257 or 260 or conferring a behenic acid level
increase after increasing the activity of the polypeptide sequences
indicated in Table IIA or IIB, columns 5 or 7, line 258.
[9009] [0122.0.0.20] to [0127.0.0.20]: see [0122.0.0.0] to
[0127.0.0.0]
[9010] [0128.0.20.20] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, columns 7,
lines 251 to 261, 627, by means of polymerase chain reaction can be
generated on the basis of a sequence shown herein, for example the
sequence as indicated in Table IA or IB, columns 5 or 7, lines 251
to 261, 627, resp. or the sequences derived from a sequences as
indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627, resp.
[9011] [0129.0.20.20] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved regions are those, which show a
very little variation in the amino acid in one particular position
of several homologs from different origin. The consensus sequence
indicated in Table IV, columns 7, lines 251 to 259, 261 and 627 is
derived from such alignments.
[9012] [0130.0.20.20] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of behenic
acid, lignoceric acid, cerotic acid or melissic acid after
increasing the expression or activity the protein comprising said
fragment.
[9013] [0131.0.0.20] to [0138.0.0.20]: see [0131.0.0.0] to
[0138.0.0.0]
[9014] [0139.0.20.20] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical level increase,
derived from other organisms, can be encoded by other DNA sequences
which hybridise to a sequence indicated in Table IA or IB, columns
5 or 7, lines 251, 255, 259, 261 or 627 for lignoceric acid or
indicated in Table IA or IB, columns 5 or 7, lines 251, 252, 253,
254 or 259 for cerotic acid or indicated in Table IA or IB, columns
5 or 7, lines 256, 257 or 260 for melissic acid or indicated in
Table IA or IB, columns 5 or 7, line 258 for benic acid under
relaxed hybridization conditions and which code on expression for
peptides having the respective fine chemical, i.e. behenic acid,
lignoceric acid, cerotic acid or melissic acid, resp.,
increasing-activity.
[9015] [0140.0.0.20] to [0146.0.0.20]: see [0140.0.0.0] to
[0146.0.0.0]
[9016] [0147.0.20.20] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
IA or IB, columns 5 or 7, lines 251 to 261, 627, preferably the
nucleic acid molecule of the invention is a functional homologue or
identical to a nucleic acid molecule indicated in Table IB, columns
5 or 7, lines 251 to 261, 627, is one which is sufficiently
complementary to one of said nucleotide sequences such that it can
hybridise to one of said nucleotide sequences, thereby forming a
stable duplex. Preferably, the hybridisation is performed under
stringent hybridization conditions. However, a complement of one of
the herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[9017] [0148.0.20.20] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table IA or IB, columns 5 or 7,
lines 251 to 261, 627 or a portion thereof and preferably has above
mentioned activity, in particular having a behenic acid, lignoceric
acid, cerotic acid or melissic acid increasing activity after
increasing the activity or an activity of a product of a gene
encoding said sequences or their homologs.
[9018] [0149.0.20.20] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table IA or IB, columns 5 or
7, lines 251 to 261, 627, preferably the nucleic acid molecule of
the invention is a functional homologue or identical to a nucleic
acid molecule indicated in Table IB, columns 5 or 7, lines 251 to
261, 627, or a portion thereof and encodes a protein having
above-mentioned activity, e.g. conferring an increase of behenic
acid, lignoceric acid, cerotic acid or melissic acid, resp., and
optionally, the activity of protein indicated in Table IIA or IIB,
column 5, lines 251 to 261, 627.
[9019] [00149.1.20.20] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table IA or IB, columns 5 or 7, lines 251 to
261, 627, preferably the nucleic acid molecule of the invention is
a functional homologue or identical to a nucleic acid molecule
indicated in Table IB, columns 5 or 7, lines 251 to 261, 627, has
further one or more of the activities annotated or known for a
protein as indicated in Table IIA or IIB, column 3, lines 251 to
261, 627.
[9020] [0150.0.20.20] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table IA or IB, columns 5 or 7, lines
251 to 261, 627 preferably the nucleic acid molecule of the
invention is a functional homologue or identical to a nucleic acid
molecule indicated in Table IB, columns 5 or 7, lines 251 to 261,
627 for example a fragment which can be used as a probe or primer
or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of behenic acid, lignoceric
acid, cerotic acid or melissic acid, resp., if its activity is
increased. The nucleotide sequences determined from the cloning of
the present protein-according-to-the-invention-encoding gene allows
for the generation of probes and primers designed for use in
identifying and/or cloning its homologues in other cell types and
organisms. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, 15 preferably about 20 or 25, more
preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one of the sequences set forth, e.g., as indicated in
Table IA or IB, columns 5 or 7, lines 251 to 261, 627, an
anti-sense sequence of one of the sequences, e.g., as indicated in
Table IA or IB, columns 5 or 7, lines 251 to 261, 627, or naturally
occurring mutants thereof. Primers based on a nucleotide of
invention can be used in PCR reactions to clone homologues of the
polypeptide of the invention or of the polypeptide used in the
process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primer pairs indicated in I Table III, column 7, lines
251 to 261, 627 will result in a fragment of a polynucleotide
sequence as indicated in Table IA or IB, columns 5 or 7, lines 251
to 261, 627 or its gene product.
[9021] [0151.0.0.20]: see [0151.0.0.0]
[9022] [0152.0.20.20] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
251 to 261, 627 such that the protein or portion thereof maintains
the ability to participate in the respective fine chemical
production, in particular a lignoceric acid (lines 251, 255, 259,
261, 627) or cerotic acid (lines 251, 252, 253, 254, 259) or
melissic acid (lines 256, 257, 260) or behenic acid (line 258)
increasing activity as mentioned above or as described in the
examples in plants or microorganisms is comprised.
[9023] [0153.0.20.20] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 251 to 261, 627 such that the protein or portion thereof is
able to participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627 has for example an activity of a polypeptide indicated in Table
IIA or IIB, column 3, lines 251 to 261, 627.
[9024] [0154.0.20.20] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
251 to 261, 627 and has above-mentioned activity, e.g. conferring
preferably the increase of the respective fine chemical.
[9025] [0155.0.0.20] to [0156.0.0.20]: see [0155.0.0.0] to
[0156.0.0.0]
[9026] [0157.0.20.20] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences as
indicated in Table IA or IB, columns 5 or 7, lines 251 to 261, 627
(and portions thereof) due to degeneracy of the genetic code and
thus encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
polypeptides comprising the sequence as indicated in Table IV,
column 7, lines 251 to 261, 627 or as polypeptides depicted in
Table IIA or IIB, columns 5 or 7, lines 251 to 261, 627 or the
functional homologues. Advantageously, the nucleic acid molecule of
the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, an amino acid sequence of a consensus sequences
as indicated in Table IV, column 7, lines 251 to 259 and 261 or of
the polypeptide as indicated in Table IIA or IIB, columns 5 or 7,
lines 251 to 261, 627, resp., or the functional homologues. In a
still further embodiment, the nucleic acid molecule of the
invention encodes a full length protein which is substantially
homologous to an amino acid sequence comprising a consensus
sequence as indicated in Table IV, column 7, lines 251 to 259 and
261, 627 or of a polypeptide as indicated in Table IIA or IIB,
columns 5 or 7, lines 251 to 261, 627 or the functional homologues.
However, in a preferred embodiment, the nucleic acid molecule of
the present invention does not consist of a sequence as indicated
in Table IA or IB, columns 5 or 7, lines 251 to 261, 627, resp.
[9027] [0158.0.0.20] to [0160.0.0.20]: see [0158.0.0.0] to
[0160.0.0.0]
[9028] [0161.0.20.20] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table IA or IB, columns 5 or 7, lines 251
to 261, 627. The nucleic acid molecule is preferably at least 20,
30, 50, 100, 250 or more nucleotides in length.
[9029] [0162.0.0.20] see [0162.0.0.0]
[9030] [0163.0.20.20] Preferably, a nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table IA or IB, columns 5 or 7, lines 251 to 261,
627 corresponds to a naturally-occurring nucleic acid molecule of
the invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the increase of
the amount of the respective fine chemical in an organism or a part
thereof, e.g. a tissue, a cell, or a compartment of a cell, after
increasing the expression or activity thereof or the activity of a
protein of the invention or used in the process of the
invention.
[9031] [0164.0.0.20] see [0164.0.0.0]
[9032] [0165.0.20.20] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table IA or IB, columns 5 or 7, lines 251 to 261, 627,
resp.
[9033] [0166.0.0.20] to [0167.0.0.20]: see [0166.0.0.0] to
[0167.0.0.0]
[9034] [0168.0.20.20] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627, resp., yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table IIA or IIB, columns 5 or
7, lines 251 to 261, 627, resp., and is capable of participation in
the increase of production of the respective fine chemical after
increasing its activity, e.g. its expression. Preferably, the
protein encoded by the nucleic acid molecule is at least about 60%
identical to a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627, resp., more preferably at least about
70% identical to one of the sequences as indicated in Table IIA or
IIB, columns 5 or 7, lines 251 to 261, 627, resp., even more
preferably at least about 80%, 90%, 95% homologous to a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627, resp., and most preferably at least about 96%, 97%, 98%, or
99% identical to the sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 251 to 261, 627.
[9035] [0169.0.0.20] to [0172.0.0.20]: see [0169.0.0.0] to
[0172.0.0.0]
[9036] [0173.0.20.20] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 25525 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 25525 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[9037] [0174.0.0.20]: see [0174.0.0.0]
[9038] [0175.0.20.20] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 25526 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 25526 by the above program algorithm with the
above parameter set, has a 80% homology.
[9039] [0176.0.20.20] Functional equivalents derived from one of
the polypeptides as indicated in Table IIA or IIB, columns 5 or 7,
lines 251 to 261, 627, resp., according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as indicated in Table IIA
or IIB, columns 5 or 7, lines 251 to 261, 627, resp., according to
the invention and are distinguished by essentially the same
properties as a polypeptide as indicated in Table IIA or IIB,
columns 5 or 7, lines 251 to 261, 627, resp.
[9040] [0177.0.20.20] Functional equivalents derived from a nucleic
acid sequence as indicated in Table IA or IB, columns 5 or 7, lines
251 to 261, 627, resp., according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table IIA or
IIB, columns 5 or 7, lines 251 to 261, 627, resp., according to the
invention and encode polypeptides having essentially the same
properties as a polypeptide as indicated in Table IIA or IIB,
columns 5 or 7, lines 251 to 261, 627, resp.
[9041] [0178.0.0.20] see [0178.0.0.0]
[9042] [0179.0.20.20] A nucleic acid molecule encoding a homologue
to a protein sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627, resp., can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table IA or IB, columns 5
or 7, lines 251 to 261, 627, resp., such that one or more amino
acid substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced into the encoding
sequences as indicated in Table IA or IB, columns 5 or 7, lines 251
to 261, 627, resp., by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis.
[9043] [0180.0.0.20] to [0183.0.0.20]: see [0180.0.0.0] to
[0183.0.0.0]
[9044] [0184.0.20.20] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table IA or IB, columns 5 or
7, lines 251 to 261, 627, resp., or of the nucleic acid sequences
derived from a sequences as indicated in Table IIA or IIB, columns
5 or 7, lines 251 to 261, 627, resp., comprise also allelic
variants with at least approximately 30%, 35%, 40% or 45% homology,
by preference at least approximately 50%, 60% or 70%, more
preferably at least approximately 90%, 91%, 92%, 93%, 94% or 95%
and even more preferably at least approximately 96%, 97%, 98%, 99%
or more homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
IA or IB, columns 5 or 7, lines 251 to 261, 627, resp., or from the
derived nucleic acid sequences, the intention being, however, that
the enzyme activity or the biological activity of the resulting
proteins synthesized is advantageously retained or increased.
[9045] [0185.0.20.20] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table IA or IB, columns 5 or 7, lines 251 to 261, 627, resp. In one
embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotides not shown in any
one of sequences as indicated in Table IA or IB, columns 5 or 7,
lines 251 to 261, 627, resp. In one embodiment, the nucleic acid
molecule comprises less than 500, 400, 300, 200, 100, 90, 80, 70,
60, 50 or 40 further nucleotides. In a further embodiment, the
nucleic acid molecule comprises less than 30, 20 or 10 further
nucleotides. In one embodiment, a nucleic acid molecule used in the
process of the invention is identical to a sequence as indicated in
Table IA or IB, columns 5 or 7, lines 251 to 261, 627, resp.
[9046] [0186.0.20.20] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 251 to 261, 627, resp. In one embodiment, the
nucleic acid molecule encodes less than 150, 130, 100, 80, 60, 50,
40 or 30 further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table IIA or IIB, columns 5 or 7, lines 251 to 261, 627,
resp.
[9047] [0187.0.20.20] In one embodiment, a nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627, resp., comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a sequences as indicated in Table IIA or IIB,
columns 5 or 7, lines 251 to 261, 627, resp.
[9048] [0188.0.20.20] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity.
Advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
IIA or IIB, columns 5 or 7, lines 251 to 261, 627, resp., and is
expressed under identical conditions.
[9049] [0189.0.20.20] Homologues of a sequences as indicated in
Table IA or IB, columns 5 or 7, lines 251 to 261, 627, resp., or of
derived sequences as indicated in Table IIA or IIB, columns 5 or 7,
lines 251 to 261, 627, resp., also mean truncated sequences, cDNA,
single-stranded DNA or RNA of the coding and noncoding DNA
sequence. Homologues of said sequences are also understood as
meaning derivatives, which comprise noncoding regions such as, for
example, UTRs, terminators, enhancers or promoter variants. The
promoters upstream of the nucleotide sequences stated can be
modified by one or more nucleotide substitution(s), insertion(s)
and/or deletion(s) without, however, interfering with the
functionality or activity either of the promoters, the open reading
frame (=ORF) or with the 3'-regulatory region such as terminators
or other 3' regulatory regions, which are far away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[9050] [0190.0.0.20]: see [0190.0.0.0]
[9051] [0191.0.20.20] The organisms or part thereof produce
according to the herein mentioned process of the invention an
increased level of free and/or bound the respective fine chemical
compared to said control or selected organisms or parts
thereof.
[9052] [0192.0.0.20] to [0203.0.0.20]: see [0192.0.0.0] to
[0203.0.0.0]
[9053] [0204.0.20.20] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [9054] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
251 to 261, 627, resp.; or a fragment thereof conferring an
increase in the amount of the respective fine chemical, i.e.
lignoceric acid (lines 251, 255, 259, 261, 627) or cerotic acid
(lines 251, 252, 253, 254, 259) or melissic acid (lines 256, 257,
260) or behenic acid (line 258), resp., in an organism or a part
thereof, [9055] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule as indicated in
Table IA or IB, columns 5 or 7, lines 251 to 261, 627, resp., or a
fragment thereof conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [9056]
c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [9057] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [9058] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [9059]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c),
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [9060] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[9061] h) nucleic acid molecule comprising a nucleic acid molecule
which is obtained by amplifying a cDNA library or a genomic library
using primers or primer pairs as indicated in I Table III, columns
5 or 7, lines 251 to 261, 627 and conferring an increase in the
amount of the respective fine chemical, i.e. lignoceric acid (lines
251, 255, 259, 261, 627) or cerotic acid (lines 251, 252, 253, 254,
259) or melissic acid (lines 256, 257, 260) or behenic acid (line
258), resp., in an organism or a part thereof; [9062] i) nucleic
acid molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [9063] j) nucleic acid molecule which encodes a
polypeptide comprising a consensus sequence as indicated in Table
IV, column 7, lines 252 to 259, 261 and 627 and conferring an
increase in the amount of the respective fine chemical, i.e.
lignoceric acid (lines 251, 255, 259, 261, 627) or cerotic acid
(lines 251, 252, 253, 254, 259) or melissic acid (lines 256, 257,
260) or behenic acid (line 258), resp., in an organism or a part
thereof; [9064] k) nucleic acid molecule encoding the amino acid
sequence of a polypeptide encoding a domaine of a polypeptide as
indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627, resp., and conferring an increase in the amount of the
respective fine chemical, i.e. lignoceric acid (lines 251, 255,
259, 261, 627) or cerotic acid (lines 251, 252, 253, 254, 259) or
melissic acid (lines 256, 257, 260) or behenic acid (line 258)
resp., in an organism or a part thereof; and [9065] l) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment of at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (h) or of a nucleic acid molecule as
indicated in
[9066] Table IA or IB, columns 5 or 7, lines 251 to 261, 627,
resp., or a nucleic acid molecule encoding, preferably at least the
mature form of, a polypeptide as indicated in Table IIA or IIB,
columns 5 or 7, lines 251 to 261, 627, resp., and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; or which encompasses a sequence which
is complementary thereto;
whereby, preferably, the nucleic acid molecule according to (a) to
(l) distinguishes over a sequence as indicated in Table IA or IB,
columns 5 or 7, lines 251 to 261, 627, resp., by one or more
nucleotides. In one embodiment, the nucleic acid molecule of the
invention does not consist of the sequence as indicated in Table IA
or IB, columns 5 or 7, lines 251 to 261, 627, resp. In another
embodiment, the nucleic acid molecule of the present invention is
at least 30% identical and less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence as indicated in Table IA or IB,
columns 5 or 7, lines 251 to 261, 627, resp. In a further
embodiment the nucleic acid molecule does not encode a polypeptide
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
251 to 261, 627, resp. Accordingly, in one embodiment, the nucleic
acid molecule of the present invention encodes in one embodiment a
polypeptide which differs at least in one or more amino acids from
a polypeptide indicated in Table IIA or IIB, columns 5 or 7, lines
251 to 261, 627 does not encode a protein of a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627. Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627. In a further embodiment, the protein
of the present invention is at least 30% identical to a protein
sequence indicated in Table IIA or IIB, columns 5 or 7, lines 251
to 261, 627 and less than 100%, preferably less than 99.999%,
99.99% or 99.9%, more preferably less than 99%, 985, 97%, 96% or
95% identical to a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 251 to 261, 627.
[9067] [0205.0.0.20] to [0206.0.0.20]: see [0205.0.0.0] to
[0206.0.0.0]
[9068] [0207.0.20.20] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the glutamic acid metabolism, the
phosphoenolpyruvate metabolism, the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[9069] [0208.0.0.20] to [0226.0.0.20]: see [0208.0.0.0] to
[0226.0.0.0]
[9070] [0227.0.20.20] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[9071] In addition to a sequence indicated in Table IA or IB,
columns 5 or 7, lines 251 to 261, 627 or its derivatives, it is
advantageous to express and/or mutate further genes in the
organisms. Especially advantageously, additionally at least one
further gene of the acetyl-CoA or malonyl-CoA metabolic pathway or
a polypeptide having a very long chain fatty acid acyl (VLCFA) CoA
synthase activity, is expressed in the organisms such as plants or
microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the fine chemicals desired since, for example,
feedback regulations no longer exist to the same extent or not at
all. In addition it might be advantageously to combine one or more
of the sequences indicated in Table IA or
[9072] IB, columns 5 or 7, lines 251 to 261, 627, resp., with genes
which generally support or enhance to growth or yield of the target
organisms, for example genes which lead to faster growth rate of
microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[9073] [0228.0.20.20] %
[9074] [0229.0.20.20] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences indicated
in Table IA or IB, columns 5 or 7, lines 251 to 261, 627 used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the fatty acid pathway, such as
acetyl-CoA or malonyl-CoA or a polypeptide having a very long chain
fatty acid acyl (VLCFA) CoA synthase activity. These genes can lead
to an increased synthesis of the VLCFAs.
[9075] [0230.0.0.20] see [230.0.0.0]
[9076] [0231.0.20.20] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a behenic acid, lignoceric acid,
cerotic acid or melissic acid degrading protein is attenuated, in
particular by reducing the rate of expression of the corresponding
gene. A person skilled in the art knows for example, that the
inhibition or repression of a behenic acid, lignoceric acid,
cerotic acid or melissic acid degrading enzyme will result in an
increased accumulation of behenic acid, lignoceric acid, cerotic
acid or melissic acid in plants.
[9077] [0232.0.0.20] to [0276.0.0.20]: see [0232.0.0.0] to
[0276.0.0.0]
[9078] [0277.0.20.20] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously.
[9079] [0278.0.0.20] to [0282.0.0.20]: see [0278.0.0.0] to
[0282.0.0.0]
[9080] [0283.0.20.20] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells, for example using the antibody
of the present invention as described below, e.g. an antibody
against a protein as indicated in Table IIA or IIB, column 3, lines
251 to 261, 627, resp., or an antibody against a polypeptide as
indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627, resp., which can be produced by standard techniques utilizing
the polypeptid of the present invention or fragment thereof.
Preferred are monoclonal antibodies.
[9081] [0284.0.0.20] see [0284.0.0.0]
[9082] [0285.0.20.20] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 251 to 261, 627, resp., or as
coded by a nucleic acid molecule as indicated in Table IA or IB,
columns 5 or 7, lines 251 to 261, 627, resp., or functional
homologues thereof.
[9083] [0286.0.20.20] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 251 to 259 and 261 and 627 and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table IV, column 7, lines 251 to 259 and 261 and 627 whereby 20 or
less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred
5 or 4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table IV, column 7, lines 251 to 259, 261 and 627,
[9084] [0287.0.0.20] to [0289.0.0.20]: see [0287.0.0.0] to
[0289.0.0.0]
[9085] [00290.0.20.20] The alignment was performed with the
Software AlignX (Sep. 25, 2002) a component of Vector NTI Suite
8.0, InforMax.TM., Invitrogen.TM. life science software, U.S. Main
Office, 7305 Executive Way, Frederick, Md. 21704, USA with the
following settings: For pairwise alignments: gap opening penalty:
10.0; gap extension penalty 0.1. For multiple alignments: Gap
opening penalty: 10.0; Gap extension penalty: 0.1; Gap separation
penalty range: 8; Residue substitution matrix: blosum62;
Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0.5;
Consensus calculation options: Residue fraction for consensus: 0.9.
Presettings were selected to allow also for the alignment of
conserved amino acids
[9086] [0291.0.20.20] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[9087] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 251 to 261, 627, resp., by one or more amino
acids. In one embodiment, polypeptide distinguishes from a sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627, resp., by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids and, preferably, the sequence
of the polypeptide of the invention distinguishes from a sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627, resp., by not more than 80% or 70% of the amino acids,
preferably not more than 60% or 50%, more preferred not more than
40% or 30%, even more preferred not more than 20% or 10%. In
another embodiment, said polypeptide of the invention does not
consist of a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627.
[9088] [0292.0.0.20] see [0292.0.0.0]
[9089] [0293.0.20.20] In one embodiment, the invention relates to a
polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or by a nucleic acid molecule used in the process of the
invention. In one embodiment, the polypeptide of the invention has
a sequence which distinguishes from a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 251 to 261, 627, resp., by
one or more amino acids. In another embodiment, said polypeptide of
the invention does not consist of the sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 251 to 261, 627, resp. In a
further embodiment, said polypeptide of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical. In one
embodiment, said polypeptide does not consist of the sequence
encoded by a nucleic acid molecules as indicated in Table IA or IB,
columns 5 or 7, lines 251 to 261, 627, resp.
[9090] [0294.0.20.20] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table IIA or IIB, column 3, lines 251 to 261, 627,
resp., which distinguishes over a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 251 to 261, 627, resp., by one or
more amino acids, preferably by more than 5, 6, 7, 8 or 9 amino
acids, preferably by more than 10, 15, 20, 25 or 30 amino acids,
even more preferred are more than 40, 50, or 60 amino acids but
even more preferred by less than 70% of the amino acids, more
preferred by less than 50%, even more preferred my less than 30% or
25%, more preferred are 20% or 15%, even more preferred are less
than 10%.
[9091] [0295.0.0.20] to [0296.0.0.20]: see [0295.0.0.0] to
[0296.0.0.0]
[9092] [0297.0.0.20] see [0297.0.0.0]
[9093] [00297.1.0.20] %
[9094] [0298.0.20.20] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627, resp. The portion of the protein is preferably a biologically
active portion as described herein. Preferably, the polypeptide
used in the process of the invention has an amino acid sequence
identical to a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627, resp.
[9095] [0299.0.20.20] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table IA or IB, columns 5 or 7,
lines 251 to 261, 627, resp. The preferred polypeptide of the
present invention preferably possesses at least one of the
activities according to the invention and described herein. A
preferred polypeptide of the present invention includes an amino
acid sequence encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions, to a nucleotide
sequence as indicated in Table IA or IB, columns 5 or 7, lines 251
to 261, 627, resp., or which is homologous thereto, as defined
above.
[9096] [0300.0.20.20] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 251 to 261, 627, resp., in amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of as indicated in Table IIA or IIB,
columns 5 or 7, lines 251 to 261, 627, resp.
[9097] [0301.0.0.20] see [0301.0.0.0]
[9098] [0302.0.20.20] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 251 to 261, 627, resp., or
the amino acid sequence of a protein homologous thereto, which
include fewer amino acids than a full length polypeptide of the
present invention or used in the process of the present invention
or the full length protein which is homologous to an polypeptide of
the present invention or used in the process of the present
invention depicted herein, and exhibit at least one activity of
polypeptide of the present invention or used in the process of the
present invention.
[9099] [0303.0.0.20] see [0303.0.0.0]
[9100] [0304.0.20.20] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
IIA or IIB, column 3, lines 251 to 261, 627 but having differences
in the sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[9101] [0305.0.0.20] to [0308.0.0.20]: see [0305.0.0.0] to
[0308.0.0.0]
[9102] [0309.0.20.20] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table IIA or
IIB, columns 5 or 7, lines 251 to 261, 627, resp., refers to a
polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas an "other polypeptide" not being indicated in
Table IIA or IIB, columns 5 or 7, lines 251 to 261, 627, resp.,
refers to a polypeptide having an amino acid sequence corresponding
to a protein which is not substantially homologous to a polypeptide
of the invention, preferably which is not substantially homologous
to a polypeptide as indicated in Table IIA or IIB, columns 5 or 7,
lines 251 to 261, 627, resp., e.g., a protein which does not confer
the activity described herein or annotated or known for as
indicated in Table IIA or IIB, column 3, lines 251 to 261, 627,
resp., and which is derived from the same or a different organism.
In one embodiment, an "other polypeptide" not being indicated in
Table IIA or IIB, columns 5 or 7, lines 251 to 261, 627, resp.,
does not confer an increase of the respective fine chemical in an
organism or part thereof.
[9103] [0310.0.0.20] to [0334.0.0.20]: see [0310.0.0.0] to
[0334.0.0.0]
[9104] [0335.0.20.20] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table IA or IB, columns
5 or 7, lines 251 to 261, 627, resp., and/or homologs thereof. As
described inter alia in WO 99/32619, dsRNAi approaches are clearly
superior to traditional antisense approaches. The invention
therefore furthermore relates to double-stranded RNA molecules
(dsRNA molecules) which, when introduced into an organism,
advantageously into a plant (or a cell, tissue, organ or seed
derived there from), bring about altered metabolic activity by the
reduction in the expression of a nucleic acid sequences as
indicated in Table IA or IB, columns 5 or 7, lines 251 to 261, 627,
resp., and/or homologs thereof. In a double-stranded RNA molecule
for reducing the expression of a protein encoded by a nucleic acid
sequence as indicated in Table IA or IB, columns 5 or 7, lines 251
to 261, 627, resp., and/or homologs thereof, one of the two RNA
strands is essentially identical to at least part of a nucleic acid
sequence, and the respective other RNA strand is essentially
identical to at least part of the complementary strand of a nucleic
acid sequence.
[9105] [0336.0.0.20] to [0342.0.0.20]: see [0336.0.0.0] to
[0342.0.0.0]
[9106] [0343.0.20.20] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table IA or IB, columns 5 or 7, lines 251
to 261, 627, resp., or its homolog is not necessarily required in
order to bring about effective reduction in the expression. The
advantage is, accordingly, that the method is tolerant with regard
to sequence deviations as may be present as a consequence of
genetic mutations, polymorphisms or evolutionary divergences. Thus,
for example, using the dsRNA, which has been generated starting
from a sequence as indicated in Table IA or IB, columns 5 or 7,
lines 251 to 261, 627, resp., or homologs thereof of the one
organism, may be used to suppress the corresponding expression in
another organism.
[9107] [0344.0.0.20] to [0350.0.0.20]: see [0344.0.0.0] to
[0350.0.0.0]
[9108] [0351.0.0.20] to [0361.0.0.20]: see [0351.0.0.0] to
[0361.0.0.0]
[9109] [0362.0.20.20] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table IIA or IIB, columns 5 or 7, lines 251 to 261, 627, resp.,
e.g. encoding a polypeptide having protein activity, as indicated
in Table IIA or IIB, columns 3, lines 251 to 261, 627, resp. Due to
the above-mentioned activity the respective fine chemical content
in a cell or an organism is increased. For example, due to
modulation or manipulation, the cellular activity of the
polypeptide of the invention or nucleic acid molecule of the
invention is increased, e.g. due to an increased expression or
specific activity of the subject matters of the invention in a cell
or an organism or a part thereof. Transgenic for a polypeptide
having an activity of a polypeptide as indicated in Table IIA or
IIB, columns 5 or 7, lines 251 to 261, 627, resp., means herein
that due to modulation or manipulation of the genome, an activity
as annotated for a polypeptide as indicated in Table IIA or IIB,
column 3, lines 251 to 261, 627, e.g. having a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627, resp., is increased in a cell or an organism or a part
thereof. Examples are described above in context with the process
of the invention.
[9110] [0363.0.0.20] see [0363.0.0.0]
[9111] [0364.0.20.20] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table IIA or IIB, column 3, lines 251 to 261, 627,
resp. with the corresponding protein-encoding sequence as indicated
in Table IA or IB, column 5, lines 251 to 261, 627, resp., becomes
a transgenic expression cassette when it is modified by
non-natural, synthetic "artificial" methods such as, for example,
mutagenization. Such methods have been described (U.S. Pat. No.
5,565,350; WO 00/15815; also see above).
[9112] [0365.0.0.20] to [0373.0.0.20]: see [0365.0.0.0] to
[0373.0.0.0]
[9113] [0374.0.20.20] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. behenic acid, lignoceric
acid, cerotic acid or melissic acid, in particular the respective
fine chemical, produced in the process according to the invention
may, however, also be isolated from the plant in the form of their
free behenic acid, lignoceric acid, cerotic acid or melissic acid,
in particular the free respective fine chemical, or bound in or to
compounds or moieties, like glucosides, e.g. diglucosides. The
respective fine chemical produced by this process can be harvested
by harvesting the organisms either from the culture in which they
grow or from the field. This can be done via expressing, grinding
and/or extraction, salt precipitation and/or ion-exchange
chromatography or other chromatographic methods of the plant parts,
preferably the plant seeds, plant fruits, plant tubers and the
like.
[9114] [0375.0.0.20] to [0376.0.0.20]: see [0375.0.0.0] to
[0376.0.0.0]
[9115] [0377.0.20.20] Accordingly, the present invention relates
also to a process whereby the produced behenic acid, lignoceric
acid, cerotic acid or melissic acid is isolated.
[9116] [0378.0.20.20] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the behenic acid,
lignoceric acid, cerotic acid or melissic acid produced in the
process can be isolated. The resulting behenic acid, lignoceric
acid, cerotic acid or melissic acid can, if appropriate,
subsequently be further purified, if desired mixed with other
active ingredients such as vitamins, amino acids, carbohydrates,
antibiotics and the like, and, if appropriate, formulated.
[9117] [0379.0.20.20] In one embodiment the product produced by the
present invention is a mixture of the respective fine chemicals
behenic acid, lignoceric acid, cerotic acid and/or melissic
acid.
[9118] [0380.0.20.20] The behenic acid, lignoceric acid, cerotic
acid or melissic acid obtained in the process by carrying out the
invention is suitable as starting material for the synthesis of
further products of value. For example, they can be used in
combination with each other or alone for the production of
pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates to a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the behenic acid, lignoceric
acid, cerotic acid or melissic acid composition produced or the
respective fine chemical produced if desired and formulating the
product with a pharmaceutical acceptable carrier or formulating the
product in a form acceptable for an application in agriculture. A
further embodiment according to the invention is the use of the
behenic acid, lignoceric acid, cerotic acid or melissic acid
produced in the process or of the transgenic organism in animal
feeds, foodstuffs, medicines, food supplements, cosmetics or
pharmaceuticals or for the production of behenic acid, lignoceric
acid, cerotic acid or melissic acid e.g. after isolation of the
respective fine chemical or without, e.g. in situ, e.g in the
organism used for the process for the production of the respective
fine chemical.
[9119] [0381.0.0.20] to [0382.0.0.20]: see [0381.0.0.0] to
[0382.0.0.0] [0383.0.20.20]
[9120] [0384.0.0.20] see [0384.0.0.0]
[9121] [0385.0.20.20] The fermentation broths obtained in this way,
containing in particular behenic acid, lignoceric acid, cerotic
acid or melissic acid in mixtures with other organic acids, amino
acids, polypeptides or polysaccarides, normally have a dry matter
content of from 1 to 70% by weight, preferably 7.5 to 25% by
weight. Sugar-limited fermentation is additionally advantageous,
e.g. at the end, for example over at least 30% of the fermentation
time. This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 10 g/l,
preferably to 0 to 3 g/l during this time. The fermentation broth
is then processed further. Depending on requirements, the biomass
can be removed or isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth or left completely in it.
[9122] The fermentation broth can then be thickened or concentrated
by known methods, such as, for example, with the aid of a rotary
evaporator, thin-film evaporator, falling film evaporator, by
reverse osmosis or by nanofiltration. This concentrated
fermentation broth can then be worked up by freeze-drying, spray
drying, spray granulation or by other processes.
[9123] [0386.0.20.20] Accordingly, it is possible to purify the
behenic acid, lignoceric acid, cerotic acid or melissic acid
produced according to the invention further. For this purpose, the
product-containing composition is subjected for example to
separation via e.g. an open column chromatography or HPLC in which
case the desired product or the impurities are retained wholly or
partly on the chromatography resin. These chromatography steps can
be repeated if necessary, using the same or different
chromatography resins. The skilled worker is familiar with the
choice of suitable chromatography resins and their most effective
use.
[9124] [0387.0.0.20] to [0392.0.0.20]: see [0387.0.0.0] to
[0392.0.0.0]
[9125] [0393.0.20.20] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [9126] a. contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [9127] b. identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table IA or IB, columns 5 or 7,
lines 251 to 261, 627, resp., and, optionally, isolating the full
length cDNA clone or complete genomic clone; [9128] c. introducing
the candidate nucleic acid molecules in host cells, preferably in a
plant cell or a microorganism, appropriate for producing the
respective fine chemical; [9129] d. expressing the identified
nucleic acid molecules in the host cells; [9130] e. assaying the
respective fine chemical level in the host cells; and [9131] f.
identifying the nucleic acid molecule and its gene product which
expression confers an increase in the respective fine chemical
level in the host cell after expression compared to the wild
type.
[9132] [0394.0.0.20] to [0398.0.0.20]: see [0394.0.0.0] to
[0398.0.0.0]
[9133] [0399.0.20.20] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increase of the respective fine chemical production in a
plant or microorganism, comprising the steps:
[9134] culturing a cell or tissue or microorganism or maintaining a
plant expressing the polypeptide according to the invention or a
nucleic acid molecule encoding said polypeptide and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and the polypeptide of the present invention or used
in the process of the invention; and
[9135] identifying if the compound is an effective agonist by
detecting the presence or absence or increase of a signal produced
by said readout system.
[9136] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in respective fine chemical
production.
[9137] [00399.1.20.20] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table IIA
or IIB, columns 5 or 7, lines 251 to 261, 627 or a homolog thereof,
e.g. comparing the phenotype of nearly identical organisms with low
and high activity of a protein as indicated in Table IIA or IIB,
columns 5 or 7, lines 251 to 261, 627 after incubation with the
drug.
[9138] [0400.0.0.20] to [0415.0.0.20]: see [0400.0.0.0] to
[0415.0.0.0]
[9139] [0416.0.0.20] see [0416.0.0.0]
[9140] [0417.0.20.20] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the behenic acid, lignoceric acid,
cerotic acid or melissic acid biosynthesis pathways. In particular,
the overexpression of the polypeptide of the present invention may
protect an organism such as a microorganism or a plant against
inhibitors, which block the behenic acid, lignoceric acid, cerotic
acid or melissic acid synthesis.
[9141] Examples for such inhibitors are oxyacetamides,
oxyacetamides or chloroacetamides which inhibit the first step in
VLCFAs biosynthesis, the condensation of acyl-CoA with malonyl-CoA
to .beta.-ketoacyl-CoA (Matthes, 2001; Schmalfuss, J., Matthes, B,
and P. Boger: Chloroacetamide mode of action. Abstr. Meeting Weed
Science Society of America, Toronto, 40, 117-118, 2000). Therefore
a plant overproducing behenic acid, lignoceric acid, cerotic acid
and/or melissic acid could result in the resistance to
chloroacetamide-type herbicides.
[9142] [0418.0.0.20] to [0423.0.0.20]: see [0418.0.0.0] to
[0423.0.0.0]
[9143] [0424.0.20.20] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other organic acids.
Accordingly, the nucleic acid of the invention, or the nucleic acid
molecule identified with the method of the present invention or the
complement sequences thereof, the polypeptide of the invention, the
nucleic acid construct of the invention, the organisms, the host
cell, the microorganisms, the plant, plant tissue, plant cell, or
the part thereof of the invention, the vector of the invention, the
antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the respective
fine chemical in a organism or part thereof, e.g. in a cell.
[9144] [0425.0.0.20] to [0434.0.0.0]: see [0425.0.0.0] to
[0434.0.0.0]
[0435.0.20.20] Example 3
In-Vivo and In-Vitro Mutagenesis
[9145] [0436.0.20.20] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of behenic acid, lignoceric
acid, cerotic acid or melissic acid can be carried out by passing a
plasmid DNA (or another vector DNA) containing the desired nucleic
acid sequence or nucleic acid sequences, e.g. the nucleic acid
molecule of the invention or the vector of the invention, through
E. coli and other microorganisms (for example Bacillus spp. or
yeasts such as Saccharomyces cerevisiae) which are not capable of
maintaining the integrity of its genetic information. Usual mutator
strains have mutations in the genes for the DNA repair system [for
example mutHLS, mutD, mutT and the like; for comparison, see Rupp,
W. D. (1996) DNA repair mechanisms in Escherichia coli and
Salmonella, pp. 2277-2294, ASM: Washington]. The skilled worker
knows these strains. The use of these strains is illustrated for
example in Greener, A. and Callahan, M. (1994) Strategies 7;
32-34.
[9146] In-vitro mutation methods such as increasing the spontaneous
mutation rates by chemical or physical treatment are well known to
the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[9147] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[9148] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.20.20] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpina
[9149] [0438.0.20.20] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology", VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[9150] [0439.0.20.20] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255f) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[9151] [0440.0.20.20] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002) teaches a homolog recombination system dependent on
rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this system
for homologous recombination are vectors derived from the Ylp
series. Plasmid vectors derived for example from the 2.mu.-Vector
are known by the skilled worker and used for the expression in
yeasts. Other preferred vectors are for example pART1, pCHY21 or
pEVP11 as they have been described by McLeod et al. (EMBO J. 1987,
6:729-736) and Hoffman et al. (Genes Dev. 5, 1991: :561-571.) or
Russell et al. (J. Biol. Chem. 258, 1983: 143-149.). Other
beneficial yeast vectors are plasmids of the REP, REP-X, pYZ or RIP
series.
[9152] [0441.0.0.20] see [0441.0.0.0]
[9153] [0442.0.0.20] see [0442.0.0.0]
[9154] [0443.0.0.20] see [0443.0.0.0]
[0444.0.20.20] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[0445.0.20.20] Example 8
[9155] Genetically modified Yeast, Mortierella or Escherichia coli
are grown in synthetic or natural growth media known by the skilled
worker. A number of different growth media for Yeast, Mortierella
or Escherichia coli are well known and widely available. A method
for culturing Mortierella is disclosed by Jang et al. [Bot. Bull.
Acad. Sin. (2000) 41:41-48]. Mortierella can be grown at 20.degree.
C. in a culture medium containing: 10 g/l glucose, 5 g/l yeast
extract at pH 6.5. Furthermore Jang et al. teaches a submerged
basal medium containing 20 g/l soluble starch, 5 g/l Bacto yeast
extract, 10 g/l KNO.sub.3, 1 g/l KH.sub.2PO.sub.4, and 0.5 g/l
MgSO.sub.4.7H.sub.2O, pH 6.5.
[9156] [0446.0.0.20] to [0450.0.0.20]: see [0446.0.0.0] to
[0450.0.0.0]
[9157] [0451.0.0.20] see [0451.0.5.5]
[9158] [0452.0.0.20] to [0453.0.0.20]: see [0452.0.0.0] to
[0453.0.0.0]
[0454.0.20.20] Example 8
Analysis of the Effect of the Nucleic Acid Molecule on the
Production of Behenic Acid, Lignoceric Acid, Cerotic Acid or
Melissic Acid
[9159] [0455.0.20.20] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a behenic acid, lignoceric acid, cerotic acid or
melissic acid) can be determined by growing the modified
microorganisms or the modified plant under suitable conditions
(such as those described above) and analyzing the medium and/or the
cellular components for the elevated production of desired product
(i.e. of behenic acid, lignoceric acid, cerotic acid or melissic
acid). These analytical techniques are known to the skilled worker
and comprise spectroscopy, thin-layer chromatography, various types
of staining methods, enzymatic and microbiological methods and
analytical chromatography such as high-performance liquid
chromatography (see, for example, Ullman, Encyclopedia of
Industrial Chemistry, Vol. A2, p. 89-90 and p. 443-613, VCH:
Weinheim (1985); Fallon, A., et al., (1987) "Applications of HPLC
in Biochemistry" in: Laboratory Techniques in Biochemistry and
Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol.
3, Chapter III: "Product recovery and purification", p. 469-714,
VCH: Weinheim; Better, P. A., et al. (1988) Bioseparations:
downstream processing for Biotechnology, John Wiley and Sons;
Kennedy, J. F., and Cabral, J. M. S. (1992) Recovery processes for
biological Materials, John Wiley and Sons; Shaeiwitz, J. A., and
Henry, J. D. (1988) Biochemical Separations, in: Ullmann's
Encyclopedia of Industrial Chemistry, Vol. B3; Chapter 11, p. 1-27,
VCH: Weinheim; and Dechow, F. J. (1989) Separation and purification
techniques in biotechnology, Noyes Publications).
[9160] [0456.0.0.20]: see [0456.0.0.0]
[0457.0.20.20] Example 9
Purification of Behenic Acid, Lignoceric Acid, Cerotic Acid or
Melissic Acid
[9161] [0458.0.20.20] Abbreviations; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[9162] The unambiguous detection for the presence of behenic acid,
lignoceric acid, cerotic acid or melissic acid can be obtained by
analyzing recombinant organisms using analytical standard methods:
LC, LC-MSMS, GC-MS or TLC, as described. The total amount produced
in the organism for example in yeasts used in the inventive process
can be analysed for example according to the following
procedure:
[9163] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods.
[9164] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[9165] A typical sample pretreatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography.
[9166] For analysis, solvent delivery and aliquot removal can be
accomplished with a robotic system comprising a single injector
valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000
W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of
50% potassium hydroxide hydro-ethanolic solution (4 water--1
ethanol) can be added to each vial, followed by the addition of 3
ml of octanol. The saponification treatment can be conducted at
room temperature with vials maintained on an IKA HS 501 horizontal
shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours
at 250 movements/minute, followed by a stationary phase of
approximately one hour.
[9167] Following saponification, the supernatant can be diluted
with 0.20 ml of methanol. The addition of methanol can be conducted
under pressure to ensure sample homogeneity. Using a 0.25 ml
syringe, a 0.1 ml aliquot can be removed and transferred to HPLC
vials for analysis.
[9168] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete
with a quaternary pump, vacuum degassing system, six-way injection
valve, temperature regulated autosampler, column oven and
Photodiode Array detector can be used [Agilent Technologies
available through Ultra Scientific Inc., 250 Smith Street, North
Kingstown, R.I.]. The column can be a Waters YMC30, 5-micron,
4.6.times.250 mm with a guard column of the same material [Waters,
34 Maple Street, Milford, Mass.]. The solvents for the mobile phase
can be 81 methanol: 4 water: 15 tetrahydrofuran (THF) stabilized
with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injections were
20 .mu.l. Separation can be isocratic at 30.degree. C. with a flow
rate of 1.7 ml/minute. The peak responses can be measured by
absorbance at 447 nm.
[9169] [0459.0.20.20] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the behenic acid, lignoceric acid, cerotic acid or
melissic acid can be further purified with a so-called RTHPLC. As
eluent acetonitrile/water or chloroform/acetonitrile mixtures can
be used. If necessary, these chromatography steps may be repeated,
using identical or other chromatography resins. The skilled worker
is familiar with the selection of suitable chromatography resin and
the most effective use for a particular molecule to be
purified.
[9170] [0460.0.0.20] see [0460.0.0.0]
[0461.0.20.20] Example 10
Cloning SEQ ID NO: 25525, 25617, 26065, 26455, 26631, 26659, 26795,
26837, 26983, 27031, 27035 and 97565 for the Expression in
Plants
[9171] [0462.0.0.20] see [0462.0.0.0]
[9172] [0463.0.20.20] SEQ ID NO: 25525, 25617, 26065, 26455, 26631,
26659, 26795, 26837, 26983, 27031, 27035 and 97565 is amplified by
PCR as described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[9173] [0464.0.0.0.20] to [0466.0.0.20]: see [0464.0.0.0] to
[0466.0.0.0]
[9174] [0466.1.0.20] In case the Herculase enzyme can be used for
the amplification, the PCR amplification cycles were as follows: 1
cycle of 2-3 minutes at 94.degree. C., followed by 25-30 cycles of
in each case 30 seconds at 94.degree. C., 30 seconds at
55-60.degree. C. and 5-10 minutes at 72.degree. C., followed by 1
cycle of 10 minutes at 72.degree. C., then 4.degree. C.
[9175] [0467.0.20.20] The following primer sequences were selected
for the gene SEQ ID: 25525
TABLE-US-00098 i) forward primer (SEQ ID NO: 25615) atgaaacatc
tgcatcgatt ctttag ii) reverse primer (SEQ ID NO: 25616) ttaaactgat
ggacgcaaac gaacg
[9176] The following primer sequences were selected for the gene
SEQ ID NO: 25617:
TABLE-US-00099 i) forward primer (SEQ ID NO: 26063) atggaaaagg
gtactgttaa gtg ii) reverse primer (SEQ ID NO: 26064) ttatgcgact
gccgcttcta cttc
[9177] The following primer sequences were selected for the gene
SEQ ID NO: 26065:
TABLE-US-00100 i) forward primer (SEQ ID NO: 26453) atgattaacc
gtatccgcgt agtc ii) reverse primer (SEQ ID NO: 26454 ) ttaaaatgtt
tcccagtttg gatcttg
[9178] The following primer sequences were selected for the gene
SEQ ID NO: 26455:
TABLE-US-00101 i) forward primer (SEQ ID NO: 26629) atgagtcgtt
tagtcgtagt atcta ii) reverse primer (SEQ ID NO: 26630) ttacgcaagc
tttggaaagg tagc
[9179] The following primer sequences were selected for the gene
SEQ ID NO: 26631:
TABLE-US-00102 i) forward primer (SEQ ID NO: 26657) atggctgaat
ggagcggcga ii) reverse primer (SEQ ID NO: 26658) ttagtattcc
cacgtctccg gg
[9180] The following primer sequences were selected for the gene
SEQ ID NO: 26659:
TABLE-US-00103 i) forward primer (SEQ ID NO: 26793) atggagacca
atttttcctt cgact ii) reverse primer (SEQ ID NO: 26794) ctattgaaat
accggcttca atattt
[9181] The following primer sequences were selected for the gene
SEQ ID NO: 26795:
TABLE-US-00104 i) forward primer (SEQ ID NO: 26835) atgaatagcg
taaaaagagt aaagct ii) reverse primer (SEQ ID NO: 26836) ctaggccaaa
gacatcttag cca
[9182] The following primer sequences were selected for the gene
SEQ ID NO: 26837:
TABLE-US-00105 i) forward primer (SEQ ID NO: 26981) atgacgagac
gtactactat taatc ii) reverse primer (SEQ ID NO: 26982) tcacttgttt
attttcgata aaatttcc
[9183] The following primer sequences were selected for the gene
SEQ ID NO: 26983:
TABLE-US-00106 i) forward primer (SEQ ID NO: 27029) atgtcgcctt
tgagaaagac ggtt ii) reverse primer (SEQ ID NO: 27030) tcactcttcc
aggtttgagt acg
[9184] The following primer sequences were selected for the gene
SEQ ID NO: 27031:
TABLE-US-00107 i) forward primer (SEQ ID NO: 27033) atggcggttg
cgatcaaaaa gga ii) reverse primer (SEQ ID NO: 27034) tcaattgata
aatgtacttt caatgatg
[9185] The following primer sequences were selected for the gene
SEQ ID NO: 27035:
TABLE-US-00108 i) forward primer (SEQ ID NO: 27295) atggctcggg
gtgacggaca t (q) reverse primer (SEQ ID NO: 27296) tcatgcttct
tttgcgtgat gcaat
[9186] The following primer sequences were selected for the gene
SEQ ID NO: 97565:
TABLE-US-00109 i) forward primer (SEQ ID NO: 97767) atgatatgct
cacctcagaa caaca ii) reverse primer (SEQ ID NO: 97768) ttaaaaacag
aggctttttc ctctgc
[9187] [0468.0.20.20] to [0470.0.20.20]: see [0468.0.0.0] to
[0470.0.0.0]
[9188] [0470.1.20.20] The PCR-products, produced by Pfu Turbo DNA
polymerase, were phosphorylated using a T4 DNA polymerase using a
standard protocol (e.g. MBI Fermentas) and cloned into the
processed binary vector.
[9189] [0471.0.20.20] see [0471.0.0.0]
[9190] [0471.1.20.20] The DNA termini of the PCR-products, produced
by Herculase DNA polymerase, were blunted in a second synthesis
reaction using Pfu Turbo DNA polymerase. The composition for the
protocol of the blunting the DNA-termini was as follows: 0.2 mM
blunting dTTP and 1.25 u Pfu Turbo DNA polymerase. The reaction was
incubated at 72.degree. C. for 30 minutes. Then the PCR-products
were phosphorylated using a T4 DNA polymerase using a standard
protocol (e.g. MBI Fermentas) and cloned into the processed vector
as well.
[9191] [0472.0.20.20] to [0479.0.20.20]: see [0472.0.0.0] to
[0479.0.0.0]
[0480.0.20.20] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 25525,
25617, 26065, 26455, 26631, 26659, 26795, 26837, 26983, 27031,
27035 or 97565
[9192] [0481.0.0.20] to [0513.0.0.20]: see [0481.0.0.0] to
[0513.0.0.0]
[9193] [0514.0.20.20] As an alternative, lignoceric acid, cerotic
acid, melissic acid or behenic acid can be detected as described in
Beerthuis, R. K., Keppler, J. G., Nature. 1957 Apr. 6;
179(4562):731-2.
[9194] The results of the different plant analyses can be seen from
the table 1 which follows:
TABLE-US-00110 TABLE 1 ORF Metabolite Method Min Max b0019 Cerotic
Acid GC 1.40 2.50 (C26:0) b0019 Lignoceric acid GC 1.46 3.24
(C24:0) b0880 Cerotic Acid GC 2.72 3.94 (C26:0) b1886 Cerotic Acid
GC 1.39 4.35 (C26:0) b1896 Cerotic Acid GC 1.39 1.75 (C26:0) b3938
Lignoceric acid GC 1.37 1.89 (C24:0) YDR513W Melissic Acid GC 1.31
2.08 (C30:0) YER156C Melissic Acid GC 1.33 1.73 (C30:0) YGL205W
Behenic acid GC 1.74 2.32 (C22:0) YHR201C Cerotic Acid GC 1.46 3.27
(C26:0) YHR201C Lignoceric acid GC 1.29 3.71 (C24:0) YLR255C
Melissic Acid GC 1.34 1.88 (C30:0) YPR138C Lignoceric acid GC 1.92
2.60 (C24:0) b0255 Lignoceric acid GC 1.35 2.15 (C24:0)
[9195] [0515.0.20.20] Column 2 shows the metabolite behenic acid,
lignoceric acid, cerotic acid or melissic acid analyzed. Columns 4
and 5 shows the ratio of the analyzed metabolite between the
transgenic plants and the wild type; Increase of the metabolite:
Max: maximal x-fold (normalised to wild type)-Min: minimal x-fold
(normalised to wild type). Decrease of the metabolite: Max: maximal
x-fold (normalised to wild type) (minimal decrease), Min: minimal
x-fold (normalised to wild type) (maximal decrease). Column 3
indicates the analytical method.
[9196] [0516.0.0.20] to [0552.0.0.20]: see [0516.0.0.0] to
[0552.0.0.0]
[0552.1.20.20] Example 15
Metabolite Profiling Info from Zea mays
[9197] Zea mays plants were engineered, grown and analysed as
described in Example 14c.
[9198] The results of the different Zea mays plants analysed can be
seen from Table 2 which follows:
TABLE-US-00111 TABLE 2 ORF_NAME Metabolite Min Max YHR201C Cerotic
acid 1.33 1.68 YHR201C Lignoceric acid 1.42 1.55
[9199] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in cerotic acid and/or
lignoceric acid in genetically modified corn plants expressing the
Saccharomyces cerevisiae nucleic acid sequence YHR201C resp.
[9200] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YHR201C or its homologs, e.g. "the activity of
an exopolyphosphatase", is increased in corn plants, preferably, an
increase of the fine chemical cerotic acid between 33% and 68% is
conferred and/or an increase of the fine chemical lignoceric acid
between 42% and 55% is conferred.
[0552.2.0.20] Example 16
Preparation of Homologous Sequences from Plants
[9201] Different plants can be grown under standard or varying
conditions in the greenhouse. RNA can be extracted following the
protocol of Jones, Dunsmuir and Bedbrook (1985) EMBO J. 4:
2411-2418. Approx. 1 gram of tissue material from various organs is
ground in liquid nitrogen. The powder is transferred to a 13 ml
Falcon tube containing 4.5 ml NTES buffer (100 mM NaCl, 10 mM
Tris/HCl pH 7.5, 1 mM EDTA, 1% SDS; in RNase-free water) and 3 ml
phenol/chloroform/isoamylalcohol (25/24/1), immediately mixed and
stored on ice. The mixture is spun for 10 minutes at 7000 rpm using
a centrifuge (Sorval; SM24 or SS34 rotor). The supernatant is
transferred to a new tube, 1/10th volume of 3 M NaAcetate (pH 5.2;
in RNase-free water) and 1 volume of isopropanol is added, mixed at
stored for 1 hour or overnight at -20.degree. C. The mixture is
spun for 10 minutes at 7000 rpm. The supernatant is discarded and
the pellet washed with 70% ethanol (v/v). The mixture is spun for 5
minutes at 7000 rpm, the supernatant is discarded and the pellet is
air-dried. 1 ml RNase-free water is added and allow the DNA/RNA
pellet to dissolve on ice at 4 C. The nucleic acid solution is
transferred to a 2 ml Eppendorf tube and 1 ml of 4 M LiAcetate is
added. After mixing the solution is kept for at least 3 hours, or
overnight, at 4 C. The mixture is spun for 10 minutes at 14000 rpm,
the supernatant discarded, the pellet washed with 70% Ethanol,
air-dried and dissolved in 200 .mu.l of RNase-free water.
[9202] Total RNA can be used to construct a cDNA-library according
to the manufacturer's protocol (for example using the ZAP-cDNA
synthesis and cloning kit of Stratagene, La Jolla, USA). Basically,
messenger RNA (mRNA) is primed in the first strand synthesis with a
oligo(dT) linker-primer and is reverse-transcribed using reverse
transcriptase. After second strand cDNA synthesis, the
double-stranded cDNA is ligated into the Uni-ZAP XR vector. The
Uni-ZAP XR vector allows in vivo excision of the pBluescript
phagemid. The polylinker of the pBluescript phagemid has 21 unique
cloning sites flanked by T3 and T7 promoters and a choice of 6
different primer sites for DNA sequencing. Systematic single run
sequencing of the expected 5 prime end of the clones can allow
preliminary annotation of the sequences for example with the help
of the pedant pro Software package (Biomax, Munchen). Clones for
the nucleic acids of the invention or used in the process according
to the invention can be identified based on homology search with
standard algorithms like blastp or gap. Identified putative full
length clones with identity or high homology can be subjected to
further sequencing in order to obtain the complete sequence.
[9203] Additional new homologous sequences can be identified in a
similar manner by preparing respective cDNA libraries from various
plant sources as described above. Libraries can then be screened
with available sequences of the invention under low stringency
conditions for example as described in Sambrook et al., Molecular
Cloning: A laboratory manual, Cold Spring Harbor 1989, Cold Spring
Harbor Laboratory Press. Purified positive clones can be subjected
to the in vivo excision and complete sequencing. A pairwise
sequence alignment of the original and the new sequence using the
blastp or gap program allows the identification of orthologs,
meaning homologous sequences from different organisms, which should
have a sequence identity of at least 30%. Furthermore the
conservation of functionally important amino acid residues or
domains, which can be identified by the alignment of several
already available paralogs, can identify a new sequence as a new
orthologs. Alternatively libraries can be subjected to mass
sequencing and obtained sequences can be stored in a sequence
database, which then can be screened for putative orthologs by
different search algorithms, for example the tbastn algorithm to
search the obtained nucleic acid sequences with a amino acid
sequence of the invention. Clones with the highest sequence
identity are used for a complete sequence determination and
orthologs can be identified as described above.
[9204] [0553.0.20.20] [9205] 1. A process for the production of
behenic acid, lignoceric acid, cerotic acid or melissic acid, which
comprises [9206] (a) increasing or generating the activity of a
protein as indicated in Table IIA or IIB, columns 5 or 7, lines 251
to 261, 627, 627 or a functional equivalent thereof in a non-human
organism or in one or more parts thereof; and [9207] (b) growing
the organism under conditions which permit the production of
behenic acid, lignoceric acid, cerotic acid or melissic acid in
said organism. [9208] 2. A process for the production of behenic
acid, lignoceric acid, cerotic acid or melissic acid, comprising
the increasing or generating in an organism or a part thereof the
expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [9209]
a) nucleic acid molecule encoding a polypeptide as indicated in
Table IIA or IIB, columns 5 or 7, lines 251 to 261, 627, 627 or a
fragment thereof, which confers an increase in the amount of
behenic acid, lignoceric acid, cerotic acid or melissic acid in an
organism or a part thereof; [9210] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table IA or
IB, columns 5 or 7, lines 251 to 261, 627, 627; [9211] c) nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as a
result of the degeneracy of the genetic code and conferring an
increase in the amount of behenic acid, lignoceric acid, cerotic
acid or melissic acid in an organism or a part thereof; [9212] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of behenic acid, lignoceric acid, cerotic
acid or melissic acid in an organism or a part thereof; [9213] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of behenic acid, lignoceric
acid, cerotic acid or melissic acid in an organism or a part
thereof; [9214] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, column 7, lines
251 to 261, 627, 627 and conferring an increase in the amount of
behenic acid, lignoceric acid, cerotic acid or melissic acid in an
organism or a part thereof; [9215] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
behenic acid, lignoceric acid, cerotic acid or melissic acid in an
organism or a part thereof; [9216] h) nucleic acid molecule
encoding a polypeptide comprising a consensus as indicated in Table
IV, column 7, lines 251 to 259 and 261, 627 and conferring an
increase in the amount of behenic acid, lignoceric acid, cerotic
acid or melissic acid in an organism or a part thereof; and [9217]
i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of behenic acid,
lignoceric acid, cerotic acid or melissic acid in an organism or a
part thereof. [9218] or comprising a sequence which is
complementary thereto. [9219] 3. The process of claim 1 or 2,
comprising recovering of the free or bound behenic acid, lignoceric
acid, cerotic acid or melissic acid. [9220] 4. The process of any
one of claims 1 to 3, comprising the following steps: [9221] a)
selecting an organism or a part thereof expressing a polypeptide
encoded by the nucleic acid molecule characterized in claim 2;
[9222] b) mutagenizing the selected organism or the part thereof;
[9223] c) comparing the activity or the expression level of said
polypeptide in the mutagenized organism or the part thereof with
the activity or the expression of said polypeptide of the selected
organisms or the part thereof; [9224] d) selecting the mutated
organisms or parts thereof, which comprise an increased activity or
expression level of said polypeptide compared to the selected
organism or the part thereof; [9225] e) optionally, growing and
cultivating the organisms or the parts thereof; and [9226] f)
recovering, and optionally isolating, the free or bound behenic
acid, lignoceric acid, cerotic acid or melissic acid produced by
the selected mutated organisms or parts thereof. [9227] 5. The
process of any one of claims 1 to 4, wherein the activity of said
protein or the expression of said nucleic acid molecule is
increased or generated transiently or stably. [9228] 6. An isolated
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: [9229] a) nucleic acid molecule
encoding a polypeptide as indicated in Table IIA or IIB, columns 5
or 7, lines 251 to 261, 627, 627 or a fragment thereof, which
confers an increase in the amount of behenic acid, lignoceric acid,
cerotic acid or melissic acid in an organism or a part thereof;
[9230] b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table IA or IB, columns 5 or 7, lines 251
to 261, 627, 627; [9231] c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as a result of the degeneracy of the
genetic code and conferring an increase in the amount of behenic
acid, lignoceric acid, cerotic acid or melissic acid in an organism
or a part thereof; [9232] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of behenic
acid, lignoceric acid, cerotic acid or melissic acid in an organism
or a part thereof; [9233] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under stringent
hybridisation conditions and conferring an increase in the amount
of behenic acid, lignoceric acid, cerotic acid or melissic acid in
an organism or a part thereof; [9234] f) nucleic acid molecule
which encompasses a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers or primer pairs as indicated in Table
III, column 7, lines 251 to 261, 627, 627 and conferring an
increase in the amount of behenic acid, lignoceric acid, cerotic
acid or melissic acid in an organism or a part thereof; [9235] g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of behenic acid, lignoceric acid, cerotic
acid or melissic acid in an organism or a part thereof; [9236] h)
nucleic acid molecule encoding a polypeptide comprising a consensus
as indicated in Table IV, column 7, lines 251 to 259 and 261, 627
and conferring an increase in the amount of behenic acid,
lignoceric acid, cerotic acid or melissic acid in an organism or a
part thereof; and [9237] i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of behenic acid, lignoceric acid, cerotic acid or melissic
acid in an organism or a part thereof. [9238] whereby the nucleic
acid molecule distinguishes over the sequence as indicated in Table
IA or IB, columns 5 or 7, lines 251 to 261, 627, 627 by one or more
nucleotides. [9239] 7. A nucleic acid construct which confers the
expression of the nucleic acid molecule of claim 6, comprising one
or more regulatory elements. [9240] 8. A vector comprising the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7. [9241] 9. The vector as claimed in claim 8,
wherein the nucleic acid molecule is in operable linkage with
regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic, host. [9242] 10. A
host cell, which has been transformed stably or transiently with
the vector as claimed in claim 8 or 9 or the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in any one of claims 2 to 5. [9243] 11. The
host cell of claim 10, which is a transgenic host cell. [9244] 12.
The host cell of claim 10 or 11, which is a plant cell, an animal
cell, a microorganism, or a yeast cell, a fungus cell, a
prokaryotic cell, an eukaryotic cell or an archaebacterium. [9245]
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. [9246] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 251 to 261,
627, 627 by one or more amino acids. [9247] 15. An antibody, which
binds specifically to the polypeptide as claimed in claim 14.
[9248] 16. A plant tissue, propagation material, harvested material
or a plant comprising the host cell as claimed in claim 12 which is
plant cell or an Agrobacterium. [9249] 17. A method for screening
for agonists and antagonists of the activity of a polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of behenic acid, lignoceric acid, cerotic
acid or melissic acid in an organism or a part thereof comprising:
(a) contacting cells, tissues, plants or microorganisms which
express the a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of behenic acid,
lignoceric acid, cerotic acid or melissic acid in an organism or a
part thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; (b) assaying the behenic acid, lignoceric acid,
cerotic acid or melissic acid level or the polypeptide expression
level in the cell, tissue, plant or microorganism or the media the
cell, tissue, plant or microorganisms is cultured or maintained in;
and (c) identifying a agonist or antagonist by comparing the
measured behenic acid, lignoceric acid, cerotic acid or melissic
acid level or polypeptide expression level with a standard behenic
acid, lignoceric acid, cerotic acid or melissic acid or polypeptide
expression level measured in the absence of said candidate compound
or a sample comprising said plurality of compounds, whereby an
increased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an agonist and
a decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[9250] 18. A process for the identification of a compound
conferring increased behenic acid, lignoceric acid, cerotic acid or
melissic acid production in a plant or microorganism, comprising
the steps: (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of behenic acid, lignoceric acid, cerotic acid or melissic
acid in an organism or a part thereof and a readout system capable
of interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of behenic acid, lignoceric
acid, cerotic acid or melissic acid in an organism or a part
thereof; (b) identifying if the compound is an effective agonist by
detecting the presence or absence or increase of a signal produced
by said readout system. [9251] 19. A method for the identification
of a gene product conferring an increase in behenic acid,
lignoceric acid, cerotic acid or melissic acid production in a
cell, comprising the following steps: (a) contacting the nucleic
acid molecules of a sample, which can contain a candidate gene
encoding a gene product conferring an increase in behenic acid,
lignoceric acid, cerotic acid or melissic acid after expression
with the nucleic acid molecule of claim 6; (b) identifying the
nucleic acid molecules, which hybridise under relaxed stringent
conditions with the nucleic acid molecule of claim 6; (c)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing behenic acid, lignoceric acid, cerotic
acid or melissic acid; (d) expressing the identified nucleic acid
molecules in the host cells; (e) assaying the behenic acid,
lignoceric acid, cerotic acid or melissic acid level in the host
cells; and (f) identifying nucleic acid molecule and its gene
product which expression confers an increase in the behenic acid,
lignoceric acid, cerotic acid or melissic acid level in the host
cell in the host cell after expression compared to the wild type.
[9252] 20. A method for the identification of a gene product
conferring an increase in behenic acid, lignoceric acid, cerotic
acid or melissic acid production in a cell, comprising the
following steps: [9253] a) identifying in a data bank nucleic acid
molecules of an organism; which can contain a candidate gene
encoding a gene product conferring an increase in the behenic acid,
lignoceric acid, cerotic acid or melissic acid amount or level in
an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; [9254]
b) introducing the candidate nucleic acid molecules in host cells
appropriate for producing behenic acid, lignoceric acid, cerotic
acid or melissic acid; [9255] c) expressing the identified nucleic
acid molecules in the host cells; [9256] d) assaying the behenic
acid, lignoceric acid, cerotic acid or melissic acid level in the
host cells; and
[9257] e) identifying nucleic acid molecule and its gene product
which expression confers an increase in the behenic acid,
lignoceric acid, cerotic acid or melissic acid level in the host
cell after expression compared to the wild type. [9258] 21. A
method for the production of an agricultural composition comprising
the steps of the method of any one of claims 17 to 20 and
formulating the compound identified in any one of claims 17 to 20
in a form acceptable for an application in agriculture. [9259] 22.
A composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [9260] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
behenic acid, lignoceric acid, cerotic acid or melissic acid after
expression. [9261] 24. Use of the polypeptide of claim 14 or the
nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of behenic acid, lignoceric
acid, cerotic acid or melissic acid levels in an organism. [9262]
25. Agrochemical, pharmaceutical, food or feed composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20. [9263] 26. The method of any one of
claims 1 to 5, the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20, wherein the fine chemical is behenic
acid, lignoceric acid, cerotic acid or melissic acid. [9264] 27. A
host cell or plant according to any of the claims 10 to 12 which is
resistant to a herbicide inhibiting the biosynthesis of behenic
acid, lignoceric acid, cerotic acid or melissic acid.
[9265] [0554.0.0.20] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[9266] [0000.0.0.21] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[9267] [0001.0.0.21] for the disclosure of this paragraph see
[0001.0.0.0].
[9268] [0002.0.21.21] Plants produce glycerol and
glycerol-3-phosphate. Importantly lipids derived from glycerol are
the major components of eukaryotic cells. In terms of dry weight
they account for anywhere between 10% and 90% of the total mass of
the cell. Triglycerol are the major source of store energy in
eucaryotic organisms.
[9269] Glycerol-3-phosphate can be synthesized via two different
routes in plants. In one route, it is formed from dihydroxyacetone
phosphate (DHAP), an intermediate of glycolysis, by the sequential
action of triosephosphate isomerase, glyceraldehyd-phosphate
phosphatase, glyeraldehyde reductase and glycerol kinase. The last
enzyme of this pathway has been suggested as the rate-limiting step
of this route for glycerol-3-phosphate synthesis. In an second
pathway glycerol-3-phosphate dehydrogenase (NAD(+)-G-3-P
oxidoreductase, EC 1.1.1.8) (GPDH) catalyses the reduction of
dihydroxyacetone phosphate (DHAP) to form glycerol-3-phosphate
(G-3-P). Based on enzymatic studies is has been suggested that this
enzyme activity is probably the primary source of
glycerol-3-phosphate at least in Brassica campestris seeds (Sharma
et al., 2001, Plant Sci 160, 603-610).
[9270] [0003.0.0.21] In plants, at least two types of GPDH, a
cytoplasmatic and a plastidial exist, which also differ in their
reducing cosubstrate. The cytsolic GPDH uses NADH as the
cosubstrate. The mitochondrial FAD-dependent glycerol-3-phosphate
dehydrogenase (FAD-GPDH) of Arabidopsis forms a G-3-P shuttle, as
previously established in other eukaryotic organisms, and links
cytosolic G-3-P metabolism to carbon source utilization and energy
metabolism in plants--also see Shen, W. et al., FEBS Lett. 2003
Feb. 11; 536(1-3): 92-6.
[9271] Glycerol-insensitive Arabidopsis mutants: glil seedlings
lack glycerol kinase, accumulate glycerol and are more resistant to
abiotic stress, see Eastmond P. J., HYPERLINK
"http://www.ingentaconnect.com/content/bsc/tpj" \o "The Plant
Journal" The Plant Journal, 2004, 37(4), 617-625. These data show
that glycerol kinase is required for glycerol catabolism in
Arabidopsis and that the accumulation of glycerol can enhance
resistance to a variety of abiotic stresses associated with
dehydration.
[9272] [0004.0.0.21] The major storage lipids (or oils) of seeds
occur in the form of triacylglycerols (TAG), or three fatty acids
linked to glycerol by ester bonds. Triacylglycerol synthesis
involves diverse cellular compartments, including the cytoplasm,
the mitochondria, the plastids, and the endoplasmic reticulum (ER).
Glycerol-3-phosphate enters the ER for the final step in
triacylglycerol synthesis. The newly formed triacylglycerols
accumulate between the two layers of the double membrane of the ER,
forming an oil body surrounded by a single (or half) unit
membrane.
[9273] [0005.0.0.21] Glycerol-3-phosphate acyltransferase (GPAT) is
one of the most important enzymes in TAG biosynthesis, since it
initiates TAG synthesis by catalyzing the acylation of the Sn-1
position of Sn-glycerol-3-phosphate, producing
Sn-1-acyl-glycerol-3-phosphate. Lyso-phosphatidic acid (LPA) is
then acetylated by LPA acyltransferases to produce phosphatidic
acid (PA). Then diacylglycerol (DAG) is released through the
dephosphorylation of PA by PA phosphohydrolase. Finally DAG becomes
acylated by the activity of the DAG acyltransferase. In a second
pathway phosphatidylcholine (PC) is formed and its acyl residues
are desaturated further. The choline phosphate residue is then
liberated by hydrolysis and the correspondinbg DAG acylated. This
second pathway operates frequently in the synthesis of highly
unsaturated TAG (Heldt 1997, Plant biochemistry and molecular
biology. Oxford University Press, New York).
[9274] Additionally at present, many researches have proved that
the GPAT is related to plant chilling-resistance, see Liu, Ji-Mei
et al., Plant Physiol. 120(1999): 934.
[9275] Glycerol-3-phosphate is a primary substrate for
triacylglycerol synthesis. Vigeolas and Geigenberger (Planta
219(2004): 827-835) have shown that injection of developing seeds
with glycerol leads to increased glycerol-3-phosphate levels. These
increased levels of glycerol-3-phosphate were accompanied by an
increase in the flux of sucrose into total lipids and
triacylglycerol providing evidence that the prevailing levels of
glycerol-3-phosphate co-limit triacylglycerol production in
developing seeds.
[9276] The direct acylation of glycerol by a glycerol: acyl-CoA
acyltransferase to form mono-acyl-glycerol and, subsequently,
diacylglycerol and triacylglycerol has been shown in myoblast and
hepatocytes (Lee, D. P. et al. J. Lipid res. 42 (2001): 1979-1986).
This direct acylation became more prominent when the
glycerol-3-phosphate pathway was attenuated or when glycerol levels
become elevated.
[9277] [0006.0.0.21] Glycerol is used together with water and
alcohol (ethyl alcohol) in glycerinated water/alcohol plant
extracts and phytoaromatic compounds. These products are used as
food supplements, providing concentrates of the minerals, trace
elements, active ingredients (alkaloids, polyphenols, pigments,
etc.) and aromatic substances to be found in plants. Glycerin acts
as a carrier for plant extracts. It is found in the end product
(the fresh plant extract) in concentrations of up to 24% or
25%.
[9278] Raw glycerol is a by-product of the transesterification
process of rape oil to rape methyl ester (RME) and used edible oil
to used edible methyl ester (AME), both better known as
Biodiesel.
[9279] Glycerol world production is estimated to be around 750.000
t/year. Around 90% is manufactured on the basis of natural oils and
fats.
[9280] [0007.0.0.21] The green alga Dunaliella, for example,
recently has been established in mass culture as a commercial
source for glycerol. Dunaliella withstands extreme salinities while
maintaing a low intracellular salt concentration. Osmotic
adjustment is achieved by intracellular accumulation of glycerol to
a level counterbalancing the external osmoticum.
[9281] The osmoregulatory isoform of dihydroxyacetone phosphate
(DHAP) reductase (Osm-DHAPR) is an enzyme unique to Dunaliella
tertiolecta and is the osmoregulatory isoform involved in the
synthesis of free glycerol for osmoregulation in extreme
environments, such as high salinity, see Ghoshal, D., et al.,
HYPERLINK
"http://www.ingentaconnect.com/content/ap/ptjsessionid=708pg3rt74t81.vict-
oria" "Protein Expression and Purification" Protein Expression and
Purification, 2002, 24, (3), 404-411.
[9282] A unsolved problem in plant biochemistry is the
understanding of metabolic regulation of glycerol-3-phosphate
synthesis and its use in modifying glyceride metabolism or glycerol
production. Practically it will have significance for rationally
genetically engineering of plants for increased synthesis of
triacylglycerols or for other value added products, and for
introducing the glycerol synthesis capability into plants of
economic importance for an elevated environmental stress
tolerance--see: Durba, G. et al., J. Plant Biochemistry &
Biotechnology 10(2001), 113-120.
[9283] [0008.0.21.21] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce glycerol and/or glycerol-3-phosphate
in plants. That type of production permits control over quality,
quantity and selection of the most suitable and efficient producer
organisms. The latter is especially important for commercial
production economics and therefore availability to consumers. In
addition it is desirable to produce glycerol and/or
glycerol-3-phosphate in plants in order to increase plant
productivity and resistance against biotic and abiotic stress as
discussed before.
[9284] Methods of recombinant DNA technology have been used for
some years to improve the production of fine chemicals in
microorganisms and plants by amplifying individual biosynthesis
genes and investigating the effect on production of fine chemicals.
It is for example reported, that the xanthophyll astaxanthin could
be produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. Those results indicated
that about 75 percent of the carotenoids found in the flower of the
transformed plant contained a keto group.
[9285] [0009.0.21.21] Thus, it would be advantageous if an algae,
plant or other microorganism were available which produce large
amounts of glycerol and/or glycerol-3-phosphate. The invention
discussed hereinafter relates in some embodiments to such
transformed prokaryotic or eukaryotic microorganisms.
[9286] It would also be advantageous if plants were available whose
roots, leaves, stem, fruits or flowers produced large amounts of
glycerol and/or glycerol-3-phosphate. The invention discussed
hereinafter relates in some embodiments to such transformed
plants.
[9287] Furthermore it would be advantageous if plants were
available whose seed produced larger amounts of total lipids. The
invention discussed hereinafter relates in some embodiments to such
transformed plants.
[9288] [0010.0.21.21] Therefore improving the quality of foodstuffs
and animal feeds is an important task of the food-and-feed
industry. This is necessary since, for example glycerol and/or
glycerol-3-phosphate, as mentioned above, which occur in plants and
some microorganisms are limited with regard to the supply of
mammals. Especially advantageous for the quality of foodstuffs and
animal feeds is as balanced as possible a specific glycerol and/or
glycerol-3-phosphate profile in the diet since an excess of
glycerol and/or glycerol-3-phosphate above a specific concentration
in the food has a positive effect. A further increase in quality is
only possible via addition of further glycerol and/or
glycerol-3-phosphate, which are limiting.
[9289] [0011.0.21.21] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add glycerol and/or
glycerol-3-phosphate in a balanced manner to suit the organism.
[9290] [0012.0.21.21] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
proteins which participate in the biosynthesis of glycerol and/or
glycerol-3-phosphate and make it possible to produce them
specifically on an industrial scale without unwanted byproducts
forming. In the selection of genes for biosynthesis two
characteristics above all are particularly important. On the one
hand, there is as ever a need for improved processes for obtaining
the highest possible contents of glycerol and/or
glycerol-3-phosphate; on the other hand as less as possible
byproducts should be produced in the production process.
[9291] Furthermore there is still a great demand for new and more
suitable genes, which encode enzymes or other proteins, which
participate in the biosynthesis of total lipids and make it
possible to produce them specifically on an industrial scale
without unwanted byproducts forming. In the selection of genes for
biosynthesis two characteristics above all are particularly
important. On the one hand, there is as ever a need for improved
processes for obtaining the highest possible contents of total
lipids; on the other hand as less as possible byproducts should be
produced in the production process.
[9292] Glycerol or glycerol-3-phosphate is biosynthetic precusor
for the biosynthesis of monoacylglycerols, diacylglycerols,
triacylglycerols, phosphatidylglycerols and other glycerolipids
(e.g. glycosylglycerides, diphosphatidylglycerols, phosphonolipids,
phosphatidylcholines, phosphatidylethanolamines,
phosphatidylinositols, phytoglycolipids). Therefore the analysis of
the glycerol content in cells, tissues or plant parts like seeds
and leaves after total lipid extraction and lipid hydrolysis
directly correlates with the analysis of the total lipid content.
For example if the overexpression of a gene participating in the
biosynthesis of triacylglycerols in the seed results in an increase
in total lipid content in the seed or leaf this seed will also show
an increased glycerol content after total lipid extraction and
hydrolysis of the lipids.
[9293] Therefore the method as described below which leads to an
increase in glycerol in the lipid fraction after cleavage of the
ester functions for example with a mixture of methanol and
hydrochloric acid clearly represents a method for an increased
production of triacylglycerol or total lipids.
[9294] [0013.0.0.21] for the disclosure of this paragraph see
[0013.0.0.0].
[9295] [0014.0.21.21] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is glycerol and/or
glycerol-3-phosphate. Accordingly, in the present invention, the
term "the fine chemical" as used herein relates to glycerol and/or
glycerol-3-phosphate. Further, the term "the fine chemicals" as
used herein also relates to fine chemicals comprising glycerol
and/or glycerol-3-phosphate.
[9296] [0015.0.21.21] In one embodiment, the term "the fine
chemical" means glycerol. In one embodiment, the term "the fine
chemical" means glycerol-3-phosphate depending on the context in
which the term is used. Throughout the specification the term "the
fine chemical" means glycerol and/or glycerol-3-phosphate, its
salts, ester, thioester or in free form or bound to other compounds
such as sugars or sugarpolymers, like glucoside or polyols like
myo-inositol.
[9297] In one embodiment, the term "the fine chemical" means
monoacylglycerols, diacylglycerols, triacylglycerols,
phosphatidylglycerols and/or other glycerolipids (e.g. but not
limited to glycosylglycerides, diphosphatidylglycerols,
phosphonolipids, phosphatidylcholines, phosphatidylethanolamines,
phosphatidylinositols or phytoglycolipids) and is hereinafter
referred to as "total lipids".
[9298] [0016.0.21.21] Accordingly, the present invention relates to
a process comprising [9299] (a) increasing or generating the
activity of one or more YDR065W, b2441, b3457, YBR084W, YDR513W,
YGL237C, YIL150C, YLR082C, YLR224W, YLR255C, [9300] YMR015C,
YOR344C, YPL099C, YPL268W, b3644, YHR072W, b2710, b3498 or b4073
protein(s) in a non-human organism in one or more parts thereof;
and [9301] (b) growing the organism under conditions which permit
the production of the fine chemical, thus glycerol and/or
glycerol-3-phosphate in said organism.
[9302] Accordingly, the present invention relates to a process
comprising [9303] (a) increasing or generating the activity of one
or more proteins having the activity of a protein indicated in
Table II, column 3, lines 173, 262 to 274 and 628 to 632, resp. or
having the sequence of a polypeptide encoded by a nucleic acid
molecule indicated in Table I, column 5 or 7, lines 173, 262 to 274
and 628 to 632, resp. in a non-human organism in one or more parts
thereof; and growing the organism under conditions which permit the
production of the fine chemical, thus, glycerol and/or
glycerol-3-phosphate, in said organism.
[9304] [0016.1.21.21] Accordingly, the term "the fine chemical"
means "glycerol" in relation to all sequences listed in Table I,
lines 262 to 274 and lines 628 and 629 or homologs thereof, means
"glycerol-3-phosphate" in relation to the sequence listed in Table
I, line 173 and lines 630 to 632 or homologs thereof. The term "the
fine chemical" could in addition mean "total lipids" comprising but
not limited to "monoacylglycerols, diacylglycerols,
triacylglycerols, phosphatidylglycerols and/or other glycerolipids
(e.g. glycosylglycerides, diphosphatidylglycerols, phosphonolipids,
phosphatidylcholines, phosphatidylethanolamines,
phosphatidylinositols or phytoglycolipids)" in relation to all
sequences listed in Table I, lines 265, 266, 267, 269, 271, 272,
263 or 628 to 632 or homologs thereof. Accordingly, the term "the
fine chemical" can mean "glycerol", "glycerol-3-phosphate" or
"total lipids", owing to circumstances and the context.
[9305] [0017.0.0.21] and [0018.0.0.21] for the disclosure of the
paragraphs [0017.0.0.21] and [0018.0.0.21] see paragraphs
[0017.0.0.0] and [0018.0.0.0] above.
[9306] [0019.0.21.21] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the respective fine chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table II, column 3, lines 173, 262 to 274 and 628 to
632 or encoded by nucleic acid molecule indicated in Table I,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632.
[9307] [0020.0.21.21] Surprisingly it was found, that the
transgenic expression of the Escherichia coli K12 protein b2441,
b3457, b3644, b2710, b3498 or b4073 or Saccharomyces cerevisiae
protein YDR065W, YBR084W, YDR513W, YGL237C, YIL150C, YLR082C,
YLR224W, YLR255C, YMR015C, YOR344C, YPL099C,
[9308] YPL268W or YHR072W in Arabidopsis thaliana conferred an
increase in glycerol and/or glycerol-3-phosphate and/or total lipid
("the fine chemical" or "the fine respective chemical") in respect
to said proteins and their homologs as wells as the encoding
nucleic acid molecules, in particular as indicated in Table II,
column 3, lines 173, 262 to 274 and 628 to 632 content of the
transformed plants.
[9309] [0021.0.0.21] for the disclosure of this paragraph see
[0021.0.0.0] above.
[9310] [0022.0.21.21] The sequence of YDR065W from Saccharomyces
cerevisiae has been published in Jacq, C. et al., Nature 387 (6632
Suppl), 75-78 (1997) and its activity has not been characterized
yet. Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein YDR065W from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of glycerol-3-phosphate, preferably in free or bound form in
an organism or a part thereof, as mentioned. In one embodiment, in
the process of the present invention the activity of the protein
YDR065W is increased.
[9311] The sequence of b2441 from Escherichia coli K12 has been
published in Blattner, F. R. et al., Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a protein having
ethanolamine ammonia-lyase activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein b2441 from Escherichia coli K12 or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of glycerol,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein b2441 is increased.
[9312] The sequence of b3457 from Escherichia coli K12 has been
published in Blattner, F. R. et al, Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a protein having
branched-chain amino acid transport activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein b3457 from Escherichia coli K12 or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of glycerol
and/or total lipids, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein b3457
is increased.
[9313] The sequence of YBR084W from Saccharomyces cerevisiae has
been published in Goffeau, A. et al., Science 274 (5287), 546-547
(1996) and its activity is being defined as a protein having
Cl-tetrahydrofolate synthase activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein YBR084W from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of glycerol,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein YBR084W is increased.
[9314] The sequence of YDR513W from Saccharomyces cerevisiae has
been published in Jacq, C. et al., Nature 387 (6632 Suppl), 75-78
(1997) and its activity is being defined as a protein having
gluthatione reductase activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
YDR513W from Saccharomyces cerevisiae or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, in
particular for increasing the amount of glycerol and/or total
lipid, preferably in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of the protein YDR513W is
increased.
[9315] The sequence of YGL237C from Saccharomyces cerevisiae has
been published in Tettelin, H. et al. Nature 387 (6632 Suppl),
81-84 (1997) and its activity is being defined as transcriptional
activator and global regulator of respiratory gene expression.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein YGL237C from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of glycerol and/or total lipid, preferably in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the protein YGL237C is increased.
[9316] The sequence of YIL150C from Saccharomyces cerevisiae has
been published in Churcher, C. et al., Nature 387 (6632 Suppl),
84-87 (1997) and its activity is being defined as a protein having
chromatin binding activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
YIL150C, e.g. a protein required for S-phase iniation or completion
from Saccharomyces cerevisiae or its homolog, e.g. as shown herein,
for the production of the respective fine chemical, in particular
for increasing the amount of glycerol and/or total lipid,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein YIL150C is increased.
[9317] The sequence of YLR082C from Saccharomyces cerevisiae has
been published in Johnston, M. et al., Nature 387 (6632 Suppl),
87-90 (1997) and its activity has been characterized as a
suppressor of Rad53 null lethality. Accordingly, in one embodiment,
the process of the present invention comprises the use of a protein
YLR082C from Saccharomyces cerevisiae or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, in
particular for increasing the amount of glycerol, preferably in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of the protein YLR082C is increased.
[9318] The sequence of YLR224W from Saccharomyces cerevisiae has
been published in Johnston, M. et al., Nature 387 (6632 Suppl),
87-90 (1997) and its activity has not been characterized yet.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein YLR224W from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of glycerol and/or total lipid, preferably in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
the protein YLR224W is increased.
[9319] The sequence of YLR255C from Saccharomyces cerevisiae has
been published in the EMBL Data Library, February 1995.
[9320] MIPS_Reference: HYPERLINK
"http://gcont01.basf-ag.de:8000/biors/searchtool/searchtool.cgi?request=l-
ookup_entry&file=entry&kind=foreign&db_name=PIR&ref_id=S59386&ref_word=MIP-
S_Reference&use_sql=0¤t_query_id=%401092226732&total=3&_db=PIR&_db=S-
PTREMBL&_db=TREMBL_NEW&S59386, Accession: S69301 and its
activity has not been characterized yet. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein YLR255C from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of glycerol,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein YLR255C is increased.
[9321] The sequence of YMR015C from Saccharomyces cerevisiae has
been published Bowman, S. et al., Nature 387 (6632 Suppl), 90-93
(1997) and its activity. is being defined as a protein having C-22
sterol desaturase activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
YMR015C from Saccharomyces cerevisiae or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, in
particular for increasing the amount of glycerol and/or total
lipid, preferably in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of the protein YMR015C is
increased.
[9322] The sequence of YOR344C from Saccharomyces cerevisiae has
been published in Dujon, B. et al., Nature 387 (6632 Suppl), 98-102
(1997) and its activity is being defined as a serine-rich protein
being involved in glycolytic gene expression. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein YOR344C from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of glycerol
and/or total lipid, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of the protein YOR344C is
increased.
[9323] The sequence of YPL099C from Saccharomyces cerevisiae has
been published in Bussey, H. et al., Nature 387 (6632 Suppl),
103-105 (1997) and its activity is being defined as a putative
membrane protein. Accordingly, in one embodiment, the process of
the present invention comprises the use of a protein YPL099C from
Saccharomyces cerevisiae or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, in particular for
increasing the amount of glycerol, preferably in free or bound form
in an organism or a part thereof, as mentioned. In one embodiment,
in the process of the present invention the activity of the YPL099C
protein is increased.
[9324] The sequence of YPL268W from Saccharomyces cerevisiae has
been published in Bussey, H. et al., Nature 387 (6632 Suppl),
103-105 (1997) and its activity is being defined as a protein
having phosphoinositide phospholipase activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein YPL268W from Saccharomyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of glycerol,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the YPL268W protein is increased.
[9325] The sequence of b2710 (Accession number NP.sub.--417190)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a flavorubredoxin (FIRd) bifunctional NO and O.sub.2
reductase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a flavorubredoxin (FIRd)
bifunctional NO and O.sub.2 reductase protein from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glycerol-3-phosphate and/or glycerides,
lipids, oils and/or fats containing glycerol-3-phosphate, in
particular for increasing the amount of glycerol-3-phosphate,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein b2710 is increased. In one
embodiment, in the process of the present invention the activity of
a flavorubredoxin (FIRd) bifunctional NO and O.sub.2 reductase
protein is increased or generated, e.g. from E. coli or a homolog
thereof.
[9326] The sequence of b3498 (Accession number NP.sub.--417955)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a oligopeptidase A. Accordingly, in one embodiment, the
process of the present invention comprises the use of a
oligopeptidase A protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
glycerol-3-phosphate and/or glycerides, lipids, oils and/or fats
containing glycerol-3-phosphate, in particular for increasing the
amount of glycerol-3-phosphate, preferably in free or bound form in
an organism or a part thereof, as mentioned. In one embodiment, in
the process of the present invention the activity of the protein
b3498 is increased. In one embodiment, in the process of the
present invention the activity of an oligopeptidase A protein is
increased or generated, e.g. from E. coli or a homolog thereof.
[9327] The sequence of b3644 (Accession number NP.sub.--418101)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as an uncharacterized stress-induced protein. Accordingly,
in one embodiment, the process of the present invention comprises
the use of an uncharacterized stress-induced protein from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of glycerol and/or tryglycerides, lipids,
oils and/or fats containing glycerol, in particular for increasing
the amount of glycerol, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of an uncharacterized
stress-induced protein is increased or generated, e.g. from E. coli
or a homolog thereof.
[9328] The sequence of b4073 (Accession number NP.sub.--418497)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a formate-dependent nitrate reductase. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a formate-dependent nitrate reductase protein from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of glycerol-3-phosphate and/or glycerides,
lipids, oils and/or fats containing glycerol-3-phosphate, in
particular for increasing the amount of glycerol-3-phosphate,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein b4073 is increased. In one
embodiment, in the process of the present invention the activity of
a formate-dependent nitrate reductase is increased or generated,
e.g. from E. coli or a homolog thereof.
[9329] The sequence of YHR072W (Accession number NP.sub.--011939)
from Escherichia coli K12 has been published in Goffeau et al.,
Science 274 (5287), 546-547 (1996) and Johnston et al. Science 265
(5181), 2077-2082 (1994), and its activity is being defined as a
2,3-oxidosqualene-lanosterol cyclase protein. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a 2,3-oxidosqualene-lanosterol cyclase protein from E. coli or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of glycerol and/or tryglycerides, lipids, oils
and/or fats containing glycerol, in particular for increasing the
amount of glycerol, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a
2,3-oxidosqualene-lanosterol cyclase protein is increased or
generated, e.g. from E. coli or a homolog thereof.
[9330] [0023.0.21.21] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[9331] In one embodiment, the homolog of any one of the
polypeptides indicated in Table II, column 3, line 173 and lines
630 to 632 is a homolog having the same or a similar activity. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms
preferablyglycerol-3-phosphate.
[9332] In one embodiment, the homolog of the polypeptides indicated
in Table II, column 3, lines 262 to 274, 628 and 629 is a homolog
having the same or a similar activity. In particular an increase of
activity confers an increase in the content of the respective fine
chemical in the organisms preferablyglycerol and/or total
lipid.
[9333] Homologs of the polypeptides indicated in Table II, column
3, lines 173 and lines 630 to 632 may be the polypeptides encoded
by the nucleic acid molecules indicated in Table I, column 7, line
173 and lines 630 to 632 or may be the polypeptides indicated in
Table II, column 7, line 173 and lines 630 to 632 having a
glycerol-3-phosphate content and/or amount increasing activity.
[9334] Homologs of the polypeptides indicated in Table II, column
3, lines 262 to 267, 269, 271 to 274 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 262 to 267, 269, 271 to 274 and 628 to 632, respectively
or may be the polypeptides indicated in Table II, column 7, lines
262 to 267, 269, 271 to 274 and 628 to 632 having a glycerol and/or
total lipid content and/or amount increasing activity.
[9335] [0023.1.0.21] Homologs of the polypeptides polypeptide
indicated in Table II, column 3, lines 173, 262 to 274 and 628 to
632 may be the polypeptides encoded by the nucleic acid molecules
polypeptide indicated in Table I, column 7, lines 173, 262 to 274
and 628 to 632 or may be the polypeptides indicated in Table II,
column 7, lines 173, 262 to 274 and 628 to 632.
[9336] Homologs of the polypeptides polypeptide indicated in Table
II, column 3, lines 173, 262 to 274 and 628 to 632 may be the
polypeptides encoded by the nucleic acid molecules polypeptide
indicated in Table I, column 7, lines 173, 262 to 274 and 628 to
632 or may be the polypeptides indicated in Table II, column 7,
lines 173, 262 to 274 and 628 to 632.
[9337] [0024.0.0.21] for the disclosure of this paragraph see
[0024.0.0.0] above.
[9338] [0025.0.21.21] In accordance with the invention, a protein
or polypeptide has the "activity of a protein of the invention",
e.g. the activity of a protein indicated in Table II, column 3,
lines 173, 262 to 274 and 628 to 632 if its de novo activity, or
its increased expression directly or indirectly leads to an
increased glycerol-3-phosphate and/or glycerol and/or total lipid
level, resp., in the organism or a part thereof, preferably in a
cell of said organism. In a preferred embodiment, the protein or
polypeptide has the above-mentioned additional activities of a
protein indicated in Table II, column 3, lines 173, 262 to 274 and
628 to 632. Throughout the specification the activity or preferably
the biological activity of such a protein or polypeptide or an
nucleic acid molecule or sequence encoding such protein or
polypeptide is identical or similar if it still has the biological
or enzymatic activity of any one of the proteins indicated in Table
II, column 3, lines 173, 262 to 274 and 628 to 632 or which has at
least 10% of the original enzymatic activity, preferably 20%,
particularly preferably 30%, most particularly preferably 40% in
comparison to any one of the proteins indicated in Table II, column
3, line 262 and 263 and 628 and 630 to 632 of Escherichia coli K12
or line 173, 264 to 274 and 629 of Saccharomyces cerevisiae
respectively.
[9339] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[9340] [0025.1.0.21] and [0025.2.0.21] for the disclosure of the
paragraphs [0025.1.0.21] and [0025.2.0.21] see [0025.1.0.0] and
[0025.2.0.0] above.
[9341] [0026.0.0.17] to [0033.0.0.17] for the disclosure of the
paragraphs [0026.0.0.17] to [0033.0.0.17] see [0026.0.0.0] to
[0033.0.0.0] above.
[9342] [0034.0.21.21] Preferably, the reference, control or wild
type differs from the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of a protein having the activity of a
protein as indicated in Table II, column 3, lines 173, 262 to 274
and 628 to 632 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, lines 173, 262 to 274 and 628 to
632 or its homologs, e.g. as indicated in Table I, column 7, lines
173, 262 to 274 and 628 to 632, its biochemical or genetic causes.
It therefore shows the increased amount of the respective fine
chemical.
[9343] [0035.0.0.21] to [0044.0.0.21] for the disclosure of the
paragraphs [0035.0.0.21] to [0044.0.0.21] see paragraphs
[0035.0.0.0] to [0044.0.0.0] above.
[9344] [0045.0.21.21] In one embodiment, in case the activity of
the Saccharomyces cerevisae protein YDR065W or its homologs, e.g.
as indicated in Table II, columns 5 or 7, line 173 is increased,
preferably, in one embodiment the increase of the respective fine
chemical, preferably of glycerol-3-phosphate between 18% and 114%
or more is conferred.
[9345] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2441 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 262 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of glycerol in the polar fraction between 41% and 81% or more is
conferred.
[9346] In one embodiment, in case the activity of the Escherichia
coli K12 proteinb3457 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 263 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of glycerol in the lipid fraction between 18% and 44% or more is
conferred.
[9347] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YBR084W or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 264 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of glycerol in the polar fraction between 102% and 253% or more is
conferred.
[9348] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YDR513W or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 265 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of glycerol in the lipid fraction between 17% and 48% or more is
conferred.
[9349] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YDR513W or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 265 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of glycerol in the polar fraction between 40% and 200% or more is
conferred.
[9350] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YGL237C or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 266 is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of glycerol in the lipid fraction between 23% and 73% or more is
conferred.
[9351] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YIL150C or its homologs as indicated in Table II,
columns 5 or 7, line 267, is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of glycerol lipid fraction between 94% and 219% or more is
conferred.
[9352] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YLR082C or its homologs as indicated in Table
II, columns 5 or 7, line 268, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of glycerol in the polar fraction between 41% and 173% or more is
conferred.
[9353] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YRL224W or its homologs as indicated in Table
II, columns 5 or 7, line 269, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of glycerol in the lipid fraction between 17% and 43% or more is
conferred.
[9354] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YLR255C or its homologs as indicated in Table
II, columns 5 or 7, line 270, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of glycerol in the polar fraction between 44% and 73% or more is
conferred.
[9355] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YMR015C or its homologs as indicated in Table
II, columns 5 or 7, line 271, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of glycerol in the lipid fraction between 18% and 23% or more is
conferred.
[9356] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YOR344C or its homologs as indicated in Table
II, columns 5 or 7, line 272, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of glycerol in the lipid fraction between 18% and 104% or more is
conferred.
[9357] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YPL099C or its homologs as indicated in Table
II, columns 5 or 7, line 273, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of glycerol in the polar fraction between 55% and 104% or more is
conferred.
[9358] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YPL268W or its homologs as indicated in Table
II, columns 5 or 7, line 274, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of glycerol in the polar fraction between 38% and 115% or more is
conferred.
[9359] In case the activity of the Escherichia coli K12 protein
b3644 or its homologs e.g. an uncharacterized stress-induced
protein e.g. as indicated in Table II, columns 5 or 7, line 628, is
increased, preferably of glycerol in the polar fraction between 17%
and 44% or more is conferred.
[9360] In case the activity of the Saccharomyces cerevisiae protein
YHR072W or its homologs e.g. a 2,3-oxidosqualene-lanosterol cyclase
protein e.g. as indicated in Table II, columns 5 or 7, line 629, is
increased, preferably of glycerol in the polar fraction between 19%
and 109% or more is conferred.
[9361] In case the activity of the Escherichia coli K12 protein
b2710 or its homologs e.g. a flavorubredoxin (FIRd) bifunctional NO
and O.sub.2 reductase e.g. as indicated in Table II, columns 5 or
7, line 630, is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glycerol-3-phosphate
between 20% and 95% or more is conferred.
[9362] In case the activity of the Escherichia coli K12 protein
b3498 or its homologs e.g. a oligopeptidase A e.g. as indicated in
Table II, columns 5 or 7, line 631, is increased, preferably, in
one embodiment the increase of the fine chemical, preferably of
glycerol-3-phosphate between 20% and 56% or more is conferred.
[9363] In case the activity of the Escherichia coli K12 protein
b4073 or its homologs e.g. a formate-dependent nitrate reductase
e.g. as indicated in Table II, columns 5 or 7, line 632, is
increased, preferably, in one embodiment the increase of the fine
chemical, preferably of glycerol-3-phosphate between 49% and 145%
or more is conferred.
[9364] In addition in case the activity of the Saccharomyces
cerevisiae protein YDR513W, YGL237C, YIL150C, YLR224W, YMR015C,
YOR344C or YHR072W or the Escherichia coli K12 protein b3457,
b3644, b2710, b3498 or b4073 or its homologs as indicated in Table
II, columns 5 or 7, lines 265, 266, 267, 269, 271, 272 or 263 or
628 to 632 is increased in plant cells or especially in the seed of
plants an increase in the total lipid content is conferred.
[9365] [0046.0.0.21] In one embodiment, in case the activity of the
Saccharomyces cerevisiae protein YDR065W or its homologs is
increased, preferably an increase of the fine chemical
glycerol-3-phosphate is conferred.
[9366] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2441 or its homologs, e.g. a protein having
ethanolamine ammonia-lyase activity increased, preferably an
increase of the fine chemical glycerol is conferred.
[9367] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3457 or its homologs, e.g. a protein having
branched-chain amino acid transport activity is increased,
preferably an increase of the fine chemical glycerol is
conferred.
[9368] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YBR084W or its homologs, e.g. a protein having
Cl-tetrahydrofolate synthase activity is increased, preferably an
increase of the fine chemical glycerol is conferred.
[9369] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein
[9370] YDR513W or its homologs, e.g. a gluthatione reductase is
increased, preferably an increase of the fine chemical glycerol is
conferred.
[9371] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YGL237C or its homologs, e.g. a transcriptional
activator protein and global regulator of respiratory gene
expression activity is increased, preferably an increase of the
fine chemical glycerol is conferred.
[9372] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein
[9373] YIL150C or its homologs, e.g. a chromatin binding protein,
required for S-phase (DNA synthesis) initiation or completion is
increased, preferably an increase of the fine chemical glycerol is
conferred.
[9374] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YLR082C or its homologs e.g. a suppressor of
Rad53 null lethality is increased, preferably an increase of the
fine chemical glycerol is conferred.
[9375] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YLR224W or its homologs is increased, preferably
an increase of the fine chemical glycerol is conferred.
[9376] In one embodiment, in case the activity of the Saccharomyces
cerevisiae protein YLR255C or its homologs is increased, preferably
an increase of the fine chemical glycerol is conferred.
[9377] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YMR015C or its homologs, e.g. a protein having
C-22 sterol desaturase activity is increased, preferably an
increase of the fine chemical glycerol is conferred.
[9378] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YOR344C or its homologs, e.g. a serine-rich
protein being involved in glycolytic gene expression is increased,
preferably an increase of the fine chemical glycerol is
conferred.
[9379] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YPL099C or its homologs, e.g. a putative membrane
protein is increased; preferably an increase of the fine chemical
glycerol is conferred.
[9380] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YPL268W or its homologs, e.g. a protein having
phosphoinositide phospholipase activity is increased, preferably an
increase of the fine chemical glycerol is conferred.
[9381] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3644 or its homologs, e.g. an uncharacterized
stress-induced protein increased, preferably an increase of the
fine chemical glycerol is conferred.
[9382] In one embodiment, in case the activity of the Saccharomyces
cerevisae protein YHR072W or its homologs, e.g. an
2,3-oxidosqualene-lanosterol cyclase protein is increased,
preferably an increase of the fine chemical glycerol is
conferred.
[9383] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2710 or its homologs, e.g. a flavorubredoxin
(FIRd) bifunctional NO and O.sub.2 reductase is increased,
preferably an increase of the fine chemical glycerol-3-phosphate is
conferred.
[9384] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3498 or its homologs, e.g. a oligopeptidase A is
increased, preferably an increase of the fine chemical
glycerol-3-phosphate is conferred.
[9385] In one embodiment, in case the activity of the Escherichia
coli K12 protein b4073 or its homologs, e.g. a formate-dependent
nitrate reductase is increased, preferably an increase of the fine
chemical glycerol-3-phosphate is conferred.
[9386] [0047.0.0.21] and [0048.0.0.21] for the disclosure of the
paragraphs [0047.0.0.21] and [0048.0.0.21] see paragraphs
[0047.0.0.0] and [0048.0.0.0] above.
[9387] [0049.0.21.21] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
glycerol-3-phosphate preferably has the structure of the
polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence as shown in SEQ ID NO: 15671 to 15676, or 97963
to 97965, or 98284 to 98287 or 98529 to 98532 or as indicated in
Table IV, columns 7, lines 173 or 630 to 632 or of a polypeptide as
indicated in Table II, columns 5 or 7, lines 173 or 630 to 632 or
of a functional homologue thereof as described herein, or of a
polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, lines 173 or 630 to 632 or its herein described functional
homologues and has the herein mentioned activity conferring an
increase in the glycerol-3-phosphate level.
[9388] A protein having an activity conferring an increase in the
amount or level of the respective fine chemical glycerol and/or
total lipid preferably has the structure of the polypeptide
described herein. In a particular embodiment, the polypeptides used
in the process of the present invention or the polypeptide of the
present invention comprises the sequence of a consensus sequence as
shown in SEQ ID NO: 28565 to 28575 or 28576, 28577 or 28578 or
28579 to 28581 or 28582 to 28584 or 28585, 28586 or 28587 to 28590
or 28591 to 28594 or 28595 to 28597 or 28598 to 28600 or 28601 to
28605 or 98450 to 98452 or 98703 to 98713 or as indicated in Table
IV, columns 7, lines 262 to 274 or 628 and 629 or of a polypeptide
as indicated in Table II, columns 5 or 7, lines 262 to 274 or 628
and 629 or of a functional homologue thereof as described herein,
or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 262 to 274 or 628 and 629 or its
herein described functional homologues and has the herein mentioned
activity conferring an increase in the glycerol level.
[9389] [0050.0.21.21] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of glycerol-3-phosphate, resp., or their esters, e.g.
monoacyl or diacyl fatty acids thereof.
[9390] [0051.0.21.21] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free or
bound, e.g compositions comprising glycerol and/or
glycerol-3-phosphate and or total lipid.
[9391] Depending on the choice of the organism used for the process
according to the present invention, for example a microorganism or
a plant, compositions or mixtures of glycerol and/or
glycerol-3-phosphate and/or total lipid can be produced.
[9392] [0052.0.0.21] for the disclosure of this paragraph see
paragraph [0052.0.0.0] above.
[9393] [0053.0.21.21] In one embodiment, the process of the present
invention comprises one or more of the following steps [9394] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines
173, 262 to 274 and 628 to 632 or its homologs, e.g. as indicated
in Table II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
activity having herein-mentioned the respective fine chemical
increasing activity; [9395] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention, e.g. of a polypeptide having an activity
of a protein as indicated in Table II, column 3, lines 173, 262 to
274 and 628 to 632 or its homologs activity, e.g. as indicated in
Table II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632, or
of a mRNA encoding the polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity;
[9396] c) increasing the specific activity of a protein conferring
the increased expression of a protein encoded by the nucleic acid
molecule of the invention or of the polypeptide of the present
invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 173, 262 to 274
and 628 to 632 or its homologs activity, e.g. as indicated in Table
II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632, or
decreasing the inhibitory regulation of the polypeptide of the
invention; [9397] d) generating or increasing the expression of an
endogenous or artificial transcription factor mediating the
expression of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention having herein-mentioned the
respective fine chemical increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 173, 262 to 274 and 628 to 632 or its homologs activity, e.g.
as indicated in Table II, columns 5 or 7, lines 173, 262 to 274 and
628 to 632; [9398] e) stimulating activity of a protein conferring
the increased expression of a protein encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 173, 262 to 274
and 628 to 632 or its homologs activity, e.g. as indicated in Table
II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632, by adding
one or more exogenous inducing factors to the organism or parts
thereof; [9399] f) expressing a transgenic gene encoding a protein
conferring the increased expression of a polypeptide encoded by the
nucleic acid molecule of the present invention or a polypeptide of
the present invention, having herein-mentioned the respective fine
chemical increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines
173, 262 to 274 and 628 to 632 or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 173, 262 to 274 and
628 to 632, and/or [9400] g) increasing the copy number of a gene
conferring the increased expression of a nucleic acid molecule
encoding a polypeptide encoded by the nucleic acid molecule of the
invention or the polypeptide of the invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 173, 262 to 274 and 628 to 632 or its
homologs, e.g. as indicated in Table II, columns 5 or 7, lines 173,
262 to 274 and 628 to 632. [9401] h) Increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 173, 262 to 274 and 628 to 632 or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 173,
262 to 274 and 628 to 632, by adding positive expression or
removing negative expression elements, e.g. homologous
recombination can be used to either introduce positive regulatory
elements like for plants the 35S enhancer into the promoter or to
remove repressor elements form regulatory regions. Further gene
conversion methods can be used to disrupt repressor elements or to
enhance to activity of positive elements. Positive elements can be
randomly introduced in plants by T-DNA or transposon mutagenesis
and lines can be identified in which the positive elements have be
integrated near to a gene of the invention, the expression of which
is thereby enhanced; and/or [9402] i) Modulating growth conditions
of an organism in such a manner, that the expression or activity of
the gene encoding the protein of the invention or the protein
itself is enhanced for example microorganisms or plants can be
grown for example under a higher temperature regime leading to an
enhanced expression of heat shock proteins, which can lead to an
enhanced respective fine chemical production; and/or [9403] j)
selecting of organisms with especially high activity of the
proteins of the invention from natural or from mutagenized
resources and breeding them into the target organisms, e.g. the
elite crops.
[9404] [0054.0.21.21] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table II, columns 3 or 5,
lines 173, 262 to 274 and 628 to 632, resp., or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 173,
262 to 274 and 628 to 632, resp.
[9405] [0055.0.0.21] to [0067.0.0.21] for the disclosure of the
paragraphs [0055.0.0.21] to [0067.0.0.21] see paragraphs
[0055.0.0.0] to [0067.0.0.0] above.
[9406] [0068.0.21.21] The mutation is introduced in such a way that
the production of the finde chemical, meaning glycerol and/or
glycerol-3-phosphate and or total lipid is not adversely
affected.
[9407] [0069.0.0.21] for the disclosure of this paragraph see
paragraph [0069.0.0.0] above.
[9408] [0070.0.21.21] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolite composition in the organism, e.g. an advantageous
composition of glycerol-3-phosphate and glycerol or their
biochemical derivatives, e.g. comprising a higher content of (from
a viewpoint of nutritional physiology limited) glycerol-3-phosphate
and glycerol or their derivatives including total lipids.
[9409] [0071.0.0.21] for the disclosure of this paragraph see
paragraph [0071.0.0.0] above.
[9410] [0072.0.0.21] %
[9411] [0073.0.21.21] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[9412] (a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [9413] (b) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table II, columns 5 or 7, lines 173, 262 to 274 and
628 to 632, or of a polypeptide being encoded by the nucleic acid
molecule of the present invention and described below, e.g.
conferring an increase of the respective fine chemical in an
organism, preferably in a microorganism, a non-human animal, a
plant or animal cell, a plant or animal tissue or a plant, [9414]
(c) growing an organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a plant
under conditions which permit the production of the respective fine
chemical in the organism, preferably the microorganism, the plant
cell, the plant tissue or the plant; and [9415] (d) if desired,
recovering, optionally isolating, the free and/or bound respective
fine chemical synthesized by the organism, the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[9416] [0074.0.21.21] for the disclosure of this paragraph see
paragraph [0071.0.0.17] above.
[9417] [0075.0.0.21] to [0077.0.0.21] for the disclosure of the
paragraphs [0075.0.0.21] to [0077.0.0.21] see paragraphs
[0075.0.0.0] to [0077.0.0.0] above.
[9418] [0078.0.21.21] The organism such as microorganisms or plants
or the recovered, and if desired isolated, respective fine chemical
can then be processed further directly into foodstuffs or animal
feeds or for other applications. The fermentation broth,
fermentation products, plants or plant products can be purified
with methods known to the person skilled in the art. Products of
these different work-up procedures are glycerol and/or
glycerol-3-phosphate and/or glycerol as a component of lipids or
comprising compositions of glycerol and/or glycerol-3-phosphate
and/or glycerol as a component of lipids still comprising
fermentation broth, plant particles and cell components in
different amounts, advantageously in the range of from 0 to 99% by
weight, preferably below 80% by weight, especially preferably below
50% by weight.
[9419] [0079.0.0.21] to [0084.0.0.21] for the disclosure of the
paragraphs [0079.0.0.21] to [0084.0.0.21] see paragraphs
[0079.0.0.0] to [0084.0.0.0] above.
[9420] [0085.0.21.21] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [9421] a) a nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 173, 262 to 274 and 628
to 632, or a derivative thereof, or [9422] b) a genetic regulatory
element, for example a promoter, which is functionally linked to
the nucleic acid sequence as indicated in Table I, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, or a derivative thereof, or
[9423] c) (a) and (b) is/are not present in its/their natural
genetic environment or has/have been modified by means of genetic
manipulation methods, it being possible for the modification to be,
by way of example, a substitution, addition, deletion, inversion or
insertion of one or more nucleotide. "Natural genetic environment"
means the natural chromosomal locus in the organism of origin or
the presence in a genomic library. In the case of a genomic
library, the natural, genetic environment of the nucleic acid
sequence is preferably at least partially still preserved. The
environment flanks the nucleic acid sequence at least on one side
and has a sequence length of at least 50 bp, preferably at least
500 bp, particularly preferably at least 1000 bp, very particularly
preferably at least 5000 bp.
[9424] [0086.0.0.21] and [0087.0.0.21] for the disclosure of the
paragraphs [0086.0.0.21] and [0087.0.0.21] see paragraphs
[0086.0.0.0] and [0087.0.0.0] above.
[9425] [0088.0.21.21] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned fine
chemicals or the plants are more resistant to biotic and abiotic
stress and the yield is increased.
[9426] [0088.1.0.21] for the disclosure of this paragraph see
paragraph [0088.1.0.0] above.
[9427] [0089.0.0.21] to [0094.0.0.21] for the disclosure of the
paragraphs [0089.0.0.21] to [0094.0.0.21] see paragraphs
[0088.0.0.0] to [0094.0.0.0] above.
[9428] [0095.0.21.21] It may be advantageous to increase the pool
of glycerol and/or glycerol-3-phosphate and/or total lipid in the
transgenic organisms by the process according to the invention in
order to isolate high amounts of the pure respective fine chemical
and/or to obtain increased resistance against biotic and abiotic
stresses and to obtain higher yield.
[9429] [0096.0.21.21] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example in
the organism, is useful to increase the production of the
respective fine chemical.
[9430] [0097.0.0.21] for the disclosure of this paragraph see
paragraph [0097.0.0.0] above.
[9431] [0098.0.21.21] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated.
[9432] [0099.0.21.21] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[9433] [0100.0.21.21] Transgenic plants which comprise the fine
chemicals such as glycerol and/or glycerol-3-phosphate and/or total
lipids synthesized in the process according to the invention can
advantageously be marketed directly without there being any need
for the fine chemicals synthesized to be isolated. Plants for the
process according to the invention are listed as meaning intact
plants and all plant parts, plant organs or plant parts such as
leaf, stem, seeds, root, tubers, anthers, fibers, root hairs,
stalks, embryos, calli, cotelydons, petioles, harvested material,
plant tissue, reproductive tissue and cell cultures which are
derived from the actual transgenic plant and/or can be used for
bringing about the transgenic plant. In this context, the seed
comprises all parts of the seed such as the seed coats, epidermal
cells, seed cells, endosperm or embryonic tissue.
[9434] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, as extracts, e.g. ether, alcohol,
or other organic solvents or water containing extract and/or free
fine chemicals. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pretreated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[9435] Because glycerol and/or glycerol-3-phosphate and/or total
lipids in microorganisms are localized intracellular, their
recovery essentially comes down to the isolation of the biomass.
Well-established approaches for the harvesting of cells include
filtration, centrifugation and coagulation/flocculation as
described herein. Of the residual hydrocarbon, adsorbed on the
cells, has to be removed. Solvent extraction or treatment with
surfactants have been suggested for this purpose.
[9436] [0101.0.21.21] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michel, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[9437] [0102.0.21.21] Glycerol and/or glycerol-3-phosphate can for
example be analyzed advantageously via HPLC, LC or GC separation
and MS (masspectrometry) detection methods. The unambiguous
detection for the presence of glycerol and/or glycerol-3-phosphate
containing products can be obtained by analyzing recombinant
organisms using analytical standard methods: LC, LC-MS, MS or TLC).
The material to be analyzed can be disrupted by sonication,
grinding in a glass mill, liquid nitrogen and grinding, cooking, or
via other applicable methods.
[9438] Total lipids in the seed of plants can for example be
analyzed advantageously by lipid extraction as described by Bligh
and Dyer (Can J Biochem Phys 1959, 37, 911-917) followed by the
determination of the lipid content by gaschromatography as
described by Benning and Sommerville (J Bacteriol 1992, 174,
6479-6487).
[9439] [0103.0.21.21] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [9440] a) nucleic acid molecule encoding, preferably
at least the mature form, of the polypeptide having a sequence as
indicated in Table II, columns 5 or 7, lines 173, 262 to 274 and
628 to 632, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [9441] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table I, columns 5 or 7, lines 173, 262 to 274 and
628 to 632; [9442] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [9443] d) nucleic
acid molecule encoding a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [9444] e) nucleic acid molecule which hybridizes with
a nucleic acid molecule of (a) to (c) under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [9445]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [9446] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [9447] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primer pairs
having a sequence as indicated in Table III, column 7, lines 173,
262 to 274 and 628 to 632, and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[9448] i) nucleic acid molecule encoding a polypeptide which is
isolated, e.g. from an expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (h), preferably to (a) to (c), and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [9449] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having sequences as indicated in Table IV, column 7, lines
173, 262 to 267, 269 and 271 to 274 and 628 to 632 and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [9450] k) nucleic acid molecule
comprising one or more of the nucleic acid molecule encoding the
amino acid sequence of a polypeptide encoding a domain of the
polypeptide indicated in Table II, columns 5 or 7, lines 173, 262
to 274 and 628 to 632, and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof; and
[9451] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[9452] [00103.1.21.21] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 173, 262 to
274 and 628 to 632, by one or more nucleotides. In one embodiment,
the nucleic acid molecule used in the process of the invention does
not consist of the sequence shown in indicated in Table I A,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632. In one
embodiment, the nucleic acid molecule used in the process of the
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I A, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II A, columns 5 or 7, lines 173, 262 to 274 and
628 to 632.
[9453] [00103.2.21.21] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, lines 173, 262 to
274 and 628 to 632, by one or more nucleotides. In one embodiment,
the nucleic acid molecule used in the process of the invention does
not consist of the sequence shown in indicated in Table I B,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632. In one
embodiment, the nucleic acid molecule used in the process of the
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I B, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II B, columns 5 or 7, lines 173, 262 to 274 and
628 to 632.
[9454] [0104.0.21.21] In one embodiment, the nucleic acid molecule
used in the process of the present invention distinguishes over the
sequence indicated in Table I, columns 5 or 7, lines 173, 262 to
274 and 628 to 632, preferably over the sequences as shown in Table
I A, columns 5 or 7, lines 173, 262 to 274 and 628 to 632 by one or
more nucleotides. In one embodiment, the nucleic acid molecule used
in the process of the invention does not consist of the sequence
indicated in Table I, columns 5 or 7, lines 173, 262 to 274 and 628
to 632. In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence indicated in Table I, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, preferably the nucleic acid
molecule of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to the sequence as shown in Table I
A, columns 5 or 7, lines 173, 262 to 274 and 628 to 632. In another
embodiment, the nucleic acid molecule does not encode a polypeptide
of a sequence indicated in Table II, columns 5 or 7, lines 173, 262
to 274 and 628 to 632, preferably as indicated in Table IIA,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632.
[9455] [0105.0.0.21] to [0107.0.0.21] for the disclosure of the
paragraphs [0105.0.0.21] to [0107.0.0.21] see paragraphs
[0105.0.0.0] to [0107.0.0.0] above.
[9456] [0108.0.21.21] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 173, 262 to 274 and 628
to 632, nucleic acid molecules which are derived from an amino acid
sequences as indicated in Table II, columns 5 or 7, lines 173, 262
to 274 and 628 to 632 or from polypeptides comprising the consensus
sequence as indicated in Table IV, column 7, lines 173, 262 to 267,
269 and 271 to 274 and 628 to 632, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of an activity of a polypeptide as indicated in Table II,
column 3, 5 or 7, lines 173, 262 to 274 and 628 to 632, e.g.
conferring the increase of the respective fine chemical, meaning
glycerol and/or glycerol-3-phosphate and/or total lipid, resp.,
after increasing its expression or activity, are advantageously
increased in the process according to the invention.
[9457] [0109.0.21.21] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular glycerol
and/or glycerol-3-phosphate and/or total lipids, produced in the
process according to the invention.
[9458] [0110.0.0.21] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide used in the method
of the invention or used in the process of the invention, e.g. of a
protein as shown in Table II, columns 5 or 7, lines 173, 262 to 274
and 628 to 632 or being encoded by a nucleic acid molecule
indicated in Table I, columns 5 or 7, lines 173, 262 to 274 and 628
to 632 or of its homologs, e.g. as indicated in Table II, columns 5
or 7, lines 173, 262 to 274 and 628 to 632 can be determined from
generally accessible databases.
[9459] [0111.0.0.21] for the disclosure of this paragraph see
[0111.0.0.0] above.
[9460] [0112.0.21.21] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table II, column 3, lines 173, 262
to 274 and 628 to 632 or having the sequence of a polypeptide as
indicated in Table II, columns 5 and 7, lines 173, 262 to 274 and
628 to 632 and conferring an increase in the glycerol and/or
glycerol-3-phosphate and/or total lipid level.
[9461] [0113.0.0.21] to [0120.0.0.21] for the disclosure of the
paragraphs [0113.0.0.21] to [0120.0.0.21] see paragraphs
[0113.0.0.0] and [0120.0.0.0] above.
[9462] [0121.0.21.21] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, line 173 and lines 630 to 632 or the functional
homologues thereof as described herein, preferably conferring
above-mentioned activity, i.e. conferring a glycerol-3-phosphate
increase after increasing the activity of the polypeptide sequences
indicated in Table II, columns 5 or 7, line 173 and lines 630 to
632 and conferring a glycerol and/or total lipid level increase
after increasing the activity of the polypeptide sequences
indicated in Table II, columns 5 or 7, lines 262 to 274 and lines
628 and 629.
[9463] [0122.0.0.21] to [0127.0.0.21] for the disclosure of the
paragraphs [0122.0.0.21] to [0127.0.0.21] see paragraphs
[0122.0.0.0] and [0127.0.0.0] above.
[9464] [0128.0.21.21] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, column 7,
lines 173, 262 to 274 and 628 to 632, by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table I, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, resp. or the sequences
derived from a sequences as indicated in Table II, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, resp.
[9465] [0129.0.21.21] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved regions are those, which show a
very little variation in the amino acid in one particular position
of several homologs from different origin. The consensus sequence
indicated in Table IV, columns 7, lines 173, 262 to 267, 269 and
271 to 274 and 628 to 632 is derived from such alignments.
[9466] [0130.0.21.21] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of glycerol
and/or glycerol-3-phosphate and/or total lipid after increasing the
expression or activity the protein comprising said fragment.
[9467] [0131.0.0.21] to [0138.0.0.21] for the disclosure of the
paragraphs [0131.0.0.21] to [0138.0.0.21] see paragraphs
[0131.0.0.0] to [0138.0.0.0] above.
[9468] [0139.0.21.21] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical level increase,
derived from other organisms, can be encoded by other DNA sequences
which hybridise to a sequence indicated in Table I, columns 5 or 7,
line 173 and lines 630 to 632 for glycerol-3-phosphate or indicated
in Table I, columns 5 or 7, lines 262 to 274 and lines 628 and 629
for glycerol, preferably of Table I B, columns 5 or 7, line 173 and
lines 630 to 632 or lines 262 to 274 and lines 628 and 629
respectively and/or total lipid under relaxed hybridization
conditions and which code on expression for peptides having the
respective fine chemical, i.e. glycerol and/or glycerol-3-phosphate
and/or total lipids, resp., increasing-activity.
[9469] [0140.0.0.21] to [0146.0.0.21] for the disclosure of the
paragraphs [0140.0.0.21] to [0146.0.0.21] see paragraphs
[0140.0.0.0] to [0146.0.0.0] above.
[9470] [0147.0.21.21] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 173, 262 to 274 and 628 to 632, preferably
of Table I B, columns 5 or 7, lines 173, 262 to 274 and 628 to 632
is one which is sufficiently complementary to one of said
nucleotide sequences such that it can hybridise to one of said
nucleotide sequences, thereby forming a stable duplex. Preferably,
the hybridisation is performed under stringent hybridization
conditions. However, a complement of one of the herein disclosed
sequences is preferably a sequence complement thereto according to
the base pairing of nucleic acid molecules well known to the
skilled person. For example, the bases A and G undergo base pairing
with the bases T and U or C, resp. and visa versa. Modifications of
the bases can influence the base-pairing partner.
[9471] [0148.0.21.21] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines
173, 262 to 274 and 628 to 632, preferably of Table I B, columns 5
or 7, lines 173, 262 to 274 and 628 to 632 or a portion thereof and
preferably has above mentioned activity, in particular having a
glycerol and/or glycerol-3-phosphate and/or total lipid increasing
activity after increasing the activity or an activity of a product
of a gene encoding said sequences or their homologs.
[9472] [0149.0.21.21] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, preferably of Table I B,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632 or a portion
thereof and encodes a protein having above-mentioned activity, e.g.
conferring an increase of glycerol-3-phosphate and/or glycerol,
resp., and optionally, the activity of protein indicated in Table
II, column 5, lines 173, 262 to 274 and 628 to 632, preferably of
Table II B, columns 5 or 7, lines 173, 262 to 274 and 628 to
632.
[9473] [00149.1.21.21] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table I, columns 5 or 7, lines 173, 262 to
274 and 628 to 632, preferably of Table I B, columns 5 or 7, lines
173, 262 to 274 and 628 to 632 has further one or more of the
activities annotated or known for a protein as indicated in Table
II, column 3, lines 173, 262 to 274 and 628 to 632, preferably of
Table II B, columns 5 or 7, lines 173, 262 to 274 and 628 to
632.
[9474] [0150.0.21.21] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table I, columns 5 or 7, lines 173,
262 to 274 and 628 to 632, preferably of Table I B, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632 for example a fragment which
can be used as a probe or primer or a fragment encoding a
biologically active portion of the polypeptide of the present
invention or of a polypeptide used in the process of the present
invention, i.e. having above-mentioned activity, e.g. conferring an
increase of gglycerol-3-phosphate and/or glycerol, resp., if its
activity is increased. The nucleotide sequences determined from the
cloning of the present protein-according-to-the-invention-encoding
gene allows for the generation of probes and primers designed for
use in identifying and/or cloning its homologues in other cell
types and organisms. The probe/primer typically comprises
substantially purified oligonucleotide. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 15 preferably
about 20 or 25, more preferably about 40, 50 or 75 consecutive
nucleotides of a sense strand of one of the sequences set forth,
e.g., as indicated in Table I, columns 5 or 7, lines 173, 262 to
274 and 628 to 632, an anti-sense sequence of one of the sequences,
e.g., as indicated in Table I, columns 5 or 7, lines 173, 262 to
274 and 628 to 632, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table III, column 7, lines 173, 262 to 274 and 628 to 632 will
result in a fragment of a polynucleotide sequence as indicated in
Table I, columns 5 or 7, lines 173, 262 to 274 and 628 to 632 or
its gene product. Preferred is Table I B, column 7, lines 173, 262
to 274 and 628 to 632
[9475] [0151.0.0.21]: for the disclosure of this paragraph see
paragraph [0151.0.0.0] above.
[9476] [0152.0.21.21] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 173, 262
to 274 and 628 to 632 such that the protein or portion thereof
maintains the ability to participate in the respective fine
chemical production, in particular a glycerol-3-phosphate (line 173
and lines 630 to 632) or glycerol (lines 262 to 274 and lines 628
and 629) and total lipid (lines 265, 266, 267, 269, 271, 272 and
263) increasing activity as mentioned above or as described in the
examples in plants or microorganisms is comprised.
[9477] [0153.0.21.21] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 173,
262 to 274 and 628 to 632 such that the protein or portion thereof
is able to participate in the increase of the respective fine
chemical production. In one embodiment, a protein or portion
thereof as indicated in Table II, columns 5 or 7, lines 173, 262 to
274 and 628 to 632 has for example an activity of a polypeptide
indicated in Table II, column 3, lines 173, 262 to 274 and 628 to
632.
[9478] [0154.0.21.21] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 173, 262
to 274 and 628 to 632 and has above-mentioned activity, e.g.
conferring preferably the increase of the respective fine
chemical.
[9479] [0155.0.0.21] and [0156.0.0.21] for the disclosure of the
paragraphs [0155.0.0.21] and [0156.0.0.21] see paragraphs
[0155.0.0.0] and [0156.0.0.0] above.
[9480] [0157.0.21.21] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences as
indicated in Table I, columns 5 or 7, lines 173, 262 to 274 and 628
to 632 (and portions thereof) due to degeneracy of the genetic code
and thus encode a polypeptide of the present invention, in
particular a polypeptide having above mentioned activity, e.g.
conferring an increase in the respective fine chemical in a
organism, e.g. as polypeptides comprising the sequence as indicated
in Table IV, column 7, lines 173, 262 to 274 and 628 to 632 or as
polypeptides depicted in Table II, columns 5 or 7, lines 173, 262
to 274 and 628 to 632 or the functional homologues. Advantageously,
the nucleic acid molecule of the invention comprises, or in an
other embodiment has, a nucleotide sequence encoding a protein
comprising, or in an other embodiment having, an amino acid
sequence of a consensus sequences as indicated in Table IV, column
7, lines 173, 262 to 267, 269 and 271 to 274 or of the polypeptide
as indicated in Table II, columns 5 or 7, lines 173, 262 to 274 and
628 to 632, resp., or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
IV, column 7, lines 173, 262 to 267, 269 and 271 to 274 or of a
polypeptide as indicated in Table II, columns 5 or 7, lines 173,
262 to 274 and 628 to 632 or the functional homologues. However, in
a preferred embodiment, the nucleic acid molecule of the present
invention does not consist of a sequence as indicated in Table I,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, preferably as
indicated in Table I A, columns 5 or 7, lines 173, 262 to 274 and
628 to 632, resp. Preferably the nucleic acid molecule of the
invention is a functional homologue or identical to a nucleic acid
molecule indicated in Table I B, column 7, lines 173, 262 to 274
and 628 to 632.
[9481] [0158.0.0.21] to [0160.0.0.21] for the disclosure of the
paragraphs [0158.0.0.21] to [0160.0.0.21] see paragraphs
[0158.0.0.0] to [0160.0.0.0] above.
[9482] [0161.0.21.21] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 173, 262 to
274 and 628 to 632. The nucleic acid molecule is preferably at
least 20, 30, 50, 100, 250 or more nucleotides in length.
[9483] [0162.0.0.21] for the disclosure of this paragraph see
paragraph [0162.0.0.0] above.
[9484] [0163.0.21.21] Preferably, a nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 173, 262 to 274 and
628 to 632 corresponds to a naturally-occurring nucleic acid
molecule of the invention. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein). Preferably, the nucleic acid molecule encodes a natural
protein having above-mentioned activity, e.g. conferring the
increase of the amount of the respective fine chemical in an
organism or a part thereof, e.g. a tissue, a cell, or a compartment
of a cell, after increasing the expression or activity thereof or
the activity of a protein of the invention or used in the process
of the invention.
[9485] [0164.0.0.21] for the disclosure of this paragraph see
paragraph [0164.0.0.0] above.
[9486] [0165.0.21.21] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 173, 262 to 274 and 628
to 632, resp.
[9487] [0166.0.0.21] and [0167.0.0.21] for the disclosure of the
paragraphs [0166.0.0.21] and [0167.0.0.21] see paragraphs
[0166.0.0.0] and [0167.0.0.0] above.
[9488] [0168.0.21.21] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, resp., yet retain said
activity described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table II, columns 5 or 7, lines
173, 262 to 274 and 628 to 632, resp., and is capable of
participation in the increase of production of the respective fine
chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table II,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp., more
preferably at least about 70% identical to one of the sequences as
indicated in Table II, columns 5 or 7, lines 173, 262 to 274 and
628 to 632, resp., even more preferably at least about 80%, 90%,
95% homologous to a sequence as indicated in Table II, columns 5 or
7, lines 173, 262 to 274 and 628 to 632, resp., and most preferably
at least about 96%, 97%, 98%, or 99% identical to the sequence as
indicated in Table II, columns 5 or 7, lines 173, 262 to 274 and
628 to 632.
[9489] Accordingly, the invention relates to nucleic acid molecules
encoding a polypeptide having above-mentioned activity, e.g.
conferring an increase in the the respective fine chemical in an
organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, preferably of Table II B,
column 7, lines 173, 262 to 274 and 628 to 632 yet retain said
activity described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table II, columns 5 or 7, lines
173, 262 to 274 and 628 to 632, preferably of Table II B, column 7,
lines 173, 262 to 274 and 628 to 632 and is capable of
participation in the increase of production of the respective fine
chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table II,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, preferably of
Table II B, column 7, lines 173, 262 to 274 and 628 to 632, more
preferably at least about 70% identical to one of the sequences as
indicated in Table II, columns 5 or 7, lines 173, 262 to 274 and
628 to 632, preferably of Table II B, column 7, lines 173, 262 to
274 and 628 to 632, even more preferably at least about 80%, 90%,
or 95% homologous to a sequence as indicated in Table II, columns 5
or 7, lines 173, 262 to 274 and 628 to 632, preferably of Table II
B, column 7, lines 173, 262 to 274 and 628 to 632, and most
preferably at least about 96%, 97%, 98%, or 99% identical to the
sequence as indicated in Table II, columns 5 or 7, lines 173, 262
to 274 and 628 to 632, preferably of Table II B, column 7, lines
173, 262 to 274 and 628 to 632.
[9490] [0169.0.0.21] to [0172.0.0.21] for the disclosure of the
paragraphs [0169.0.0.21] to [0172.0.0.21] see paragraphs
[0169.0.0.0] to [0172.0.0.0] above.
[9491] [0173.0.21.21] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 27329 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 27329 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[9492] [0174.0.0.21]: for the disclosure of this paragraph see
paragraph [0174.0.0.0] above.
[9493] [0175.0.21.21] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 27330 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 27330 by the above program algorithm with the
above parameter set, has a 80% homology.
[9494] [0176.0.21.21] Functional equivalents derived from one of
the polypeptides as indicated in Table II, columns 5 or 7, lines
173, 262 to 274 and 628 to 632, resp., according to the invention
by substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp.,
according to the invention and are distinguished by essentially the
same properties as a polypeptide as indicated in Table II, columns
5 or 7, lines 173, 262 to 274 and 628 to 632, resp.
[9495] [0177.0.21.21] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 173,
262 to 274 and 628 to 632, preferably of Table I B, lines 173, 262
to 274 and 628 to 632 resp., according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp.,
according to the invention and encode polypeptides having
essentially the same properties as a polypeptide as indicated in
Table II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
preferably of Table II B, lines 173, 262 to 274 and 628 to 632
resp.
[9496] [0178.0.0.21] for the disclosure of this paragraph see
[0178.0.0.0] above.
[9497] [0179.0.21.21] A nucleic acid molecule encoding a homologue
to a protein sequence as indicated in Table II, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, preferably as indicated in
Table II B, lines 173, 262 to 274 and 628 to 632, resp., can be
created by introducing one or more nucleotide substitutions,
additions or deletions into a nucleotide sequence of the nucleic
acid molecule of the present invention, in particular as indicated
in Table I, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
preferably as indicated in Table I B, lines 173, 262 to 274 and 628
to 632 resp., such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences as
indicated in Table I, columns 5 or 7, lines 173, 262 to 274 and 628
to 632, preferably as indicated in Table I B, lines 173, 262 to 274
and 628 to 632, resp., by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
[9498] [0180.0.0.21] to [0183.0.0.21] for the disclosure of the
paragraphs [0180.0.0.21] to [0183.0.0.21] see paragraphs
[0180.0.0.0] to [0183.0.0.0] above.
[9499] [0184.0.21.21] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table I, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, preferably as indicated in
Table I B, lines 173, 262 to 274 and 628 to 632, resp., or of the
nucleic acid sequences derived from a sequences as indicated in
Table II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
preferably as indicated in Table II B, lines 173, 262 to 274 and
628 to 632, resp., comprise also allelic variants with at least
approximately 30%, 35%, 40% or 45% homology, by preference at least
approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
I, columns 5 or 7, lines 173, 262 to 274 and 628 to 632, preferably
as indicated in Table I B, lines 173, 262 to 274 and 628 to 632,
resp., or from the derived nucleic acid sequences, the intention
being, however, that the enzyme activity or the biological activity
of the resulting proteins synthesized is advantageously retained or
increased.
[9500] [0185.0.21.21] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table I, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
preferably as indicated in Table I B, lines 173, 262 to 274 and 628
to 632, resp. In one embodiment, it is preferred that the nucleic
acid molecule comprises as little as possible other nucleotides not
shown in any one of sequences as indicated in Table I, columns 5 or
7, lines 173, 262 to 274 and 628 to 632, preferably as indicated in
Table I B, lines 173, 262 to 274 and 628 to 632, resp. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequence as indicated in Table I, columns 5 or 7, lines 173, 262 to
274 and 628 to 632, preferably as indicated in Table I B, lines
173, 262 to 274 and 628 to 632, resp.
[9501] [0186.0.21.21] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 173, 262 to 274 and 628 to 632, preferably as
indicated in Table II B, lines 173, 262 to 274 and 628 to 632,
resp. In one embodiment, the nucleic acid molecule encodes less
than 150, 130, 100, 80, 60, 50, 40 or 30 further amino acids. In a
further embodiment, the encoded polypeptide comprises less than 20,
15, 10, 9, 8, 7, 6 or 5 further amino acids. In one embodiment, the
encoded polypeptide used in the process of the invention is
identical to the sequences as indicated in Table II, columns 5 or
7, lines 173, 262 to 274 and 628 to 632, preferably as indicated in
Table II B, lines 173, 262 to 274 and 628 to 632, resp.
[9502] [0187.0.21.21] In one embodiment, a nucleic acid molecule of
the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, preferably as indicated in
Table II B, lines 173, 262 to 274 and 628 to 632, resp., comprises
less than 100 further nucleotides. In a further embodiment, said
nucleic acid molecule comprises less than 30 further nucleotides.
In one embodiment, the nucleic acid molecule used in the process is
identical to a coding sequence encoding a sequences as indicated in
Table II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
preferably as indicated in Table II B, lines 173, 262 to 274 and
628 to 632, resp.
[9503] [0188.0.21.21] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity.
Advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp.,
and is expressed under identical conditions. In one embodiment, the
polypeptide of the invention is a homolog consisting of or
comprising the sequence as indicated in Table II B, columns 7,
lines 173, 262 to 274 and 628 to 632.
[9504] [0189.0.21.21] Homologues of a sequences as indicated in
Table I, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
resp., or of derived sequences as indicated in Table II, columns 5
or 7, lines 173, 262 to 274 and 628 to 632, resp., also mean
truncated sequences, cDNA, single-stranded DNA or RNA of the coding
and noncoding DNA sequence. Homologues of said sequences are also
understood as meaning derivatives, which comprise noncoding regions
such as, for example, UTRs, terminators, enhancers or promoter
variants. The promoters upstream of the nucleotide sequences stated
can be modified by one or more nucleotide substitution(s),
insertion(s) and/or deletion(s) without, however, interfering with
the functionality or activity either of the promoters, the open
reading frame (=ORF) or with the 3'-regulatory region such as
terminators or other 3' regulatory regions, which are far away from
the ORF. It is furthermore possible that the activity of the
promoters is increased by modification of their sequence, or that
they are replaced completely by more active promoters, even
promoters from heterologous organisms. Appropriate promoters are
known to the person skilled in the art and are mentioned herein
below.
[9505] [0190.0.0.21]: for the disclosure of this paragraph see
[0190.0.0.0] above.
[9506] [0191.0.21.21] In one embodiment, the organisms or part
thereof produce according to the herein mentioned process of the
invention an increased level of free and/or bound the respective
fine chemical compared to said control or selected organisms or
parts thereof.
[9507] [0191.1.0.21]: for the disclosure of this paragraph see
[0191.1.0.0] above.
[9508] [0192.0.0.21] to [0203.0.0.21] for the disclosure of the
paragraphs [0192.0.0.21] to [0203.0.0.21] see paragraphs
[0192.0.0.0] to [0203.0.0.0] above.
[9509] [0204.0.21.21] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [9510] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, columns 5 or 7, lines 173,
262 to 274 and 628 to 632, preferably as indicated in Table II B,
lines 173, 262 to 274 and 628 to 632, resp.; or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, i.e. glycerol-3-phosphate (line 173 and lines 630 to 632)
or glycerol (lines 262 to 274 and lines 628 and 629), and
accordingly glycerol as a component of total lipid (lines 265, 266,
267, 269, 271, 272 and 263), resp., in an organism or a part
thereof; [9511] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
preferably as indicated in Table I B, lines 173, 262 to 274 and 628
to 632, resp., or a fragment thereof conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [9512] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [9513] d) nucleic
acid molecule encoding a polypeptide whose sequence has at least
50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [9514] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [9515] f) nucleic acid molecule encoding a polypeptide,
the polypeptide being derived by substituting, deleting and/or
adding one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[9516] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [9517] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table III, columns 5 or 7, lines 173, 262 to 274
and 628 to 632 and conferring an increase in the amount of the
respective fine chemical, i.e. glycerol-3-phosphate (line 173 and
lines 630 to 632) or glycerol (lines 262 to 274 and lines 628 and
629) and accordingly glycerol as a component of total lipid (lines
265, 266, 267, 269, 271, 272 and 263) resp., in an organism or a
part thereof; [9518] i) nucleic acid molecule encoding a
polypeptide which is isolated, e.g. from a expression library, with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (g), preferably to (a)
to (c) and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [9519] j) nucleic
acid molecule which encodes a polypeptide comprising a consensus
sequence as indicated in Table IV, column 7, lines 173, 262 to 267,
269 and 271 to 274 and lines 628 to 632 and conferring an increase
in the amount of the respective fine chemical, i.e.
glycerol-3-phosphate (line 173 and lines 630 to 632) or glycerol
(lines 262 to 274 and lines 628 and 629) and accordingly glycerol
as a component of total lipid (lines 265, 266, 267, 269, 271, 272
and 263) resp., in an organism or a part thereof; [9520] k) nucleic
acid molecule encoding the amino acid sequence of a polypeptide
encoding a domaine of a polypeptide as indicated in Table II,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, preferably as
indicated in Table II B, lines 173, 262 to 274 and 628 to 632,
resp., and conferring an increase in the amount of the respective
fine chemical, i.e. glycerol-3-phosphate (line 173 and lines 630 to
632) or glycerol (lines 262 to 274 and lines 628 and 629) and
accordingly glycerol as a component of total lipid (lines 265, 266,
267, 269, 271, 272 and 263) resp., in an organism or a part
thereof; and [9521] l) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, preferably as
indicated in Table I B, lines 173, 262 to 274 and 628 to 632,
resp., or a nucleic acid molecule encoding, preferably at least the
mature form of, a polypeptide as indicated in Table II, columns 5
or 7, lines 173, 262 to 274 and 628 to 632, preferably as indicated
in Table II B, lines 173, 262 to 274 and 628 to 632, resp., and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which encompasses a
sequence which is complementary thereto; whereby, preferably, the
nucleic acid molecule according to (a) to (l) distinguishes over a
sequence as indicated in Table IA or IB, columns 5 or 7, lines 173,
262 to 274 and 628 to 632, resp., by one or more nucleotides. In
one embodiment, the nucleic acid molecule of the invention does not
consist of the sequence as indicated in Table IA or IB, columns 5
or 7, lines 173, 262 to 274 and 628 to 632, resp. In another
embodiment, the nucleic acid molecule of the present invention is
at least 30% identical and less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence as indicated in Table IA or IB,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp. In a
further embodiment the nucleic acid molecule does not encode a
polypeptide sequence as indicated in Table IIA or IIB, columns 5 or
7, lines 173, 262 to 274 and 628 to 632, resp. Accordingly, in one
embodiment, the nucleic acid molecule of the present invention
encodes in one embodiment a polypeptide which differs at least in
one or more amino acids from a polypeptide indicated in Table IIA
or IIB, columns 5 or 7, lines 173, 262 to 274 and 628 to 632 does
not encode a protein of a sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 173, 262 to 274 and 628 to 632.
Accordingly, in one embodiment, the protein encoded by a sequences
of a nucleic acid according to [9522] (a) to (l) does not consist
of a sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632. In a further embodiment, the
protein of the present invention is at least 30% identical to a
protein sequence indicated in Table IIA or IIB, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632 and less than 100%, preferably
less than 99.999%, 99.99% or 99.9%, more preferably less than 99%,
985, 97%, 96% or 95% identical to a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 173, 262 to 274 and 628 to
632.
[9523] [0205.0.0.21] to [0206.0.0.21]: see [0205.0.0.0] to
[0206.0.0.0]
[9524] [0207.0.21.21] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway.
[9525] These genes can be of heterologous or homologous origin.
Moreover, further biosynthesis genes may advantageously be present,
or else these genes may be located on one or more further nucleic
acid constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the glutamic acid metabolism, the
phosphoenolpyruvate metabolism, the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[9526] [0208.0.0.21] to [0226.0.0.21] for the disclosure of the
paragraphs [0208.0.0.21] to [0226.0.0.21] see paragraphs
[0208.0.0.0] to [0226.0.0.0] above.
[9527] [0227.0.21.21] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[9528] In addition to a sequence indicated in Table I, columns 5 or
7, lines 173, 262 to 274 and 628 to 632 or its derivatives, it is
advantageous to express and/or mutate further genes in the
organisms. Especially advantageously, additionally at least one
further gene of the acetyl-CoA or malonyl-CoA metabolic pathway or
a polypeptide having a very long chain fatty acid acyl (VLCFA) CoA
synthase activity, is expressed in the organisms such as plants or
microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the fine chemicals desired since, for example,
feedback regulations no longer exist to the same extent or not at
all. In addition it might be advantageously to combine one or more
of the sequences indicated in Table I, columns 5 or 7, lines 173,
262 to 274 and 628 to 632, resp., with genes which generally
support or enhance to growth or yield of the target organisms, for
example genes which lead to faster growth rate of microorganisms or
genes which produces stress-, pathogen, or herbicide resistant
plants.
[9529] [0228.0.21.21] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the
glycerol-3-phosphate and/or glycerol metabolism, in particular in
synthesis of glycerol-3-phosphate or glycerol.
[9530] [0229.0.21.21] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences indicated
in Table I, columns 5 or 7, lines 173, 262 to 274 and 628 to 632
used in the process and/or the abovementioned biosynthesis genes
are the sequences encoding further genes of the fatty acid pathway,
such as acetyl-CoA or malonyl-CoA or a polypeptide having a very
long chain fatty acid acyl (VLCFA) CoA synthase activity. These
genes can lead to an increased synthesis of the VLCFAs.
[9531] [0230.0.0.21] for the disclosure of this paragraph see
paragraph [0230.0.0.0] above.
[9532] [0231.0.21.21] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a glycerol and/or
glycerol-3-phosphate and/or a lipid degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene. A person skilled in the art knows for example,
that the inhibition or repression of a glycerol and/or
glycerol-3-phosphate or lipid degrading enzyme will result in an
increased accumulation of glycerol and/or glycerol-3-phosphate
and/or total lipids in plants.
[9533] [0232.0.0.21] to [0276.0.0.21] for the disclosure of the
paragraphs [0232.0.0.21] to [0276.0.0.21] see paragraphs
[0232.0.0.0] to [0276.0.0.0] above.
[9534] [0277.0.21.21] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously.
[9535] [0278.0.0.21] to [0282.0.0.21] for the disclosure of the
paragraphs [0278.0.0.21] to [0282.0.0.21] see paragraphs
[0278.0.0.0] to [0282.0.0.0] above.
[9536] [0283.0.21.21] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells, for example using the antibody
of the present invention as described below, e.g. an antibody
against a protein as indicated in Table II, column 3, lines 173,
262 to 274 and 628 to 632, resp., or an antibody against a
polypeptide as indicated in Table II, columns 5 or 7, lines 173,
262 to 274 and 628 to 632, resp., which can be produced by standard
techniques utilizing the polypeptid of the present invention or
fragment thereof.
[9537] Preferred are monoclonal antibodies specifically binding to
polypeptides as indicated in Table II, columns 5 or 7, lines 173,
262 to 274 and 628 to 632, more preferred specifically binding to
polypeptides as indicated in Table II, column 5, lines 173, 262 to
274 and 628 to 632.
[9538] [0284.0.0.21] for the disclosure of this paragraph see
[0284.0.0.0] above.
[9539] [0285.0.21.21] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp., or
as coded by a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp., or
functional homologues thereof.
[9540] [0286.0.21.21] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 173, 262 to 267, 269 and 271 to 274 and
lines 628 to 632 and in one another embodiment, the present
invention relates to a polypeptide comprising or consisting of a
consensus sequence as indicated in Table IV, column 7, lines 173,
262 to 267, 269 and 271 to 274 and lines 628 to 632 whereby 20 or
less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred
5 or 4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table IV, column 7, lines 173, 262 to 267, 269 and 271 to 274 and
lines 628 to 632.
[9541] [0287.0.0.21] to [0290.0.0.21] for the disclosure of the
paragraphs [0287.0.0.21] to [0290.0.0.21] see paragraphs
[0287.0.0.0] to [0290.0.0.0] above.
[9542] [0291.0.21.21] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[9543] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp., by one
or more amino acids. In one embodiment, polypeptide distinguishes
from a sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, resp., by more than 5, 6, 7,
8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or 30
amino acids, even more preferred are more than 40, 50, or 60 amino
acids and, preferably, the sequence of the polypeptide of the
invention distinguishes from a sequence as indicated in Table IIA
or IIB, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
resp., by not more than 80% or 70% of the amino acids, preferably
not more than 60% or 50%, more preferred not more than 40% or 30%,
even more preferred not more than 20% or 10%. In an other
embodiment, said polypeptide of the invention does not consist of a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
173, 262 to 274 and 628 to 632.
[9544] [0292.0.0.21] for the disclosure of this paragraph see
[0292.0.0.0] above.
[9545] [0293.0.21.21] In one embodiment, the invention relates to a
polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or by a nucleic acid molecule used in the process of the
invention. In one embodiment, the polypeptide of the invention has
a sequence which distinguishes from a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 173, 262 to 274 and 628 to
632, resp., by one or more amino acids. In an other embodiment,
said polypeptide of the invention does not consist of the sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 173, 262 to
274 and 628 to 632, resp. In a further embodiment, said polypeptide
of the present invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical. In one embodiment, said polypeptide does not
consist of the sequence encoded by a nucleic acid molecules as
indicated in Table IA or IB, columns 5 or 7, lines 173, 262 to 274
and 628 to 632, resp.
[9546] [0294.0.21.21] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 173, 262 to 274 and 628 to
632, resp., which distinguishes over a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 173, 262 to 274 and 628 to
632, resp., by one or more amino acids, preferably by more than 5,
6, 7, 8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or
30 amino acids, even more preferred are more than 40, 50, or 60
amino acids but even more preferred by less than 70% of the amino
acids, more preferred by less than 50%, even more preferred my less
than 30% or 25%, more preferred are 20% or 15%, even more preferred
are less than 10%.
[9547] [0295.0.0.21] to [0297.0.0.21] for the disclosure of the
paragraphs [0295.0.0.21] to [0297.0.0.21] see paragraphs
[0295.0.0.0] to [0297.0.0.0] above.
[9548] [00297.1.21.21] Non polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table II, columns 3, 5 or 7, lines 173, 262 to 274 and
628 to 632, resp.
[9549] [0298.0.21.21] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table II, columns 5 or 7, lines 173, 262 to 274 and
628 to 632, resp. The portion of the protein is preferably a
biologically active portion as described herein.
[9550] Preferably, the polypeptide used in the process of the
invention has an amino acid sequence identical to a sequence as
indicated in Table II, columns 5 or 7, lines 173, 262 to 274 and
628 to 632, resp.
[9551] [0299.0.21.21] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
173, 262 to 274 and 628 to 632, resp. The preferred polypeptide of
the present invention preferably possesses at least one of the
activities according to the invention and described herein.
[9552] A preferred polypeptide of the present invention includes an
amino acid sequence encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions, to a
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
173, 262 to 274 and 628 to 632, resp., or which is homologous
thereto, as defined above.
[9553] [0300.0.21.21] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp., in
amino acid sequence due to natural variation or mutagenesis, as
described in detail herein. Accordingly, the polypeptide comprise
an amino acid sequence which is at least about 35%, 40%, 45%, 50%,
55%, 60%, 65% or 70%, preferably at least about 75%, 80%, 85% or
90, and more preferably at least about 91%, 92%, 93%, 94% or 95%,
and most preferably at least about 96%, 97%, 98%, 99% or more
homologous to an entire amino acid sequence of as indicated in
Table IIA or IIB, columns 5 or 7, lines 173, 262 to 274 and 628 to
632, resp.
[9554] [0301.0.0.21] for the disclosure of this paragraph see
[0301.0.0.0] above.
[9555] [0302.0.21.21] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
resp., or the amino acid sequence of a protein homologous thereto,
which include fewer amino acids than a full length polypeptide of
the present invention or used in the process of the present
invention or the full length protein which is homologous to an
polypeptide of the present invention or used in the process of the
present invention depicted herein, and exhibit at least one
activity of polypeptide of the present invention or used in the
process of the present invention.
[9556] [0303.0.0.21] for the disclosure of this paragraph see
[0303.0.0.0] above.
[9557] [0304.0.21.21] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 173, 262 to 274 and 628 to 632 but having
differences in the sequence from said wild-type protein. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity in relation to the wild type protein.
[9558] [0305.0.0.21] to [0308.0.0.21] for the disclosure of the
paragraphs [0305.0.0.21] to [0308.0.0.21] see paragraphs
[0305.0.0.0] to [0308.0.0.0] above.
[9559] [0306.1.0.21] %
[9560] [0309.0.21.21] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table II,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp., refers
to a polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas an "other polypeptide" not being indicated in
Table II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
resp., refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to
a polypeptide of the invention, preferably which is not
substantially homologous to a polypeptide as indicated in Table II,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp., e.g.,
a protein which does not confer the activity described herein or
annotated or known for as indicated in Table II, column 3, lines
173, 262 to 274 and 628 to 632, resp., and which is derived from
the same or a different organism. In one embodiment, an "other
polypeptide" not being indicated in Table II, columns 5 or 7, lines
173, 262 to 274 and 628 to 632, resp., does not confer an increase
of the respective fine chemical in an organism or part thereof.
[9561] [0310.0.0.21] to [0334.0.0.21] for the disclosure of the
paragraphs [0310.0.0.21] to [0334.0.0.21] see paragraphs
[0310.0.0.0] to [0334.0.0.0] above.
[9562] [0335.0.21.21] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table I, columns 5 or
7, lines 173, 262 to 274 and 628 to 632, resp., and/or homologs
thereof. As described inter alia in WO 99/32619, dsRNAi approaches
are clearly superior to traditional antisense approaches. The
invention therefore furthermore relates to double-stranded RNA
molecules (dsRNA molecules) which, when introduced into an
organism, advantageously into a plant (or a cell, tissue, organ or
seed derived there from), bring about altered metabolic activity by
the reduction in the expression of a nucleic acid sequences as
indicated in Table I, columns 5 or 7, lines 173, 262 to 274 and 628
to 632, resp., and/or homologs thereof. In a double-stranded RNA
molecule for reducing the expression of a protein encoded by a
nucleic acid sequence as indicated in Table I, columns 5 or 7,
lines 173, 262 to 274 and 628 to 632, resp., and/or homologs
thereof, one of the two RNA strands is essentially identical to at
least part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[9563] [0336.0.0.21] to [0342.0.0.21] for the disclosure of the
paragraphs [0336.0.0.21] to [0342.0.0.21] see paragraphs
[0336.0.0.0] to [0342.0.0.0] above.
[9564] [0343.0.21.21] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 173, 262 to
274 and 628 to 632, resp., or its homolog is not necessarily
required in order to bring about effective reduction in the
expression. The advantage is, accordingly, that the method is
tolerant with regard to sequence deviations as may be present as a
consequence of genetic mutations, polymorphisms or evolutionary
divergences. Thus, for example, using the dsRNA, which has been
generated starting from a sequence as indicated in Table I, columns
5 or 7, lines 173, 262 to 274 and 628 to 632, resp., or homologs
thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[9565] [0344.0.0.21] to [0361.0.0.21] for the disclosure of the
paragraphs [0344.0.0.21] to [0361.0.0.21] see paragraphs
[0344.0.0.0] to [0361.0.0.0] above.
[9566] [0362.0.21.21] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, columns 5 or 7, lines 173, 262 to 274 and 628 to 632,
resp., e.g. encoding a polypeptide having protein activity, as
indicated in Table II, columns 3, lines 173, 262 to 274 and 628 to
632, resp. Due to the above-mentioned activity the respective fine
chemical content in a cell or an organism is increased. For
example, due to modulation or manipulation, the cellular activity
of the polypeptide of the invention or nucleic acid molecule of the
invention is increased, e.g. due to an increased expression or
specific activity of the subject matters of the invention in a cell
or an organism or a part thereof. Transgenic for a polypeptide
having an activity of a polypeptide as indicated in Table II,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632, resp., means
herein that due to modulation or manipulation of the genome, an
activity as annotated for a polypeptide as indicated in Table II,
column 3, lines 173, 262 to 274 and 628 to 632, e.g. having a
sequence as indicated in Table II, columns 5 or 7, lines 173, 262
to 274 and 628 to 632, resp., is increased in a cell or an organism
or a part thereof. Examples are described above in context with the
process of the invention.
[9567] [0363.0.0.21] for the disclosure of this paragraph see
paragraph [0363.0.0.0] above.
[9568] [0364.0.21.21] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table II, column 3, lines 173, 262 to 274 and 628 to
632, resp. with the corresponding protein-encoding sequence as
indicated in Table I, column 5, lines 173, 262 to 274 and 628 to
632, resp., becomes a transgenic expression cassette when it is
modified by non-natural, synthetic "artificial" methods such as,
for example, mutagenization. Such methods have been described (U.S.
Pat. No. 5,565,350; WO 00/15815; also see above).
[9569] [0365.0.0.21] to [0373.0.0.21] for the disclosure of the
paragraphs [0365.0.0.21], to [0373.0.0.21] see paragraphs
[0365.0.0.0] to [0373.0.0.0] above.
[9570] [0374.0.21.21] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. Glycerol and/or
glycerol-3-phosphate, in particular the respective fine chemical,
produced in the process according to the invention may, however,
also be isolated from the plant in the form of their free glycerol
and/or glycerol-3-phosphate, in particular the free respective fine
chemical, or bound in or to compounds or moieties, as for example
but not limited to in mono-, di- or triacylglyceroles,
phosphoglycerides, monoacylglycerol phosphate or diacylglycerol
phosphate. The respective fine chemical produced by this process
can be harvested by harvesting the organisms either from the
culture in which they grow or from the field. This can be done via
expressing, grinding and/or extraction, salt precipitation and/or
ion-exchange chromatography or other chromatographic methods of the
plant parts, preferably the plant seeds, plant fruits, plant tubers
and the like.
[9571] [0375.0.0.21] and [0376.0.0.21] for the disclosure of the
paragraphs [0375.0.0.21] and [0376.0.0.21] see paragraphs
[0375.0.0.0] and [0376.0.0.0] above.
[9572] [0377.0.21.21] Accordingly, the present invention relates
also to a process whereby the produced glycerol and/or
glycerol-3-phosphate and/or total lipid is isolated.
[9573] [0378.0.21.21] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the glycerol
and/or glycerol-3-phosphate and/or total lipid produced in the
process can be isolated. The resulting glycerol and/or
glycerol-3-phosphate and/or total lipid can, if appropriate,
subsequently be further purified, if desired mixed with other
active ingredients such as vitamins, amino acids, carbohydrates,
antibiotics and the like, and, if appropriate, formulated.
[9574] [0379.0.21.21] In one embodiment the product produced by the
present invention is a mixture of the respective fine chemicals
glycerol, glycerol-3-phosphate and total lipids.
[9575] [0380.0.21.21] The glycerol and/or glycerol-3-phosphate or
total lipids obtained in the process by carrying out the invention
is suitable as starting material for the synthesis of further
products of value. For example, they can be used in combination
with each other or alone for the production of pharmaceuticals,
foodstuffs, animal feeds or cosmetics. Accordingly, the present
invention relates to a method for the production of
pharmaceuticals, food stuff, animal feeds, nutrients or cosmetics
comprising the steps of the process according to the invention,
including the isolation of the glycerol and/or glycerol-3-phosphate
composition produced or the respective fine chemical produced if
desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the glycerol and/or
glycerol-3-phosphate and/or total lipid produced in the process or
of the transgenic organism in animal feeds, foodstuffs, medicines,
food supplements, cosmetics or pharmaceuticals or for the
production of glycerol and/or glycerol-3-phosphate e.g. after
isolation of the respective fine chemical or without, e.g. in situ,
e.g in the organism used for the process for the production of the
respective fine chemical.
[9576] [0381.0.0.21] and [0382.0.0.21] for the disclosure of the
paragraphs [0381.0.0.21] and [0382.0.0.21] see paragraphs
[0381.0.0.0] and [0382.0.0.0] above.
[9577] [0383.0.21.21] %
[9578] [0384.0.0.21] for the disclosure of this paragraph see
[0384.0.0.0] above.
[9579] [0385.0.21.21] The fermentation broths obtained in this way,
containing in particular glycerol and/or glycerol-3-phosphate
and/or total lipids in mixtures with other organic acids, amino
acids, polypeptides or polysaccarides, normally have a dry matter
content of from 1 to 70% by weight, preferably 7.5 to 25% by
weight. Sugar-limited fermentation is additionally advantageous,
e.g. at the end, for example over at least 30% of the fermentation
time. This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 10 g/l,
preferably to 0 to 3 g/l during this time. The fermentation broth
is then processed further. Depending on requirements, the biomass
can be removed or isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth or left completely in it.
[9580] The fermentation broth can then be thickened or concentrated
by known methods, such as, for example, with the aid of a rotary
evaporator, thin-film evaporator, falling film evaporator, by
reverse osmosis or by nanofiltration. This concentrated
fermentation broth can then be worked up by freeze-drying, spray
drying, spray granulation or by other processes.
[9581] [0386.0.21.21] Accordingly, it is possible to purify the
glycerol and/or glycerol-3-phosphate produced according to the
invention further. For this purpose, the product-containing
composition is subjected for example to separation via e.g. an open
column chromatography or HPLC in which case the desired product or
the impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use.
[9582] [0387.0.0.21] to [0392.0.0.21] for the disclosure of the
paragraphs [0387.0.0.21] to [0392.0.0.21] see paragraphs
[0387.0.0.0] to [0392.0.0.0] above.
[9583] [0393.0.21.21] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [9584] a. contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [9585] b. identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table I, columns 5 or 7, lines
173, 262 to 274 and 628 to 632, preferably as indicated in Table
IB, columns 5 or 7, lines 173, 262 to 274 and 628 to 632 resp.,
and, optionally, isolating the full length cDNA clone or complete
genomic clone; [9586] c. introducing the candidate nucleic acid
molecules in host cells, preferably in a plant cell or a
microorganism, appropriate for producing the respective fine
chemical; [9587] d. expressing the identified nucleic acid
molecules in the host cells; [9588] e. assaying the respective fine
chemical level in the host cells; and [9589] f. identifying the
nucleic acid molecule and its gene product which expression confers
an increase in the respective fine chemical level in the host cell
after expression compared to the wild type.
[9590] [0394.0.0.21] to [0398.0.0.21] for the disclosure of the
paragraphs [0394.0.0.21] to [0398.0.0.21] see paragraphs
[0394.0.0.0] to [0398.0.0.0] above.
[9591] [0399.0.21.21] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increase of the respective fine chemical production in a
plant or microorganism, comprising the steps: [9592] (a) culturing
a cell or tissue or microorganism or maintaining a plant expressing
the polypeptide according to the invention or a nucleic acid
molecule encoding said polypeptide and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and the
polypeptide of the present invention or used in the process of the
invention; and [9593] (b) identifying if the compound is an
effective agonist by detecting the presence or absence or increase
of a signal produced by said readout system.
[9594] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in respective fine chemical
production.
[9595] [00399.1.21.21] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632 or a homolog
thereof, e.g. comparing the phenotype of nearly identical organisms
with low and high activity of a protein as indicated in Table II,
columns 5 or 7, lines 173, 262 to 274 and 628 to 632 after
incubation with the drug.
[9596] [0400.0.0.21] to [0416.0.0.21] for the disclosure of the
paragraphs [0400.0.0.21] to [0416.0.0.21] see paragraphs
[0400.0.0.0] to [0416.0.0.0] above.
[9597] [0417.0.21.21] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the glycerol and/or glycerol-3-phosphate
biosynthesis pathways. In particular, the overexpression of the
polypeptide of the present invention may protect an organism such
as a microorganism or a plant against inhibitors, which block the
glycerol and/or glycerol-3-phosphate synthesis.
[9598] [0418.0.0.21] to [0423.0.0.21] for the disclosure of the
paragraphs [0418.0.0.21] to [0423.0.0.21] see paragraphs
[0418.0.0.0] to [0423.0.0.0] above.
[9599] [0424.0.21.21] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other organic acids.
Accordingly, the nucleic acid of the invention, or the nucleic acid
molecule identified with the method of the present invention or the
complement sequences thereof, the polypeptide of the invention, the
nucleic acid construct of the invention, the organisms, the host
cell, the microorganisms, the plant, plant tissue, plant cell, or
the part thereof of the invention, the vector of the invention, the
antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the respective
fine chemical in a organism or part thereof, e.g. in a cell.
[9600] [0425.0.0.21] to [0434.0.0.21] for the disclosure of the
paragraphs [0425.0.0.21] to [0434.0.0.21] see paragraphs
[0425.0.0.0] to [0434.0.0.0] above.
[0435.0.21.21] Example 3
In-Vivo and In-Vitro Mutagenesis
[9601] [0436.0.21.21] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of glycerol and/or
glycerol-3-phosphate can be carried out by passing a plasmid DNA
(or another vector DNA) containing the desired nucleic acid
sequence or nucleic acid sequences, e.g. the nucleic acid molecule
of the invention or the vector of the invention, through E. coli
and other microorganisms (for example Bacillus spp. or yeasts such
as Saccharomyces cerevisiae) which are not capable of maintaining
the integrity of its genetic information. Usual mutator strains
have mutations in the genes for the DNA repair system [for example
mutHLS, mutD, mutT and the like; for comparison, see Rupp, W. D.
(1996) DNA repair mechanisms in Escherichia coli and Salmonella,
pp. 2277-2294, ASM: Washington]. The skilled worker knows these
strains. The use of these strains is illustrated for example in
Greener, A. and Callahan, M. (1994) Strategies 7; 32-34.
[9602] [0436.1.21.21] In-vitro mutation methods such as increasing
the spontaneous mutation rates by chemical or physical treatment
are well known to the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[9603] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new
[9604] DNA synthesis to create a complementary full-length sequence
containing the desired mutation. Another site-directed mutagensis
method is the PCR mismatch primer mutagensis method also known to
the skilled person. Dpnl site-directed mutagensis is a further
known method as described for example in the Stratagene
Quickchange.TM. site-directed mutagenesis kit protocol. A huge
number of other methods are also known and used in common
practice.
[9605] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.21.21] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpina
[9606] [0438.0.21.21] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology", VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[9607] [0439.0.21.21] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255f) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[9608] [0440.0.21.21] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002) teaches a homolog recombination system dependent on
rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this system
for homologous recombination are vectors derived from the Ylp
series. Plasmid vectors derived for example from the 2.mu.-Vector
are known by the skilled worker and used for the expression in
yeasts. Other preferred vectors are for example pART1, pCHY21 or
pEVP11 as they have been described by McLeod et al. (EMBO J. 1987,
6:729-736) and Hoffman et al. (Genes Dev. 5, 1991: :561-571.) or
Russell et al. (J. Biol. Chem. 258, 1983: 143-149.). Other
beneficial yeast vectors are plasmids of the REP, REP-X, pYZ or RIP
series.
[9609] [0441.0.0.21] to [0443.0.0.21] for the disclosure of the
paragraphs [0441.0.0.21] to [0443.0.0.21] see [0441.0.0.0] to
[0443.0.0.0] above.
[0444.0.21.21] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[9610] [0445.0.21.21] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[9611] [0446.0.0.21] to [0450.0.0.21] for the disclosure of the
paragraphs [0446.0.0.21] to [0450.0.0.21] see [0446.0.0.0] to
[0450.0.0.0] above.
[9612] [0451.0.5.21] for the disclosure of this paragraph see
paragraph [0451.0.5.5] above.
[9613] [0452.0.0.21] and [0453.0.0.21] for the disclosure of the
paragraphs [0452.0.0.21] and [0453.0.0.21] see [0452.0.0.0] and
[0453.0.0.0] above.
[0454.0.21.21] Example 8
Analysis of the Effect of the Nucleic Acid Molecule on the
Production of Glycerol and/or Glycerol-3-Phosphate
[9614] [0455.0.21.21] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a glycerol and/or glycerol-3-phosphate) can be
determined by growing the modified microorganisms or the modified
plant under suitable conditions (such as those described above) and
analyzing the medium and/or the cellular components for the
elevated production of desired product (i.e. of glycerol and/or
glycerol-3-phosphate). These analytical techniques are known to the
skilled worker and comprise spectroscopy, thin-layer
chromatography, various types of staining methods, enzymatic and
microbiological methods and analytical chromatography such as
high-performance liquid chromatography (see, for example, Ullman,
Encyclopedia of Industrial Chemistry, Vol. A2, p. 89-90 and p.
443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987)
"Applications of HPLC in Biochemistry" in: Laboratory Techniques in
Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993)
Biotechnology, Vol. 3, Chapter III: "Product recovery and
purification", p. 469-714, VCH: Weinheim; Belter, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[9615] [0456.0.0.21]: for the disclosure of this paragraph see
paragraph [0456.0.0.0] above.
[0457.0.21.21] Example 9
Purification of Glycerol and/or Glycerol-3-Phosphate
[9616] [0458.0.21.21] Abbreviations; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[9617] The unambiguous detection for the presence of glycerol
and/or glycerol-3-phosphate can be obtained by analyzing
recombinant organisms using analytical standard methods: LC,
LC-MSMS, GC-MS or TLC, as described. The total amount produced in
the organism for example in yeasts used in the inventive process
can be analysed for example according to the following
procedure:
[9618] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods.
[9619] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[9620] A typical sample pretreatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography.
[9621] For analysis, solvent delivery and aliquot removal can be
accomplished with a robotic system comprising a single injector
valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000
W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of
50% potassium hydroxide hydro-ethanolic solution (4 water--1
ethanol) can be added to each vial, followed by the addition of 3
ml of octanol. The saponification treatment can be conducted at
room temperature with vials maintained on an IKA HS 501 horizontal
shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours
at 250 movements/minute, followed by a stationary phase of
approximately one hour.
[9622] Following saponification, the supernatant can be diluted
with 0-20 ml of methanol. The addition of methanol can be conducted
under pressure to ensure sample homogeneity. Using a 0.25 ml
syringe, a 0.1 ml aliquot can be removed and transferred to HPLC
vials for analysis.
[9623] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete
with a quaternary pump, vacuum degassing system, six-way injection
valve, temperature regulated autosampler, column oven and
Photodiode Array detector can be used [Agilent Technologies
available through Ultra Scientific Inc., 250 Smith Street, North
Kingstown,
[9624] RI]. The column can be a Waters YMC30, 5-micron,
4.6.times.250 mm with a guard column of the same material [Waters,
34 Maple Street, Milford, Mass.]. The solvents for the mobile phase
can be 81 methanol: 4 water: 15 tetrahydrofuran (THF) stabilized
with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injections were
20 .mu.l. Separation can be isocratic at 30.degree. C. with a flow
rate of 1.7 ml/minute. The peak responses can be measured by
absorbance at 447 nm.
[9625] [0459.0.21.21] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the glycerol and/or glycerol-3-phosphate can be
further purified with a so-called RTHPLC. As eluent
acetonitrile/water or chloroform/acetonitrile mixtures can be used.
If necessary, these chromatography steps may be repeated, using
identical or other chromatography resins. The skilled worker is
familiar with the selection of suitable chromatography resin and
the most effective use for a particular molecule to be
purified.
[9626] [0460.0.0.21] for the disclosure of this paragraph see
paragraph [0460.0.0.0] above.
[0461.0.21.21] Example 10
Cloning SEQ ID NO: 14364, 27329, 27377, 27857, 28009, 28145, 28179,
28197, 28201, 28207, 28211, 28341, 28363, 28369, 98288, 98533,
97771, 97966 or 98453 for the Expression in Plants
[9627] [0462.0.0.21] for the disclosure of this paragraph see
paragraph [0462.0.0.0] above.
[9628] [0463.0.21.21] SEQ ID NO: 14364, 27329, 27377, 27857, 28009,
28145, 28179, 28197, 28201, 28207, 28211, 28341, 28363, 28369,
98288, 98533, 97771, 97966 or 98453 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[9629] [0464.0.0.21] to [0466.0.0.21] for the disclosure of the
paragraphs [0464.0.0.21] to [0466.0.0.21] see paragraphs
[0464.0.0.0] to [0466.0.0.0] above.
[9630] [0466.1.0.21] In case the Herculase enzyme can be used for
the amplification, the PCR amplification cycles were as follows: 1
cycle of 2-3 minutes at 94.degree. C., followed by 25-30 cycles of
in each case 30 seconds at 94.degree. C., 30 seconds at
55-60.degree. C. and 5-10 minutes at 72.degree. C., followed by 1
cycle of 10 minutes at 72.degree. C., then 4.degree. C.
[9631] [0467.0.21.21] The following primer sequences were selected
for the gene SEQ ID NO: 14364
TABLE-US-00112 i) forward primer (SEQ ID NO: 14370) atggctcaaa
attttggaaa gattcc ii) reverse primer (SEQ ID NO: 14371) ttaaaaaccg
tatttcgcca aaacac
[9632] The following primer sequences were selected for the gene
SEQ ID NO:27329:
TABLE-US-00113 i) forward primer (SEQ ID NO: 27375) atgaaaaatg
ccaatcatcg attcttc ii) reverse primer (SEQ ID NO: 27376) ttagaagaac
agtgacggat cgcc
[9633] The following primer sequences were selected for the gene
SEQ ID NO:27377:
TABLE-US-00114 i) forward primer (SEQ ID NO: 27855) atgtctgagc
agtttttgta tttctt ii) reverse primer (SEQ ID NO: 27856) ttatactttc
tctacctccg ggcg
[9634] The following primer sequences were selected for the gene
SEQ ID NO: 27857:
TABLE-US-00115 i) forward primer (SEQ ID NO: 28007) atgttgtcga
gactatcttt attgag ii) reverse primer (SEQ ID NO: 28008) ttaaaataga
ccttcaattt caccgt
[9635] The following primer sequences were selected for the gene
SEQ ID NO:28009:
TABLE-US-00116 i) forward primer (SEQ ID NO: 28143) atggagacca
atttttcctt cgact ii) reverse primer (SEQ ID NO: 28144) ctattgaaat
accggcttca atattt
[9636] The following primer sequences were selected for the gene
SEQ ID NO:28145:
TABLE-US-00117 i) forward primer (SEQ ID NO: 28177) atgtcagcag
acgaaacgga tgc ii) reverse primer (SEQ ID NO: 28178) ttatgttttt
ttgtctgctg cagct
[9637] The following primer sequences were selected for the gene
SEQ ID NO:28179:
TABLE-US-00118 i) forward primer (SEQ ID NO: 28195) atgaatgatc
ctcgtgaaat tttagc ii) reverse primer (SEQ ID NO: 28196) ttatattatc
tcaagatcgc tggca
[9638] The following primer sequences were selected for the gene
SEQ ID NO:28197:
TABLE-US-00119 i) forward primer (SEQ ID NO: 28199) atgtccaact
ttaagaattt tactttaa ii) reverse primer (SEQ ID NO: 28200)
tcatttgttt atcagtgtaa caagca
[9639] The following primer sequences were selected for the gene
SEQ ID NO:28201:
TABLE-US-00120 i) forward primer (SEQ ID NO: 28205) atgaatcaga
gcgatagcag cttg ii) reverse primer (SEQ ID NO: 28206) tcatcttcga
agataagggg tattc
[9640] The following primer sequences were selected for the gene
SEQ ID NO:28207:
TABLE-US-00121 i) forward primer (SEQ ID NO: 28209) atggcggttg
cgatcaaaaa gga ii) reverse primer (SEQ ID NO: 28210) tcaattgata
aatgtacttt caatgatg
[9641] The following primer sequences were selected for the gene
SEQ ID NO:28211:
TABLE-US-00122 i) forward primer (SEQ ID NO: 28339) atgagttctg
tcgcagaaaa tataat ii) reverse primer (SEQ ID NO: 28340) ttattcgaag
acttctccag taattg
[9642] The following primer sequences were selected for the gene
SEQ ID NO: 28341:
TABLE-US-00123 i) forward primer (SEQ ID NO: 28361) atgaactcta
ttttagacag aaatgtt ii) reverse primer (SEQ ID NO: 28362) ttatttttgg
tcttgtttca aagtgtc
[9643] The following primer sequences were selected for the gene
SEQ ID NO: 28363:
TABLE-US-00124 i) forward primer (SEQ ID NO: 28367) atgttaaaaa
gacgctctaa tgctc ii) reverse primer (SEQ ID NO: 28368) tcaccaaaac
catctcctat accaa
[9644] The following primer sequences were selected for the gene
SEQ ID NO: 28369:
TABLE-US-00125 i) forward primer (SEQ ID NO: 28563) atgactgaaa
gtgctataga tgacc ii) reverse primer (SEQ ID NO: 28564) ctataaaatt
tgtgtatgaa taaataaag
[9645] The following primer sequences were selected for the gene
SEQ ID NO: 98288:
TABLE-US-00126 i) forward primer (SEQ ID NO: 98448) atgatccgca
gtatgaccgc ct ii) reverse primer (SEQ ID NO: 98449) ttattcgatg
ttctgaatct gctc
[9646] The following primer sequences were selected for the gene
SEQ ID NO: 98533:
TABLE-US-00127 i) forward primer (SEQ ID NO: 98701) ttaaagcgta
tgtgtttcat atgcc ii) reverse primer (SEQ ID NO: 98702) atgacagaat
tttattctga cacaatc
[9647] The following primer sequences were selected for the gene
SEQ ID NO: 97771:
TABLE-US-00128 i) forward primer (SEQ ID NO: 97961) atgtctattg
tggtgaaaaa taacatt ii) reverse primer (SEQ ID NO: 97962) ttattttgcc
tccgatgcca gttct
[9648] The following primer sequences were selected for the gene
SEQ ID NO: 97966:
TABLE-US-00129 i) forward primer (SEQ ID NO: 98282) atgacgaatc
cgttactgac tcc ii) reverse primer (SEQ ID NO: 98283) ttagccctta
atgccgtaat gctcc
[9649] The following primer sequences were selected for the gene
SEQ ID NO: 98453:
TABLE-US-00130 i) forward primer (SEQ ID NO: 98527) atgacgcaga
cttccgcatt tca ii) reverse primer (SEQ ID NO: 98528) ttacgccacc
gtcaactgtc cg
[9650] [0468.0.0.21] to [0470.0.0.21] for the disclosure of the
paragraphs [0468.0.0.21] to [0470.0.0.21] see paragraphs
[0468.0.0.0] to [0470.0.0.0] above.
[9651] [0470.1.21.21] The PCR-products, produced by Pfu Turbo DNA
polymerase, were phosphorylated using a T4 DNA polymerase using a
standard protocol (e.g. MBI Fermentas) and cloned into the
processed binary vector.
[9652] [0471.0.21.21] for the disclosure of this paragraph see
[0471.0.0.0] above.
[9653] [0471.1.21.21] The DNA termini of the PCR-products, produced
by Herculase DNA polymerase, were blunted in a second synthesis
reaction using Pfu Turbo DNA polymerase. The composition for the
protocol of the blunting the DNA-termini was as follows: 0.2 mM
blunting dTTP and 1.25 u Pfu Turbo DNA polymerase. The reaction was
incubated at 72.degree. C. for 30 minutes. Then the PCR-products
were phosphorylated using a T4 DNA polymerase using a standard
protocol (e.g. MBI Fermentas) and cloned into the processed vector
as well.
[9654] [0472.0.0.21] to [0479.0.0.21] for the disclosure of the
paragraphs [0472.0.0.21] to [0479.0.0.21] see paragraphs
[0472.0.0.0] to [0479.0.0.0] above.
[0480.0.21.21] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 14364,
27329, 27377, 27857, 28009, 28145, 28179, 28197, 28201, 28207,
28211, 28341, 28363, 28369, 98288, 98533, 97771, 97966 or 98453
[9655] [0481.0.0.21] to [0513.0.0.21] for the disclosure of the
paragraphs [0481.0.0.21] to [0513.0.0.21] see paragraphs
[0482.0.0.0] to [0513.0.0.0] above.
[9656] [0514.0.21.21] As an alternative, glycerol-3-phosphate can
be detected as described in Yaqoob Metal., J Biolumin Chemilumin.
1997 January-February; 12 (1): 1-5. Glycerol can be detected as
described in Foglia, T. A. et al., 2004. Chromatographia.
60:305-311.
[9657] The results of the different plant analyses can be seen from
the table 1 which follows:
TABLE-US-00131 TABLE 1 ORF Metabolite Method Min Max YDR065
glycerol-3-phosphate GC 1.18 2.14 YBR084W glycerol, polar fraction
GC 2.02 3.53 YDR513W glycerol, lipid fraction GC 1.17 1.48 YDR513W
glycerol, polar fraction GC 1.40 3.00 YGL237C glycerol, lipid
fraction GC 1.23 1.73 YIL150C glycerol, lipid fraction GC 1.94 3.19
YLR082C glycerol, polar fraction GC 1.41 2.73 YLR224W glycerol,
lipid fraction GC 1.17 1.43 YLR255C glycerol, polar fraction GC
1.44 1.73 YMR015C glycerol, lipid fraction GC 1.18 1.23 YOR344C
glycerol, lipid fraction GC 1.18 2.04 YPL099C glycerol, polar
fraction GC 1.55 2.04 YPL268W glycerol, polar fraction GC 1.38 2.15
b2441 glycerol, polar fraction GC 1.41 1.81 b3457 glycerol, lipid
fraction GC 1.18 1.44 YHR072W Glycerol, lipid fraction GC 1.19 2.09
b3644 Glycerol, lipid fraction GC 1.17 1.44 b3498
Glycerol-3-phosphate, GC 1.20 1.56 lipid fraction b2710
Glycerol-3-phosphate, GC 1.20 1.95 lipid fraction b4073
Glycerol-3-phosphate, GC 1.49 2.45 lipid fraction
[9658] [0515.0.21.21] Column 2 shows the metabolite glycerol or
glycerol-3-phosphate analyzed. According to its solubility the free
glycerol of the cells is present and measured in the polar fraction
(glycerol, polar fraction). The non polar extract (=lipid fraction)
is evaporated and the residue is treated with a mixture of methanol
and hydrochloric acid in order to cleave ester functions of the
analytes as described above in example 13. The glycerol, bonded
with up to three ester functions, i.e. in lipids, is converted to
free glycerol by this reaction, measured as the glycerol in the
lipid fraction. Columns 4 and 5 shows the ratio of the analyzed
metabolite between the transgenic plants and the wild type;
Increase of the metabolite: Max: maximal x-fold (normalised to wild
type)-Min: minimal x-fold (normalised to wild type). Decrease of
the metabolite: Max: maximal x-fold (normalised to wild type)
(minimal decrease), Min: minimal x-fold (normalised to wild type)
(maximal decrease). Column 3 indicates the analytical method.
[9659] [0516.0.0.21] to [0530.0.0.21] for the disclosure of the
paragraphs [0516.0.0.21] to [0530.0.0.21] see paragraphs
[0516.0.0.0] to [0530.0.0.0] above.
[9660] [0530.1.0.21] to [0530.6.0.21] for the disclosure of the
paragraphs [0530.1.0.21] to [0530.6.0.21] see paragraphs
[0530.1.0.0] to [0530.6.0.0] above.
[9661] [0531.0.0.21] to [0552.0.0.21] for the disclosure of the
paragraphs [0531.0.0.21] to [0552.0.0.21] see paragraphs
[0531.0.0.0] to [0552.0.0.0] above.
[0552.1.21.21]: Example 15
Metabolite Profiling Info from Zea mays
[9662] Zea mays plants were engineered, grown and analysed as
described in Example 14c.
[9663] The results of the different Zea mays plants analysed can be
seen from Table 2, which follows:
TABLE-US-00132 TABLE 2 ORF_NAME Metabolite Min Max YIL150C
Glycerol, lipid fraction 2.12 2.66 b3644 Glycerol, lipid fraction
1.22 1.33 YBR084W Glycerol, polar fraction 1.36 1.44 YLR082C
Glycerol, polar fraction 1.12 1.35
[9664] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in glycerol in genetically
modified corn plants expressing the Saccharomyces cerevisiae
nucleic acid sequence YIL150C, YBR084W or YLR082C or E. coli
nucleic acid sequence b3644 resp.
[9665] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YIL150C or its homologs, e.g. a "chromatin
binding protein, required for S-phase (DNA synthesis) initiation or
completion", is increased in corn plants, preferably, an increase
of the fine chemical glycerol between 112% and 166% is
conferred.
[9666] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YBR084W or its homologs, e.g. a protein with
C1-tetrahydrofolate synthase activity or its homologs, is increased
in corn plants, preferably, an increase of the fine chemical
glycerol between 36% and 44% is conferred.
[9667] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YLR082C or its homologs, e.g. a suppressor of
Rad53 null lethality protein or its homologs, is increased in corn
plants, preferably, an increase of the fine chemical glycerol
between 12% and 35% is conferred.
[9668] In one embodiment, in case the activity of the E. coli
protein b3644 or its homologs, e.g. "the activity of an
uncharacterized stress-induced protein", is increased in corn
plants, preferably, an increase of the fine chemical glycerol
between 22% and 33% is conferred.
[9669] [0552.2.0.21] for the disclosure of this paragraph see
[0552.2.0.0] above.
[9670] [0553.0.21.21] [9671] 1. A process for the production of
glycerol-3-phosphate or glycerol, which comprises [9672] (a)
increasing or generating the activity of a protein as indicated in
Table II, columns 5 or 7, lines 173,262 to 274 and 628 to 632 or a
functional equivalent thereof in a non-human organism or in one or
more parts thereof; and [9673] (b) growing the organism under
conditions which permit the production of glycerol-3-phosphate or
glycerol in said organism. [9674] 2. A process for the production
of glycerol-3-phosphate or glycerol, comprising the increasing or
generating in an organism or a part thereof the expression of at
least one nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [9675] a) nucleic acid
molecule encoding a polypeptide as indicated in Table II, columns 5
or 7, lines 173,262 to 274 and 628 to 632 or a fragment thereof,
which confers an increase in the amount of glycerol-3-phosphate or
glycerol in an organism or a part thereof; [9676] b) nucleic acid
molecule comprising of a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 173,262 to 274 and 628 to 632;
[9677] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of glycerol-3-phosphate or
glycerol in an organism or a part thereof; [9678] d) nucleic acid
molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of glycerol-3-phosphate or glycerol in an organism or
a part thereof; [9679] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under stringent
hybridisation conditions and conferring an increase in the amount
of glycerol-3-phosphate or glycerol in an organism or a part
thereof; [9680] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, column 7, lines
173, 262 to 274 and 628 to 632 and conferring an increase in the
amount of glycerol-3-phosphate or glycerol in an organism or a part
thereof; [9681] g) nucleic acid molecule encoding a polypeptide
which is isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of
glycerol-3-phosphate or glycerol in an organism or a part thereof;
[9682] h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table IV, column 7, lines 173, 262 to 274
and 628 to 632 and conferring an increase in the amount of
glycerol-3-phosphate or glycerol in an organism or a part thereof;
and [9683] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of glycerol-3-phosphate or glycerol in an organism or a part
thereof. [9684] or comprising a sequence which is complementary
thereto. [9685] 3. The process of claim 1 or 2, comprising
recovering of the free or bound glycerol-3-phosphate or glycerol.
[9686] 4. The process of any one of claims 1 to 3, comprising the
following steps: [9687] a) selecting an organism or a part thereof
expressing a polypeptide encoded by the nucleic acid molecule
characterized in claim 2; [9688] b) mutagenizing the selected
organism or the part thereof; [9689] c) comparing the activity or
the expression level of said polypeptide in the mutagenized
organism or the part thereof with the activity or the expression of
said polypeptide of the selected organisms or the part thereof;
[9690] d) selecting the mutated organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
[9691] e) optionally, growing and cultivating the organisms or the
parts thereof; and [9692] f) recovering, and optionally isolating,
the free or bound glycerol-3-phosphate or glycerol produced by the
selected mutated organisms or parts thereof. [9693] 5. The process
of any one of claims 1 to 4, wherein the activity of said protein
or the expression of said nucleic acid molecule is increased or
generated transiently or stably. [9694] 6. An isolated nucleic acid
molecule comprising a nucleic acid molecule selected from the group
consisting of: [9695] a) nucleic acid molecule encoding a
polypeptide as indicated in Table II, columns 5 or 7, lines 173,262
to 274 and 628 to 632 or a fragment thereof, which confers an
increase in the amount of glycerol-3-phosphate or glycerol in an
organism or a part thereof; [9696] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 173,262 to 274 and 628 to 632; [9697] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of glycerol-3-phosphate or
glycerol in an organism or a part thereof; [9698] d) nucleic acid
molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of glycerol-3-phosphate or glycerol in an organism or
a part thereof; [9699] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under stringent
hybridisation conditions and conferring an increase in the amount
of glycerol-3-phosphate or glycerol in an organism or a part
thereof; [9700] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table III, column 7, lines
173, 262 to 274 and 628 to 632 and conferring an increase in the
amount of glycerol-3-phosphate or glycerol in an organism or a part
thereof; [9701] g) nucleic acid molecule encoding a polypeptide
which is isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of
glycerol-3-phosphate or glycerol in an organism or a part thereof;
[9702] h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table IV, column 7, lines 173, 262 to 274
and 628 to 632 and conferring an increase in the amount of
glycerol-3-phosphate or glycerol in an organism or a part thereof;
and [9703] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of glycerol-3-phosphate or glycerol in an organism or a part
thereof. [9704] whereby the nucleic acid molecule distinguishes
over the sequence as indicated in Table I, columns 5 or 7, lines
173, 262 to 274 and 628 to 632 by one or more nucleotides. [9705]
7. A nucleic acid construct which confers the expression of the
nucleic acid molecule of claim 6, comprising one or more regulatory
elements. [9706] 8. A vector comprising the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7.
[9707] 9. The vector as claimed in claim 8, wherein the nucleic
acid molecule is in operable linkage with regulatory sequences for
the expression in a prokaryotic or eukaryotic, or in a prokaryotic
and eukaryotic, host. [9708] 10. A host cell, which has been
transformed stably or transiently with the vector as claimed in
claim 8 or 9 or the nucleic acid molecule as claimed in claim 6 or
the nucleic acid construct of claim 7 or produced as described in
any one of claims 2 to 5. [9709] 11. The host cell of claim 10,
which is a transgenic host cell. [9710] 12. The host cell of claim
10 or 11, which is a plant cell, an animal cell, a microorganism,
or a yeast cell, a fungus cell, a prokaryotic cell, an eukaryotic
cell or an archaebacterium. [9711] 13. A process for producing a
polypeptide, wherein the polypeptide is expressed in a host cell as
claimed in any one of claims 10 to 12. [9712] 14. A polypeptide
produced by the process as claimed in claim 13 or encoded by the
nucleic acid molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table II, columns 5
or 7, lines 173,262 to 274 and 628 to 632 by one or more amino
acids. [9713] 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. [9714] 16. A plant tissue,
propagation material, harvested material or a plant comprising the
host cell as claimed in claim 12 which is plant cell or an
Agrobacterium. [9715] 17. A method for screening for agonists and
antagonists of the activity of a polypeptide encoded by the nucleic
acid molecule of claim 6 conferring an increase in the amount of
glycerol-3-phosphate or glycerol in an organism or a part thereof
comprising: [9716] (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of glycerol-3-phosphate or glycerol in an organism or a part
thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; [9717] (b) assaying the glycerol-3-phosphate or
glycerol level or the polypeptide expression level in the cell,
tissue, plant or microorganism or the media the cell, tissue, plant
or microorganisms is cultured or maintained in; and [9718] (c)
identifying a agonist or antagonist by comparing the measured
glycerol-3-phosphate or glycerol level or polypeptide expression
level with a standard glycerol-3-phosphate or glycerol or
polypeptide expression level measured in the absence of said
candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. [9719] 18. A process for the
identification of a compound conferring increased
glycerol-3-phosphate or glycerol production in a plant or
microorganism, comprising the steps: [9720] a) culturing a plant
cell or tissue or microorganism or maintaining a plant expressing
the polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of glycerol-3-phosphate or
glycerol in an organism or a part thereof and a readout system
capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
glycerol-3-phosphate or glycerol in an organism or a part thereof;
[9721] b) identifying if the compound is an effective agonist by
detecting the presence or absence or increase of a signal produced
by said readout system. [9722] 19. A method for the identification
of a gene product conferring an increase in glycerol-3-phosphate or
glycerol production in a cell, comprising the following steps:
[9723] a) contacting the nucleic acid molecules of a sample, which
can contain a candidate gene encoding a gene product conferring an
increase in glycerol-3-phosphate or glycerol after expression with
the nucleic acid molecule of claim 6; [9724] b) identifying the
nucleic acid molecules, which hybridise under relaxed stringent
conditions with the nucleic acid molecule of claim 6; [9725] c)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing glycerol-3-phosphate or glycerol; [9726]
d) expressing the identified nucleic acid molecules in the host
cells; [9727] e) assaying the glycerol-3-phosphate or glycerol
level in the host cells; and [9728] f) identifying nucleic acid
molecule and its gene product which expression confers an increase
in the glycerol-3-phosphate or glycerol in the host cell in the
host cell after expression compared to the wild type. [9729] 20. A
method for the identification of a gene product conferring an
increase in glycerol-3-phosphate or glycerol production in a cell,
comprising the following steps: [9730] a) identifying in a data
bank nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the glycerol-3-phosphate or glycerol amount or level in an organism
or a part thereof after expression, and which are at least 20%
homolog to the nucleic acid molecule of claim 6; [9731] b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing glycerol-3-phosphate or glycerol; [9732]
c) expressing the identified nucleic acid molecules in the host
cells; [9733] d) assaying the glycerol-3-phosphate or glycerol
level in the host cells; and [9734] e) identifying nucleic acid
molecule and its gene product which expression confers an increase
in the glycerol-3-phosphate or glycerol level in the host cell
after expression compared to the wild type. [9735] 21. A method for
the production of an agricultural composition comprising the steps
of the method of any one of claims 17 to 20 and formulating the
compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. [9736] 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [9737] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
glycerol-3-phosphate or glycerol after expression.
[9738] 24. Use of the polypeptide of claim 14 or the nucleic acid
construct claim 7 or the gene product identified according to the
method of claim 19 or 20 for identifying compounds capable of
conferring a modulation of glycerol-3-phosphate or glycerol levels
in an organism. [9739] 25. Agrochemical, pharmaceutical, food or
feed composition comprising the nucleic acid molecule of claim 6,
the polypeptide of claim 14, the nucleic acid construct of claim 7,
the vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. [9740] 26. The method of any one of
claims 1 to 5, the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20, wherein the fine chemical is
glycerol-3-phosphate or glycerol. [9741] 27. A host cell or plant
according to any of the claims 10 to 12 which is resistant to a
herbicide inhibiting the biosynthesis of glycerol-3-phosphate or
glycerol. [9742] 28. A process for the increased production of
total lipids, comprising the increasing or generating in an
organism or a part thereof the expression of at least one nucleic
acid molecule comprising a nucleic acid molecule selected from the
group consisting of: [9743] a) nucleic acid molecule encoding a
polypeptide as indicated in Table II, columns 5 or 7, lines 173,262
to 274 and 628 to 632 or a fragment thereof, which confers an
increase in the amount of total lipids in an organism or a part
thereof; [9744] b) nucleic acid molecule comprising of a nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines
173,262 to 274 and 628 to 632; [9745] c) nucleic acid molecule
whose sequence can be deduced from a polypeptide sequence encoded
by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of total lipids in an organism or a part thereof; [9746] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of total lipids in an organism or a part
thereof; [9747] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of total lipids
in an organism or a part thereof; [9748] f) nucleic acid molecule
which encompasses a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers or primer pairs as indicated in Table
III, column 7, lines 173, 262 to 274 and 628 to 632 and conferring
an increase in the amount of total lipids in an organism or a part
thereof; [9749] g) nucleic acid molecule encoding a polypeptide
which is isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of total lipids in an
organism or a part thereof; [9750] h) nucleic acid molecule
encoding a polypeptide comprising a consensus as indicated in Table
IV, column 7, lines 173, 262 to 274 and 628 to 632 and conferring
an increase in the amount of total lipids in an organism or a part
thereof; and [9751] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of total lipids in an organism or a part thereof. [9752] or
comprising a sequence which is complementary thereto. [9753] 29.
The use of the nucleic acid molecule encoding a polypeptide as
indicated in Table II, columns 5 or 7, lines 173, 262 to 274 and
628 to 632 or a fragment thereof, which confers an increase in the
amount of total lipids in an organism or a part thereof for the
identification of a nucleic acid molecule conferring an increase in
the amount of total lipids after expression.
[9754] [0554.0.0.21] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[9755] [0000.0.0.22] In a further embodiment, the present invention
relates to a further process for the production of fine chemicals
as defined below and the corresponding embodiments as described
herein as follows.
[9756] [0001.0.0.22] for the disclosure of this paragraph see
[0001.0.0.0].
[9757] [0002.0.22.22] Lipids differ markedly from other groups of
biomolecules and metabolites. By definition, lipids are
water-insoluble biomolecules that are highly soluble in organic
solvents such as chloroform. Lipids have a variety of biological
roles: they serve as fuel molecules, highly concentrated energy
stores, signal molecules, and components of membranes.
[9758] The major kinds of membrane lipids are phospholipids,
glycolipids, and cholesterol. Glycolipids are sugar-containing
lipids. The term glycolipid designates any compound containing one
or more monosaccharide residues bound by a glycosidic linkage to a
hydrophobic moiety such as an acylglycerol, a sphingoid, a ceramide
(N-acylsphingoid) or a prenyl phosphate.
[9759] [0003.0.22.22] Galactose-containing lipids are the
predominant nonproteinaceous components of photosynthetic membranes
in plants, algae, and a variety of bacteria. In higher plants, the
galactolipids contain a high proportion of polyunsaturated fatty
acids, up to 95% of which can be linolenic acid (18:3(n-3)). In
non-photosynthetic tissues, such as tubers or roots, the C.sub.18
fatty acids are usually more saturated.
[9760] In plants, especially photosynthetic tissues, a substantial
proportion of the lipids consists of 1,2-diacyl-sn-glycerols joined
by a glycosidic linkage at position sn-3 to a carbohydrate moiety.
The two most common galactolipids are monogalactosyl diacylglycerol
and digalactosyl diacylglycerol. Up to 80% of all lipids in plants
are associated with photosynthetic membranes, and monogalactosyl
diacylglycerol is widely considered to be the most abundant
membrane lipid on earth.
[9761] Monogalactosyldiacylglycerols are not solely plant lipids as
they have been found in small amounts in brain and nervous tissue
in some animal species.
[9762] Related compounds of those main components of plant
glycolipids, e.g. mono- and digalactosyldiacylglycerols, have been
found with up to four galactose units, or in which one or more of
these is replaced by glucose moieties. In addition, a
6-O-acyl-monogalactosyldiacylglycerol is occasionally a component
of plant tissues.
[9763] [0004.0.22.4] The final step in monogalactosyl
diacylglycerol biosynthesis occurs in the plastid envelope and is
catalyzed by monogalactosyl diacylglycerol synthase (EC 2.4.1.46).
This enzyme transfers D-galactose from UDP-galactose to
sn-1,2-diacylglycerol (DAG) (Joyard, J. & Douce, R. Stumpf, P.
K., ed. (1987) in Biochemistry of Plants (Academic, New York)).
[9764] Digalactosyl diacylglycerol synthase catalyzes the transfer
of galactose from one molecule of monogalactosyl diacylglycerol to
another, producing digalactosyl diacylglycerol and DAG in equimolar
amounts.
[9765] [0005.0.22.22] Even if some details are known, galactolipid
biosynthesis in plants is highly complex. It involves multiple
pathways giving rise to different molecular species.
[9766] Recent studies indicate that the amounts of the lipids
sulfolipid sulfoquinovosyl-diacylglycerol (SQDG) and
digalactosyldiacylglycerol and DGDG increase strongly during
phosphate deprivation (Hartel et al., Proc. Natl. Acad. Sci., 97,
10649-10654, 2000). When phosphate is limiting, phospholipids in
plant membranes are reduced and at least in part replaced by
glycolipids (i.e., SQDG and DGDG).
[9767] In addition to serving as a surrogate lipid for
phospholipids, galactolipids were found to be critical for the
stabilization of photosynthetic complexes in the thylakoids
(Dormann and Benning, Trends Plant Sci. 7, 112-118, 2002).
[9768] [0006.0.22.22] In contrast to plants, which contain high
amounts of glycolipids, which carry a sugar moiety in the head
group, in animals and yeast phospholipids are very abundant.
Nevertheless, one type of glycolipids to be found in mammalians are
galactosylceramide (cerebroside), which is prevalent in brain and
the central nervous system. The cerebrosides have been localized to
the outer leaflet of the plasma membrane, exposed on the cell
surface. They seem to be responsible for the different blood types.
Blood group antigens include cerebrosides with multiple sugars
attached.
[9769] [0007.0.22.22.] It has long been recognized that many
complex glycolipid antigens are involved in the binding of lectins
and antibodies at the cell surface glycolipids, containing only a
single sugar headgroup, may play a combination of immunological,
regulatory and structural roles in the membrane (Varki et al.,
Essentials of Glycobiology. Cold Spring Harbor Laboratory Press,
New York, 1999).
[9770] The glycosphingolipid, galactosylceramide, has been shown to
be a key activator of triggered cell death (Zhao et al., Cancer
Res. 59, 482-486,1999) and may play a role in the inhibition of
virus replication (Kakimi et al., J. Exp. Med. 192, 921-930,
2000).
[9771] It has recently been demonstrated that galactolipids are
also responsible for preventing cell damage and the high resistance
to oxidation and heat in the membranes of some microorganisms
(Nakata, J. Biochem. 127, 731-737, 2000).
[9772] [0008.0.22.22.] Other glycoglycerolipids, such as the
1,2-di-Oacyl-3-O-(D-galactopyranosyl)-sn-glycerols, are found
widely in nature as structural components of the photosynthetic
membranes of higher plants in the cell membranes of prokaryotic
blue-green algae and several other microorganisms and in the seeds
of cereals, such as wheat and oats.
[9773] [0009.0.22.22.] Galactolipids are one of the more abundant
lipid classes in nature. Sources for the galactolipids are
foodstuffs, such as certain grains (oat, wheat, barley, and maize),
which have been a significant part of the human diet since the
beginning of time.
[9774] In addition galactolipids contain important fatty acids like
linoleic acid, linolenic acids and others which have numerous
applications in the food and feed industry, in cosmetics and in the
drug sector. For example for cyanobacterium and marine green algae
fermentation it has been described that the gamma-linolenic acid
(GLA) was restricted the the galactolipid fraction (Cohen et al.,
J. Appl. Phycol.; (1993) 5, 1, 109-15; FEMS-Microbiol. Lett.;
(1993) 107, 2-3, 163-67), meaning that increasing the concentration
of galactolipids can be another method for increasing the
concentration of interesting fatty acids like linoleic acid,
linolenic acid, stearic acid and palmitic acid in some production
systems.
[9775] [0010.0.22.22] Most vegetables and fruits in human and
animal diets contain galactolipids, and their breakdown products
represent an important dietary source of galactose and
polyunsaturated fatty acids.
[9776] [0011.0.22.22] On account of the positive properties and
interesting physiological roles potential of galactose and
galactose comprising lipids there is a need to produce those
compounds in large amounts and well defined quality and
composition.
[9777] [0012.0.22.22.] Thus, it would be desirable to produce
galactolipids, in a defined proportion in microorganisms or plants.
This should be in way, which is not dependent on the availability
of phosphate, in particular on phosphate deprivation. One way to
increase the productive capacity of biosynthesis is to apply
recombinant DNA technology. That type of production permits control
over quality, quantity and selection of the most suitable and
efficient producer organisms. The latter is especially important
for commercial production economics and therefore availability to
consumers.
[9778] [0013.0.22.22] Methods of recombinant DNA technology have
been used for some years to improve the production of fine
chemicals in microorganisms and plants by amplifying individual
biosynthesis genes and investigating the effect on production of
fine chemicals.
[9779] There is a constant need for providing novel enzyme
activities or direct or indirect regulators and thus alternative
methods with advantageous properties for producing compounds
containing galactose and fatty acids, preferably galactolipids or
cerebrosid, in a defined proportion or its precursor in organisms,
e.g. in transgenic organisms.
[9780] [0014.0.22.22] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a lipid, preferably a
glycolipide, a glycolipid containing galactose, more preferably a
galactolipid or cerebrosid.
[9781] Accordingly, in the present invention, the term "the fine
chemical" as used herein relates to "lipid, preferably a
glycolipid, a glycolipide containing galactose, more preferably a
galactolipide and/or cerebroside".
[9782] Further, the term "the fine chemicals" as used herein also
relates to fine chemicals comprising lipid, preferably a
glycolipid, a glycolipide containing galactose, more preferably a
galactolipide and/or cerebroside.
[9783] [0015.0.22.22] In one embodiment, the term "the fine
chemical" means lipid, preferably a glycolipid, a glycolipide
containing galactose, more preferably a galactolipide and/or
cerebroside.
[9784] Throughout the specification the term "the fine chemical"
means lipid, preferably a glycolipid, a glycolipide containing
galactose, more preferably a galactolipide and/or cerebroside in
free form or bound to other compounds, such as sulfonated salts. In
a preferred embodiment, the term "the fine chemical" means
galactolipid, in free form or its salts or bound to sulfate.
[9785] [0015.1.22.22] A measure for the content of the lipid,
preferably a glycolipid, a glycolipide containing galactose, more
preferably a galactolipide and/or cerebroside of the invention can
be the content of galactopyranoside, preferably
methyl-galactopyranosid. This compound is the analyte which
indicates the presence of the lipid, preferably a glycolipid, a
glycolipide containing galactose, more preferably a galactolipide
and/or cerebroside of the invention if the samples are prepared and
measured as described in the examples
[9786] [0016.0.22.22] Accordingly, the present invention relates to
a process comprising [9787] (a) increasing or generating the
activity of of one or more YLR224W, YLR255C, YER173W, b2699, b3129
and/or YHR072W protein(s), in a non-human organism in one or more
parts thereof and [9788] (b) growing the organism under conditions
which permit the production of the fine chemical, thus, lipid,
preferably a glycolipid, a glycolipide containing galactose, more
preferably a galactolipide and/or cerebroside in said organism.
[9789] Accordingly, the present invention relates to a process for
the production of the respective fine chemical comprising [9790]
(a) increasing or generating the activity of one or more protein(s)
having the activity of a protein indicated in Table II, column 3,
lines 186 to 189 and/or lines 633 and 634 or having the sequence of
a polypeptide encoded by a nucleic acid molecule indicated in Table
I, columns 5 or 7, and/or lines 633 and 634, in a non-human
organism in one or more parts thereof and [9791] (b) growing the
organism under conditions which permit the production of the fine
chemical, thus, lipid, preferably a glycolipid, a glycolipide
containing galactose, more preferably a galactolipide and/or
cerebroside.
[9792] [0017.0.0.22] and [0018.0.0.22] for the disclosure of the
paragraphs [0017.0.0.22] and [0018.0.0.22] see paragraphs
[0017.0.0.0] and [0018.0.0.0] above.
[9793] [0019.0.22.22] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the fine respective chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table II, column 3, lines 186 to 189 and/or lines 633
and 634 or encoded by nucleic acid molecule indicated in Table I,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634.
[9794] [0020.0.22.22] Surprisingly it was found, that the
transgenic expression of the Saccaromyces cerevisiae protein(s)
YLR224W, YLR255C YER173W and/or YHR072W as indicated in Table II,
columns 3 or 5, lines 186 to 188 and 634 respectively and/or the
Escherichia coli K12 protein(s) b2699 and/or b3129 as indicated in
Table I, columns 3 or 5, lines 189 and 633 in Arabidopsis thaliana
conferred an increase in the content of lipid, preferably a
glycolipid, a glycolipide containing galactose, more preferably a
galactolipide and/or cerebroside of the transformed plants. Thus,
in one embodiment, said protein(s) or its homologs as indicated in
Table II, column 7, lines 186 to 189 and/or lines 633 and 634 are
used for the production of lipid, preferably a glycolipid, a
glycolipide containing galactose, more preferably a galactolipide
and/or cerebroside.
[9795] [0021.0.0.22] for the disclosure of this paragraph see
[0021.0.0.0] above.
[9796] [0022.0.22.22] The sequence of YLR224W from Saccharomyces
cerevisiae has been published in Johnston et al., Nature 387 (6632
Suppl), 87-90 (1997) and in Goffeau et al., Science 274 (5287),
546-547, 1996 and its cellular activity has not been characterized
yet. It seems to have a activity of the Saccharomyces cerevisiae
hypothetical protein YLR224w superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the Saccharomyces cerevisiae
hypothetical protein YLR224w superfamily, preferably a protein with
a YLR224w protein activity or its homolog, e.g. as shown herein,
from Saccharomyces cerevisiae or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of lipid,
preferably a glycolipid, a glycolipide containing galactose, more
preferably a galactolipide and/or cerebroside in free or bound form
in an organism or a part thereof, as mentioned. In one further
embodiment the YLR224w protein expression is increased together
with the increase of another gene of the galactose pathway,
preferably with a gene encoding a protein being involved in the
production of glycolipide containing galactose, or encoding a
galactose transporter protein or a compound, which functions as a
sink for the respective lipid, preferably a glycolipid, a
glycolipide containing galactose, more preferably a galactolipide
and/or cerebroside. In one embodiment, in the process of the
present invention the activity of a protein of the Saccharomyces
cerevisiae hypothetical protein YLR224w superfamily, preferably of
a YLR224w protein is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[9797] The sequence of YLR255C from Saccharomyces cerevisiae has
been submitted by Johnson et al. to the EMBL Data Library,
(February 1995), and its activity has not been characterized yet,
but having preferably an activity of Saccharomyces hypothetical
protein YLR255c superfamily. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of a protein of the Saccharomyces
hypothetical protein YLR255c superfamily, preferably of a gene
product with an activity of a YLR255C protein from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, meaning of lipid, preferably a
glycolipid, a glycolipide containing galactose, more preferably a
galactolipide and/or cerebroside in free or bound form in an
organism or a part thereof, as mentioned. In one further embodiment
the YLR255C protein expression is increased together with the
increase of another gene of the galactose pathway, preferably with
a gene encoding a protein being involved in the production of
glycolipid or encoding a galactose transporter protein or a
compound, which functions as a sink for the resprective lipid,
preferably a glycolipid, a glycolipide containing galactose, more
preferably a galactolipide and/or cerebroside. In one embodiment,
in the process of the present invention the activity of a
Saccharomyces hypothetical protein YLR255c superfamily protein,
preferably YLR255C protein, is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[9798] The sequence of YER173W from Saccharomyces cerevisiae has
been published in Dietrich et al., Nature 387 (6632 Suppl), 78-81,
1997, and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is being defined as a checkpoint protein, involved in
the activation of the DNA damage and meiotic pachytene checkpoints.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
Checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
oflipid, preferably a glycolipid, a glycolipide containing
galactose, more preferably a galactolipide and/or cerebroside in
free or bound form in an organism or a part thereof, as mentioned.
In one further embodiment the YER173w protein expression is
increased together with the increase of another gene of the
galactose pathway, preferably with a gene encoding a protein being
involved in the production of lipid, preferably a glycolipid, a
glycolipide containing galactose, more preferably a galactolipide
and/or cerebroside. In one embodiment, in the process of the
present invention the activity of a Checkpoint protein, involved in
the activation of the DNA damage and meiotic pachytene checkpoints
is increased or generated, e.g. from Saccharomyces cerevisiae or a
homolog thereof.
[9799] The sequence of b2699 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a DNA strand exchange and
recombination protein with protease and nuclease activity of the
superfamily of the recombination protein recA. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein with a DNA recombination and DNA repair activity, a
pheromone response activity, a mating-type determination activity,
a sex-specific protein activity, a nucleotide binding activity
and/or a protease and nuclease activity, in particular a DNA strand
exchange and recombination protein with protease and nuclease
activity, in particular of the superfamily of the recombination
protein recA from E. coli or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, meaning lipid,
preferably a glycolipid, a glycolipide containing galactose, more
preferably a galactolipide and/or cerebroside, preferably in free
or bound form in an organism or a part thereof, as mentioned. In
one further embodiment the b2699 protein expression is increased
together with the increase of another gene of the galactose
pathway, preferably with a gene encoding a protein being involved
in the production of lipid, preferably a glycolipid, a glycolipide
containing galactose, more preferably a galactolipide and/or
cerebroside. In one embodiment, in the process of the present
invention said activity, e.g. the activity of a protease and
nuclease activity, in particular a DNA strand exchange and
recombination protein with protease and nuclease activity, in
particular of the superfamily of the recombination protein recA is
increased or generated, e.g. from E. coli or a homolog thereof.
[9800] The sequence of YHR072W (Accession number NP.sub.--011939)
from Saccharomyces cerevisiae has been published in Goffeau et al.,
Science 274 (5287), 546-547 (1996) and Johnston et al. Science 265
(5181), 2077-2082 (1994), and its activity is being defined as a
2,3-oxidosqualene-lanosterol cyclase protein. Accordingly, in one
embodiment, the process of the present invention comprises the use
of an uncharacterized stress-induced protein from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the fine chemical, meaning lipid, preferably a glycolipid, a
glycolipide containing galactose, more preferably a galactolipide
and/or cerebroside, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the activity of a
2,3-oxidosqualene-lanosterol cyclase protein is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[9801] The sequence of b3129 (Accession number NP.sub.--417598)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a putative protease; htrA suppressor protein.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a putative protease; htrA suppressor
protein from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning lipid, preferably a
glycolipid, a glycolipide containing galactose, more preferably a
galactolipide and/or cerebroside, preferably in free or bound form
in an organism or a part thereof, as mentioned. In one embodiment,
in the process of the present invention the activity of a putative
protease; htrA suppressor protein is increased or generated, e.g.
from E. coli or a homolog thereof.
[9802] [0023.0.22.22] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[9803] Further, in the present invention, the term "homologue"
relates to the sequence of an organism having the highest sequence
homology to the herein mentioned or listed sequences of all
expressed sequences of said organism. However, the person skilled
in the art knows, that, preferably, the homologue has said
the--fine-chemical-increasing activity and, if known, the same
biological function or activity in the organism as at least one of
the protein(s) indicated in Table I, Column 3, lines 186 to 188 and
634, e.g. having the sequence of a polypeptide encoded by a nucleic
acid molecule comprising the sequence indicated in indicated in
Table I, Column 5 or 7, lines 186 to 188 and 634. In one
embodiment, the homolog of any one of the polypeptides indicated in
Table II, lines 186 to 188 and 634 is a homolog having the same or
a similar activity, in particular an increase of activity confers
an increase in the content of the fine chemical in the organisms
and being derived from eukaryot.
[9804] In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, line 189 and 633 is a homolog having the same
or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from bacteria.
[9805] In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, lines 186 to 188 and 634 is a homolog having
the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
an organisms or part thereof, and being derived from Fungi.
[9806] In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, line 189 and 633 is a homolog having the same
or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof and being derived from
Proteobacteria.
[9807] In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, lines 186 to 188 and 634 is a homolog having
the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms or a part thereof and being derived from
Ascomycota.
[9808] In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, line 189 and 633 is a homolog having the same
or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from
Gammaproteobacteria.
[9809] In one embodiment, the homolog of a polypeptide polypeptide
indicated in Table II, column 3, lines 186 to 188 and 634 is a
homolog having the same or a similar activity, in particular an
increase of activity confers an increase in the content of the fine
chemical in the organisms or part thereof, and being derived from
Saccharomycotina.
[9810] In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, line 189 and 633 is a homolog having the same
or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or part thereof, and being derived from
Enterobacteriales.
[9811] In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, lines 186 to 188 and 634 is a homolog having
the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms or a part thereof, and being derived from
Saccharomycetes.
[9812] In one embodiment, the homolog of the a polypeptide
indicated in Table II, column 3, line 189 and 633 is a homolog
having the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms or part thereof, and being derived from
Enterobacteriaceae.
[9813] In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, lines 186 to 188 and 634 is a homolog having
the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms, and being derived from Saccharomycetales.
[9814] In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, lines 189 and 633 is a homolog having the same
or a similar activity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or a part thereof, and being derived from
Escherichia.
[9815] In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, lines 186 to 188 and 634 is a homolog having
the same or a similar activity, in particular an increase of
activity confers an increase in the content of the fine chemical in
the organisms or a part thereof, and being derived from
Saccharomycetaceae.
[9816] In one embodiment, the homolog of a polypeptide indicated in
Table II, column 3, line 186 to 188 and 634 is a homolog having the
same or a similar acivity, in particular an increase of activity
confers an increase in the content of the fine chemical in the
organisms or a part thereof, and being derived from
Saccharomycetes.
[9817] [0023.1.22.22] Homologs of the polypeptides polypeptide
indicated in Table II, column 3, lines 186 to 189 and/or lines 633
and 634 may be the polypeptides encoded by the nucleic acid
molecules polypeptide indicated in Table I, column 7, lines 186 to
189 and/or lines 633 and/or 634 or may be the polypeptides
indicated in Table II, column 7, lines 186 to 189. Homologs of the
polypeptides indicated in Table II, column 3, lines 186 to 189
and/or lines 633 and/or 634 may be the polypeptides encoded by the
nucleic acid molecules polypeptide indicated in Table I, column 7,
lines 186 to 189 and/or lines 633 and/or 634 or may be the
polypeptides indicated in Table II, column 7, lines 186 to 189
and/or lines 633 and/or 634.
[9818] [0024.0.0.22] for the disclosure of this paragraph see
[0024.0.0.0] above.
[9819] [0025.0.22.22] In accordance with the invention, a protein
or polypeptide has the "activity of an protein of the invention",
e.g. the activity of a protein indicated in Table II, column 3,
lines 186 to 189 and/or lines 633 and 634 if its de novo activity,
or its increased expression directly or indirectly leads to an
increased level of lipid, preferably a glycolipid, a glycolipide
containing galactose, more preferably a galactolipide and/or
cerebroside in the organism or a part thereof, preferably in a cell
of said organism. In a preferred embodiment, the protein or
polypeptide has the above-mentioned additional activities of a
protein indicated in Table II, column 3, lines 186 to 189 and/or
lines 633 and 634. During the specification the activity or
preferably the biological activity of such a protein or polypeptide
or an nucleic acid molecule or sequence encoding such protein or
polypeptide is identical or similar if it still has the biological
or enzymatic activity of any one of the proteins indicated in Table
II, column 3, lines 186 to 189 and/or lines 633 and 634, i.e. if it
has at least 10% of the original enzymatic activity, preferably
20%, particularly preferably 30%, most particularly preferably 40%
in comparison to an any one of the proteins indicated in Table II,
column 3, lines 186 to 188 and/or 634 of Saccharomyces cerevisiae
and/or any one of the proteins indicated in Table II, column 3,
line 189 and/or 633 of E. coli K12.
[9820] [0025.1.0.22] and [0025.2.0.22] for the disclosure of the
paragraphs [0025.1.0.22] and [0025.2.0.22] see [0025.1.0.0] and
[0025.2.0.0] above.
[9821] [0026.0.0.22] to [0033.0.0.22] for the disclosure of the
paragraphs [0026.0.0.22] to [0033.0.0.22] see [0026.0.0.0] to
[0033.0.0.0] above.
[9822] [0034.0.22.22] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of an protein having the activity of a
protein as indicated in Table II, column 3, lines 186 to 189 and/or
lines 633 and/or 634 or being encoded by a nucleic acid molecule
indicated in Table I, column 5, lines 186 to 189 and/or lines 633
and/or 634 or its homologs, e.g. as indicated in Table I, column 7,
lines 186 to 189 and/or lines 633 and/or 634, its biochemical or
genetical causes and therefore shows the increased amount of the
fine chemical.
[9823] [0035.0.0.22] and [0036.0.0.22] for the disclosure of the
paragraphs [0035.0.0.22] and [0036.0.0.22] see paragraphs
[0035.0.0.0] to [0036.0.0.0] above.
[9824] [0037.0.22.22] A series of mechanisms exists via which a
modification of the a protein, e.g. the polypeptide of the
invention can directly or indirectly affect the yield, production
and/or production efficiency of the lipid, preferably a glycolipid,
a glycolipide containing galactose, more preferably a galactolipide
and/or cerebroside.
[9825] [0038.0.0.22] to [0044.0.0.22] for the disclosure of the
paragraphs [0038.0.0.22] to [0044.0.0.22] see paragraphs
[0038.0.0.0] to [0044.0.0.0] above.
[9826] [0045.0.22.22] In one embodiment, in case the activity of
the Saccaromyces cerevisiae protein YLR224W or its homologs, e.g.
as indicated in Table II, columns 5 or 7, line 186, is increased,
preferably, an increase of the fine chemical between 15% and 46% or
more is conferred.
[9827] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YLR255C or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 187, is increased, preferably, in
one embodiment the increase of the fine chemical between 16% and
30% or more is conferred.
[9828] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YER173w or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 188, e.g. a checkpoint protein,
involved in the activation of the DNA damage and meiotic pachytene
checkpoints; is increased, preferably, the increase of the fine
chemical between 20% and 70% or more is conferred.
[9829] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2699 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 189, is increased, e.g. the activity of a
DNA strand exchange and recombination protein with protease and
nuclease activity of the superfamily of the recombination protein
recA or its homolog is increased preferably, the increase of the
fine chemical between 15% and 64% or more is conferred.
[9830] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3129 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 633, is increased, e.g. the activity of a
putative protease; htrA suppressor protein or its homolog is
increased preferably, the increase of the fine chemical between 16%
and 32% or more is conferred.
[9831] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YHR072W or its homologs, e.g. as indicated in
Table II, columns 5 or 7, line 634, is increased, e.g. the activity
of a 2,3-oxidosqualene-lanosterol cyclase protein or its homolog is
increased, preferably, the increase of the fine chemical between
17% and 35% or more is conferred.
[9832] [0046.0.22.22] In one embodiment, the activity of the
Saccaromyces cerevisiae protein YLR224W or its homologs, e.g. an
activity of a YLR224W protein, e.g. as indicated in Table II,
columns 5 or 7, line 186, confers an increase of the respective
fine chemical and of further lipids, preferably glycolipids,
sphingolipids and/or galactolipids, or their precursors.
[9833] In one embodiment, the activity of the Saccaromyces
cerevisiae protein YLR255C or its homologs, e.g. an activity of a
YLR255C protein, e.g. as indicated in Table II, columns 5 or 7,
line 187, confers an increase of the respective fine chemical and
of further lipids, preferably glycolipids, sphingolipids and/or
galactolipids, or their precursors.
[9834] In one embodiment, the activity of the Saccaromyces
cerevisiae protein YER173W or its homologs, e.g. an activity of a
checkpoint protein, involved in the activation of the DNA damage
and meiotic pachytene checkpoints, e.g. as indicated in Table II,
columns 5 or 7, line 188, confers an increase of the respective
fine chemical and of further lipids, preferably glycolipids,
sphingolipids and/or galactolipids, or their precursors.
[9835] In one embodiment, the activity of the Escherichia coli K12
protein b2699 or its homologs, e.g. an activity of a DNA strand
exchange and recombination protein with protease and nuclease
activity of the superfamily of the recombination protein recA, e.g.
as indicated in Table II, columns 5 or 7, line 189, confers an
increase of the respective fine chemical and of further lipids,
preferably glycolipids, sphingolipids and/or galactolipids,
compounds or their precursors.
[9836] In one embodiment, the activity of the Escherichia coli K12
protein b3129 or its homologs, e.g. a protein having the activity
of a putative protease; htrA suppressor protein, e.g. as indicated
in Table II, columns 5 or 7, line 633, confers an increase of the
respective fine chemical and of further lipids, preferably
glycolipids, sphingolipids and/or galactolipids, compounds or their
precursors.
[9837] In one embodiment, the activity of the Saccaromyces
cerevisiae protein YHR072W or its homologs, e.g. a
2,3-oxidosqualene-lanosterol cyclase protein, e.g. as indicated in
Table II, columns 5 or 7, line 634, confers an increase of the
respective fine chemical and of further lipids, preferably
glycolipids, sphingolipids and/or galactolipids, or their
precursors.
[9838] [0047.0.0.22] and [0048.0.0.22] for the disclosure of the
paragraphs [0047.0.0.22] and [0048.0.0.22] see paragraphs
[0047.0.0.0] and [0048.0.0.0] above.
[9839] [0049.0.22.22] A protein having an activity conferring an
increase in the amount or level of the fine chemical preferably has
the structure of the polypeptide described herein, in particular of
a polypeptides comprising a consensus sequence as indicated in
Table IV, column 7, lines 186 to 189 and/or lines 633 and/or 634 or
of a polypeptide as indicated in Table II, columns 5 or 7, lines
186 to 189 and/or lines 633 and/or 634 or the functional homologues
thereof as described herein, or is encoded by the nucleic acid
molecule characterized herein or the nucleic acid molecule
according to the invention, for example by a nucleic acid molecule
as indicated in Table I, columns 5 or 7, lines 186 to 189 and/or
lines 633 and/or 634 or its herein described functional homologues
and has the herein mentioned activity.
[9840] [0050.0.22.22] For the purposes of the present invention,
the term "lipid, preferably a glycolipid, a glycolipide containing
galactose, more preferably a galactolipide and/or cerebroside" also
encompasses monogalactosyl diacylglycerol and/or digalactosyl
diacylglycerol and/or galactolipids comprising 3, 4 or more
galactosyl units.
[9841] [0051.0.22.22] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
fine chemical, i.e. an increased amount of the fine chemical free
or bound, e.g compositions comprising lipid, preferably a
glycolipid, a glycolipide containing galactose, more preferably a
galactolipide and/or cerebroside. Depending on the choice of the
organism used for the process according to the present invention,
for example a microorganism or a plant, compositions or mixtures of
various fatty acids can be produced.
[9842] [0052.0.0.22] for the disclosure of this paragraph see
paragraph [0052.0.0.0] above.
[9843] [0053.0.22.22] In one embodiment, the process of the present
invention comprises one or more of the following steps [9844] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 186
to 189 and/or lines 633 and 634 or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634, having herein-mentioned the respective fine
chemical-increasing activity; [9845] b) stabilizing a mRNA
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 186 to 189 and/or lines 633 and 634, or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 186
to 189 and/or lines 633 and 634, or of a mRNA encoding the
polypeptide of the present invention having herein-mentioned the
respective fine chemical increasing activity; [9846] c) increasing
the specific activity of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptide of the present invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 186 to 189 and/or lines 633 and 634 or
its homologs activity, e.g. as indicated in Table II, columns 5 or
7, lines 186 to 189 and/or lines 633 and 634, or decreasing the
inhibitory regulation of the polypeptide of the invention; [9847]
d) generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 186 to 189 and/or
lines 633 and 6349, or its homologs activity, e.g. as indicated in
Table II, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634; [9848] e) stimulating activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table II, column 3, lines 186 to 189 and/or
lines 633 and 634, or its homologs activity, e.g. as indicated in
Table II, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634, by adding one or more exogenous inducing factors to the
organism or parts thereof; [9849] f) expressing a transgenic gene
encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 186 to 189 and/or lines 633 and 634,
or its homologs activity, e.g. as indicated in Table II, columns 5
or 7, lines 186 to 189 and/or lines 633 and 634; [9850] g)
increasing the copy number of a gene conferring the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or the
polypeptide of the invention having herein-mentioned the respective
fine chemical increasing activity, e.g. of a polypeptide having an
an activity of a protein as indicated in Table II, column 3, lines
186 to 189 and/or lines 633 and 634, or its homologs activity, e.g.
as indicated in Table II, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634; [9851] h) Increasing the expression of the
endogenous gene encoding the polypeptide of the invention, e.g. a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 186 to 189 and/or lines 633 and 634, or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634 by adding positive
expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; [9852] i)
Modulating growth conditions of an organism in such a manner, that
the expression or activity of the gene encoding the protein of the
invention or the protein itself is enhanced for example
microorganisms or plants can be grown under a higher temperature
regime leading to an enhanced expression of heat shock proteins,
e.g. the heat shock protein of the invention, which can lead an
enhanced the fine chemical production; and/or [9853] j) selecting
of organisms with especially high activity of the proteins of the
invention from natural or from mutagenized resources and breeding
them into the target organisms, eg the elite crops.
[9854] [0054.0.22.22] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein according to Table II, column 3, lines 186 to
189 and/or lines 633 and 634 or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634.
[9855] [0055.0.0.22] to [0067.0.0.22] for the disclosure of the
paragraphs [0055.0.0.22] to [0067.0.0.22] see paragraphs
[0055.0.0.0] to [0067.0.0.0] above.
[9856] [0068.0.22.22] The mutation is introduced in such a way that
the production of lipid, preferably a glycolipid, a glycolipide
containing galactose, more preferably a galactolipide and/or
cerebroside is not adversely affected.
[9857] [0069.0.22.22] Less influence on the regulation of a gene or
its gene product is understood as meaning a reduced regulation of
the enzymatic activity leading to an increased specific or cellular
activity of the gene or its product. An increase of the enzymatic
activity is understood as meaning an enzymatic activity, which is
increased by at least 10%, advantageously at least 20, 30 or 40%,
especially advantageously by at least 50, 60 or 70% in comparison
with the starting organism. This leads to an increased productivity
of the desired lipid, preferably a glycolipid, a glycolipide
containing galactose, more preferably a galactolipide and/or
cerebroside containing compound(s).
[9858] [0070.0.22.22] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below into an organism
alone or in combination with other genes, it is possible not only
to increase the biosynthetic flux towards the end product, but also
to increase, modify or create de novo an advantageous, preferably
novel metabolites composition in the organism, e.g. an advantageous
lipid, preferably a glycolipid, a glycolipide containing galactose,
more preferably a galactolipide and/or cerebroside containing
composition comprising a higher content of (from a viewpoint of
nutritonal physiology limited) lipid, preferably a glycolipid, a
glycolipide containing galactose, more preferably a galactolipide
and/or cerebroside.
[9859] [0071.0.0.22] for the disclosure of this paragraph see
paragraph [0071.0.0.0] above.
[9860] [0072.0.22.22] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to lipid, preferably a glycolipid, a glycolipide
containing galactose, more preferably a galactolipide and/or
cerebroside compounds like galacturonic acid, oligo- and/or
polysaccharides containing galactose or galacturonic acid,
galactosamines, UDP-galactose, psychosine,
UDP-N-Acyl-galactosamines, UDP-galacturonates, further
glycoproteins, gangliosides, mucins, blood group substances and/or
ceramides.
[9861] [0073.0.22.22] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[9862] a. providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [9863] b. increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table II, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634 resp., or of a polypeptide being encoded by the
nucleic acid molecule of the present invention and described below,
i.e. conferring an increase of the respective fine chemical in the
organism, preferably in a microorganism, a non-human animal, a
plant or animal cell, a plant or animal tissue or a plant, [9864]
c. growing the organism, preferably the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant under conditions which permit the production of
the fine chemical in the organism, preferably the microorganism,
the plant cell, the plant tissue or the plant; and if desired,
recovering, optionally isolating, the free and/or bound the fine
chemical and, optionally further free and/or bound lipid,
preferably a glycolipid, a glycolipide containing galactose, more
preferably a galactolipide and/or cerebroside synthesized by the
organism, the microorganism, the non-human animal, the plant or
animal cell, the plant or animal tissue or the plant.
[9865] [0074.0.22.22] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the fine chemical or the free and bound the fine
chemical but as option it is also possible to produce, recover and,
if desired isolate, other free or/and bound lipid, preferably a
glycolipid, a glycolipide containing galactose, more preferably a
galactolipide and/or cerebroside galactose containing compound.
[9866] [0075.0.0.22] to [0077.0.0.22] for the disclosure of the
paragraphs [0075.0.0.22] to [0077.0.0.22] see paragraphs
[0075.0.0.0] to [0077.0.0.0] above.
[9867] [0078.0.22.22] The organism such as microorganisms or plants
or the recovered, and if desired isolated, fine chemical can then
be processed further directly into foodstuffs or food supplements
or animal feeds or for other applications, for example
pharmaceutical composition.
[9868] [0079.0.0.22] to [0084.0.0.22] for the disclosure of the
paragraphs [0079.0.0.22] to [0084.0.0.22] see paragraphs
[0079.0.0.0] to [0084.0.0.0] above.
[9869] [0085.0.22.22] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [9870] a) the nucleic acid sequence as
shown in table I, columns 5 or 7, lines 186 to 189 and/or lines 633
and 634 or a derivative thereof, or [9871] b) a genetic regulatory
element, for example a promoter, which is functionally linked to
the nucleic acid sequence as shown table I, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634 or a derivative thereof, or
[9872] c) (a) and (b) is/are not present in its/their natural
genetic environment or has/have been modified by means of genetic
manipulation methods, it being possible for the modification to be,
by way of example, a substitution, addition, deletion, inversion or
insertion of one or more nucleotide. "Natural genetic environment"
means the natural chromosomal locus in the organism of origin or
the presence in a genomic library. In the case of a genomic
library, the natural, genetic environment of the nucleic acid
sequence is preferably at least partially still preserved. The
environment flanks the nucleic acid sequence at least on one side
and has a sequence length of at least 50 bp, preferably at least
500 bp, particularly preferably at least 1000 bp, very particularly
preferably at least 5000 bp.
[9873] [0086.0.0.22] and [0087.0.0.22] for the disclosure of the
paragraphs [0086.0.0.22] and [0087.0.0.22] see paragraphs
[0086.0.0.0] and [0087.0.0.0] above.
[9874] [0088.0.22.22] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose whose
lipid, preferably a glycolipid, a glycolipide containing galactose,
more preferably a galactolipide and/or cerebrosid content is
modified advantageously owing to the nucleic acid molecule of the
present invention expressed.
[9875] This is important for plant breeders since, for example, the
nutritional value of plants for animals is dependent on the
abovementioned lipids and the general amount of glycolipids
containing galactose as energy source in feed. After the above
mentioned protein activity has been increased or generated, or
after the expression of nucleic acid molecule or polypeptide
according to the invention has been generated or increased, the
transgenic plant generated thus is grown on or in a nutrient medium
or else in the soil and subsequently harvested.
[9876] [0088.1.0.22] for the disclosure of this paragraph see
paragraph [0088.1.0.0] above.
[9877] [0089.0.0.22] to [0094.0.0.22] for the disclosure of the
paragraphs [0089.0.0.22] to [0094.0.0.22] see paragraphs
[0089.0.0.0] to [0094.0.0.0] above.
[9878] [0095.0.22.22] It may be advantageous to increase the pool
of lipids, preferably glycolipids, glycolipids containing
galactose, more preferably galactolipids and/or cerebrosids in the
transgenic organisms by the process according to the invention in
order to isolate high amounts of the pure respective fine chemical
and/or to obtain increased resistance against biotic and abiotic
stresses and to obtain higher yield.
[9879] [0096.0.22.22] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptide for example a fatty acid
transporter protein or a compound, which functions as a sink for
the desired lipid or compound containing galactose in the organism
is useful to increase the production of the fine chemical.
[9880] [0097.0.0.22] for the disclosure of this paragraph see
paragraph [0097.0.0.0] above.
[9881] [0098.0.22.22] In a preferred embodiment, the fine chemical
(lipid, preferably a glycolipid, a glycolipide containing
galactose, more preferably a galactolipide and/or cerebrosid) is
produced in accordance with the invention and, if desired, is
isolated. The production of further lipids, fatty acids and/or
oligosaccharides and/or mixtures thereof or mixtures of other fatty
acids by the process according to the invention is
advantageous.
[9882] [0099.0.22.22] In the case of the fermentation of
microorganisms, the above mentioned lipid, preferably a glycolipid,
a glycolipide containing galactose, more preferably a galactolipide
and/or cerebrosid may accumulate in the medium and/or the cells. If
microorganisms are used in the process according to the invention,
the fermentation broth can be processed after the cultivation.
Depending on the requirement, all or some of the biomass can be
removed from the fermentation broth by separation methods such as,
for example, centrifugation, filtration, decanting or a combination
of these methods, or else the biomass can be left in the
fermentation broth. The fermentation broth can subsequently be
reduced, or concentrated, with the aid of known methods such as,
for example, rotary evaporator, thin-layer evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. Afterwards
advantageously further compounds for formulation can be added such
as corn starch or silicates. This concentrated fermentation broth
advantageously together with compounds for the formulation can
subsequently be processed by lyophilization, spray drying, spray
granulation or by other methods. Preferably the fine chemical of
the invention compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[9883] [0100.0.22.22] Transgenic plants which comprise the lipid
synthesized in the process according to the invention can
advantageously be marketed directly without there being any need
for the oils, lipids or fatty acids synthesized to be isolated.
Plants for the process according to the invention are listed as
meaning intact plants and all plant parts, plant organs or plant
parts such as leaf, stem, seeds, root, tubers, anthers, fibers,
root hairs, stalks, embryos, calli, cotelydons, petioles, harvested
material, plant tissue, reproductive tissue and cell cultures which
are derived from the actual transgenic plant and/or can be used for
bringing about the transgenic plant. In this context, the seed
comprises all parts of the seed such as the seed coats, epidermal
cells, seed cells, endosperm or embryonic tissue. However, the fine
chemical produced in the process according to the invention can
also be isolated from the organisms, advantageously plants, in the
form of their lipids, oligosaccharides and/or free galactolipid.
Galactolipids produced by this process can be obtained by
harvesting the organisms, either from the crop in which they grow,
or from the field. This can be done via pressing or extraction of
the plant parts, preferably the plant seeds. To allow for greater
ease of disruption of the plant parts, specifically the seeds, they
are previously comminuted, steamed or roasted. The seeds or plants,
which have been pretreated in this manner can subsequently be
pressed or extracted with solvents or hydrolysed under acidic or
basic conditions or via enzymatic cleavage. The solvent is
subsequently removed. In the case of microorganisms, the latter
are, after harvesting, for example extracted directly without
further processing steps or else, after disruption, extracted via
various methods with which the skilled worker is familiar. In this
manner, more than 96% of the compounds produced in the process can
be isolated. Thereafter, the resulting products are processed
further, i.e. refined. If desired the resulting product can be
washed thoroughly with water to remove traces of acid or alkali
remaining in the product and then dried. To remove the pigment
remaining in the product, the products can be subjected to
bleaching, for example using filler's earth or active charcoal. At
the end, the product can be deodorized, for example using steam
distillation under vacuum. These chemically pure lipids, preferably
glycolipids, or glycolipids containing galactose, more preferably a
galactolipids and/or cerebrosids compositions are advantageous for
applications in the food industry sector, the cosmetic sector and
especially the pharmacological industry sector.
[9884] [0101.0.22.22] for the disclosure of this paragraph see
paragraph [0101.0.0.0] above.
[9885] [0102.0.22.22] Lipids, preferably glycolipids, or
glycolipids containing galactose, more preferably galactolipids
and/or cerebrosides can for example be detected advantageously by
the means of GC separation methods following a suitable sample
preparation. The unambiguous detection for the presence of lipids,
preferably glycolipids, or glycolipids containing galactose, more
preferably galactolipids and/or cerebrosides can be obtained by
analyzing recombinant organisms using analytical standard methods:
GC, GC-MS or TLC, as described on several occasions by Christie and
the references therein (1997, in: Advances on Lipid Methodology,
Fourth Edition: Christie, Oily Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). The
material to be analyzed can be disrupted by sonication, grinding in
a glass mill, liquid nitrogen and grinding or via other applicable
methods. After disruption, the material must be centrifuged. The
sediment is resuspended in distilled water, heated for 10 minutes
at 100.degree. C., cooled on ice and recentrifuged, followed by
extraction for one hour at 90.degree. C. in 0.5 M sulfuric acid in
methanol with 2% dimethoxypropane, which leads to hydrolyzed oil
and lipid compounds, which give transmethylated lipids (fatty acid
methyl esters and the respective complements as glycerol,
carbohydrates, phosphates). These fatty acid methyl esters are
extracted in petroleum ether and finally subjected to a GC analysis
using a capillary column (Chrompack, WCOT Fused Silica, CP-Wax-52
CB, 25 .mu.m, 0.32 mm) at a temperature gradient of between
170.degree. C. and 240.degree. C. for 20 minutes and 5 minutes at
240.degree. C. The identity of the resulting fatty acid methyl
esters must be defined using standards, which are available from
commercial sources (i.e. Sigma).
[9886] [0103.0.22.22] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: [9887]
a) nucleic acid molecule encoding, preferably at least the mature
form, of the polypeptide shown in Table II, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634 or a fragment thereof, which
confers an increase in the amount of the fine chemical in an
organism or a part thereof; [9888] b) nucleic acid molecule
comprising, preferably at least the mature form, of the nucleic
acid molecule shown in Table I, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634; [9889] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
fine chemical in an organism or a part thereof; [9890] d) nucleic
acid molecule encoding a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; [9891] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under stringent hybridization
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [9892] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c) and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [9893] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
and conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [9894] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers shown in Table III, column 7, lines 186 to 189
and/or lines 633 and 634 and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [9895] i)
nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [9896] j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence shown in Table IV,
column 7, lines 186 to 189 and/or lines 633 and 634 and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [9897] k) nucleic acid molecule comprising one or
more of the nucleic acid molecule encoding the amino acid sequence
of a polypeptide encoding a domain of the polypeptide shown in
Table II, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634
and conferring an increase in the amount of the fine chemical in an
organism or a part thereof; and [9898] l) nucleic acid molecule
which is obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably to (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; or which comprises a sequence which is complementary
thereto.
[9899] [00103.1.22.22] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634, by one or more nucleotides. In one
embodiment, the nucleic acid molecule used in the process of the
invention does not consist of the sequence shown in indicated in
Table I A, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634. In one embodiment, the nucleic acid molecule used in the
process of the invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence indicated in Table I A, columns 5 or
7, lines 186 to 189 and/or lines 633 and 634. In another
embodiment, the nucleic acid molecule does not encode a polypeptide
of a sequence indicated in Table II A, columns 5 or 7, lines 186 to
189 and/or lines 633 and 634.
[9900] [00103.2.22.22] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7 lines 186 to 189
and/or lines 633 and 634, by one or more nucleotides. In one
embodiment, the nucleic acid molecule used in the process of the
invention does not consist of the sequence shown in indicated in
Table I B, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634. In one embodiment, the nucleic acid molecule used in the
process of the invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence indicated in Table I B, columns 5 or
7, lines 186 to 189 and/or lines 633 and 634. In another
embodiment, the nucleic acid molecule does not encode a polypeptide
of a sequence indicated in Table II B, columns 5 or 7, lines 186 to
189 and/or lines 633 and 634.
[9901] [0104.0.22.22] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence shown in Table
I, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably over the sequences as shown in Table IA, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634 by one or more
nucleotides or does not consist of the sequence shown in Table I,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably of the sequences as shown in Table IA, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634. In one embodiment, the
nucleic acid molecule of the present invention is less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to the sequence shown in
Table I, columns 5 or 7, lines 1186 to 189, preferably to the
sequences as shown in Table IA, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of the sequence shown in
Table II, columns 5 or 7, lines 186 to 189, preferably of the
sequences as shown in Table IIA, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634.
[9902] [0105.0.0.22] to [0107.0.0.22] for the disclosure of the
paragraphs [0105.0.0.22] to [0107.0.0.22] see paragraphs
[0105.0.0.0] to [0107.0.0.0] above.
[9903] [0108.0.22.22] Nucleic acid molecules with the sequence
shown in Table I, columns 5 or 7, lines 186 to 189 and/or lines 633
and 634, nucleic acid molecules which are derived from the amino
acid sequences shown in Table II, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634 or from polypeptides comprising the
consensus sequence shown in Table IV, column 7, lines 186 to 189
and/or lines 633 and 634, or their derivatives or homologues
encoding polypeptides with the enzymatic or biological activity of
a polypeptide as indicated in Table I, column 3, 5 or 7, lines 186
to 189 and/or lines 633 and 634, or e.g. conferring an increase in
the respective fine chemical after increasing its expression or
activity are advantageously increased in the process according to
the invention.
[9904] [0109.0.0.22] for the disclosure of this paragraph see
[0109.0.0.0] above.
[9905] [0110.0.0.22] Nucleic acid molecules, which are advantageous
for the process according to the invention and which encode
polypeptides with an activity of a polypeptide used in the method
of the invention or used in the process of the invention, e.g. of a
protein as shown in Table II, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634 or being encoded by a nucleic acid
molecule indicated in Table I, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634 or of its homologs, e.g. as indicated in
Table II, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634
can be determined from generally accessible databases.
[9906] [0111.0.0.22] for the disclosure of this paragraph see
[0111.0.0.0] above.
[9907] [0112.0.22.22] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table I, column 3, lines 186 to
189 and/or lines 633 and 634, or having the sequence of a
polypeptide as indicated in Table II, columns 5 and 7, lines 186 to
189 and/or lines 633 and 634, and conferring an increase of the
respective fine chemical.
[9908] [0113.0.0.22] to [0120.0.0.22] for the disclosure of the
paragraphs [0113.0.0.22] to [0120.0.0.22] see paragraphs
[0113.0.0.0] and [0120.0.0.0] above.
[9909] [0121.0.22.22] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634 or the
functional homologues thereof as described herein, preferably
conferring above-mentioned activity, i.e. conferring a increase of
the fine chemical after increasing its activity.
[9910] [0122.0.0.22] to [0127.0.0.22] for the disclosure of the
paragraphs [0122.0.0.22] to [0127.0.0.22] see paragraphs
[0122.0.0.0] and [0127.0.0.0] above.
[9911] [0128.0.22.22] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, columns 7,
lines 186 to 189 and/or lines 633 and 634, by means of polymerase
chain reaction can be generated on the basis of a sequence shown
herein, for example the sequence as indicated in Table I, columns 5
or 7, lines 186 to 189 and/or lines 633 and 634, or the sequences
derived from sequences as indicated in Table II, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634.
[9912] [0129.0.22.22] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequences indicated in Table IV,
column 7, lines 186 to 189 and/or lines 633 and 634, are derived
from said alignments.
[9913] [0130.0.0.22] to [0138.0.0.22] for the disclosure of the
paragraphs [0130.0.0.22] to [0138.0.0.22] see paragraphs
[0130.0.0.0] to [0138.0.0.0] above.
[9914] [0139.0.22.22] Polypeptides having above-mentioned activity,
i.e. conferring an increase of the respective fine chemical level,
derived from other organisms, can be encoded by other DNA sequences
which hybridize to a sequences indicated in Table I, columns 5 or
7, lines 186 to 189 and/or lines 633 and 634, preferably of Table
IB, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634 under
relaxed hybridization conditions and which code on expression for
peptides having the lipid, preferably a glycolipid, glycolipid
containing galactose, more preferably a galactolipide and/or
cerebroside resp., increasing activity.
[9915] [0140.0.0.22] to [0146.0.0.22] for the disclosure of the
paragraphs [0140.0.0.22] to [0146.0.0.22] see paragraphs
[0140.0.0.0] to [0146.0.0.0] above.
[9916] [0147.0.22.22] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably of Table IB, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634, is one which is sufficiently complementary to
one of said nucleotide sequences s such that it can hybridize to
one of said nucleotide sequences thereby forming a stable duplex.
Preferably, the hybridisation is performed under stringent
hybridization conditions. However, a complement of one of the
herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[9917] [0148.0.22.22] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence shown in Table I, columns 5 or 7, lines 186
to 189 and/or lines 633 and 634, preferably of Table IB, columns 5
or 7, lines 186 to 189 and/or lines 633 and 634, or a portion
thereof and preferably has above mentioned activity, in particular
having a lipid, preferably a glycolipid, glycolipid containing
galactose, more preferably a galactolipide and/or
cerebroside-increasing activity after increasing its activity or an
activity of a product of a gene encoding said sequence or its
homologs.
[9918] [0149.0.22.22] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, preferably of Table IB,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634, or a
portion thereof and encodes a protein having above-mentioned
activity, e.g. conferring an increase of the respective fine
chemical, e.g. of lipid, preferably a glycolipid, glycolipid
containing galactose more preferably a galactolipide and/or
cerebroside and optionally the activity of a protein indicated in
Table II, column 5, lines 186 to 189 and/or lines 633 and 634,
preferably of Table IB, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634.
[9919] [00149.1.22.22] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably of Table IB, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634, has further one or more of the activities
annotated or known for the a protein as indicated in Table II,
column 3, lines 186 to 189 and/or lines 633 and 634, preferably of
Table IIB, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634.
[9920] [0150.0.22.22] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences shown in Table I, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634, preferably of Table IB, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, for example a fragment
which can be used as a probe or primer or a fragment encoding a
biologically active portion of the polypeptide of the present
invention or of a polypeptide used in the process of the present
invention, i.e. having above-mentioned activity, e.g. conferring an
increase of lipid, preferably a glycolipid, glycolipid containing
galactose, more preferably a galactolipide and/or cerebroside if
its activity is increased. The nucleotide sequences determined from
the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., in Table I, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634, an anti-sense sequence of one
of the sequences, e.g., set forth in Table I, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634, or naturally occurring mutants
thereof. Primers based on a nucleotide of invention can be used in
PCR reactions to clone homologues of the polypeptide of the
invention or of the polypeptide used in the process of the
invention, e.g. as the primers described in the examples of the
present invention, e.g. as shown in the examples. A PCR with the
primers shown in table III, column 7, lines 186 to 189., will
result in a fragment of a polynucleotide sequence as indicated in
Table I, columns 5 or 7, lines 186 to 189. Preferably is Table IB,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634.
[9921] [0151.0.0.22] for the disclosure of this paragraph see
paragraph [0151.0.0.0] above.
[9922] [0152.0.22.22] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634, such that the protein or portion thereof
maintains the ability to participate in the respective fine
chemical production, in particular an activity increasing the level
of lipids, preferably a glycolipid, glycolipid containing
galactose, more preferably a galactolipide and/or cerebrosideas
mentioned above or as described in the examples in plants or
microorganisms is comprised.
[9923] [0153.0.22.22] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 186
to 189 and/or lines 633 and 634, such that the protein or portion
thereof is able to participate in the increase of the respective
fine chemical production. In one embodiment, a protein or portion
thereof as indicated in Table II, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634, has for example an activity of a
polypeptide indicated in Table II, column 3, lines 186 to 189
and/or lines 633 and 634.
[9924] [0154.0.22.22] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634, and has above-mentioned activity, e.g.
conferring preferably the increase of the respective fine
chemical.
[9925] [0155.0.0.22] and [0156.0.0.22] for the disclosure of the
paragraphs [0155.0.0.22] and [0156.0.0.22] see paragraphs
[0155.0.0.0] and [0156.0.0.0] above.
[9926] [0157.0.22.22] The invention further relates to nucleic acid
molecules that differ from one of a nucleotide sequences as
indicated in Table I, columns 5 or 7, lines 186 to 189 and/or lines
633 and 634, (and portions thereof) due to degeneracy of the
genetic code and thus encode a polypeptide of the present
invention, in particular a polypeptide having above mentioned
activity, e.g. conferring an increase in the respective fine
chemical in a organism, e.g. as polypeptides comprising the
sequence as indicated in
[9927] Table IV, columns 5 or 7, lines 186 to 189 and/or lines 633
and 634 or of the polypeptide as indicated in Table II, columns 5
or 7, lines 186 to 189 and/or lines 633 and 634 or their functional
homologues. Advantageously, the nucleic acid molecule of the
invention comprises, or in an other embodiment has, a nucleotide
sequence encoding a protein comprising, or in an other embodiment
having, a consensus sequences as indicated in Table IV, column 7,
lines 186 to 189 and/or lines 633 and 634, or of the polypeptide as
as indicated in Table II, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634, or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
IV, column 7, lines 186 to 189 and/or lines 633 and 634, or of a
polypeptide as indicated in Table II, columns 5 or 7, lines 186 to
189 and/or lines 633 and 634, or the functional homologues thereof.
However, in a preferred embodiment, the nucleic acid molecule of
the present invention does not consist of a sequence as indicated
in Table I, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634, preferably as indicated in Table I A, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634, resp. Preferably the nucleic
acid molecule of the invention is a functional homologue or
identical to a nucleic acid molecule indicated in Table I B, column
7, lines 186 to 189 and/or lines 633 and 634.
[9928] [0158.0.0.22] to [0160.0.0.22] for the disclosure of the
paragraphs [0158.0.0.22] to [0160.0.0.22] see paragraphs
[0158.0.0.0] to [0160.0.0.0] above.
[9929] [0161.0.22.22] Accordingly, in another embodiment, a nucleic
acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634. The nucleic acid molecule is preferably
at least 20, 30, 50, 100, 250 or more nucleotides in length.
[9930] [0162.0.0.22] for the disclosure of this paragraph see
paragraph [0162.0.0.0] above.
[9931] [0163.0.22.22] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634, corresponds to a naturally-occurring nucleic
acid molecule of the invention. As used herein, a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA
molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein). Preferably, the nucleic acid molecule
encodes a natural protein having above-mentioned activity, e.g.
conferring the respective fine chemical increase after increasing
the expression or activity thereof or the activity of a protein of
the invention or used in the process of the invention.
[9932] [0164.0.0.22] for the disclosure of this paragraph see
paragraph [0164.0.0.0] above.
[9933] [0165.0.22.22] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 186 to 189 and/or lines
633 and 634.
[9934] [0166.0.0.22] and [0167.0.0.22] for the disclosure of the
paragraphs [0166.0.0.22] and [0167.0.0.22] see paragraphs
[0166.0.0.0] and [0167.0.0.0] above.
[9935] [0168.0.22.22] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table II, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634, and is capable of
participation in the increase of production of the respective fine
chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table II,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634 more
preferably at least about 70% identical to one of the sequences as
indicated in Table II, columns 5 or 7, lines 186 to 189, even more
preferably at least about 80%, 90%, or 95% homologous to a sequence
as indicated in Table II, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634 and most preferably at least about 96%, 97%, 98%,
or 99% identical to the sequence as indicated in Table II, columns
5 or 7, lines 186 to 189 and/or lines 633 and 634.
[9936] Accordingly, the invention relates to nucleic acid molecules
encoding a polypeptide having above-mentioned activity, e.g.
conferring an increase in the the respective fine chemical in an
organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, preferably of Table II
B, column 7, lines 186 to 189 and/or lines 633 and 634 yet retain
said activity described herein. The nucleic acid molecule can
comprise a nucleotide sequence encoding a polypeptide, wherein the
polypeptide comprises an amino acid sequence at least about 50%
identical to an amino acid sequence as indicated in Table II,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably of Table II B, column 7, lines 186 to 189 and/or lines
633 and 634 and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table II, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634, preferably of Table II B, column 7, lines
186 to 189 and/or lines 633 and 634, more preferably at least about
70% identical to one of the sequences as indicated in Table II,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably of Table II B, column 7 lines 186 to 189 and/or lines
633 and 634, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table II, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, preferably of Table II
B, column 7, lines 186 to 189 and/or lines 633 and 634, and most
preferably at least about 96%, 97%, 98%, or 99% identical to the
sequence as indicated in Table II, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634, preferably of Table II B, column 7, lines
186 to 189 and/or lines 633 and 634.
[9937] [0169.0.0.22] to [0172.0.0.22] for the disclosure of the
paragraphs [0169.0.0.22] to [0172.0.0.22] see paragraphs
[0169.0.0.0] to [0172.0.0.0] above.
[9938] [0173.0.22.22] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 14358 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 14358 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[9939] [0174.0.0.22] for the disclosure of this paragraph see
paragraph [0174.0.0.0] above.
[9940] [0175.0.22.22] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 14355 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 14355 by the above program algorithm with the
above parameter set, has a 80% homology.
[9941] [0176.0.22.22] Functional equivalents derived from one of
the polypeptides as indicated in Table II, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634, according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
according to the invention and are distinguished by essentially the
same properties as a polypeptide as indicated in Table II, columns
5 or 7, lines 186 to 189 and/or lines 633 and 634.
[9942] [0177.0.22.22] Functional equivalents derived from a nucleic
acid sequence as indicated in Table I, columns 5 or 7, lines 186 to
189 and/or lines 633 and 634, lines 186 to 189 and/or lines 633 and
634, preferably of Table I B, lines 186 to 189 and/or lines 633 and
634 resp. according to the invention by substitution, insertion or
deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at
least 55%, 60%, 65% or 70% by preference at least 80%, especially
preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very
especially preferably at least 95%, 97%, 98% or 99% homology with
one of a polypeptides as indicated in Table II, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, according to the
invention and encode polypeptides having essentially the same
properties as a polypeptide as indicated in Table II, columns 5 or
7, lines 186 to 189 and/or lines 633 and 634, preferably of Table
II B, lines 186 to 189 and/or lines 633 and 634 resp.
[9943] [0178.0.0.22] for the disclosure of this paragraph see
[0178.0.0.0] above.
[9944] [0179.0.22.22] A nucleic acid molecule encoding a homologous
to a protein sequence of as indicated in Table II, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, preferably of Table II
B, lines 186 to 189 and/or lines 633 and 634 resp., can be created
by introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of the nucleic acid molecule
of the present invention, in particular as indicated in Table I,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably of Table I B, lines 186 to 189 and/or lines 633 and 634
resp., such that one or more amino acid substitutions, additions or
deletions are introduced into the encoded protein. Mutations can be
introduced into the encoding sequence of a sequence as indicated in
Table I, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably of Table I B, lines 186 to 189 and/or lines 633 and 634
resp., by standard techniques, such as site-directed mutagenesis
and PCR-mediated mutagenesis.
[9945] [0180.0.0.22] to [0183.0.0.22] for the disclosure of the
paragraphs [0180.0.0.22] to [0183.0.0.22] see paragraphs
[0180.0.0.0] to [0183.0.0.0] above.
[9946] [0184.0.22.22] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table I, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, preferably of Table I B,
lines 186 to 189 and/or lines 633 and 634 resp., or of the nucleic
acid sequences derived from a sequences as indicated in Table II,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably of Table II B, lines 186 to 189 and/or lines 633 and 634
resp., comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table I, columns 5 or 7, lines 186
to 189 and/or lines 633 and 634, preferably of Table I B, lines 186
to 189 and/or lines 633 and 634 resp., or from the derived nucleic
acid sequences, the intention being, however, that the enzyme
activity or the biological activity of the resulting proteins
synthesized is advantageously retained or increased.
[9947] [0185.0.22.22] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table I, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably of Table I B, lines 186 to 189 and/or lines 633 and 634
resp. In one embodiment, it is preferred that the nucleic acid
molecule comprises as little as possible other nucleotide sequences
not shown in any one of sequences as indicated in Table I, columns
5 or 7, lines 186 to 189 and/or lines 633 and 634, preferably of
Table I B, lines 186 to 189 and/or lines 633 and 634 resp. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequence as indicated in Table I, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634, preferably of Table I B, lines 186 to 189
and/or lines 633 and 634 resp.
[9948] [0186.0.22.22] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encode a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 186 to 189 and/or lines 633 and 634, preferably of
Table II B, lines 186 to 189 and/or lines 633 and 634 resp. In one
embodiment, the nucleic acid molecule encodes less than 150, 130,
100, 80, 60, 50, 40 or 30 further amino acids. In a further
embodiment, the encoded polypeptide comprises less than 20, 15, 10,
9, 8, 7, 6 or 5 further amino acids. In one embodiment, the encoded
polypeptide used in the process of the invention is identical to
the sequences as indicated in Table II, columns 5 or 7, lines 186
to 189 and/or lines 633 and 634, preferably of Table II B, lines
186 to 189 and/or lines 633 and 634 resp.
[9949] [0187.0.22.22] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in
[9950] Table II, columns 5 or 7, lines 186 to 189 and/or lines 633
and 634, preferably of Table II B, lines 186 to 189 and/or lines
633 and 634 resp., comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence
encoding a sequences as indicated in Table II, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, preferably of Table II
B, lines 186 to 189 and/or lines 633 and 634 resp.
[9951] [0188.0.22.22] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably compared to a sequence as indicated in Table II, column
3 and 5, lines 186 to 189 and/or lines 633 and 634, and expressed
under identical conditions. In one embodiment, the polypeptide of
the invention is a homolog consisting of or comprising the sequence
as indicated in Table II B, columns 7, lines 186 to 189 and/or
lines 633 and 634.
[9952] [0189.0.22.22] Homologues of a sequences as indicated in
Table I, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
or of a derived sequences as indicated in Table II, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, also mean truncated
sequences, cDNA, single-stranded DNA or RNA of the coding and
noncoding DNA sequence. Homologues of said sequences are also
understood as meaning derivatives, which comprise noncoding regions
such as, for example, UTRs, terminators, enhancers or promoter
variants. The promoters upstream of the nucleotide sequences stated
can be modified by one or more nucleotide substitution(s),
insertion(s) and/or deletion(s) without, however, interfering with
the functionality or activity either of the promoters, the open
reading frame (=ORF) or with the 3'-regulatory region such as
terminators or other 3' regulatory regions, which are far away from
the ORF. It is furthermore possible that the activity of the
promoters is increased by modification of their sequence, or that
they are replaced completely by more active promoters, even
promoters from heterologous organisms. Appropriate promoters are
known to the person skilled in the art and are mentioned herein
below.
[9953] [0190.0.0.22] and [0191.0.0.22] for the disclosure of the
paragraphs [0190.0.0.22] and [0191.0.0.22] see paragraphs
[0190.0.0.0] and [0191.0.0.0] above.
[9954] [0191.1.0.22]: for the disclosure of this paragraph see
[0191.1.0.0] above.
[9955] [0192.0.0.22] to [0203.0.0.22] for the disclosure of the
paragraphs [0192.0.0.22] to [0203.0.0.22] see paragraphs
[0192.0.0.0] to [0203.0.0.0] above.
[9956] [0204.0.22.22] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule, which comprises a nucleic acid
molecule selected from the group consisting of: [9957] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table II, columns 5 or 7, lines 186 to
189 and/or lines 633 and 634, preferably of Table II B, lines 186
to 189 and/or lines 633 and 634 resp.; or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, i.e. lipid, preferably a glycolipid, glycolipid
containing galactose, more preferably a galactolipide and/or
cerebroside in an organism or a part thereof [9958] b) nucleic acid
molecule comprising, preferably at least the mature form, of a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, preferably of Table I B,
lines 186 to 189 and/or lines 633 and 634 resp., or a fragment
thereof conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [9959] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[9960] d) nucleic acid molecule encoding a polypeptide whose
sequence has at least 50% identity with the amino acid sequence of
the polypeptide encoded by the nucleic acid molecule of (a) to (c)
and conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [9961] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[9962] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [9963] g)
nucleic acid molecule encoding a fragment or an epitope of a
polypeptide which is encoded by one of the nucleic acid molecules
of (a) to (e), preferably to (a) to (c) and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; [9964] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying a cDNA library or a
genomic library using primers or primer pairs as indicated in Table
III, column 7, lines 186 to 189 and/or lines 633 and 634, and
conferring an increase in the amount of the respective fine
chemical, i.e. lipid, preferably a glycolipid, glycolipid
containing galactose, more preferably a galactolipide and/or
cerebrosidein an organism or a part thereof; [9965] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[9966] j) nucleic acid molecule which encodes a polypeptide
comprising a consensus sequence as indicated in Table IV, column 7,
lines 186 to 189, and conferring an increase in the amount of the
respective fine chemical i.e. lipid, preferably a glycolipid,
glycolipid containing galactose, more preferably a galactolipide
and/or cerebroside in an organism or a part thereof; [9967] k)
nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of a polypeptide as indicated in
Table II, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634, preferably of Table II B, lines 186 to 189 and/or lines 633
and 634 resp., and conferring an increase in the amount of the
respective fine chemical i.e. lipid, preferably a glycolipid,
glycolipid containing galactose, more preferably a galactolipide
and/or cerebroside in an organism or a part thereof; and [9968] l)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (h) or of a nucleic
acid molecule as indicated in Table I, columns 5 or 7, lines 186 to
189 and/or lines 633 and 634 resp., or a nucleic acid molecule
encoding, preferably at least the mature form of, the polypeptide
as indicated in Table II, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; whereby,
preferably, the nucleic acid molecule according to (a) to (l)
distinguishes over a sequence depicted in as indicated in Table IA
or IB, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
by one or more nucleotides. In one embodiment, the nucleic acid
molecule of the invention does not consist of a sequence as
indicated in Table IA or IB, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634. In an other embodiment, the nucleic acid
molecule of the present invention is at least 30 identical and less
than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table IA or IB, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634.
[9969] In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634.
Accordingly, in one embodiment, the nucleic acid molecule of the
present invention encodes in one embodiment a polypeptide which
differs at least in one or more amino acids from a polypeptide
indicated in Table IIA or IIB, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634. In another embodiment, a nucleic acid
molecule indicated in Table IA or IB, columns 5 or 7, lines 186 to
189 and/or lines 633 and 634, does not encode a protein of a
sequence indicated in Table IIA or IIB, columns 5 or 7, lines 186
to 189 and/or lines 633 and 634. Accordingly, in one embodiment,
the protein encoded by a sequences of a nucleic acid according to
(a) to (l) does not consist of a sequence as indicated in Table IIA
or IIB, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634.
In a further embodiment, the protein of the present invention is at
least 30 identical to a protein sequence indicated in Table IIA or
IIB, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634, and
less than 100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 98%, 97%, 96% or 95% identical to a
sequence as indicated in Table IA or IB, columns 5 or 7, lines 186
to 189 and/or lines 633 and 634.
[9970] [0205.0.0.22] and [0206.0.0.22] for the disclosure of the
paragraphs [0205.0.0.22] and [0206.0.0.22] see paragraphs
[0205.0.0.0] and [0206.0.0.0] above.
[9971] [0207.0.22.22] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes are genes of the glycolipid, preferably
galactolipid metabolism, galactose metabolism, fatty acid
metabolism, of glycolysis, of the tricarboxylic acid metabolism, of
triacylglycerol or lipid, preferably glycerophospholipids,
sphingolipids and/or galactolipids biosynthesis or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[9972] [0208.0.0.22] to [0226.0.0.22] for the disclosure of the
paragraphs [0208.0.0.22] to [0226.0.0.22] see paragraphs
[0208.0.0.0] to [0226.0.0.0] above.
[9973] [0227.0.22.22] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[9974] In addition to the sequence mentioned in Table I, columns 5
or 7, lines 186 to 189 and/or lines 633 and 634, or its
derivatives, it is advantageous additionally to express and/or
mutate further genes in the organisms. Especially advantageously,
additionally at least one further gene of the galyctolipid
synthesis, of the galactose pathway or of the of triacylglycerol or
lipid, preferably glycerolipids, sphingolipids, ceramid and/or
cerebroside biosynthetic pathway is expressed in the organisms such
as plants or microorganisms. It is also possible that the
regulation of the natural genes has been modified advantageously so
that the gene and/or its gene product is no longer subject to the
regulatory mechanisms which exist in the organisms. This leads to
an increased synthesis of the amino acids desired since, for
example, feedback regulations no longer exist to the same extent or
not at all. In addition it might be advantageously to combine the
sequences shown in Table I, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634, with genes which generally support or enhances
to growth or yield of the target organism, for example genes which
lead to faster growth rate of microorganisms or genes which
produces stress-, pathogen, or herbicide resistant plants.
[9975] [0228.0.22.22] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the glycoliopid,
preferably galactolipid synthesis or galactose pathway.
[9976] [0229.0.22.22] for the disclosure of this paragraph see
[0229.0.5.5] above.
[9977] [0230.0.0.22] for the disclosure of this paragraph see
[0230.0.0.0] above.
[9978] [0231.0.22.22] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a lipid, preferably a glycolipid,
glycolipid containing galactose, more preferably a galactolipide
and/or cerebroside degrading protein is attenuated, in particular
by reducing the rate of expression of the corresponding gene.
[9979] [0232.0.0.22] to [0276.0.0.22] for the disclosure of the
paragraphs [0232.0.0.22] to [0276.0.0.22] see paragraphs
[0232.0.0.0] to [0276.0.0.0] above.
[9980] [0277.0.22.22] Glycolipids, preferably galactolipids or
galactose containing compounds produced according to the process of
the invention can be isolated from the organism by methods with
which the skilled worker is familiar, for example via extraction,
salt precipitation and/or different chromatography methods. The
process according to the invention can be conducted batchwise,
semibatchwise or continuously. The fine chemical produced in the
process according to the invention can be isolated as mentioned
above from the organisms, advantageously plants, in the form of
their glycolipids, preferably galactolipids and/or free fatty
acids, oils, fats. Glycolipids, preferably galactolipids or
galactose containing compounds produced by this process can be
obtained by harvesting the organisms, either from the crop in which
they grow, or from the field. This can be done via pressing or
extraction of the plant parts, preferably the plant seeds. Hexane
is preferably used as solvent in the process, in which more than
96% of the compounds produced in the process can be isolated. The
galactolipids are easily separated from phospholipids by adsorption
chromatography, usually by making use of the fact that they, unlike
phospholipids, are soluble in acetone. Useful are also other
organic solvents such as hydrocarbons, chloroform, benzene, ethers
and alcohols. Thereafter, the resulting products are processed
further, i.e. degummed, refined, bleached and/or deodorized.
[9981] [0278.0.0.22] to [0282.0.0.22] for the disclosure of the
paragraphs [0278.0.0.22] to [0282.0.0.22] see paragraphs
[0278.0.0.0] to [0282.0.0.0] above.
[9982] [0283.0.22.22] Moreover, a native polypeptide conferring the
increase of the fine chemical in an organism or part thereof can be
isolated from cells (e.g., endothelial cells), for example using
the antibody of the present invention as described below, in
particular, an antibody against a protein as indicated in Table II,
column 3, lines 186 to 189 and/or lines 633 and 634, e.g. an
antibody against a polypeptide as indicated in Table II, columns 5
or 7, lines 186 to 189 and/or lines 633 and 634, which can be
produced by standard techniques utilizing polypeptides comprising
or consisting of above mentioned sequences, e.g. the polypeptide of
the present invention or fragment thereof. Preferred are monoclonal
antibodies specifically binding to polypeptides as indicated in
Table II, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634, more preferred specifically binding to polypeptides as
indicated in Table II, column 5, lines 186 to 189 and/or lines 633
and 634.
[9983] [0284.0.0.22] for the disclosure of this paragraph see
[0284.0.0.0] above.
[9984] [0285.0.22.22] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634, or
as coded by a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634, or
functional homologues thereof.
[9985] [0286.0.22.22] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table IV, column 7, lines 186 to 189 and/or lines 633 and 634, and
in one another embodiment, the present invention relates to a
polypeptide comprising or consisting of a consensus sequence as
indicated in Table IV, column 7, lines 186 to 189 and/or lines 633
and 634, whereby 20 or less, preferably 15 or 10, preferably 9, 8,
7, or 6, more preferred 5 or 4, even more preferred 3, even more
preferred 2, even more preferred 1, most preferred 0 of the amino
acids positions indicated can be replaced by any amino acid or, in
an further embodiment, can be replaced and/or absent. In one
embodiment, the present invention relates to the method of the
present invention comprising a polypeptide or to a polypeptide
comprising more than one consensus sequences (of an individual
line) as indicated in Table IV, column 7, lines 186 to 189 and/or
lines 633 and 634.
[9986] [0287.0.0.22] to [0290.0.0.22] for the disclosure of the
paragraphs [0287.0.0.22] to [0290.0.0.22] see paragraphs
[0287.0.0.0] to [0290.0.0.0] above.
[9987] [0291.0.22.22] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. In one embodiment, said polypeptide of the
invention distinguishes over a sequence as indicated in Table IIA
or IIB, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
by one or more amino acids. In one embodiment, polypeptide
distinguishes form a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634, by more
than 5, 6, 7, 8 or 9 amino acids, preferably by more than 10, 15,
20, 25 or 30 amino acids, even more preferred are more than 40, 50,
or 60 amino acids and, preferably, the sequence of the polypeptide
of the invention distinguishes from a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 186 to 189 and/or lines 633
and 634, by not more than 80% or 70% of the amino acids, preferably
not more than 60% or 50%, more preferred not more than 40% or 30%,
even more preferred not more than 20% or 10%. In an other
embodiment, said polypeptide of the invention does not consist of a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634.
[9988] [0292.0.0.22] for the disclosure of this paragraph see
[0292.0.0.0] above.
[9989] [0293.0.22.22] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the process
of the invention. In one embodiment, the polypeptide of the
invention has a sequence which distinguishes from a sequence as
indicated in Table IIA or IIB, lines 186 to 189 and/or lines 633
and 634, by one or more amino acids. In an other embodiment, said
polypeptide of the invention does not consist of the sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634. In a further embodiment, said polypeptide
of the present invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical. In one embodiment, said polypeptide does not
consist of the sequence encoded by a nucleic acid molecules as
indicated in Table IA or IB, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634.
[9990] [0294.0.22.22] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table IIA or IIB, column 3, lines 186 to 189 and/or
lines 633 and 634, which distinguishes over a sequence as indicated
in Table IIA or IIB, columns 5 or 7, lines 186 to 189 and/or lines
633 and 634 by one or more amino acids, preferably by more than 5,
6, 7, 8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or
30 amino acids, even more preferred are more than 40, 50, or 60
amino acids but even more preferred by less than 70% of the amino
acids, more preferred by less than 50%, even more preferred my less
than 30% or 25%, more preferred are 20% or 15%, even more preferred
are less than 10%.
[9991] [0295.0.0.22] to [0297.0.0.22] for the disclosure of the
paragraphs [0295.0.0.22] to [0297.0.0.22] see paragraphs
[0295.0.0.0] to [0297.0.0.0] above.
[9992] [00297.1.22.22] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table II, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634.
[9993] [0298.0.22.22] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence, which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634, such that the protein or portion thereof
maintains the ability to confer the activity of the present
invention. The portion of the protein is preferably a biologically
active portion as described herein. Preferably, the polypeptide
used in the process of the invention has an amino acid sequence
identical to a sequence as indicated in Table II, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634.
[9994] [0299.0.22.22] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences as shown in Table II, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634. The preferred polypeptide of
the present invention preferably possesses at least one of the
activities according to the invention and described herein. A
preferred polypeptide of the present invention includes an amino
acid sequence encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions, to a nucleotide
sequence of Table I, columns 5 or 7, lines 186 to 189 and/or lines
633 and 634 or which is homologous thereto, as defined above.
[9995] [0300.0.22.22] Accordingly the polypeptide of the present
invention can vary from the sequences shown in table II, columns 5
or 7, lines 186 to 189 and/or lines 633 and 634, in amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence shown in table IIA or IIB, columns 5
or 7, lines 186 to 189 and/or lines 633 and 634.
[9996] [0301.0.0.22] for the disclosure of this paragraph see
[0301.0.0.0] above.
[9997] [0302.0.22.22] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., the amino acid sequence shown in table II,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634, or the
amino acid sequence of a protein homologous thereto, which include
fewer amino acids than a full length polypeptide of the present
invention or used in the process of the present invention or the
full length protein which is homologous to an polypeptide of the
present invention or used in the process of the present invention
depicted herein, and exhibit at least one activity of polypeptide
of the present invention or used in the process of the present
invention.
[9998] [0303.0.0.22] for the disclosure of this paragraph see
[0303.0.0.0] above.
[9999] [0304.0.22.22] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines lines 186 to 189 and/or lines 633 and 634, but
having differences in the sequence from said wild-type protein.
These proteins may be improved in efficiency or activity, may be
present in greater numbers in the cell than is usual, or may be
decreased in efficiency or activity in relation to the wild type
protein.
[10000] [0305.0.0.22] to [0308.0.0.22] for the disclosure of the
paragraphs [0305.0.0.22] to [0308.0.0.22] see paragraphs
[0305.0.0.0] to [0308.0.0.0] above.
[10001] [0306.1.0.22] %
[10002] [0309.0.22.22] In one embodiment, an reference to a
"protein (=polypeptide)" of the invention or as indicated in Table
II, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
refers to a polypeptide having an amino acid sequence corresponding
to the polypeptide of the invention or used in the process of the
invention, whereas a "non-polypeptide of the invention" or "other
polypeptide" not being indicated in Table II, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634, refers to a polypeptide having
an amino acid sequence corresponding to a protein which is not
substantially homologous a polypeptide of the invention, preferably
which is not substantially homologous to a as indicated in Table
II, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
e.g., a protein which does not confer the activity described herein
or annotated or known for as indicated in Table II, column 3, lines
186 to 189 and/or lines 633 and 634 and which is derived from the
same or a different organism. In one embodiment, a "non-polypeptide
of the invention" or "other polypeptide" not being indicated in
Table II, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634, does not confer an increase of the respective fine chemical in
an organism or part thereof.
[10003] [0310.0.0.22] to [0334.0.0.22] for the disclosure of the
paragraphs [0310.0.0.22] to [0334.0.0.22] see paragraphs
[0310.0.0.0] to [0334.0.0.0] above.
[10004] [0335.0.22.22] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of the nucleic acid sequences of the table I, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634, and/or homologs thereof. As
described inter alia in WO 99/32619, dsRNAi approaches are clearly
superior to traditional antisense approaches. The invention
therefore furthermore relates to double-stranded RNA molecules
(dsRNA molecules) which, when introduced into an organism,
advantageously into a plant (or a cell, tissue, organ or seed
derived therefrom), bring about altered metabolic activity by the
reduction in the expression of the nucleic acid sequences of the
table I, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
and/or homologs thereof. In a double-stranded RNA molecule for
reducing the expression of an protein encoded by a nucleic acid
sequence of one of the table I, columns 5 or 7, lines 186 to 189
and/or lines 633 and 634, and/or homologs thereof, one of the two
RNA strands is essentially identical to at least part of a nucleic
acid sequence, and the respective other RNA strand is essentially
identical to at least part of the complementary strand of a nucleic
acid sequence.
[10005] [0336.0.0.22] to [0342.0.0.22] for the disclosure of the
paragraphs [0336.0.0.22] to [0342.0.0.22] see paragraphs
[0336.0.0.0] to [0342.0.0.0] above.
[10006] [0343.0.22.22] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence of one of the sequences shown in table I, columns 5 or 7,
lines 186 to 189 and/or lines 633 and 634, or its homolog is not
necessarily required in order to bring about effective reduction in
the expression. The advantage is, accordingly, that the method is
tolerant with regard to sequence deviations as may be present as a
consequence of genetic mutations, polymorphisms or evolutionary
divergences. Thus, for example, using the dsRNA, which has been
generated starting from a sequence of one of sequences shown in
table I, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
or homologs thereof of the one organism, may be used to suppress
the corresponding expression in another organism.
[10007] [0344.0.0.22] to [0361.0.0.22] for the disclosure of the
paragraphs [0344.0.0.22] to [0361.0.0.22] see paragraphs
[0344.0.0.0] to [0361.0.0.0] above.
[10008] [0362.0.22.22] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634. Due to the above mentioned activity the fine chemical content
in a cell or an organism is increased. For example, due to
modulation or manipulation, the cellular activity of the
polypeptide of the invention or nucleic acid molecule of the
invention is increased, e.g. due to an increased expression or
specific activity of the subject matters of the invention in a cell
or an organism or a part thereof. In one embodiment, transgenic for
a polypeptide having an activity of a polypeptide as indicated in
Table II, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634, means herein that due to modulation or manipulation of the
genome, an activity as annotated for a polypeptide as indicated in
Table II, column 3, lines 186 to 189 and/or lines 633 and 634, e.g.
having a sequence as indicated in Table II, columns 5 or 7, lines
186 to 189 and/or lines 633 and 634, is increased in a cell or an
organism or a part thereof. Examples are described above in context
with the process of the invention.
[10009] [0363.0.0.22] for the disclosure of this paragraph see
paragraph [0363.0.0.0] above.
[10010] [0364.0.22.22] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a polypeptide of the invention as indicated in Table
II, column 3 and/or 5, lines 186 to 189 and/or lines 633 and 634,
with the corresponding protein-encoding sequence as indicated in
Table I, column 3 and/or 5, lines 186 to 189 and/or lines 633 and
634.
[10011] [0365.0.0.22] to [0373.0.0.22] for the disclosure of the
paragraphs [0365.0.0.22] to [0373.0.0.22] see paragraphs
[0365.0.0.0] to [0373.0.0.0] above.
[10012] [0374.0.22.22] Transgenic plants comprising the lipid,
preferably a glycolipid, glycolipid containing galactose, more
preferably a galactolipide and/or cerebroside synthesized in the
process according to the invention can be marketed directly without
isolation of the compounds synthesized. In the process according to
the invention, plants are understood as meaning all plant parts,
plant organs such as leaf, stalk, root, tubers or seeds or
propagation material or harvested material or the intact plant. In
this context, the seed encompasses all parts of the seed such as
the seed coats, epidermal cells, seed cells, endosperm or embryonic
tissue. Lipid, preferably a glycolipid, glycolipid containing
galactose, more preferably a galactolipide and/or cerebroside
produced by this process can be harvested by harvesting the
organisms either from the culture in which they grow or from the
field. This can be done via expressing, grinding and/or extraction,
salt precipitation and/or ion-exchange chromatography or other
chromatographic methods of the plant parts, preferably the plant
seeds, plant fruits, plant tubers and the like.
[10013] [0375.0.0.22] and [0376.0.0.22] for the disclosure of the
paragraphs [0375.0.0.22] and [0376.0.0.22] see paragraphs
[0375.0.0.0] and [0376.0.0.0] above.
[10014] [0377.0.22.22] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced lipid, preferably a glycolipid, glycolipid containing
galactose, more preferably a galactolipide and/or cerebroside or
the produced fine chemical is isolated.
[10015] [0378.0.22.22] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the lipid,
preferably a glycolipid, glycolipid containing galactose, more
preferably a galactolipide and/or cerebrosideproduced in the
process can be isolated. The resulting lipid, preferably a
glycolipid, glycolipid containing galactose, more preferably a
galactolipide and/or cerebroside can, if appropriate, subsequently
be further purified, if desired mixed with other active ingredients
such as vitamins, amino acids, carbohydrates, antibiotics and the
like, and, if appropriate, formulated.
[10016] [0379.0.22.22] In one embodiment, the fine chemical, in
particular, lipid, preferably a glycolipid, glycolipid containing
galactose, more preferably a galactolipide and/or cerebroside, is a
mixture comprising of one or more the respective fine chemicals. In
one embodiment, the respective fine chemical means here lipid,
preferably a glycolipid, glycolipid containing galactose, more
preferably a galactolipide and/or cerebroside. In one embodiment,
lipid, preferably a glycolipid, glycolipid containing galactose,
more preferably a galactolipide and/or cerebroside means here a
mixture of the respective fine chemicals.
[10017] [0380.0.22.22] The lipid, preferably a glycolipid,
glycolipid containing galactose, more preferably a galactolipide
and/or cerebroside obtained in the process are suitable as starting
material for the synthesis of further products of value. For
example, they can be used in combination with each other or alone
for the production of pharmaceuticals, foodstuffs, animal feeds or
cosmetics. The fine chemicals of the invention can be used as
liposomes and drug carrier in pharmaceutical preparations, called
Galactosomes.RTM. (U.S. Pat. Nos. 5,688,528; 5,716,639, 6,022,561
and 6,068,860).
[10018] Galactopyranosyl is used for production of
oligosaccharides, which themselves have no antigenic action, but
have antigenic properties if they are bonded to a suitable
high-molecular carrier. For example as "haptens" which show a
sialyl-galacto pyranosyl linkageas disclosed in U.S. Pat. Nos.
5,296,594 and 5,854,218 or 4,686,193.
[10019] Galactose or galactose derivatives are supplements in
animal feed composition for reducing colonization of animal
intestines by Salmonella and other bacterial pathogens (U.S. Pat.
No. 6,126,961). Oligosaccharides containing galactose for
preventing the invasion and infection of mammal cells by pathogens
and for fighting diseases caused by such pathogens are disclosed in
US 20050004070.
[10020] Accordingly, the present invention relates to a method for
the production of a pharmaceuticals, food stuff, animal feeds,
nutrients or cosmetics comprising the steps of the process
according to the invention, including the isolation of the lipid,
preferably a glycolipid, glycolipid containing galactose, more
preferably a galactolipide and/or cerebroside composition produced
or the fine chemical produced if desired and formulating the
product with a pharmaceutical acceptable carrier or formulating the
product in a form acceptable for an application in agriculture. A
further embodiment according to the invention is the use of the
lipid, preferably a glycolipid, glycolipid containing galactose,
more preferably a galactolipide and/or cerebroside produced in the
process or of the transgenic organisms in animal feeds, foodstuffs,
medicines, food supplements, cosmetics or pharmaceuticals.
[10021] [0381.0.0.22] and [0382.0.0.22] for the disclosure of the
paragraphs [0381.0.0.22] and [0382.0.0.22] see paragraphs
[0381.0.0.0] and [0382.0.0.0] above.
[10022] [0383.0.22.22] For preparing lipid, preferably a
glycolipid, glycolipid containing galactose, more preferably a
galactolipide and/or cerebrosidecompound-containing fine chemicals,
in particular the fine chemical, it is possible to use as lipid,
preferably a glycolipid, glycolipid containing galactose, more
preferably a galactolipide and/or cerebrosidesource organic
compounds such as, for example, and/or lipids comprising fatty
acids such as fatty acids having a carbon back bone between
C.sub.10- to C.sub.16-carbon atoms and/or small organic acids such
acetic acid, propionic acid or butanoic acid as precursor
compounds.
[10023] [0384.0.0.22] for the disclosure of this paragraph see
[0384.0.0.0] above.
[10024] [0385.0.22.22] The fermentation broths obtained in this
way, containing in particular lipid, preferably a glycolipid,
glycolipid containing galactose, more preferably a galactolipide
and/or cerebroside in mixtures with other lipids, fats and/or oils,
normally have a dry matter content of from 7.5 to 25% by weight.
Depending on requirements, the biomass can be removed entirely or
partly by separation methods, such as, for example, centrifugation,
filtration, decantation or a combination of these methods, from the
fermentation broth or left completely in it. The fermentation broth
can then be thickened or concentrated by known methods, such as,
for example, with the aid of a rotary evaporator, thin-film
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. This concentrated fermentation broth can then be
worked up by freeze-drying, spray drying, spray granulation or by
other processes.
[10025] [0386.0.22.22] However, it is also possible to purify the
lipid, preferably a glycolipid, glycolipid containing galactose,
more preferably a galactolipide and/or cerebroside produced
further. For this purpose, the product-containing composition is
subjected for example to a thin layer chromatography on silica gel
plates or to a chromatography such as a Florisil column (Bouhours
J. F., J. Chromatrogr. 1979, 169, 462), in which case the desired
product or the impurities are retained wholly or partly on the
chromatography resin. These chromatography steps can be repeated if
necessary, using the same or different chromatography resins. The
skilled worker is familiar with the choice of suitable
chromatography resins and their most effective use. Alternatively
the lipid purfication can be performed as described by Dormann et
al., 1999, Science 284, 2181-2184.
[10026] [0387.0.0.22] to [0392.0.0.22] for the disclosure of the
paragraphs [0387.0.0.22] to [0392.0.0.22] see paragraphs
[0387.0.0.0] to [0392.0.0.0] above.
[10027] [0393.0.22.22] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [10028] a. contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [10029] b. identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence shown in table
I, columns 5 or 7, lines 186 to 189 and/or lines 633 and 634,
preferably in Table I B, columns 5 or 7, lines 186 to 189 and/or
lines 633 and 634, and, optionally, isolating the full length cDNA
clone or complete genomic clone; [10030] c. introducing the
candidate nucleic acid molecules in host cells, preferably in a
plant cell or a microorganism, appropriate for producing the fine
chemical; [10031] d. expressing the identified nucleic acid
molecules in the host cells; [10032] e. assaying the fine chemical
level in the host cells; and [10033] f. identifying the nucleic
acid molecule and its gene product which expression confers an
increase in the fine chemical level in the host cell after
expression compared to the wild type.
[10034] [0394.0.0.22] to [0399.0.0.22] for the disclosure of the
paragraphs [0394.0.0.22] to [0399.0.0.22] see paragraphs
[0394.0.0.0] to [0399.0.0.0] above.
[10035] [00399.1.22.22] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the fine
chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 186 to 189 and/or lines 633 and 634, or a
homolog thereof, e.g. comparing the phenotype of nearly identical
organisms with low and high activity of a protein as indicated in
Table II, columns 5 or 7, lines 186 to 189 and/or lines 633 and
634, after incubation with the drug.
[10036] [0400.0.0.22] to [0416.0.0.22] for the disclosure of the
paragraphs [0400.0.0.22] to [0416.0.0.22] see paragraphs
[0400.0.0.0] to [0416.0.0.0] above.
[10037] [0417.0.22.22] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the fatty acid production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect an organism such as a
microorganism or a plant against inhibitors, which block the fine
chemical synthesis in said organism. Examples of inhibitors or
herbicides blocking the fine chemical synthesis in organism such as
microorganism or plants might for example be phenylpyridazinones,
such as Norflurazon
[10038] [0418.0.0.22] to [0423.0.0.22] for the disclosure of the
paragraphs [0418.0.0.22] to [0423.0.0.22] see paragraphs
[0418.0.0.0] to [0423.0.0.0] above.
[10039] [0424.0.22.22] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the fine chemical or of the fine
chemical and one or more other lipid, preferably a glycolipid,
glycolipid containing galactose, more preferably a galactolipide
and/or cerebroside and mixtures thereof or mixtures of other
glycolipides. Accordingly, the nucleic acid of the invention, or
the nucleic acid molecule identified with the method of the present
invention or the complement sequences thereof, the polypeptide of
the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the fine chemical in a organism or part thereof, e.g.
in a cell.
[10040] [0425.0.0.22] to [0435.0.0.22] for the disclosure of the
paragraphs [0425.0.0.22] to [0435.0.0.22] see paragraphs
[0425.0.0.0] to [0435.0.0.0] above.
[10041] [0436.0.22.22] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of lipid, preferably a
glycolipid, glycolipid containing galactose, more preferably a
galactolipide and/or cerebroside can be carried out by passing a
plasmid DNA (or another vector DNA) containing the desired nucleic
acid sequence or nucleic acid sequences through E. coli and other
microorganisms (for example Bacillus spp. or yeasts such as
Saccharomyces cerevisiae) which are not capable of maintaining the
integrity of its genetic information. Usual mutator strains have
mutations in the genes for the DNA repair system [for example
mutHLS, mutD, mutT and the like; for comparison, see Rupp, W. D.
(1996) DNA repair mechanisms in Escherichia coli and Salmonella,
pp. 2277-2294, ASM: Washington]. The skilled worker knows these
strains. The use of these strains is illustrated for example in
Greener, A. and Callahan, M. (1994) Strategies 7; 32-34.
[10042] [0436.1.22.22] In-vitro mutation methods such as increasing
the spontaneous mutation rates by chemical or physical treatment
are well known to the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[10043] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[10044] Positive mutation events can be selected by screening the
organisms for the production of the desired fine chemical.
[10045] [0437.0.0.22] to [0453.0.0.22] and [0451.1.0.22] for the
disclosure of the paragraphs [0437.0.0.22] to [0453.0.0.22] and
[0451.1.0.22] see paragraphs [0437.0.0.0] to [0453.0.0.0] and
[0451.1.0.0] above.
[0454.0.22.22] Example 8
Analysis of the Effect of the Nucleic Acid Molecule on the
Production of the Lipid, Preferably a Glycolipid, Glycolipid
Containing Galactose, More Preferably a Galactolipide and/or
Cerebroside
[10046] [0455.0.0.22] and [0456.0.0.22] for the disclosure of the
paragraphs [0455.0.0.22] and [0456.0.0.22] see [0455.0.5.5] and
[0456.0.0.0] above.
[0457.0.22.22] Example 9
Purification of Lipid, Preferably a Glycolipid, Glycolipid
Containing Galactose, More Preferably a Galactolipide and/or
Cerebroside can be Carried Out by Thin Layer Chromatographie as for
Example Described by Dormann et al., 1999, Science 284,
2181-2184
[10047] [0458.0.22.22] One example is the analysis of lipid,
preferably a glycolipid, glycolipid containing galactose, more
preferably a galactolipide and/or cerebroside (abbreviations: FAME,
fatty acid methyl ester; GC-MS, gas liquid chromatography/mass
spectrometry; TAG, triacylglycerol; TLC, thin-layer
chromatography).
[10048] The unambiguous detection for the presence of fatty acid
products can be obtained by analyzing recombinant organisms using
analytical standard methods: GC, GC-MS or TLC, as described on
several occasions by Christie and the references therein (1997, in:
Advances on Lipid Methodology, Fourth Edition: Christie, Oily
Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). The
total fatty acids produced in the organism for example in yeasts
used in the inventive process can be analysed for example according
to the following procedure: The material such as yeasts, E. coli or
plants to be analyzed can be disrupted by sonication, grinding in a
glass mill, liquid nitrogen and grinding or via other applicable
methods. After disruption, the material must be centrifuged
(1000.times.g, 10 min., 4.degree. C.) and washed once with 100 mM
NaHCO.sub.3, pH 8.0 to remove residual medium and fatty acids. For
preparation of the fatty acid methyl esters (FAMES) the sediment is
resuspended in distilled water, heated for 10 minutes at
100.degree. C., cooled on ice and recentrifuged, followed by
extraction for one hour at 90.degree. C. in 0.5 M sulfuric acid in
methanol with 2% dimethoxypropane, which leads to hydrolyzed oil
and lipid compounds, which give transmethylated lipids.
[10049] The FAMES are then extracted twice with 2 ml petrolether,
washed once with 100 mM NaHCO.sub.3, pH 8.0 and once with distilled
water and dried with Na.sub.2SO.sub.4. The organic solvent can be
evaporated under a stream of Argon and the FAMES were dissolved in
50 .mu.l of petrolether. The samples can be separated on a ZEBRON
ZB-Wax capillary column (30 m, 0.32 mm, 0.25 .mu.m; Phenomenex) in
a Hewlett Packard 6850 gas chromatograph with a flame ionisation
detector. The oven temperature is programmed from 70.degree. C. (1
min. hold) to 200.degree. C. at a rate of 20.degree. C./min., then
to 250.degree. C. (5 min. hold) at a rate of 5.degree. C./min and
finally to 260.degree. C. at a rate of 5.degree. C./min. Nitrogen
is used as carrier gas (4.5 ml/min. at 70.degree. C.). The identity
of the resulting fatty acid methyl esters can be identified by
comparison with retention times of FAME standards, which are
available from commercial sources (i.e. Sigma).
[10050] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[10051] This is followed by heating at 100.degree. C. for 10
minutes and, after cooling on ice, by resedimentation. The cell
sediment is hydrolyzed for one hour at 90.degree. C. with 1 M
methanolic sulfuric acid and 2% dimethoxypropane, and the lipids
are transmethylated. The resulting fatty acid methyl esters (FAMEs)
are extracted in petroleum ether. The extracted FAMEs are analyzed
by gas liquid chromatography using a capillary column (Chrompack,
WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm) and a temperature
gradient of from 170.degree. C. to 240.degree. C. in 20 minutes and
5 minutes at 240.degree. C. The identity of the fatty acid methyl
esters is confirmed by comparison with corresponding FAME standards
(Sigma). The identity and position of the double bond can be
analyzed further by suitable chemical derivatization of the FAME
mixtures, for example to give 4,4-dimethoxyoxazoline derivatives
(Christie, 1998) by means of GC-MS.
[10052] The methodology is described for example in Napier and
Michaelson, 2001, Lipids. 36(8):761-766; Sayanova et al., 2001,
Journal of Experimental Botany. 52(360):1581-1585, Sperling et al.,
2001, Arch. Biochem. Biophys. 388(2): 293-298 and Michaelson et
al., 1998, FEBS Letters. 439(3): 215-218.
[10053] [0459.0.22.22] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously the lipid, preferably a glycolipid, glycolipid
containing galactose, more preferably a galactolipide and/or
cerebroside can be further purified with a so-called RTHPLC. As
eluent different an acetonitrile/water or chloroform/acetonitrile
mixtures are advantageously is used. For the analysis of the fatty
acids an ELSD detector (evaporative light-scattering detector) is
used. MPLC, dry-flash chromatography or thin layer chromatography
are other beneficial chromatography methods for the purification of
glycolipids. If necessary, these chromatography steps may be
repeated, using identical or other chromatography resins. The
skilled worker is familiar with the selection of suitable
chromatography resin and the most effective use for a particular
molecule to be purified.
[10054] [0460.0.22.22] In addition depending on the produced fine
chemical purification is also possible with cristalisation or
destilation. Both methods are well known to a person skilled in the
art.
[0461.0.22.22] Example 10
Cloning SEQ ID NO: 14354 for the Expression in Plants
[10055] [0462.0.0.22] to [0466.0.0.22] for the disclosure of the
paragraphs [0462.0.0.22] to [0466.0.0.22] see [0462.0.0.0] to
[0466.0.0.0] above.
[10056] [0467.0.22.22] The following primer sequences were selected
for the gene SEQ ID NO: 14354:
TABLE-US-00133 i) forward primer (SEQ ID NO: 14356) atggcggttg
cgatcaaaaa gga ii) reverse primer (SEQ ID NO: 14357) tcaattgata
aatgtacttt caatgatg
[10057] [0468.0.0.22] to [0479.0.0.22] for the disclosure of the
paragraphs [0468.0.0.22] to [0479.0.0.22] see [0468.0.0.0] to
[0479.0.0.0] above.
[0480.0.22.22] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 14354
[10058] [0481.0.0.22] to [0513.0.0.22] for the disclosure of the
paragraphs [0481.0.0.22] to [0513.0.0.22] see paragraphs
[0482.0.0.0] to [0513.0.0.0] above.
[10059] [0514.0.22.22] In order to analyze glycolipids being
present in the transgenic organism by the means of gas
chromatography-mass spectrometry, material from the transgenic
organisms have to be extracted and the extracts subsequently being
hydrolyzed in the presence of methanol and an inorganic acid,
yielding the corresponding fatty acid methyl esters and the
respective monosaccharid moietye as its methylhexopyranoside.
[10060] Primary and secondary amino functions, hydroxy groups and
free carboxylic functions eventually will be trimethylsilylated by
reaction with N-Methyl-N-trimethylsilyltrifluoroacetamide, yielding
the trimethylsilyl (TMS) derivatives of the methylhexopyranosides
formed in the previous hydrolysis step (eg methylgalactopyranoside
4TMS in the case of a galactolipid). These compounds are accessible
to gas chromatographic-mass spectrometric analysis.
[10061] Therefore, an increased content of the trimethylsilylated
methylhexopyranosides directly correlates to an increased content
of glycolipids in the transgenic organism.
[10062] The results of the different plant analyses can be seen
from the table, which follows:
TABLE-US-00134 TABLE 1 ORF Metabolites Analyte Method Min Max
YER173W Galactolipids Methylgalacto- GC 1.20 1.70 pyranosid 4 TMS
YLR224W Galactolipids Methylgalacto- GC 1.15 1.46 pyranosid 4 TMS
YLR255C Galactolipids Methylgalacto- GC 1.16 1.30 pyranosid 4 TMS
b2699 Galactolipids Methylgalacto- GC 1.15 1.64 pyranosid 4 TMS
YHR072W Galactolipids Methylgalacto- GC 1.17 1.35 pyranosid 4 TMS
b3129 Galactolipids Methylgalacto- GC 1.16 1.32 pyranosid 4 TMS
[10063] [0515.0.22.22] Column 2 shows the metabolite Galactolipids
and column 3 the analyte analyzed. Columns 5 and 6 shows the ratio
of the analyzed metabolite/alnalyte between the transgenic plants
and the wild type; Increase of the metabolite: Max: maximal x-fold
(normalised to wild type)-Min: minimal x-fold (normalised to wild
type).
[10064] Decrease of the metabolite: Max: maximal x-fold (normalised
to wild type) (minimal decrease), Min: minimal x-fold (normalised
to wild type) (maximal decrease). Column 4 indicates the analytical
method.
[10065] [0516.0.0.22] to [0530.0.0.22] for the disclosure of the
paragraphs [0516.0.0.22] to [0530.0.0.22] see paragraphs
[0516.0.0.0] to [0530.0.0.0] above.
[10066] [0530.1.0.22] to [0530.6.0.22] for the disclosure of the
paragraphs [0530.1.0.22] to [0530.6.0.22] see paragraphs
[0530.1.0.0] to [0530.6.0.0] above.
[10067] [0531.0.0.22] to [0552.0.0.22] for the disclosure of the
paragraphs [0531.0.0.22] to [0552.0.0.22] see paragraphs
[0531.0.0.0] to [0552.0.0.0] above.
[10068] [0552.1.0.22]: %
[10069] [0552.2.0.22] for the disclosure of this paragraph see
[0552.2.0.0] above.
[10070] [0553.0.22.22] [10071] 1. A process for the production of a
lipid, preferably a glycolipid, a glycolipide containing galactose,
more preferably a galactolipide and/or a cerebroside, which
comprises (a) increasing or generating the activity of a protein as
indicated in Table II, columns 5 or 7, lines 186 to 189 or 633 and
634, or a functional equivalent thereof in a non-human organism, or
in one or more parts thereof; and (b) growing the organism under
conditions which permit the production of a lipid, preferably a
glycolipid, a glycolipide containing galactose, more preferably a
galactolipide and/or a cerebrosidein said organism. [10072] 2. A
process for the production of a lipid, preferably a glycolipid, a
glycolipide containing galactose, more preferably a galactolipide
and/or a cerebroside, comprising the increasing or generating in an
organism or a part thereof the expression of at least one nucleic
acid molecule comprising a nucleic acid molecule selected from the
group consisting of: [10073] a) nucleic acid molecule encoding of
the polypeptide shown in table II, columns 5 or 7, lines 186 to 189
or 633 and 634, or a fragment thereof, which confers an increase in
the amount of lipids, preferably of glycolipids, of glycolipides
containing galactose, more preferably of galactolipide and/or
cerebrosides in an organism or a part thereof; [10074] b) nucleic
acid molecule comprising of the nucleic acid molecule shown in
table I, columns 5 or 7, lines 186 to 189 or 633 and 634; [10075]
c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of lipids, preferably of
glycolipids, of glycolipides containing galactose, more preferably
of galactolipides and/or cerebrosides in an organism or a part
thereof; [10076] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of lipids,
preferably of glycolipids, of glycolipides containing galactose,
more preferably of galactolipides and/or cerebrosides in an
organism or a part thereof; [10077] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to [10078] (c) under
under stringent hybridisation conditions and conferring an increase
in the amount of lipids, preferably of glycolipids, ofa
glycolipides containing galactose, more preferably of
galactolipides and/or cerebrosides in an organism or a part
thereof; [10079] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers as shown in table III, column 7, lines 186 to 189 or 633
and 634, and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [10080] g) nucleic acid
molecule encoding a polypeptide which is isolated with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (f) and conferring an increase in
the amount of lipids, preferably of glycolipids, of glycolipides
containing galactose, more preferably of galactolipides and/or
cerebrosides in an organism or a part thereof; [10081] h) nucleic
acid molecule encoding a polypeptide comprising the consensus
sequence shown in table IV, column 7, lines 186 to 189 or 633 and
634, and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; and [10082] i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof.
[10083] or comprising a sequence which is complementary thereto.
[10084] 3. The process of claim 1 or 2, comprising recovering of
the free or bound lipid, preferably of a glycolipid, of a
glycolipide containing galactose, more preferably of a
galactolipide and/or cerebroside. [10085] 4. The process of any one
of claims 1 to 3, comprising the following steps: [10086] (a)
selecting an organism or a part thereof expressing a polypeptide
encoded by the nucleic acid molecule characterized in claim 2;
[10087] (b) mutagenizing the selected organism or the part thereof;
[10088] (c) comparing the activity or the expression level of said
polypeptide in the mutagenized organism or the part thereof with
the activity or the expression of said polypeptide of the selected
organisms or the part thereof; [10089] (d) selecting the mutated
organisms or parts thereof, which comprise an increased activity or
expression level of said polypeptide compared to the selected
organism or the part thereof; [10090] (e) optionally, growing and
cultivating the organisms or the parts thereof; and [10091] (f)
recovering, and optionally isolating, the free or bound palmitic
acid produced by the selected mutated organisms or parts thereof.
[10092] 5. The process of any one of claims 1 to 4, wherein the
activity of said protein or the expression of said nucleic acid
molecule is increased or generated transiently or stably. [10093]
6. An isolated nucleic acid molecule comprising a nucleic acid
molecule selected from the group consisting of: [10094] a) nucleic
acid molecule encoding of the polypeptide shown in table II,
columns 5 or 7, lines 186 to 189 or 633 and 634, or a fragment
thereof, which confers an increase in the amount of lipids,
preferably of glycolipids, of glycolipides containing galactose,
more preferably of galactolipides and/or cerebrosides in an
organism or a part thereof; [10095] b) nucleic acid molecule
comprising of the nucleic acid molecule shown in table I, columns 5
or 7, lines 186 to 189 or 633 and 634; [10096] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of lipids, preferably of glycolipids, of glycolipides
containing galactose, more preferably of galactolipides and/or
cerebrosides in an organism or a part thereof; [10097] d) nucleic
acid molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of lipids, preferably of glycolipids, of glycolipides
containing galactose, more preferably of galactolipides and/or
cerebrosides in an organism or a part thereof; [10098] e) nucleic
acid molecule which hybridizes with a nucleic acid molecule of (a)
to [10099] (c) under stringent hybridisation conditions and
conferring an increase in the amount of lipids, preferably of
glycolipids, of glycolipides containing galactose, more preferably
of galactolipides and/or cerebrosides in an organism or a part
thereof; [10100] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers as shown in table III, column 7, lines 186 to 189 or 633
and 634, and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [10101] g) nucleic acid
molecule encoding a polypeptide which is isolated with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (f) and conferring an increase in
the amount of lipids, preferably of glycolipids, of glycolipides
containing galactose, more preferably of galactolipides and/or
cerebrosides in an organism or a part thereof; [10102] h) nucleic
acid molecule encoding a polypeptide comprising the consensus
sequence shown in table IV, column 7, lines 186 to 189 or 633 and
634, and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; and [10103] i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof.
[10104] whereby the nucleic acid molecule distinguishes over the
sequence as shown in table IA, columns 5 or 7, lines 186 to 189 or
633 and 634, by one or more nucleotides. [10105] 7. A nucleic acid
construct which confers the expression of the nucleic acid molecule
of claim 6, comprising one or more regulatory elements. [10106] 8.
A vector comprising the nucleic acid molecule as claimed in claim 6
or the nucleic acid construct of claim 7. [10107] 9. The vector as
claimed in claim 8, wherein the nucleic acid molecule is in
operable linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [10108] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [10109] 11. The host cell of claim 10, which is a
transgenic host cell. [10110] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [10111] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [10112] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over the sequence as shown in table IIA, columns 5 or
7, lines 186 to 189 or 633 and 634, by one or more amino acids 15.
An antibody, which binds specifically to the polypeptide as claimed
in claim 14. [10113] 16. A plant tissue, propagation material,
harvested material or a plant comprising the host cell as claimed
in claim 12 which is plant cell or an Agrobacterium. [10114] 17. A
method for screening for agonists and antagonists of the activity
of a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of lipids, preferably of
glycolipids, of glycolipides containing galactose, more preferably
of galactolipides and/or cerebrosides in an organism or a part
thereof comprising: (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of lipids, preferably of glycolipids, of glycolipides
containing galactose, more preferably of galactolipides and/or
cerebrosides in an organism or a part thereof with a candidate
compound or a sample comprising a plurality of compounds under
conditions which permit the expression the polypeptide; (b)
assaying the lipids, preferably of glycolipids, of glycolipides
containing galactose, more preferably of galactolipides and/or
cerebrosides level or the polypeptide expression level in the cell,
tissue, plant or microorganism or the media the cell, tissue, plant
or microorganisms is cultured or maintained in; and (c) identifying
a agonist or antagonist by comparing the measured lipid, preferably
the glycolipid, the glycolipide containing galactose, more
preferably the galactolipide and/or cerebrosidelevel or polypeptide
expression level with a standard lipid, preferably a glycolipid, a
glycolipide containing galactose, more preferably a galactolipide
and/or cerebroside or polypeptide expression level measured in the
absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. [10115] 18. A process for
the identification of a compound conferring increased lipid,
preferably a glycolipid, a glycolipide containing galactose, more
preferably a galactolipide and/or cerebrosideproduction in a plant
or microorganism, comprising the steps: a) culturing a plant cell
or tissue or microorganism or maintaining a plant expressing the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of lipids, preferably of
glycolipids, of glycolipides containing galactose, more preferably
of galactolipides and/or cerebrosides in an organism or a part
thereof and a readout system capable of interacting with the
polypeptide under suitable conditions which permit the interaction
of the polypeptide with dais readout system in the presence of a
compound or a sample comprising a plurality of compounds and
capable of providing a detectable signal in response to the binding
of a compound to said polypeptide under conditions which permit the
expression of said readout system and of the polypeptide encoded by
the nucleic acid molecule of claim 6 conferring an increase in the
amount of lipids, preferably of glycolipids, of glycolipides
containing galactose, more preferably of galactolipides and/or
cerebrosides in an organism or a part thereof; b) identifying if
the compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout system.
[10116] 19. A method for the identification of a gene product
conferring an increase in lipids, preferably in glycolipid, in
glycolipide containing galactose, more preferably in galactolipide
and/or cerebroside production in a cell, comprising the following
steps: (a) contacting the nucleic acid molecules of a sample, which
can contain a candidate gene encoding a gene product conferring an
increase in lipids, preferably in glycolipid, in glycolipide
containing galactose, more preferably in galactolipide and/or
cerebroside after expression with the nucleic acid molecule of
claim 6; (b) identifying the nucleic acid molecules, which
hybridise under relaxed stringent conditions with the nucleic acid
molecule of claim 6; (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing lipid, preferably
a glycolipid, a glycolipide containing galactose, more preferably a
galactolipide and/or cerebroside;
(d) expressing the identified nucleic acid molecules in the host
cells; (e) assaying the lipid, preferably a glycolipid, a
glycolipide containing galactose, more preferably a galactolipide
and/or cerebrosidelevel in the host cells; and (f) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the lipid, preferably in glycolipid, in glycolipide
containing galactose, more preferably in galactolipide and/or
cerebroside level in the host cell in the host cell after
expression compared to the wild type. [10117] 20. A method for the
identification of a gene product conferring an increase in lipid,
preferably in glycolipid, in glycolipide containing galactose, more
preferably in galactolipide and/or cerebroside production in a
cell, comprising the following steps: [10118] (a) identifying in a
data bank nucleic acid molecules of an organism; which can contain
a candidate gene encoding a gene product conferring an increase in
the lipid, preferably a glycolipid, a glycolipide containing
galactose, more preferably a galactolipide and/or cerebroside
amount or level in an organism or a part thereof after expression,
and which are at least 20% homolog to the nucleic acid molecule of
claim 6; [10119] (b) introducing the candidate nucleic acid
molecules in host cells appropriate for producing lipid, preferably
a glycolipid, a glycolipide containing galactose, more preferably a
galactolipide and/or cerebroside; [10120] (c) expressing the
identified nucleic acid molecules in the host cells; [10121] (d)
assaying the lipid, preferably a glycolipid, a glycolipide
containing galactose, more preferably a galactolipide and/or
cerebroside level in the host cells; and [10122] (e) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the lipid, preferably in the glycolipid, in the
glycolipide containing galactose, more preferably in the
galactolipide and/or cerebroside level in the host cell after
expression compared to the wild type. [10123] 21. A method for the
production of an agricultural composition comprising the steps of
the method of any one of claims 17 to 20 and formulating the
compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. [10124] 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [10125] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
lipid, preferably of glycolipid, of glycolipide containing
galactose, more preferably of galactolipide and/or cerebroside
after expression. [10126] 24. Use of the polypeptide of claim 14 or
the nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of lipid, preferably of
glycolipid, of glycolipide containing galactose, more preferably of
galactolipide and/or cerebroside levels in an organism. [10127] 25.
Food or feed composition comprising the nucleic acid molecule of
claim 6, the polypeptide of claim 14, the nucleic acid construct of
claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the harvested material of claim
16, the host cell of claim 10 to 12 or the gene product identified
according to the method of claim 19 or 20. [10128] 26. Use of the
nucleic acid molecule of claim 6, the polypeptide of claim 14, the
nucleic acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
host cell of claim 10 to 12 or the gene product identified
according to the method of claim 19 or 20 for the protection of a
plant against a glycolipid, of glycolipide containing galactose,
more preferably of galactolipide and/or cerebroside synthesis
inhibiting herbicide.
[10129] [0554.0.0.22] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[10130] [0000.0.0.23] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[10131] [0001.0.0.23] for the disclosure of this paragraph see
[0001.0.0.0] above.
[10132] [0002.0.23.23] Salicylic acid is common throughout the
plant kingdom and is also found in bacteria. It is an important
regulator of induced plant resistance to pathogens.
[10133] Small amounts of salicylic acid are known to be present in
plants. Originally salicylic acid was extracted from the willow
bark to make the well-known pain relief medication Aspirin.
Salicylic acid is thought to promote disease resistance, increase
flower life, inhibit seed germination, and promote ethylene
synthesis.
[10134] Salicylic acid can be synthesized from cinnamate. Previous
isotope feeding experiments in tobacco and other higher plants,
including rice, demonstrated that the direct precursor of salicylic
acid is free benzoic acid. Benzoic acid is synthesized by cinnamate
chain shortening reactions via the so-called beta-oxidation,
analogous to fatty acid beta-oxidation. Benzoic acid is then
converted to salicylic acid by benzoic acid 2-hydroxylase. Recent
studies in tobacco indicated that conjugated benzoic acid, CoA
thioesters or glucose esters, are more likely to be the precursors
of salicylic acid. More recent genetic studies in Arabidopsis have
shown that salicylic acid can also be synthesized from chorismate
and that the bulk of salicylic acid is produced from
chorismate.
[10135] Plants react to pathogen attack by activating elaborate
defense mechanisms. The defense response is activated not only at
the sites of infection, but also in neighboring and even distal
uninfected parts of the plant, leading to systemic acquired
resistance. Plant resistance is associated with activated
expression of a large number of defense-related genes, whose
products may play important roles in the restriction of pathogen
growth and spread. During the past several years, evidence has
accumulated which indicates that salicylic acid (SA) acts as an
endogenous signal for plant defense responses.
[10136] In most plants, exposure to powdery mildew and other
pathogens triggers the plant defense pathway, a series of
biochemical events that occur in succession and help the plant
resist infection. Salicylic acid governs this pathway.
[10137] Where resistance to a pathogen is associated with a
localised necrotic lesion, the plant will subsequently be
systemically "immunized" so that further infection will either
exhibit increased resistance or reduced disease symptoms (reviewed
by Ryals et al., 1996). This "systemic acquired resistance" (SAR)
is associated with the systemic expression of a subset of defence
genes, e.g. the acidic forms of pathogenesis-related PR1-5 proteins
(Ward et al., 1992). Search for a signal that may be mobilised from
the lesion to elicit systemic resistance has led to the
identification of salicylic acid (SA) as the most likely candidate.
SA is synthesised to high levels around the necrotic lesion, before
being (possibly) mobilised through the phloem to accumulate, at
much lower levels, systemically.
[10138] [0003.0.23.23] When faced with a fungus or bacteria, most
plants turn up their production of salicylic acid, which then
interacts with other molecules in the plant, eventually turning on
the genes that produce the proteins involved in fighting infection.
These infection-fighting proteins also turn off salicylic acid
production, a phenomenon known as negative feedback. In this way,
plants can turn the pathogen defense pathway on and off as
needed.
[10139] The basic idea to enhance plant disease resistance by
overproduction of salicylic acid has already been published years
ago for example by Verberne et al., Pharm. World-Sci.; (1995) 17,
6. Later on in 2000 is was published that the expression of the
Escherichia coli isochorismate-synthase and Pseudomonas
fluorescence isochorismate-pyruvate-lyase in transgenic tobacco can
lead to improved disease-resistance (Verberne, Metal., Nat.
Biotechnol.; (2000) 18, 7, 779-83. The two enzymes converted
chorismate into SA by a 2-step process. When the enzymes were
targeted to the chloroplasts, the transgenic plants showed a 500-
to 1,000-fold increased accumulation of SA and SA glucoside
compared to control plants. These plants showed a resistance to
viral (tobacco-mosaic virus) and fungal (Oidium lycopersicon)
infection resembling SAR in nontransgenic plants. As the effect was
the result of the plastidal expression of two heterologous genes,
there is the obvious need for alternative and more simple methods
for enhanced salicylic acid production in plants by the cytosolic
expression of individual genes. For individual cases or specific
plant species a more moderate salicylic acid increase may also be
useful and desired.
[10140] Additionally salicylic acid binding proteins have been
described as useful for the production of transgenic plants with
increased resistance to disease (WO2003016551).
[10141] Most plants maintain very low levels of salicylic acid in
their tissues unless they are fighting an infection. Metal
hyperaccumulators, however, have significantly elevated salicylic
acid in their tissues all the time--see:
www.newswise.com/articles/view/510423/Recent results also suggest
that in some plant species high level of endogenous salicylic acid
protects the plants from oxidative stress caused for example by
aging or biotic or abiotic stress (Yang et al., Plant J. 2004
December; 40 (6): 909-19).
[10142] [0004.0.23.23] Aspirin was introduced into clinical
practice more than 100 years ago. This unique drug belongs to a
family of compounds called the salicylates, the simplest of which
is salicylic acid, the principal metabolite of aspirin. Salicylic
acid is responsible for the anti-inflammatory action of aspirin,
and may cause the reduced risk of colorectal cancer observed in
those who take aspirin. Yet salicylic acid and other salicylates
occur naturally in fruits and plants, while diets rich in these are
believed to reduce the risk of colorectal cancer. Serum salicylic
acid concentrations are greater in vegetarians than
non-vegetarians, and there is overlap between concentrations in
vegetarians and those taking low-dose aspirin. It is proposed that
the cancer-preventive action of aspirin is due to its principal
metabolite, salicylic acid, and that dietary salicylates can have
the same effect. It is also possible that natural salicylates
contribute to the other recognized benefits of a healthy diet.
[10143] [0005.0.23.23] The hydroxyl group of salicylic acid reacts
with acetic acid to form the acetate ester, acetylsalicylic acid
(see aspirin). Several useful esters are formed by reaction of the
carboxyl group with alcohols. The methyl ester, methyl salicylate
(also called oil of wintergreen since it produces the fragrance of
wintergreen), is formed with methanol; it is used in food
flavorings and in liniments. The phenyl ester, phenyl salicylate,
or salol, is formed with phenol; it is used in medicine as an
antiseptic and antipyretic. This ester hydrolyzes, not in the
acidic stomach, but in the alkaline intestines, releasing free
salicylic acid. The menthyl ester, menthyl salicylate, which is
used in suntan lotions, is formed with menthol.
[10144] Salicylic acid possesses bacteriostatic, fungicidal, and
keratolytic actions.
[10145] [0006.0.23.23] Salicylic acid is used as a food
preservative and as an antiseptic in toothpaste. It is a peeling
agent in ointments, creams, gels, and shampoos applied to reduce
the scaling of the skin or scalp in psoriasis. It is the active
ingredient in many skin products for the treatment of acne since it
causes skin cells to slough off more readily, preventing them from
clogging up the pores.
[10146] [0007.0.23.23] Salicylic acid belongs to the group of
medicines known as keratolytics. Salicylic acid works by breaking
down keratin, a protein, which forms part of the skin structure.
This results in the shedding of skin cells from the affected area.
In the treatment of warts, calluses and verrucae the effect of
salicylic acid is to remove the affected skin over a period of
time. If successful, the new skin, which grows underneath will be
healthy.
[10147] [0008.0.23.23] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce salicylic acid and/or salicylic acid
esters in plants. That type of production permits control over
quality, quantity and selection of the most suitable and efficient
producer organisms. The latter is especially important for
commercial production economics and therefore availability to
consumers. In addition it is desirable to produce salicylic acidin
plants in order to increase plant productivity and resistance
against biotic and abiotic stress as discussed before.
[10148] Methods of recombinant DNA technology have been used for
some years to improve the production of fine chemicals in
microorganisms and plants by amplifying individual biosynthesis
genes and investigating the effect on production of fine chemicals.
It is for example reported, that the xanthophyll astaxanthin could
be produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. Those results indicated
that about 75 percent of the carotenoids found in the flower of the
transformed plant contained a keto group.
[10149] [0009.0.23.23] Thus, it would be advantageous if algae,
plant or other microorganism were available which produce large
amounts of salicylic acid. The invention discussed hereinafter
relates in some embodiments to such transformed prokaryotic or
eukaryotic microorganisms.
[10150] It would also be advantageous if plants were available
whose roots, leaves, stems, fruits or flowers produced large
amounts of salicylic acid. The invention discussed hereinafter
relates in some embodiments to such transformed plants.
[10151] Furthermore it would be advantageous if plants were
available whose seed produced larger amounts of total lipids. The
invention discussed hereinafter relates in some embodiments to such
transformed plants.
[10152] [0010.0.23.23] Therefore improving the quality of
foodstuffs and animal feeds is an important task of the
food-and-feed industry. This is necessary since, for example
salicylic acid, as mentioned above, which occur in plants and some
microorganisms are limited with regard to the supply of mammals.
Especially advantageous for the quality of foodstuffs and animal
feeds is as balanced as possible a specific salicylic acid profile
in the diet since an excess of salicylic acid above a specific
concentration in the food has a positive effect. A further increase
in quality is only possible via addition of further salicylic acid,
which are limiting.
[10153] [0011.0.23.23] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add salicylic acidand/or
salicylic acid esters in a balanced manner to suit the
organism.
[10154] [0012.0.23.23] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
proteins which participate in the biosynthesis of salicylic acidand
make it possible to produce them specifically on an industrial
scale without unwanted byproducts forming. In the selection of
genes for biosynthesis two characteristics above all are
particularly important. On the one hand, there is as ever a need
for improved processes for obtaining the highest possible contents
of salicylic acid and/or salicylic acid esters; on the other hand
as less as possible byproducts should be produced in the production
process.
[10155] Furthermore there is still a great demand for new and more
suitable genes which encode enzymes or other proteins which
participate in the biosynthesis of total lipids and make it
possible to produce them specifically on an industrial scale
without unwanted byproducts forming. In the selection of genes for
biosynthesis two characteristics above all are particularly
important. On the one hand, there is as ever a need for improved
processes for obtaining the highest possible contents of total
lipids; on the other hand as less as possible byproducts should be
produced in the production process.
[10156] [0013.0.0.23] for the disclosure of this paragraph see
[0013.0.0.0] above.
[10157] [0014.0.23.23] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is salicylic acid or salicylic
acid esters. Further, the term "the fine chemicals" as used herein
also relates to fine chemicals comprising salicylic acid and/or
salicylic acid esters.
[10158] In one embodiment, the term "the fine chemical" means
salicylic acid. Throughout the specification the term "the fine
chemical" means salicylic acid, its salts, ester, thioester or in
free form or bound to other compounds such as sugars.
[10159] [0015.0.23.23] In particular it is known to the skilled
that anionic compounds such as acids are present in the aqueous
solution in an equilibrium between the acid and its salts according
to the pH present in the respective compartment of the cell or
organism and the pK of the acid. Depending on the strength of the
acid (pK) and the pH the salt or the free acid are predominant.
Thus, the term "the fine chemical", the term "the respective fine
chemical", the term "acid" or the use of a denomination referring
to a neutralized anionic compound respectively, relates to the
anionic form as well as the neutralised status of that compound in
relation to the conditions of the aqueous solution.
[10160] [0016.0.23.23] Accordingly, the present invention relates
to a process comprising [10161] (a) increasing or generating the
activity of one or more b0161, b2664, b2796, b3116 or YLR089C
protein(s) in a non-human organism in one or more parts thereof;
and [10162] (b) growing the organism under conditions which permit
the production of the fine chemical, thus salicylic acid in said
organism.
[10163] Accordingly, the present invention relates to a process
comprising [10164] (a) increasing or generating the activity of one
or more proteins having the activity of a protein indicated in
Table II, column 3, lines 275 to 277 and/or 635 or 636, resp. or
having the sequence of a polypeptide encoded by a nucleic acid
molecule indicated in Table I, columns 5 or 7, lines 275 to 277
and/or 635 or 636, resp. in a non-human organism in one or more
parts thereof; and growing the organism under conditions which
permit the production of the fine chemical, thus, salicylic acid
and/or salicylic acid esters, in said organism.
[10165] [0016.1.23.23] Accordingly, the term "the fine chemical"
means "salicylic acid" in relation to all sequences listed in Table
I, columns 5 or 7, lines 275 to 277 and/or 635 or 636 or homologs
thereof.
[10166] [0017.0.0.23] and [0018.0.0.23] for the disclosure of the
paragraphs [0017.0.0.23] and [0018.0.0.23] see paragraphs
[0017.0.0.0] and [0018.0.0.0] above.
[10167] [0019.0.23.23] Advantageously the process for the
production of the respective fine chemical leads to an enhanced
production of the respective fine chemical. The terms "enhanced" or
"increase" mean at least a 10%, 20%, 30%, 40% or 50%, preferably at
least 60%, 70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%,
400% or 500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table II, column 3, lines 275 to 277 and/or 635 or 636
or encoded by nucleic acid molecule indicated in Table I, columns 5
or 7, lines 275 to 277 and/or 635 or 636.
[10168] [0020.0.23.23] Surprisingly it was found, that the
transgenic expression of the Escherichia coli K12 protein b0161,
b2796, b3116 or b2664 or Saccharomyces cerevisiae protein YLR089C
in Arabidopsis thaliana conferred an increase in salicylic acid
("the fine chemical" or "the fine respective chemical") in respect
to said proteins and their homologs as wells as the encoding
nucleic acid molecules, in particular as indicated in Table II,
column 3, lines 275 to 277 and/or 635 or 636 content of the
transformed plants.
[10169] Surprisingly it was found, that the transgenic expression
of the Saccaromyces cerevisiae protein YLR089C as indicated in
Table II, columns 3 or 5, line 277 and/or the Escherichia coli K12
protein(s) b0161, b2796, b3116 and/or b2664 as indicated in Table
II, columns 3 or 5, lines 275, 276, 635 and/or 636 in Arabidopsis
thaliana conferred an increase in the content of salicylic acid
and/or salicylic acid esters in the transformed plants. Thus, in
one embodiment, said protein(s) or its homologs as indicated in
Table II, column 7, lines 275 to 277 and/or 635 or 636 are used for
the production of salicylic acid and/or salicylic acid esters.
[10170] [0021.0.0.23] for the disclosure of this paragraph see
[0021.0.0.0] above.
[10171] [0022.0.23.23] The sequence of b0161 from Escherichia coli
K12 has been published in Blattner, F. R. et al., Science 277
(5331), 1453-1474 (1997) and its activity is being defined as a
protein having serine protease activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein b0161 from Escherichia coli K12 or its homolog, e.g.
as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of salicylic
acid, preferably in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of the protein b0161 is
increased.
[10172] The sequence of b2664 from Escherichia coli K12 has been
published in Blattner, F. R. et al, Science 277 (5331), 1453-1474
(1997) and its activity is being defined as a protein having
transcriptional repressor (GntR familiy) activity. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a protein b2664 from Escherichia coli K12 or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of salicylic
acid, preferably in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of the protein b2664 is
increased.
[10173] The sequence of b2796 (Accession number NP.sub.--417276)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a putative serine transport protein (HAAAP family).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
threonine-serine permease superfamily, preferably a protein with
the activity of a putative serine transport protein (HAAAP family)
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of salicylic acid,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the protein b2796 is increased.
[10174] The sequence of b3116 (Accession number NP.sub.--417586)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a L-threonine/L-serine permease, anaerobically inducible
(HAAAP family). Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of threonine-serine permease superfamily, preferably a
protein with the activity of a L-threonine/L-serine permease,
anaerobically inducible (HAAAP family) from E. coli or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of salicylic acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein b3116
is increased.
[10175] The sequence of YLR089C from Saccharomyces cerevisiae has
been published in Johnston, M. et al., Nature 387 (6632 Suppl),
87-90 (1997) and its activity is being defined as a protein having
alanine transaminase (glutamic pyruvic transaminase) activity.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein YLR089C from Saccharomyces
cerevisiae or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, in particular for increasing the
amount of salicylic acid preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein
YLR089C is increased.
[10176] [0023.0.23.23] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[10177] In one embodiment, the homolog of the polypeptides
indicated in Table II, column 3, lines 275 to 277 and/or 635 or 636
is a homolog having the same or a similar activity. In particular
an increase of activity confers an increase in the content of the
respective fine chemical in the organisms preferably salicylic
acid.
[10178] Homologs of the polypeptides indicated in Table II, column
3, lines 275 to 277 and/or 635 or 636 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table I, column
7, lines 275 to 277 and/or 635 or 636 having a salicylic acid
content and/or amount increasing activity.
[10179] [0023.1.23.23] Homologs of the polypeptides polypeptide
indicated in Table II, column 3, lines 275 to 277 and/or 635 or 636
may be the polypeptides encoded by the nucleic acid molecules
polypeptide indicated in Table I, column 7, lines 275 to 277 and/or
635 or 636 or may be the polypeptides indicated in Table II, column
7, lines 275 to 277 and/or 635 or 636.
[10180] [0024.0.0.23] for the disclosure of this paragraph see
[0024.0.0.0] above.
[10181] [0025.0.23.23] In accordance with the invention, a protein
or polypeptide has the "activity of a protein of the invention",
e.g. the activity of a protein indicated in Table II, column 3,
lines 275 to 277 and/or 635 or 636 if its de novo activity, or its
increased expression directly or indirectly leads to an increased
salicylic acid level, resp., in the organism or a part thereof,
preferably in a cell of said organism. In a preferred embodiment,
the protein or polypeptide has the above-mentioned additional
activities of a protein indicated in Table II, column 3, lines 275
to 277 and/or 635 or 636. Throughout the specification the activity
or preferably the biological activity of such a protein or
polypeptide or an nucleic acid molecule or sequence encoding such
protein or polypeptide is identical or similar if it still has the
biological or enzymatic activity of any one of the proteins
indicated in Table II, column 3, lines 275 to 277 and/or 635 or 636
or which has at least 10% of the original enzymatic activity,
preferably 20%, particularly preferably 30%, most particularly
preferably 40% in comparison to any one of the proteins indicated
in Table II, column 3, lines 275, 276, 635 and/or 636 of
Escherichia coli K12 or line 277 of Saccharomyces cerevisiae
respectively.
[10182] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[10183] [0025.1.0.23] and [0025.2.0.23] for the disclosure of the
paragraphs [0025.1.0.23] and [0025.2.0.23] see [0025.1.0.0] and
[0025.2.0.0] above.
[10184] [0026.0.0.23] to [0033.0.0.23] for the disclosure of the
paragraphs [0026.0.0.23] to [0033.0.0.23] see [0026.0.0.0] to
[0033.0.0.0] above.
[10185] [0034.0.23.23] Preferably, the reference, control or wild
type differs from the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of a protein having the activity of a
protein as indicated in Table II, column 3, lines 275 to 277 and/or
635 or 636 or being encoded by a nucleic acid molecule indicated in
Table I, column 5, lines 275 to 277 and/or 635 or 636 or its
homologs, e.g. as indicated in Table I, column 7, lines 275 to 277
and/or 635 or 636, its biochemical or genetic causes. It therefore
shows the increased amount of the respective fine chemical.
[10186] [0035.0.0.23] to [0044.0.0.23] for the disclosure of the
paragraphs [0035.0.0.23] to [0044.0.0.23] see paragraphs
[0035.0.0.0] to [0044.0.0.0] above.
[10187] [0045.0.23.23] In one embodiment, in case the activity of
the Escherichia coli K12 protein b0161 or its homologs, e.g. as
indicated in Table II, columns 5 or 7, line 275 is increased,
preferably, in one embodiment an increase of the respective fine
chemical, preferably of salicylic acid between 67% and 475% or more
is conferred.
[10188] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2664 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 276 is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of salicylic acid between 56% and 425% or more is conferred.
[10189] In one embodiment, in case the activity of the
Saccharomyces cerevisae protein YLR089C or its homologs, e.g. as
indicated in Table II, columns 5 or 7, line 277 is increased,
preferably, in one embodiment the increase of the respective fine
chemical, preferably of salicylic acid between 45% and 153% or more
is conferred.
[10190] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2796 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 635 is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of salicylic acid between 42% and 248% or more is conferred.
[10191] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3116 or its homologs, e.g. as indicated in Table
II, columns 5 or 7, line 636 is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of salicylic acid between 41% and 130% or more is conferred.
[10192] [0046.0.0.23] %
[10193] [0047.0.0.23] and [0048.0.0.23] for the disclosure of the
paragraphs [0047.0.0.23] and [0048.0.0.23] see paragraphs
[0047.0.0.0] and [0048.0.0.0] above.
[10194] [0049.0.23.23] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
salicylic acid preferably has the structure of the polypeptide
described herein. In a particular embodiment, the polypeptides used
in the process of the present invention or the polypeptide of the
present invention comprises the sequence of a consensus sequence as
shown in SEQ ID NO: 33804, 33805, 33806, 33807, 33808, 33809 or
33896, 33897, 33898 or 34219, 34220, 34221, 34222, 34223, 34224,
34225, 34226, 34227 or 99065, 99066, 99067, 99170, 99171, 99172,
99173, 99174, 99175, 99176, 99177 or as indicated in Table IV,
column 7, lines 275 to 277 and/or 635 or 636 or of a polypeptide as
indicated in Table II, columns 5 or 7, lines 275 to 277 and/or 635
or 636 or of a functional homologue thereof as described herein, or
of a polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, lines 275 to 277 and/or 635 or 636 or its herein described
functional homologues and has the herein mentioned activity
conferring an increase in the salicylic acid level.
[10195] [0050.0.23.23] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of salicylic acid, resp., or their esters, e.g. but not
limited to the methyl ester, the phenyl ester or the menthol
ester.
[10196] [0051.0.23.23] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free or
bound salicylic acid, e.g compositions comprising salicylic acid.
Depending on the choice of the organism used for the process
according to the present invention, for example a microorganism or
a plant, compositions or mixtures of salicylic acidand salicylic
acid esters can be produced.
[10197] [0052.0.0.23] for the disclosure of this paragraph see
paragraph [0052.0.0.0] above.
[10198] [0053.0.23.23] In one embodiment, the process of the
present invention comprises one or more of the following steps
[10199] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 275 to 277 and/or 635 or 636 or its homologs,
e.g. as indicated in Table II, columns 5 or 7, lines 275 to 277
and/or 635 or 636, activity having herein-mentioned the respective
fine chemical increasing activity; [10200] b) stabilizing a mRNA
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 275 to 277 and/or 635 or 636 or its homologs activity, e.g.
as indicated in Table II, columns 5 or 7, lines 275 to 277 and/or
635 or 636, or of a mRNA encoding the polypeptide of the present
invention having herein-mentioned the respective fine chemical
increasing activity; [10201] c) increasing the specific activity of
a protein conferring the increased expression of a protein encoded
by the nucleic acid molecule of the invention or of the polypeptide
of the present invention having herein-mentioned the respective
fine chemical increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 275
to 277 and/or 635 or 636 or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 275 to 277 and/or 635
or 636, or decreasing the inhibitory regulation of the polypeptide
of the invention; [10202] d) generating or increasing the
expression of an endogenous or artificial transcription factor
mediating the expression of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptide of the invention having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 275 to 277 and/or 635 or 636 or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636; [10203] e) stimulating activity
of a protein conferring the increased expression of a protein
encoded by the nucleic acid molecule of the present invention or a
polypeptide of the present invention having herein-mentioned the
respective fine chemical increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 275 to 277 and/or 635 or 636 or its homologs activity, e.g.
as indicated in Table II, columns 5 or 7, lines 275 to 277 and/or
635 or 636, by adding one or more exogenous inducing factors to the
organism or parts thereof; [10204] f) expressing a transgenic gene
encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned the respective fine chemical increasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in Table II, column 3, lines 275 to 277 and/or 635 or 636 or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, and/or [10205] g) increasing
the copy number of a gene conferring the increased expression of a
nucleic acid molecule encoding a polypeptide encoded by the nucleic
acid molecule of the invention or the polypeptide of the invention
having herein-mentioned the respective fine chemical increasing
activity, e.g. of a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 275 to 277 and/or 635 or 636
or its homologs, e.g. as indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636. [10206] h) Increasing the
expression of the endogenous gene encoding the polypeptide of the
invention, e.g. a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 275 to 277 and/or 635 or 636
or its homologs activity, e.g. as indicated in Table II, columns 5
or 7, lines 275 to 277 and/or 635 or 636, by adding positive
expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; and/or
[10207] i) Modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead to an enhanced respective fine
chemical production. [10208] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, e.g. the elite crops.
[10209] [0054.0.23.23] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table II, columns 3 or 5,
lines 275 to 277 and/or 635 or 636, resp., or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, lines 275
to 277 and/or 635 or 636, resp.
[10210] [0055.0.0.23] to [0067.0.0.23] for the disclosure of the
paragraphs [0055.0.0.23] to [0067.0.0.23] see paragraphs
[0055.0.0.0] to [0067.0.0.0] above.
[10211] [0068.0.23.23] The mutation is introduced in such a way
that the production of salicylic acid is not adversely
affected.
[10212] [0069.0.0.23] for the disclosure of this paragraph see
paragraph [0069.0.0.0] above.
[10213] [0070.0.23.23] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolite composition in the organism, e.g. an advantageous
composition of salicylic acid or their biochemical derivatives,
e.g. comprising a higher content of (from a viewpoint of
nutritional physiology limited) salicylic acid or their derivatives
including but not limited to salicylic acid esters.
[10214] [0071.0.0.23] for the disclosure of this paragraph see
paragraph [0071.0.0.0] above.
[10215] [0072.0.0.23] %
[10216] [0073.0.23.23] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
(g) providing a non-human organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant; (h) increasing an activity of a polypeptide of the
invention or a homolog thereof, e.g. as indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the respective fine chemical in an organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, (i) growing an organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant under conditions which permit the
production of the respective fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and [10217] (j) if desired, recovering, optionally
isolating, the free and/or bound respective fine chemical
synthesized by the organism, the microorganism, the non-human
animal, the plant or animal cell, the plant or animal tissue or the
plant.
[10218] [0074.0.23.23] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound respective fine chemical.
[10219] [0075.0.0.23] to [0077.0.0.23] for the disclosure of the
paragraphs [0075.0.0.23] to [0077.0.0.23] see paragraphs
[0075.0.0.0] to [0077.0.0.0] above.
[10220] [0078.0.23.23] The organism such as microorganisms or
plants or the recovered, and if desired isolated, respective fine
chemical can then be processed further directly into foodstuffs or
animal feeds or for other applications. The fermentation broth,
fermentation products, plants or plant products can be purified
with methods known to the person skilled in the art. Products of
these different work-up procedures are salicylic acidor salicylic
acid esters or comprising compositions of salicylic acid and
salicylic acid esters still comprising fermentation broth, plant
particles and cell components in different amounts, advantageously
in the range of from 0 to 99% by weight, preferably below 80% by
weight, especially preferably below 50% by weight.
[10221] [0079.0.0.23] to [0084.0.0.23] for the disclosure of the
paragraphs [0079.0.0.23] to [0084.0.0.23] see paragraphs
[0079.0.0.0] to [0084.0.0.0] above.
[10222] [0085.0.23.23] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either
a) a nucleic acid sequence as indicated in Table I, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, or a derivative thereof, or b)
a genetic regulatory element, for example a promoter, which is
functionally linked to the nucleic acid sequence as indicated in
Table I, columns 5 or 7, lines 275 to 277 and/or 635 or 636, or a
derivative thereof, or c) (a) and (b) is/are not present in
its/their natural genetic environment or has/have been modified by
means of genetic manipulation methods, it being possible for the
modification to be, by way of example, a substitution, addition,
deletion, inversion or insertion of one or more nucleotide.
"Natural genetic environment" means the natural chromosomal locus
in the organism of origin or the presence in a genomic library. In
the case of a genomic library, the natural, genetic environment of
the nucleic acid sequence is preferably at least partially still
preserved. The environment flanks the nucleic acid sequence at
least on one side and has a sequence length of at least 50 bp,
preferably at least 500 bp, particularly preferably at least 1000
bp, very particularly preferably at least 5000 bp.
[10223] [0086.0.0.23] and [0087.0.0.23] for the disclosure of the
paragraphs [0086.0.0.23] and [0087.0.0.23] see paragraphs
[0086.0.0.0] and [0087.0.0.0] above
[10224] [0088.0.23.23] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned fine
chemicals or the plants are more resistant to biotic and abiotic
stress and the yield is increased.
[10225] [0088.1.0.23] for the disclosure of this paragraph see
paragraph [0088.1.0.0] above.
[10226] [0089.0.0.23] to [0094.0.0.23] for the disclosure of the
paragraphs [0089.0.0.23] to [0094.0.0.23] see paragraphs
[0089.0.0.0] to [0094.0.0.0] above.
[10227] [0095.0.23.23] It may be advantageous to increase the pool
of salicylic acid and/or salicylic acid esters in the transgenic
organisms by the process according to the invention in order to
isolate high amounts of the pure respective fine chemical and/or to
obtain increased resistance against biotic and abiotic stresses and
to obtain higher yield.
[10228] [0096.0.23.23] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example in
the organism, is useful to increase the production of the
respective fine chemical.
[10229] [0097.0.0.23] for the disclosure of this paragraph see
paragraph [0097.0.0.0] above.
[10230] [0098.0.23.23] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated.
[10231] [0099.0.23.23] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical might
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[10232] [0100.0.23.23] Transgenic plants which comprise the fine
chemicals such as salicylic acid and/or salicylic acid esters
synthesized in the process according to the invention can
advantageously be marketed directly without there being any need
for the fine chemicals synthesized to be isolated. Plants for the
process according to the invention are listed as meaning intact
plants and all plant parts, plant organs or plant parts such as
leaf, stem, seeds, root, tubers, anthers, fibers, root hairs,
stalks, embryos, calli, cotelydons, petioles, harvested material,
plant tissue, reproductive tissue and cell cultures which are
derived from the actual transgenic plant and/or can be used for
bringing about the transgenic plant. In this context, the seed
comprises all parts of the seed such as the seed coats, epidermal
cells, seed cells, endosperm or embryonic tissue.
[10233] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, as extracts, e.g. ether, alcohol,
or other organic solvents or water containing extract and/or free
fine chemicals. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pretreated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[10234] Because salicylic acid and/or salicylic acid esters in
microorganisms are localized intracellular or extracellular, their
recovery essentially comes down to the isolation of the biomass or
the supernatant. Well-established approaches for the harvesting of
cells include filtration, centrifugation and
coagulation/flocculation as described herein. Of the residual
hydrocarbon, adsorbed on the cells, has to be removed. Solvent
extraction or treatment withsurfactants have been suggested for
this purpose.
[10235] [0101.0.23.23] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[10236] [0102.0.23.23] Salicylic acid and/or salicylic acid esters
can for example be analyzed advantageously via HPLC, LC or GC
separation and MS (masspectrometry) detection methods. The
unambiguous detection for the presence of salicylic acid and/or
salicylic acid containing products can be obtained by analyzing
recombinant organisms using analytical standard methods: LC, LC-MS,
MS or TLC). The material to be analyzed can be disrupted by
sonication, grinding in a glass mill, liquid nitrogen and grinding,
cooking, or via other applicable methods.
[10237] [0103.0.23.23] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [10238] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide having a
sequence as indicated in Table II, columns 5 or 7, lines 275 to 277
and/or 635 or 636, or a fragment thereof, which confers an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [10239] b) nucleic acid molecule comprising,
preferably at least the mature form, of a nucleic acid molecule
having a sequence as indicated in Table I, columns 5 or 7, lines
275 to 277 and/or 635 or 636, [10240] c) nucleic acid molecule
whose sequence can be deduced from a polypeptide sequence encoded
by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [10241] d) nucleic acid molecule encoding a polypeptide
which has at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [10242] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under stringent hybridization conditions and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [10243] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [10244] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [10245]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primer pairs having a
sequence as indicated in Table III, column 7, lines 275 to 277
and/or 635 or 636, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [10246]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [10247] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence having
sequences as indicated in Table IV, column 7, lines 275 to 277
and/or 635 or 636 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [10248]
k) nucleic acid molecule comprising one or more of the nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domain of the polypeptide indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; and [10249] l) nucleic acid molecule which is obtainable
by screening a suitable library under stringent conditions with a
probe comprising one of the sequences of the nucleic acid molecule
of (a) to (k), preferably to (a) to (c), or with a fragment of at
least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500
nt of the nucleic acid molecule characterized in (a) to (k),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
or which comprises a sequence which is complementary thereto.
[10250] [00103.1.23.23] In one embodiment, the nucleic acid
molecule used in the process of the invention distinguishes over
the sequence indicated in Table IA, columns 5 or 7, lines 275 to
277 and/or 635 or 636, by one or more nucleotides. In one
embodiment, the nucleic acid molecule used in the process of the
invention does not consist of the sequence shown in indicated in
Table I A, columns 5 or 7, lines 275 to 277 and/or 635 or 636. In
one embodiment, the nucleic acid molecule used in the process of
the invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I A, columns 5 or 7,
lines 275 to 277 and/or 635 or 636. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II A, columns 5 or 7, lines 275 to 277 and/or
635 or 636.
[10251] [00103.2.23.23] In one embodiment, the nucleic acid
molecule used in the process of the invention distinguishes over
the sequence indicated in Table I B, columns 5 or 7, lines 275 to
277 and/or 635 or 636, by one or more nucleotides. In one
embodiment, the nucleic acid molecule used in the process of the
invention does not consist of the sequence shown in indicated in
Table I B, columns 5 or 7, lines 275 to 277 and/or 635 or 636. In
one embodiment, the nucleic acid molecule used in the process of
the invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I B, columns 5 or 7,
lines 275 to 277 and/or 635 or 636. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II B, columns 5 or 7, lines 275 to 277 and/or
635 or 636.
[10252] [0104.0.23.23] In one embodiment, the nucleic acid molecule
used in the process of the present invention distinguishes over the
sequence indicated in Table I, columns 5 or 7, lines 275 to 277
and/or 635 or 636, preverably over the sequences as shown in Table
IA, columns 5 or 7, lines 275 to 277 and/or 635 or 636 by one or
more nucleotides. In one embodiment, the nucleic acid molecule used
in the process of the invention does not consist of the sequence
indicated in Table I, columns 5 or 7, lines 275 to 277 and/or 635
or 636, preverably of the sequences as shown in Table IA, columns 5
or 7, lines 275 to 277 and/or 635 or 636. In one embodiment, the
nucleic acid molecule of the present invention is less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to the sequence indicated
in Table I, columns 5 or 7, lines 275 to 277 and/or 635 or 636. In
another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, preverably the nucleic acid
molecule does not encode a polypeptide of a sequence as indicated
in Table IIA, columns 5 or 7, lines 275 to 277 and/or 635 or
636.
[10253] [0105.0.0.23] to [0107.0.0.23] for the disclosure of the
paragraphs [0105.0.0.23] to [0107.0.0.23] see paragraphs
[0105.0.0.0] to [0107.0.0.0] above.
[10254] [0108.0.23.23] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 275 to 277 and/or 635
or 636, nucleic acid molecules which are derived from an amino acid
sequences as indicated in Table II, columns 5 or 7, lines 275 to
277 and/or 635 or 636 or from polypeptides comprising the consensus
sequence as indicated in Table IV, column 7, lines 275 to 277
and/or 635 or 636, or their derivatives or homologues encoding
polypeptides with the enzymatic or biological activity of an
activity of a polypeptide as indicated in Table II, columns 3, 5 or
7, lines 275 to 277 and/or 635 or 636, e.g. conferring the increase
of the respective fine chemical, meaning salicylic acid and/or
salicylic acid esters, resp., after increasing its expression or
activity, are advantageously increased in the process according to
the invention.
[10255] [0109.0.23.23] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular salicylic
acid and/or salicylic acid esters, produced in the process
according to the invention.
[10256] [0110.0.0.23] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide used in the
method of the invention or used in the process of the invention,
e.g. of a protein as shown in Table II, columns 5 or 7, lines 275
to 277 and/or 635 or 636 or being encoded by a nucleic acid
molecule indicated in Table I, columns 5 or 7, lines 275 to 277
and/or 635 or 636 or of its homologs, e.g. as indicated in Table
II, columns 5 or 7, lines 275 to 277 and/or 635 or 636 can be
determined from generally accessible databases.
[10257] [0111.0.0.23] for the disclosure of this paragraph see
[0111.0.0.0] above.
[10258] [0112.0.23.23] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table II, column 3, lines 275 to
277 and/or 635 or 636 or having the sequence of a polypeptide as
indicated in Table II, columns 5 and 7, lines 275 to 277 and/or 635
or 636 and conferring an increase in the salicylic acid and/or
salicylic acid ester level.
[10259] [0113.0.0.23] to [0120.0.0.23] for the disclosure of the
paragraphs [0113.0.0.23] to [0120.0.0.23] see paragraphs
[0113.0.0.0] and [0120.0.0.0] above.
[10260] [0120.1.0.23]: %
[10261] [0121.0.23.23] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636 or the
functional homologues thereof as described herein, preferably
conferring above-mentioned activity, i.e. conferring a salicylic
acid increase after increasing the activity of the polypeptide
sequences indicated in Table II, columns 5 or 7, lines 275 to 277
and/or 635 or 636 and conferring a salicylic acid level increase
after increasing the activity of the polypeptide sequences
indicated in Table II, columns 5 or 7, lines 275 to 277 and/or 635
or 636.
[10262] [0122.0.0.23] to [0127.0.0.23] for the disclosure of the
paragraphs [0122.0.0.23] to [0127.0.0.23] see paragraphs
[0122.0.0.0] and [0127.0.0.0] above.
[10263] [0128.0.23.23] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, columns 7,
lines 275 to 277 and/or 635 or 636, by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table I, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, resp. or the sequences derived
from a sequence as indicated in Table II, columns 5 or 7, lines 275
to 277 and/or 635 or 636, resp.
[10264] [0129.0.23.23] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved regions are those, which show a
very little variation in the amino acid in one particular position
of several homologs from different origin. The consensus sequence
indicated in Table IV, columns 7, lines 275 to 277 and/or 635 or
636 is derived from such alignments.
[10265] [0130.0.23.23] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of salicylic
acid and/or salicylic acid esters after increasing the expression
or activity the protein comprising said fragment.
[10266] [0131.0.0.23] to [0138.0.0.23] for the disclosure of the
paragraphs [0131.0.0.23] to [0138.0.0.23] see paragraphs
[0131.0.0.0] to [0138.0.0.0] above.
[10267] [0139.0.23.23] Polypeptides having above-mentioned
activity, i.e. conferring the respective fine chemical level
increase, derived from other organisms, can be encoded by other DNA
sequences which hybridise to a sequence indicated in Table I,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, preverably to a
sequence as shown in Table IB, columns 5 or 7, lines 275 to 277
and/or 635 or 636 under relaxed hybridization conditions and which
code on expression for peptides having the respective fine
chemical, i.e. salicylic acid and/or salicylic acid ester,
increasing-activity.
[10268] [0140.0.0.23] to [0146.0.0.23] for the disclosure of the
paragraphs [0140.0.0.23] to [0146.0.0.23] see paragraphs
[0140.0.0.0] to [0146.0.0.0] above.
[10269] [0147.0.23.23] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 275 to 277 and/or 635 or 636, preverably
to a sequence as shown in Table IB, columns 5 or 7, lines 275 to
277 and/or 635 or 636 is one which is sufficiently complementary to
one of said nucleotide sequences such that it can hybridise to one
of said nucleotide sequences, thereby forming a stable duplex.
Preferably, the hybridisation is performed under stringent
hybridization conditions. However, a complement of one of the
herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[10270] [0148.0.23.23] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, columns 5 or 7, lines
275 to 277 and/or 635 or 636, preverably to a sequence as shown in
Table IB, columns 5 or 7, lines 275 to 277 and/or 635 or 636 or a
portion thereof and preferably has above mentioned activity, in
particular having a salicylic acid and/or salicylic acid increasing
activity after increasing the activity or an activity of a product
of a gene encoding said sequences or their homologs.
[10271] [0149.0.23.23] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, or a portion thereof and
encodes a protein having above-mentioned activity, e.g. conferring
an increase of salicylic acid and/or salicylic acid, resp., and
optionally, the activity of protein indicated in Table II, column
5, lines 275 to 277 and/or 635 or 636.
[10272] [00149.1.23.23] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table I, columns 5 or 7, lines 275 to 277
and/or 635 or 636, preverably to a sequence as shown in Table IB,
columns 5 or 7, lines 275 to 277 and/or 635 or 636 has further one
or more of the activities annotated or known for a protein as
indicated in Table II, column 3, lines 275 to 277 and/or 635 or
636, preverably one or more activities annotated or known for a
protein as indicated in Table IIB, columns 5 or 7, lines 275 to 277
and/or 635 or 636.
[10273] [00149.1.22.22] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, preferably of
Table IB, columns 5 or 7, lines 275 to 277 and/or 635 or 636, has
further one or more of the activities annotated or known for the a
protein as indicated in Table II, column 3, lines 275 to 277 and/or
lines 635 or 636, preferably of Table IIB, columns 5 or 7, lines
275 to 277 and/or 635 or 636.
[10274] [0150.0.23.23] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table I, columns 5 or 7, lines 275 to
277 and/or 635 or 636, preferably indicated in Table IB, columns 5
or 7, lines 275 to 277 and/or 635 or 636 for example a fragment
which can be used as a probe or primer or a fragment encoding a
biologically active portion of the polypeptide of the present
invention or of a polypeptide used in the process of the present
invention, i.e. having above-mentioned activity, e.g. conferring an
increase of salicylic acid and/or salicylic acid esters, resp., if
its activity is increased. The nucleotide sequences determined from
the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table I, columns
5 or 7, lines 275 to 277 and/or 635 or 636, an anti-sense sequence
of one of the sequences, e.g., as indicated in Table I, columns 5
or 7, lines 275 to 277 and/or 635 or 636, or naturally occurring
mutants thereof. Primers based on a nucleotide of invention can be
used in PCR reactions to clone homologues of the polypeptide of the
invention or of the polypeptide used in the process of the
invention, e.g. as the primers described in the examples of the
present invention, e.g. as shown in the examples. A PCR with the
primer pairs indicated in Table III, column 7, lines 275 to 277
and/or 635 or 636 will result in a fragment of a polynucleotide
sequence as indicated in Table I, columns 5 or 7, lines 275 to 277
and/or 635 or 636 or its gene product. Preferably is Table IB,
columns 5 or 7, lines 275 to 277 and/or 635 or 636.
[10275] [0151.0.0.23]: for the disclosure of this paragraph see
paragraph [0151.0.0.0] above.
[10276] [0152.0.23.23] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 275 to 277
and/or 635 or 636 such that the protein or portion thereof
maintains the ability to participate in the respective fine
chemical production, in particular a salicylic acid increasing
activity as mentioned above or as described in the examples in
plants or microorganisms is comprised.
[10277] [0153.0.23.23] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 275
to 277 and/or 635 or 636 such that the protein or portion thereof
is able to participate in the increase of the respective fine
chemical production. In one embodiment, a protein or portion
thereof as indicated in Table II, columns 5 or 7, lines 275 to 277
and/or 635 or 636 has for example an activity of a polypeptide
indicated in Table II, column 3, lines 275 to 277 and/or 635 or
636.
[10278] [0154.0.23.23] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 96%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 275 to 277
and/or 635 or 636 and has above-mentioned activity, e.g. conferring
preferably the increase of the respective fine chemical.
[10279] [0155.0.0.23] and [0156.0.0.23] for the disclosure of the
paragraphs [0155.0.0.23] and [0156.0.0.23] see paragraphs
[0155.0.0.0] and [0156.0.0.0] above.
[10280] [0157.0.23.23] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences as
indicated in Table I, columns 5 or 7, lines 275 to 277 and/or 635
or 636 (and portions thereof) due to degeneracy of the genetic code
and thus encode a polypeptide of the present invention, in
particular a polypeptide having above mentioned activity, e.g.
conferring an increase in the respective fine chemical in a
organism, e.g. as polypeptides comprising the sequence as indicated
in Table IV, column 7, lines 275 to 277 and/or 635 or 636 or as
polypeptides depicted in Table II, columns 5 or 7, lines 275 to 277
and/or 635 or 636 or the functional homologues. Advantageously, the
nucleic acid molecule of the invention comprises, or in an other
embodiment has, a nucleotide sequence encoding a protein
comprising, or in an other embodiment having, an amino acid
sequence of a consensus sequences as indicated in Table IV, column
7, lines 275 to 277 and/or 635 or 636 or of the polypeptide as
indicated in Table II, columns 5 or 7, lines 275 to 277 and/or 635
or 636, resp., or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
IV, column 7, lines 275 to 277 and/or 635 or 636 or of a
polypeptide as indicated in Table II, columns 5 or 7, lines 275 to
277 and/or 635 or 636 or the functional homologues. However, in a
preferred embodiment, the nucleic acid molecule of the present
invention does not consist of a sequence as indicated in Table I,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp.,
preferably as indicated in Table I A, columns 5 or 7, lines 275 to
277 and/or 635 or 636, resp. Preferably the nucleic acid molecule
of the invention is a functional homologue or identical to a
nucleic acid molecule indicated in Table I B, column 7, lines 275
to 277 and/or 635 or 636.
[10281] [0158.0.0.23] to [0160.0.0.23] for the disclosure of the
paragraphs [0158.0.0.23] to [0160.0.0.23] see paragraphs
[0158.0.0.0] to [0160.0.0.0] above.
[10282] [0161.0.23.23] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 275 to 277
and/or 635 or 636. The nucleic acid molecule is preferably at least
20, 30, 50, 100, 250 or more nucleotides in length.
[10283] [0162.0.0.23] for the disclosure of this paragraph see
paragraph [0162.0.0.0] above.
[10284] [0163.0.23.23] Preferably, a nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 275 to 277 and/or
635 or 636 corresponds to a naturally-occurring nucleic acid
molecule of the invention. As used herein, a "naturally-occurring"
nucleic acid molecule refers to an RNA or DNA molecule having a
nucleotide sequence that occurs in nature (e.g., encodes a natural
protein). Preferably, the nucleic acid molecule encodes a natural
protein having above-mentioned activity, e.g. conferring the
increase of the amount of the respective fine chemical in an
organism or a part thereof, e.g. a tissue, a cell, or a compartment
of a cell, after increasing the expression or activity thereof or
the activity of a protein of the invention or used in the process
of the invention.
[10285] [0164.0.0.23] for the disclosure of this paragraph see
paragraph [0164.0.0.0] above.
[10286] [0165.0.23.23] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7, lines 275 to 277 and/or 635
or 636, resp.
[10287] [0166.0.0.23] and [0167.0.0.23] for the disclosure of the
paragraphs [0166.0.0.23] and [0167.0.0.23] see paragraphs
[0166.0.0.0] and [0167.0.0.0] above.
[10288] [0168.0.23.23] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the
respective fine chemical in an organism or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., yet
retain said activity described herein. The nucleic acid molecule
can comprise a nucleotide sequence encoding a polypeptide, wherein
the polypeptide comprises an amino acid sequence at least about 50%
identical to an amino acid sequence as indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., and is
capable of participation in the increase of production of the
respective fine chemical after increasing its activity, e.g. its
expression. Preferably, the protein encoded by the nucleic acid
molecule is at least about 60% identical to a sequence as indicated
in Table II, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
resp., more preferably at least about 70% identical to one of the
sequences as indicated in Table II, columns 5 or 7, lines 275 to
277 and/or 635 or 636, resp., even more preferably at least about
80%, 90%, 95% homologous to a sequence as indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., and most
preferably at least about 96%, 97%, 98%, or 99% identical to the
sequence as indicated in Table II, columns 5 or 7, lines 275 to 277
and/or 635 or 636.
[10289] Accordingly, the invention relates to nucleic acid
molecules encoding a polypeptide having above-mentioned activity,
e.g. conferring an increase in the the respective fine chemical in
an organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, preferably of Table II B,
column 7, lines 275 to 277 and/or 635 or 636 yet retain said
activity described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table II, columns 5 or 7, lines
275 to 277 and/or 635 or 636, preferably of Table II B, column 7,
lines 275 to 277 and/or 635 or 636 and is capable of participation
in the increase of production of the respective fine chemical after
increasing its activity, e.g. its expression. Preferably, the
protein encoded by the nucleic acid molecule is at least about 60%
identical to a sequence as indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, preferably of Table II B,
column 7, lines 275 to 277 and/or 635 or 636, more preferably at
least about 70% identical to one of the sequences as indicated in
Table II, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
preferably of Table II B, column 7, lines 275 to 277 and/or 635 or
636, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, preferably of Table II B,
column 7, lines 275 to 277 and/or 635 or 636, and most preferably
at least about 96%, 97%, 98%, or 99% identical to the sequence as
indicated in Table II, columns 5 or 7, lines 275 to 277 and/or 635
or 636, preferably of Table II B, column 7, lines 275 to 277 and/or
635 or 636.
[10290] [0169.0.0.23] to [0172.0.0.23] for the disclosure of the
paragraphs [0169.0.0.23] to [0172.0.0.23] see paragraphs
[0169.0.0.0] to [0172.0.0.0] above.
[10291] [0173.0.23.23] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 33502 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 33502 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[10292] [0174.0.0.23]: for the disclosure of this paragraph see
paragraph [0174.0.0.0] above.
[10293] [0175.0.23.23] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 33503 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 33503 by the above program algorithm with the
above parameter set, has a 80% homology.
[10294] [0176.0.23.23] Functional equivalents derived from one of
the polypeptides as indicated in Table II, columns 5 or 7, lines
275 to 277 and/or 635 or 636, resp., according to the invention by
substitution, insertion or deletion have at least 30%, 35%, 40%,
45% or 50%, preferably at least 55%, 60%, 65% or 70% by preference
at least 80%, especially preferably at least 85% or 90%, 91%, 92%,
93% or 94%, very especially preferably at least 95%, 97%, 98% or
99% homology with one of the polypeptides as indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp.,
according to the invention and are distinguished by essentially the
same properties as a polypeptide as indicated in Table II, columns
5 or 7, lines 275 to 277 and/or 635 or 636, resp.
[10295] [0177.0.23.23] Functional equivalents derived from a
nucleic acid sequence as indicated in Table I, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, preferably of Table I B,
columns 5 or 7, lines 275 to 277 and/or 635 or 636 resp., according
to the invention by substitution, insertion or deletion have at
least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65%
or 70% by preference at least 80%, especially preferably at least
85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at
least 95%, 97%, 98% or 99% homology with one of the polypeptides as
indicated in Table II, columns 5 or 7, lines 275 to 277 and/or 635
or 636, resp., according to the invention and encode polypeptides
having essentially the same properties as a polypeptide as
indicated in Table II, columns 5 or 7, lines 275 to 277 and/or 635
or 636, preferably of Table II B, columns 5 or 7, lines 275 to 277
and/or 635 or 636, resp.
[10296] [0178.0.0.23] for the disclosure of this paragraph see
[0178.0.0.0] above.
[10297] [0179.0.23.23] A nucleic acid molecule encoding a homologue
to a protein sequence as indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, preferably in Table II B,
columns 5 or 7, lines 275 to 277 and/or 635 or 636 resp., can be
created by introducing one or more nucleotide substitutions,
additions or deletions into a nucleotide sequence of the nucleic
acid molecule of the present invention, in particular as indicated
in Table I, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
preferably in Table I B, columns 5 or 7, lines 275 to 277 and/or
635 or 636, resp., such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences as
indicated in Table I, columns 5 or 7, lines 275 to 277 and/or 635
or 636, preferably in Table I B, columns 5 or 7, lines 275 to 277
and/or 635 or 636, resp., by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
[10298] [0180.0.0.23] to [0183.0.0.23] for the disclosure of the
paragraphs [0180.0.0.23] to [0183.0.0.23] see paragraphs
[0180.0.0.0] to [0183.0.0.0] above.
[10299] [0184.0.23.23] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table I, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, preferably in Table I B,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., or of
the nucleic acid sequences derived from a sequences as indicated in
Table II, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
preferably in Table II B, columns 5 or 7, lines 275 to 277 and/or
635 or 636, resp., comprise also allelic variants with at least
approximately 30%, 35%, 40% or 45% homology, by preference at least
approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
I, columns 5 or 7, lines 275 to 277 and/or 635 or 636, preferably
in Table I B, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
resp., or from the derived nucleic acid sequences, the intention
being, however, that the enzyme activity or the biological activity
of the resulting proteins synthesized is advantageously retained or
increased.
[10300] [0185.0.23.23] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table I, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
preferably in Table I B, columns 5 or 7, lines 275 to 277 and/or
635 or 636, resp. In one embodiment, it is preferred that the
nucleic acid molecule comprises as little as possible other
nucleotides not shown in any one of sequences as indicated in Table
I, columns 5 or 7, lines 275 to 277 and/or 635 or 636, preferably
in Table I B, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
resp. In one embodiment, the nucleic acid molecule comprises less
than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or 40 further
nucleotides. In a further embodiment, the nucleic acid molecule
comprises less than 30, 20 or 10 further nucleotides. In one
embodiment, a nucleic acid molecule used in the process of the
invention is identical to a sequence as indicated in Table I,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, preferably in
Table I B, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
resp.
[10301] [0186.0.23.23] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 275 to 277 and/or 635 or 636, preferably in Table II
B, columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp. In one
embodiment, the nucleic acid molecule encodes less than 150, 130,
100, 80, 60, 50, 40 or 30 further amino acids. In a further
embodiment, the encoded polypeptide comprises less than 20, 15, 10,
9, 8, 7, 6 or 5 further amino acids. In one embodiment, the encoded
polypeptide used in the process of the invention is identical to
the sequences as indicated in Table II, columns 5 or 7, lines 275
to 277 and/or 635 or 636, preferably in Table II B, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, resp.
[10302] [0187.0.23.23] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, preferably in Table II B,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp.,
comprises less than 100 further nucleotides. In a further
embodiment, said nucleic acid molecule comprises less than 30
further nucleotides. In one embodiment, the nucleic acid molecule
used in the process is identical to a coding sequence encoding a
sequences as indicated in Table II, columns 5 or 7, lines 275 to
277 and/or 635 or 636, preferably in Table II B, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, resp.
[10303] [0188.0.23.23] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity.
Advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
II, columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., and
is expressed under identical conditions.
[10304] [0189.0.23.23] Homologues of a sequences as indicated in
Table I, columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp.,
or of derived sequences as indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, resp., also mean truncated
sequences, cDNA, single-stranded DNA or RNA of the coding and
noncoding DNA sequence. Homologues of said sequences are also
understood as meaning derivatives, which comprise noncoding regions
such as, for example, UTRs, terminators, enhancers or promoter
variants. The promoters upstream of the nucleotide sequences stated
can be modified by one or more nucleotide substitution(s),
insertion(s) and/or deletion(s) without, however, interfering with
the functionality or activity either of the promoters, the open
reading frame (=ORF) or with the 3'-regulatory region such as
terminators or other 3' regulatory regions, which are far away from
the ORF. It is furthermore possible that the activity of the
promoters is increased by modification of their sequence, or that
they are replaced completely by more active promoters, even
promoters from heterologous organisms. Appropriate promoters are
known to the person skilled in the art and are mentioned herein
below.
[10305] [0190.0.0.23] and [0191.0.0.23] for the disclosure of the
paragraphs [0190.0.0.23] and [0191.0.0.23] see paragraphs
[0190.0.0.0] and [0191.0.0.0] above.
[10306] [0191.1.0.23]: for the disclosure of this paragraph see
[0191.1.0.0] above.
[10307] [0192.0.0.23] to [0203.0.0.23] for the disclosure of the
paragraphs [0192.0.0.23] to [0203.0.0.23] see paragraphs
[0192.0.0.0] to [0203.0.0.0] above.
[10308] [0204.0.23.23] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule, which comprises a
nucleic acid molecule selected from the group consisting of:
[10309] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table II, columns 5
or 7, lines 275 to 277 and/or 635 or 636, preferably in Table II B,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp.; or a
fragment thereof conferring an increase in the amount of the
respective fine chemical, i.e. salicylic acid in an organism or a
part thereof, [10310] b) nucleic acid molecule comprising,
preferably at least the mature form, of a nucleic acid molecule as
indicated in Table I, columns 5 or 7, lines 275 to 277 and/or 635
or 636, preferably in Table I B, columns 5 or 7, lines 275 to 277
and/or 635 or 636 resp., or a fragment thereof conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [10311] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [10312]
d) nucleic acid molecule encoding a polypeptide whose sequence has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [10313] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under stringent hybridisation conditions and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [10314] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [10315] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [10316]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
primers or primer pairs as indicated in Table III, column 7, lines
275 to 277 and/or 635 or 636 and conferring an increase in the
amount of the respective fine chemical, i.e. salicylic acid in an
organism or a part thereof; [10317] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from a expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [10318] j) nucleic acid molecule which encodes a
polypeptide comprising a consensus sequence as indicated in Table
IV, column 7, line 275 to 277 and/or 635 or 636 and conferring an
increase in the amount of the respective fine chemical, i.e.
salicylic acid, in an organism or a part thereof; [10319] k)
nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domaine of a polypeptide as indicated in
Table II, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
resp., and conferring an increase in the amount of the respective
fine chemical, i.e. salicylic acidin an organism or a part thereof;
and [10320] l) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., or a
nucleic acid molecule encoding, preferably at least the mature form
of, a polypeptide as indicated in Table II, columns 5 or 7, lines
275 to 277 and/or 635 or 636, resp., and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over a sequence as indicated in Table
IA or IB, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
resp., by one or more nucleotides. In one embodiment, the nucleic
acid molecule of the invention does not consist of the sequence as
indicated in Table IA or
[10321] IB, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
resp. In another embodiment, the nucleic acid molecule of the
present invention is at least 30% identical and less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence as indicated
in Table IA or IB, columns 5 or 7, lines 275 to 277 and/or 635 or
636, resp. In a further embodiment the nucleic acid molecule does
not encode a polypeptide sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp.
Accordingly, in one embodiment, the nucleic acid molecule of the
present invention encodes in one embodiment a polypeptide which
differs at least in one or more amino acids from a polypeptide
indicated in Table IIA or IIB, columns 5 or 7, lines 275 to 277
and/or 635 or 636 does not encode a protein of a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 275 to 277
and/or 635 or 636. Accordingly, in one embodiment, the protein
encoded by a sequence of a nucleic acid according to (a) to (l)
does not consist of a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 275 to 277 and/or 635 or 636. In a further
embodiment, the protein of the present invention is at least 30%
identical to a protein sequence indicated in Table IIA or IIB,
columns 5 or 7, lines 275 to 277 and/or 635 or 636 and less than
100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 98%, 97%, 96% or 95% identical to a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
275 to 277 and/or 635 or 636.
[10322] [0205.0.0.23] and [0206.0.0.23] for the disclosure of the
paragraphs [0205.0.0.23] and [0206.0.0.23] see paragraphs
[0205.0.0.0] and [0206.0.0.0] above.
[10323] [0207.0.23.23] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the glutamic acid metabolism, the
phosphoenolpyruvate metabolism, the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[10324] [0208.0.0.23] to [0226.0.0.23] for the disclosure of the
paragraphs [0208.0.0.23] to [0226.0.0.23] see paragraphs
[0208.0.0.0] to [0226.0.0.0] above.
[10325] [0227.0.23.23] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[10326] In addition to a sequence indicated in Table I, columns 5
or 7, lines 275 to 277 and/or 635 or 636 or its derivatives, it is
advantageous to express and/or mutate further genes in the
organisms. Especially advantageously, additionally at least one
further gene of the cinnamate and/or chorismate biosynthetic
pathway is expressed in the organisms such as plants or
microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the fine chemicals desired since, for example,
feedback regulations no longer exist to the same extent or not at
all. In addition it might be advantageously to combine one or more
of the sequences indicated in Table I, columns 5 or 7, lines 275 to
277 and/or 635 or 636, resp., with genes which generally support or
enhance to growth or yield of the target organisms, for example
genes which lead to faster growth rate of microorganisms or genes
which produces stress-, pathogen, or herbicide resistant
plants.
[10327] [0228.0.23.23] %
[10328] [0229.0.23.23] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences indicated
in Table I, columns 5 or 7, lines 275 to 277 and/or 635 or 636 used
in the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes relating to the cinnamate or
chorismate biosynthetic anabolic or catabolic pathway.
[10329] [0230.0.0.23] for the disclosure of this paragraph see
[0230.0.0.0] above.
[10330] [0231.0.23.23] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a salicylic aciddegrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene. A person skilled in the art knows for example,
that the inhibition or repression of a salicylic aciddegrading
enzyme will result in an increased accumulation of salicylic acid
in plants.
[10331] [0232.0.0.23] to [0276.0.0.23] for the disclosure of the
paragraphs [0232.0.0.23] to [0276.0.0.23] see paragraphs
[0232.0.0.0] to [0276.0.0.0] above.
[10332] [0277.0.23.23] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously.
[10333] [0278.0.0.23] to [0282.0.0.23] for the disclosure of the
paragraphs [0278.0.0.23] to [0282.0.0.23] see paragraphs
[0278.0.0.0] to [0282.0.0.0] above.
[10334] [0283.0.23.23] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells, for example using the antibody
of the present invention as described below, e.g. an antibody
against a protein as indicated in Table II, column 3, lines 275 to
277 and/or 635 or 636, resp., or an antibody against a polypeptide
as indicated in Table II, columns 5 or 7, lines 275 to 277 and/or
635 or 636, resp., which can be produced by standard techniques
utilizing the polypeptid of the present invention or fragment
thereof.
[10335] Preferred are monoclonal antibodies specifically binding to
polypeptides as indicated in Table II, columns 5 or 7, lines 275 to
277 and/or 635 or 636, more preferred specifically binding to
polypeptides as indicated in Table II, column 5, lines 275 to 277
and/or 635 or 636.
[10336] [0284.0.0.23] for the disclosure of this paragraph see
[0284.0.0.0] above.
[10337] [0285.0.23.23] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., or
as coded by a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., or
functional homologues thereof.
[10338] [0286.0.23.23] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table IV, column 7, lines 275 to 277 and/or 635 or 636
and in one another embodiment, the present invention relates to a
polypeptide comprising or consisting of a consensus sequence as
indicated in Table IV, column 7, lines 275 to 277 and/or 635 or 636
whereby 20 or less, preferably 15 or 10, preferably 9, 8, 7, or 6,
more preferred 5 or 4, even more preferred 3, even more preferred
2, even more preferred 1, most preferred 0 of the amino acids
positions indicated can be replaced by any amino acid or, in an
further embodiment, can be replaced and/or absent. In one
embodiment, the present invention relates to the method of the
present invention comprising a polypeptide or to a polypeptide
comprising more than one consensus sequences (of an individual
line) as indicated in Table IV, column 7, lines 275 to 277 and/or
635 or 636.
[10339] [0287.0.0.23] to [0290.0.0.23] for the disclosure of the
paragraphs [0287.0.0.23] to [0290.0.0.23] see paragraphs
[0287.0.0.0] to [0290.0.0.0] above.
[10340] [0291.0.23.23] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[10341] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., by one
or more amino acids. In one embodiment, polypeptide distinguishes
from a sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, resp., by more than 5, 6, 7, 8
or 9 amino acids, preferably by more than 10, 15, 20, 25 or 30
amino acids, even more preferred are more than 40, 50, or 60 amino
acids and, preferably, the sequence of the polypeptide of the
invention distinguishes from a sequence as indicated in Table IIA
or IIB, columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp.,
by not more than 80% or 70% of the amino acids, preferably not more
than 60% or 50%, more preferred not more than 40% or 30%, even more
preferred not more than 20% or 10%. In an other embodiment, said
polypeptide of the invention does not consist of a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 275 to 277
and/or 635 or 636.
[10342] [0292.0.0.23] for the disclosure of this paragraph see
[0292.0.0.0] above.
[10343] [0293.0.23.23] In one embodiment, the invention relates to
a polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or by a nucleic acid molecule used in the process of the
invention. In one embodiment, the polypeptide of the invention has
a sequence which distinguishes from a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 275 to 277 and/or 635 or
636, resp., by one or more amino acids. In an other embodiment,
said polypeptide of the invention does not consist of the sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 275 to 277
and/or 635 or 636, resp. In a further embodiment, said polypeptide
of the present invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical. In one embodiment, said polypeptide does not
consist of the sequence encoded by a nucleic acid molecules as
indicated in Table IA or IB, columns 5 or 7, lines 275 to 277
and/or 635 or 636, resp.
[10344] [0294.0.23.23] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table IIA or IIB, column 3, lines 275 to 277 and/or
635 or 636, resp., which distinguishes over a sequence as indicated
in Table IIA or IIB, columns 5 or 7, lines 275 to 277 and/or 635 or
636, resp., by one or more amino acids, preferably by more than 5,
6, 7, 8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or
30 amino acids, even more preferred are more than 40, 50, or 60
amino acids but even more preferred by less than 70% of the amino
acids, more preferred by less than 50%, even more preferred my less
than 30% or 25%, more preferred are 20% or 15%, even more preferred
are less than 10%.
[10345] [0295.0.0.23] to [0297.0.0.23] for the disclosure of the
paragraphs [0295.0.0.23] to [0297.0.0.23] see paragraphs
[0295.0.0.0] to [0297.0.0.0] above.
[10346] [00297.1.0.23] %
[10347] [0298.0.23.23] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table II, columns 5 or 7, lines 275 to 277 and/or
635 or 636, resp. The portion of the protein is preferably a
biologically active portion as described herein. Preferably, the
polypeptide used in the process of the invention has an amino acid
sequence identical to a sequence as indicated in Table II, columns
5 or 7, lines 275 to 277 and/or 635 or 636, resp.
[10348] [0299.0.23.23] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
275 to 277 and/or 635 or 636, resp. The preferred polypeptide of
the present invention preferably possesses at least one of the
activities according to the invention and described herein. A
preferred polypeptide of the present invention includes an amino
acid sequence encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions, to a nucleotide
sequence as indicated in Table I, columns 5 or 7, lines 275 to 277
and/or 635 or 636, resp., or which is homologous thereto, as
defined above.
[10349] [0300.0.23.23] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., in amino
acid sequence due to natural variation or mutagenesis, as described
in detail herein. Accordingly, the polypeptide comprise an amino
acid sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%,
65% or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of as indicated in Table IIA or IIB,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp.
[10350] [0301.0.0.23] for the disclosure of this paragraph see
[0301.0.0.0] above.
[10351] [0302.0.23.23] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table II, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
resp., or the amino acid sequence of a protein homologous thereto,
which include fewer amino acids than a full length polypeptide of
the present invention or used in the process of the present
invention or the full length protein which is homologous to an
polypeptide of the present invention or used in the process of the
present invention depicted herein, and exhibit at least one
activity of polypeptide of the present invention or used in the
process of the present invention.
[10352] [0303.0.0.23] for the disclosure of this paragraph see
[0303.0.0.0] above.
[10353] [0304.0.23.23] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 275 to 277 and/or 635 or 636 but having
differences in the sequence from said wild-type protein. These
proteins may be improved in efficiency or activity, may be present
in greater numbers in the cell than is usual, or may be decreased
in efficiency or activity in relation to the wild type protein.
[10354] [0305.0.0.23] to [0308.0.0.23] for the disclosure of the
paragraphs [0305.0.0.23] to [0308.0.0.23] see paragraphs
[0305.0.0.0] to [0308.0.0.0] above.
[10355] [0306.1.0.22] %
[10356] [0309.0.23.23] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., refers
to a polypeptide having an amino acid sequence corresponding to the
polypeptide of the invention or used in the process of the
invention, whereas an "other polypeptide" not being indicated in
Table II, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
resp., refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous to
a polypeptide of the invention, preferably which is not
substantially homologous to a polypeptide as indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., e.g., a
protein which does not confer the activity described herein or
annotated or known for as indicated in Table II, column 3, lines
275 to 277 and/or 635 or 636, resp., and which is derived from the
same or a different organism. In one embodiment, an "other
polypeptide" not being indicated in Table II, columns 5 or 7, lines
275 to 277 and/or 635 or 636, resp., does not confer an increase of
the respective fine chemical in an organism or part thereof.
[10357] [0310.0.0.23] to [0334.0.0.23] for the disclosure of the
paragraphs [0310.0.0.23] to [0334.0.0.23] see paragraphs
[0310.0.0.0] to [0334.0.0.0] above.
[10358] [0335.0.23.23] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table I, columns 5 or
7, lines 275 to 277 and/or 635 or 636, resp., and/or homologs
thereof. As described inter alia in WO 99/32619, dsRNAi approaches
are clearly superior to traditional antisense approaches. The
invention therefore furthermore relates to double-stranded RNA
molecules (dsRNA molecules) which, when introduced into an
organism, advantageously into a plant (or a cell, tissue, organ or
seed derived there from), bring about altered metabolic activity by
the reduction in the expression of a nucleic acid sequences as
indicated in Table I, columns 5 or 7, lines 275 to 277 and/or 635
or 636, resp., and/or homologs thereof. In a double-stranded RNA
molecule for reducing the expression of a protein encoded by a
nucleic acid sequence as indicated in Table I, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, resp., and/or homologs thereof,
one of the two RNA strands is essentially identical to at least
part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[10359] [0336.0.0.23] to [0342.0.0.23] for the disclosure of the
paragraphs [0336.0.0.23] to [0342.0.0.23] see paragraphs
[0336.0.0.0] to [0342.0.0.0] above.
[10360] [0343.0.23.23] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table I, columns 5 or 7, lines 275 to 277
and/or 635 or 636, resp., or its homolog is not necessarily
required in order to bring about effective reduction in the
expression. The advantage is, accordingly, that the method is
tolerant with regard to sequence deviations as may be present as a
consequence of genetic mutations, polymorphisms or evolutionary
divergences. Thus, for example, using the dsRNA, which has been
generated starting from a sequence as indicated in Table I, columns
5 or 7, lines 275 to 277 and/or 635 or 636, resp., or homologs
thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[10361] [0344.0.0.23] to [0361.0.0.23] for the disclosure of the
paragraphs [0344.0.0.23] to [0361.0.0.23] see paragraphs
[0344.0.0.0] to [0361.0.0.0] above.
[10362] [0362.0.23.23] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, columns 5 or 7, lines 275 to 277 and/or 635 or 636,
resp., e.g. encoding a polypeptide having protein activity, as
indicated in Table II, columns 3, lines 275 to 277 and/or 635 or
636, resp. Due to the above-mentioned activity the respective fine
chemical content in a cell or an organism is increased. For
example, due to modulation or manipulation, the cellular activity
of the polypeptide of the invention or nucleic acid molecule of the
invention is increased, e.g. due to an increased expression or
specific activity of the subject matters of the invention in a cell
or an organism or a part thereof. Transgenic for a polypeptide
having an activity of a polypeptide as indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636, resp., means
herein that due to modulation or manipulation of the genome, an
activity as annotated for a polypeptide as indicated in
[10363] Table II, column 3, lines 275 to 277 and/or 635 or 636,
e.g. having a sequence as indicated in Table II, columns 5 or 7,
lines 275 to 277 and/or 635 or 636, resp., is increased in a cell
or an organism or a part thereof. Examples are described above in
context with the process of the invention.
[10364] [0363.0.0.23] for the disclosure of this paragraph see
paragraph [0363.0.0.0] above.
[10365] [0364.0.23.23] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table II, column 3, lines 275 to 277 and/or 635 or
636, resp. with the corresponding protein-encoding sequence as
indicated in Table I, column 5, lines 275 to 277 and/or 635 or 636,
resp., becomes a transgenic expression cassette when it is modified
by non-natural, synthetic "artificial" methods such as, for
example, mutagenization. Such methods have been described (U.S.
Pat. No. 5,565,350; WO 00/15815; also see above).
[10366] [0365.0.0.23] to [0373.0.0.23] for the disclosure of the
paragraphs [0365.0.0.23] to [0373.0.0.23] see paragraphs
[0365.0.0.0] to [0373.0.0.0] above.
[10367] [0374.0.23.23] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. Salicylic acid, in particular
the respective fine chemical, produced in the process according to
the invention may, however, also be isolated from the plant in the
form of their free salicylic acid in particular the free respective
fine chemical, or bound in or to compounds or moieties as for
example but not limited to esters. The respective fine chemical
produced by this process can be harvested by harvesting the
organisms either from the culture in which they grow or from the
field. This can be done via expressing, grinding and/or extraction,
salt precipitation and/or ion-exchange chromatography or other
chromatographic methods of the plant parts, preferably the plant
seeds, plant fruits, plant tubers and the like.
[10368] [0375.0.0.23] and [0376.0.0.23] for the disclosure of the
paragraphs [0375.0.0.23] and [0376.0.0.23] see paragraphs
[0375.0.0.0] and [0376.0.0.0] above.
[10369] [0377.0.23.23] Accordingly, the present invention relates
also to a process whereby the produced salicylic acid and/or
salicylic acid ester is isolated.
[10370] [0378.0.23.23] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the salicylic
acid and/or salicylic acid ester produced in the process can be
isolated. The resulting salicylic acid and/or salicylic acid ester
can, if appropriate, subsequently be further purified, if desired
mixed with other active ingredients such as vitamins, amino acids,
carbohydrates, antibiotics and the like, and, if appropriate,
formulated.
[10371] [0379.0.23.23] In one embodiment the product produced by
the present invention is a mixture of the respective fine chemicals
salicylic acid and/or salicylic acid esters.
[10372] [0380.0.23.23] The salicylic acid obtained in the process
by carrying out the invention is suitable as starting material for
the synthesis of further products of value. For example, they can
be used in combination with each other or alone for the production
of pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates to a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the salicylic acid and
salicylic acid ester composition produced or the respective fine
chemical produced if desired and formulating the product with a
pharmaceutical acceptable carrier or formulating the product in a
form acceptable for an application in agriculture. A further
embodiment according to the invention is the use of the salicylic
acid and/or salicylic acid esters produced in the process or of the
transgenic organism in animal feeds, foodstuffs, medicines, food
supplements, cosmetics or pharmaceuticals or for the production of
salicylic acid and/or salicylic acid esters e.g. after isolation of
the respective fine chemical or without, e.g. in situ, e.g in the
organism used for the process for the production of the respective
fine chemical.
[10373] [0381.0.0.23] and [0382.0.0.23] for the disclosure of the
paragraphs [0381.0.0.23] and [0382.0.0.23] see paragraphs
[0381.0.0.0] and [0382.0.0.0] above.
[10374] [0383.0.23.23] %
[10375] [0384.0.0.23] for the disclosure of this paragraph see
[0384.0.0.0] above.
[10376] [0385.0.23.23] The fermentation broths obtained in this
way, containing in particular salicylic acid and/or salicylic acid
esters in mixtures with other organic acids, amino acids,
polypeptides or polysaccarides, normally have a dry matter content
of from 1 to 70% by weight, preferably 7.5 to 25% by weight.
Sugar-limited fermentation is additionally advantageous, e.g. at
the end, for example over at least 30% of the fermentation time.
This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 10 g/l,
preferably to 0 to 3 g/l during this time. The fermentation broth
is then processed further. Depending on requirements, the biomass
can be removed or isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth or left completely in it.
[10377] The fermentation broth can then be thickened or
concentrated by known methods, such as, for example, with the aid
of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[10378] [0386.0.23.23] Accordingly, it is possible to purify the
salicylic acid and/or salicylic acid esters produced according to
the invention further. For this purpose, the product-containing
composition is subjected for example to separation via e.g. an open
column chromatography or HPLC in which case the desired product or
the impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use.
[10379] [0387.0.0.23] to [0392.0.0.23] for the disclosure of the
paragraphs [0387.0.0.23] to [0392.0.0.23] see paragraphs
[0387.0.0.0] to [0392.0.0.0] above.
[10380] [0393.0.23.23] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [10381] a. contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [10382] b. identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table I, columns 5 or 7, lines
275 to 277 and/or 635 or 636, preferably in Table I B, columns 5 or
7, lines 275 to 277 and/or 635 or 636, resp., and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[10383] c. introducing the candidate nucleic acid molecules in host
cells, preferably in a plant cell or a microorganism, appropriate
for producing the respective fine chemical; [10384] d. expressing
the identified nucleic acid molecules in the host cells; [10385] e.
assaying the respective fine chemical level in the host cells; and
[10386] f. identifying the nucleic acid molecule and its gene
product which expression confers an increase in the respective fine
chemical level in the host cell after expression compared to the
wild type.
[10387] [0394.0.0.23] to [0399.0.0.23] for the disclosure of the
paragraphs [0394.0.0.23] to [0399.0.0.23] see paragraphs
[0394.0.0.0] to [0399.0.0.0] above.
[10388] [00399.1.23.23] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636 or a homolog
thereof, e.g. comparing the phenotype of nearly identical organisms
with low and high activity of a protein as indicated in Table II,
columns 5 or 7, lines 275 to 277 and/or 635 or 636 after incubation
with the drug.
[10389] [0400.0.0.23] to [0416.0.0.23] for the disclosure of the
paragraphs [0400.0.0.23] to [0416.0.0.23] see paragraphs
[0400.0.0.0] to [0416.0.0.0] above.
[10390] [0417.0.23.23] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the salicylic acidbiosynthesis pathways.
In particular, the overexpression of the polypeptide of the present
invention may protect an organism such as a microorganism or a
plant against inhibitors, which block the salicylic
acidsynthesis.
[10391] Furthermore the overexpression of nucleic acids sequences
as characterized in Table I, columns 5 and 7, lines 275 to 277
and/or 635 or 636, by increasing the salicylic acid content in
plant cells and may also be useful for increasing the nickel
(Ni)/zinc (Zn) tolerance in plants.
[10392] [0418.0.0.23] to [0423.0.0.23] for the disclosure of the
paragraphs [0418.0.0.23] to [0423.0.0.23] see paragraphs
[0418.0.0.0] to [0423.0.0.0] above.
[10393] [0424.0.23.23] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other organic acids.
Accordingly, the nucleic acid of the invention, or the nucleic acid
molecule identified with the method of the present invention or the
complement sequences thereof, the polypeptide of the invention, the
nucleic acid construct of the invention, the organisms, the host
cell, the microorganisms, the plant, plant tissue, plant cell, or
the part thereof of the invention, the vector of the invention, the
antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the respective
fine chemical in a organism or part thereof, e.g. in a cell.
[10394] [0425.0.0.23] to [0435.0.0.23] for the disclosure of the
paragraphs [0425.0.0.23] to [0435.0.0.23] see paragraphs
[0425.0.0.0] to [0435.0.0.0] above.
[10395] [0436.0.23.23] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of salicylic acid can be
carried out by passing a plasmid DNA (or another vector DNA)
containing the desired nucleic acid sequence or nucleic acid
sequences, e.g. the nucleic acid molecule of the invention or the
vector of the invention, through E. coli and other microorganisms
(for example Bacillus spp. or yeasts such as Saccharomyces
cerevisiae) which are not capable of maintaining the integrity of
its genetic information. Usual mutator strains have mutations in
the genes for the DNA repair system [for example mutHLS, mutD, mutT
and the like; for comparison, see Rupp, W. D. (1996) DNA repair
mechanisms in Escherichia coli and Salmonella, pp. 2277-2294, ASM:
Washington]. The skilled worker knows these strains. The use of
these strains is illustrated for example in Greener, A. and
Callahan, M. (1994) Strategies 7; 32-34.
[10396] In-vitro mutation methods such as increasing the
spontaneous mutation rates by chemical or physical treatment are
well known to the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[10397] Site-directed mutagensis method such as the introduction of
desired mutations with an
[10398] M13 or phagemid vector and short oligonucleotides primers
is a well-known approach for site-directed mutagensis. The clou of
this method involves cloning of the nucleic acid sequence of the
invention into an M13 or phagemid vector, which permits recovery of
single-stranded recombinant nucleic acid sequence. A mutagenic
oligonucleotide primer is then designed whose sequence is perfectly
complementary to nucleic acid sequence in the region to be mutated,
but with a single difference: at the intended mutation site it
bears a base that is complementary to the desired mutant nucleotide
rather than the original. The mutagenic oligonucleotide is then
allowed to prime new DNA synthesis to create a complementary
full-length sequence containing the desired mutation. Another
site-directed mutagensis method is the PCR mismatch primer
mutagensis method also known to the skilled person. Dpnl
site-directed mutagensis is a further known method as described for
example in the Stratagene Quickchange.TM. site-directed mutagenesis
kit protocol. A huge number of other methods are also known and
used in common practice.
[10399] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.23.23] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpina
[10400] [0438.0.23.23] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology", VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[10401] [0439.0.23.23] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255f) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[10402] [0440.0.23.23] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002) teaches a homolog recombination system dependent on
rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this system
for homologous recombination are vectors derived from the Ylp
series. Plasmid vectors derived for example from the 2.mu.-Vector
are known by the skilled worker and used for the expression in
yeasts. Other preferred vectors are for example pART1, pCHY21 or
pEVP11 as they have been described by McLeod et al. (EMBO J. 1987,
6:729-736) and Hoffman et al. (Genes Dev. 5, 1991: :561-571.) or
Russell et al. (J. Biol. Chem. 258, 1983: 143-149.). Other
beneficial yeast vectors are plasmids of the REP, REP-X, pYZ or RIP
series.
[10403] [0441.0.0.23] to [0443.0.0.23] for the disclosure of the
paragraphs [0441.0.0.23] to [0443.0.0.23] see paragraphs
[0441.0.0.0] to [0443.0.0.0] above.
[0444.0.23.23] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[10404] [0445.0.23.23] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad.
[10405] Sin. (2000) 41:41-48]. Mortierella can be grown at
20.degree. C. in a culture medium containing: 10 g/l glucose, 5 g/l
yeast extract at pH 6.5. Furthermore Jang et al. teaches a
submerged basal medium containing 20 g/l soluble starch, 5 g/l
Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l KH.sub.2PO.sub.4, and
0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[10406] [0446.0.0.23] to [0453.0.0.23] for the disclosure of the
paragraphs [0446.0.0.23] to [0453.0.0.23] see paragraphs
[0446.0.0.0] to [0453.0.0.0] above.
[0454.0.23.23] Example 8
Analysis of the Effect of the Nucleic Acid Molecule on the
Production of Salicylic Acid
[10407] [0455.0.23.23] The effect of the genetic modification in
plants, fungi, algae or ciliates on the production of a desired
compound (such as a salicylic acidand/or salicylic acid esters) can
be determined by growing the modified microorganisms or the
modified plant under suitable conditions (such as those described
above) and analyzing the medium and/or the cellular components for
the elevated production of desired product (i.e. of salicylic acid
and/or salicylic acid esters). These analytical techniques are
known to the skilled worker and comprise spectroscopy, thin-layer
chromatography, various types of staining methods, enzymatic and
microbiological methods and analytical chromatography such as
high-performance liquid chromatography (see, for example, Ullman,
Encyclopedia of Industrial Chemistry, Vol. A2, p. 89-90 and p.
443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987)
"Applications of HPLC in Biochemistry" in: Laboratory Techniques in
Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993)
Biotechnology, Vol. 3, Chapter III: "Product recovery and
purification", p. 469-714, VCH: Weinheim; Belter, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[10408] [0456.0.0.23]: for the disclosure of this paragraph see
[0456.0.0.0] above.
[0457.0.23.23] Example 9
Purification of Salicylic Acid
[10409] [0458.0.23.23] Abbreviations; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[10410] The unambiguous detection for the presence of salicylic
acidcan be obtained by analyzing recombinant organisms using
analytical standard methods: LC, LC-MSMS,
[10411] GC-MS or TLC, as described. The total amount produced in
the organism for example in yeasts used in the inventive process
can be analysed for example according to the following
procedure:
[10412] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods.
[10413] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[10414] A typical sample pretreatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography.
[10415] For analysis, solvent delivery and aliquot removal can be
accomplished with a robotic system comprising a single injector
valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000
W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of
50% potassium hydroxide hydro-ethanolic solution (4 water--1
ethanol) can be added to each vial, followed by the addition of 3
ml of octanol. The saponification treatment can be conducted at
room temperature with vials maintained on an IKA HS 501 horizontal
shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours
at 250 movements/minute, followed by a stationary phase of
approximately one hour.
[10416] Following saponification, the supernatant can be diluted
with 0-20 ml of methanol. The addition of methanol can be conducted
under pressure to ensure sample homogeneity. Using a 0.25 ml
syringe, a 0.1 ml aliquot can be removed and transferred to HPLC
vials for analysis.
[10417] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete
with a quaternary pump, vacuum degassing system, six-way injection
valve, temperature regulated autosampler, column oven and
Photodiode Array detector can be used [Agilent Technologies
available through Ultra Scientific Inc., 250 Smith Street, North
Kingstown, R.I.]. The column can be a Waters YMC30, 5-micron,
4.6.times.250 mm with a guard column of the same material [Waters,
34 Maple Street, Milford, Mass.]. The solvents for the mobile phase
can be 81 methanol: 4 water: 15 tetrahydrofuran (THF) stabilized
with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injections were
20 .mu.l. Separation can be isocratic at 30.degree. C. with a flow
rate of 1.7 ml/minute. The peak responses can be measured by
absorbance at 447 nm.
[10418] [0459.0.23.23] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the salicylic acidcan be further purified with a
so-called RTHPLC. As eluent acetonitrile/water or
chloroform/acetonitrile mixtures can be used. If necessary, these
chromatography steps may be repeated, using identical or other
chromatography resins. The skilled worker is familiar with the
selection of suitable chromatography resin and the most effective
use for a particular molecule to be purified.
[10419] [0460.0.0.23] for the disclosure of this paragraph see
[0460.0.0.0] above.
[0461.0.23.23] Example 10
Cloning SEQ ID NO: 33502, 33810, 33899, 98917 or 99068 for the
Expression in Plants
[10420] [0462.0.0.23] for the disclosure of this paragraph see
[0462.0.0.0] above.
[10421] [0463.0.23.23] SEQ ID NO: 33502, 33810, 33899, 98917 or
99068 are amplified by PCR as described in the protocol of the Pfu
Turbo or DNA Herculase polymerase (Stratagene).
[10422] [0464.0.0.23] to [0466.0.0.23] for the disclosure of the
paragraphs [0464.0.0.23] to [0466.0.0.23] see [0464.0.0.0] to
[0466.0.0.0] above.
[10423] [0466.1.0.23] In case the Herculase enzyme can be used for
the amplification, the PCR amplification cycles were as follows: 1
cycle of 2-3 minutes at 94.degree. C., followed by 25-30 cycles of
in each case 30 seconds at 94.degree. C., 30 seconds at
55-60.degree. C. and 5-10 minutes at 72.degree. C., followed by 1
cycle of 10 minutes at 72.degree. C., then 4.degree. C.
[10424] [0467.0.23.23] The following primer sequences were selected
for the gene SEQ ID: 33502
TABLE-US-00135 i) forward primer (SEQ ID NO: 33802) atgaaaaaaa
ccacattagc actg ii) reverse primer (SEQ ID NO: 33803) ttactgcatt
aacaggtaga tggtg
[10425] The following primer sequences were selected for the gene
SEQ ID NO: 33810:
TABLE-US-00136 i) forward primer (SEQ ID NO: 33894) atgatcagga
gtcacaccat ga ii) reverse primer (SEQ ID NO: 33895) ttaattgcca
gccatcgcct g
[10426] The following primer sequences were selected for the gene
SEQ ID NO: 33899:
TABLE-US-00137 i) forward primer (SEQ ID NO: 34217) atgttatcac
tgtctgccaa aaatc ii) reverse primer (SEQ ID NO: 34218) tcagtcacgg
tattggtcaa aaaat
[10427] The following primer sequences were selected for the gene
SEQ ID NO: 98917:
TABLE-US-00138 i) forward primer (SEQ ID NO: 99063) atggaaacga
ctcaaaccag cac ii) reverse primer (SEQ ID NO: 99064) ttagctgaac
agagagtaga agatt
[10428] The following primer sequences were selected for the gene
SEQ ID NO: 99068:
TABLE-US-00139 i) forward primer (SEQ ID NO: 99168) atgagtactt
cagatagcat tgtatc ii) reverse primer (SEQ ID NO: 99169) ttaaaacagt
ttgtatacga tgttcag
[10429] [0468.0.0.23] to [0470.0.0.23] for the disclosure of the
paragraphs [0468.0.0.23] to [0470.0.0.23] see [0468.0.0.0] to
[0470.0.0.0] above.
[10430] [0470.1.23.23] The PCR-products, produced by Pfu Turbo DNA
polymerase, were phosphorylated using a T4 DNA polymerase using a
standard protocol (e.g. MBI Fermentas) and cloned into the
processed binary vector.
[10431] [0471.0.23.23] for the disclosure of this paragraph see
[0471.0.0.0] above.
[10432] [0471.1.23.23] The DNA termini of the PCR-products,
produced by Herculase DNA polymerase, were blunted in a second
synthesis reaction using Pfu Turbo DNA polymerase. The composition
for the protocol of the blunting the DNA-termini was as follows:
0.2 mM blunting dTTP and 1.25 u Pfu Turbo DNA polymerase. The
reaction was incubated at 72.degree. C. for 30 minutes. Then the
PCR-products were phosphorylated using a T4 DNA polymerase using a
standard protocol (e.g. MBI Fermentas) and cloned into the
processed vector as well.
[10433] [0472.0.0.23] to [0479.0.0.23] for the disclosure of the
paragraphs [0472.0.0.23] to [0479.0.0.23] see [0472.0.0.0] to
[0479.0.0.0] above.
[0480.0.23.23] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 33502,
33810, 33899, 98917 or 99068
[10434] [0481.0.0.23] to [0513.0.0.23] for the disclosure of the
paragraphs [0481.0.0.23] to [0513.0.0.23] see paragraphs
[0482.0.0.0] to [0513.0.0.0] above.
[10435] [0514.0.23.23] As an alternative, salicylic acid can be
detected as described in Kmetec, V., J. Pharm. Biomed. Anal. 1992
October-December; 10 (10-12):1073-6.
[10436] The results of the different plant analyses can be seen
from the table 1, which follows:
TABLE-US-00140 TABLE 1 ORF Metabolite Method Min Max b0161
Salicylic acid LC 1.67 5.75 b2664 Salicylic acid LC 1.56 5.25
YLR089C Salicylic acid LC 1.45 2.53 b2796 Salicylic acid LC 1.42
3.48 b3116 Salicylic acid LC 1.41 2.30
[10437] [0515.0.23.23] Column 2 shows the metabolite salicylic acid
analyzed. Columns 4 and 5 shows the ratio of the analyzed
metabolite between the transgenic plants and the wild type;
Increase of the metabolite: Max: maximal x-fold (normalised to wild
type)-Min: minimal x-fold (normalised to wild type). Decrease of
the metabolite: Max: maximal x-fold (normalised to wild type)
(minimal decrease), Min: minimal x-fold (normalised to wild type)
(maximal decrease). Column 3 indicates the analytical method.
[10438] [0516.0.0.23] to [0530.0.0.23] for the disclosure of the
paragraphs [0516.0.0.23] to [0530.0.0.23] see paragraphs
[0516.0.0.0] to [0530.0.0.0] above.
[10439] [0530.1.0.23] to [0530.6.0.23] for the disclosure of the
paragraphs [0530.1.0.23] to [0530.6.0.23] see paragraphs
[0530.1.0.0] to [0530.6.0.0] above.
[10440] [0531.0.0.23] to [0552.0.0.23] for the disclosure of the
paragraphs [0531.0.0.23] to [0552.0.0.23] see paragraphs
[0531.0.0.0] to [0552.0.0.0] above.
[10441] [0552.1.0.23]: %
[10442] [0552.2.0.23] for the disclosure of this paragraph see
[0552.2.0.0] above.
[10443] [0553.0.23.23] [10444] 1. A process for the production of
salicylic acid, which comprises [10445] (a) increasing or
generating the activity of a protein as indicated in Table II,
columns 5 or 7, lines 275 to 277 or 635 and 636 or a functional
equivalent thereof in a non-human organism or in one or more parts
thereof; and [10446] (b) growing the organism under conditions
which permit the production of salicylic acid in said organism.
[10447] 2. A process for the production of salicylic acid,
comprising the increasing or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [10448] a) nucleic acid molecule encoding a
polypeptide as indicated in Table II, columns 5 or 7, lines 275 to
277 or 635 and 636 or a fragment thereof, which confers an increase
in the amount of salicylic acid in an organism or a part thereof;
[10449] b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 275 to 277
or 635 and 636; [10450] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of salicylic acid in
an organism or a part thereof; [10451] d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of salicylic acid in an organism or a part thereof; [10452]
e) nucleic acid molecule which hybridizes with a nucleic acid
molecule of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of salicylic acid in an
organism or a part thereof; [10453] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table III, column
7, lines 275 to 277 or 635 and 636 and conferring an increase in
the amount of salicylic acid in an organism or a part thereof;
[10454] g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of salicylic acid in
an organism or a part thereof; [10455] h) nucleic acid molecule
encoding a polypeptide comprising a consensus as indicated in Table
IV, column 7, lines 275 to 277 or 635 and 636 and conferring an
increase in the amount of salicylic acid in an organism or a part
thereof; and [10456] i) nucleic acid molecule which is obtainable
by screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of salicylic acid in an organism or a part thereof. [10457]
or comprising a sequence which is complementary thereto. [10458] 3.
The process of claim 1 or 2, comprising recovering of the free or
bound salicylic acid. [10459] 4. The process of any one of claims 1
to 3, comprising the following steps: [10460] a) selecting an
organism or a part thereof expressing a polypeptide encoded by the
nucleic acid molecule characterized in claim 2; [10461] b)
mutagenizing the selected organism or the part thereof; [10462] c)
comparing the activity or the expression level of said polypeptide
in the mutagenized organism or the part thereof with the activity
or the expression of said polypeptide of the selected organisms or
the part thereof; [10463] d) selecting the mutated organisms or
parts thereof, which comprise an increased activity or expression
level of said polypeptide compared to the selected organism or the
part thereof; [10464] e) optionally, growing and cultivating the
organisms or the parts thereof; and [10465] f) recovering, and
optionally isolating, the free or bound salicylic acid produced by
the selected mutated organisms or parts thereof. [10466] 5. The
process of any one of claims 1 to 4, wherein the activity of said
protein or the expression of said nucleic acid molecule is
increased or generated transiently or stably. [10467] 6. An
isolated nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [10468] a) nucleic acid
molecule encoding a polypeptide as indicated in Table II, columns 5
or 7, lines 275 to 277 or 635 and 636 or a fragment thereof, which
confers an increase in the amount of salicylic acid in an organism
or a part thereof; [10469] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 275 to 277 or 635 and 636; [10470] c) nucleic acid molecule
whose sequence can be deduced from a polypeptide sequence encoded
by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of salicylic acid in an organism or a part thereof; [10471]
d) nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of salicylic acid in an organism or a
part thereof; [10472] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under stringent
hybridisation conditions and conferring an increase in the amount
of salicylic acid in an organism or a part thereof; [10473] f)
nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table III, column 7, lines 275 to 277 or 635 and 636
and conferring an increase in the amount of salicylic acid in an
organism or a part thereof; [10474] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
salicylic acid in an organism or a part thereof; [10475] h) nucleic
acid molecule encoding a polypeptide comprising a consensus as
indicated in Table IV, column 7, lines 275 to 277 or 635 and 636
and conferring an increase in the amount of salicylic acid in an
organism or a part thereof; and [10476] i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of salicylic acid in an organism or a part thereof. [10477]
whereby the nucleic acid molecule distinguishes over the sequence
as indicated in Table IA, columns 5 or 7, lines 275 to 277 or 635
and 636 by one or more nucleotides. [10478] 7. A nucleic acid
construct which confers the expression of the nucleic acid molecule
of claim 6, comprising one or more regulatory elements. [10479] 8.
A vector comprising the nucleic acid molecule as claimed in claim 6
or the nucleic acid construct of claim 7. [10480] 9. The vector as
claimed in claim 8, wherein the nucleic acid molecule is in
operable linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [10481] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in any one of claims
2 to 5. [10482] 11. The host cell of claim 10, which is a
transgenic host cell. [10483] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [10484] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [10485] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table IIA, columns 5
or 7, lines 275 to 277 or 635 and 636 by one or more amino acids.
[10486] 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. [10487] 16. A plant tissue,
propagation material, harvested material or a plant comprising the
host cell as claimed in claim 12 which is plant cell or an
Agrobacterium. [10488] 17. A method for screening for agonists and
antagonists of the activity of a polypeptide encoded by the nucleic
acid molecule of claim 6 conferring an increase in the amount of
salicylic acid in an organism or a part thereof comprising: [10489]
(a) contacting cells, tissues, plants or microorganisms which
express the a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of salicylic acid in
an organism or a part thereof with a candidate compound or a sample
comprising a plurality of compounds under conditions which permit
the expression the polypeptide; [10490] (b) assaying the salicylic
acid level or the polypeptide expression level in the cell, tissue,
plant or microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [10491] (c)
identifying a agonist or antagonist by comparing the measured
salicylic acid level or polypeptide expression level with a
standard salicylic acid or polypeptide expression level measured in
the absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. [10492] 18. A process for
the identification of a compound conferring increased salicylic
acid production in a plant or microorganism, comprising the steps:
[10493] a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of salicylic acid in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
salicylic acid in an organism or a part thereof; [10494] b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [10495] 19. A method for the identification of a
gene product conferring an increase in salicylic acid production in
a cell, comprising the following steps: [10496] a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in salicylic
acid after expression with the nucleic acid molecule of claim 6;
[10497] b) identifying the nucleic acid molecules, which hybridise
under relaxed stringent conditions with the nucleic acid molecule
of claim 6; [10498] c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing salicylic acid;
[10499] d) expressing the identified nucleic acid molecules in the
host cells; [10500] e) assaying the salicylic acid level in the
host cells; and [10501] f) identifying nucleic acid molecule and
its gene product which expression confers an increase in the
salicylic acid in the host cell in the host cell after expression
compared to the wild type. [10502] 20. A method for the
identification of a gene product conferring an increase in
salicylic acid production in a cell, comprising the following
steps: [10503] a) identifying in a data bank nucleic acid molecules
of an organism; which can contain a candidate gene encoding a gene
product conferring an increase in the salicylic acid amount or
level in an organism or a part thereof after expression, and which
are at least 20% homolog to the nucleic acid molecule of claim 6;
[10504] b) introducing the candidate nucleic acid molecules in host
cells appropriate for producing salicylic acid; [10505] c)
expressing the identified nucleic acid molecules in the host cells;
[10506] d) assaying the salicylic acid level in the host cells; and
[10507] e) identifying nucleic acid molecule and its gene product
which expression confers an increase in the salicylic acid level in
the host cell after expression compared to the wild type. [10508]
21. A method for the production of an agricultural composition
comprising the steps of the method of any one of claims 17 to 20
and formulating the compound identified in any one of claims 17 to
20 in a form acceptable for an application in agriculture. [10509]
22. A composition comprising the nucleic acid molecule of claim 6,
the polypeptide of claim 14, the nucleic acid construct of claim 7,
the vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [10510] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
salicylic acid after expression. [10511] 24. Use of the polypeptide
of claim 14 or the nucleic acid construct claim 7 or the gene
product identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of
salicylic acid levels in an organism. [10512] 25. Agrochemical,
pharmaceutical, food or feed composition comprising the nucleic
acid molecule of claim 6, the polypeptide of claim 14, the nucleic
acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20.
[10513] 26. The method of any one of claims 1 to 5, the nucleic
acid molecule of claim 6, the polypeptide of claim 14, the nucleic
acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20, wherein the fine chemical is salicylic acid. [10514] 27. A host
cell or plant according to any of the claims 10 to 12 which is
resistant to a herbicide inhibiting the biosynthesis of salicylic
acid. [10515] 28. A host cell or plant according to any of the
claims 10 to 12 showing increased resistance to pathogens in
comparison to control plants. [10516] 29. A host cell or plant
according to any of the claims 10 to 12 showing increased
resistance to biotic or abiotic stress. [10517] 30. A host cell or
plant according to any of the claims 10 to 12 showing reduced
senescence.
[10518] 31. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the host cell of claim 10 to 12 or the gene
product identified according to the method of claim 19 or 20 for
the protection of a plant against a salicylic acid synthesis
inhibiting herbicide.
[10519] [0554.0.0.23] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[10520] [0000.0.0.24] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[10521] [0001.0.0.24] see [0001.0.0.0]
[10522] [0002.0.24.24] to [0009.0.24.24] see [0002.0.24.24] to
[0009.0.10.10]
[10523] [0010.0.24.24] Therefore improving the quality of
foodstuffs and animal feeds is an important task of the
food-and-feed industry. This is necessary since, for example, as
mentioned above beta-carotene, which occur in plants and some
microorganisms are limited with regard to the supply of mammals.
Especially advantageous for the quality of foodstuffs and animal
feeds is as balanced as possible a carotenoids profile in the diet
since a great excess of some carotenoids above a specific
concentration in the food has only some positive effect. A further
increase in quality is only possible via addition of further
carotenoids, which are limiting.
[10524] [0011.0.24.24] see [0011.0.10.10]
[10525] [0012.0.24.24] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes which
participate in the biosynthesis of carotenoids, e.g. beta-carotene
or its/their precursor, e.g. isopentyl pyrophosphate (IPP), and
make it possible to produce them specifically on an industrial
scale without unwanted byproducts forming.
[10526] In the selection of genes for biosynthesis two
characteristics above all are particularly important. On the one
hand, there is as ever a need for improved processes for obtaining
the highest possible contents of carotenoids like beta-carotene; on
the other hand as less as possible byproducts should be produced in
the production process.
[10527] [0013.0.0.24] see [0013.0.0.0]
[10528] [0014.0.24.24] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a carotene or a precursor
thereof. In a preferred embodiment, the fine chemical is
beta-carotene or IPP, resp. Accordingly, in the present invention,
the term "the fine chemical" as used herein relates to a "carotene,
in particular beta-carotene, or its precursor, in particular IPP,
Further, the term "the fine chemicals" as used herein also relates
to fine chemicals comprising carotene, in particular beta-carotene,
or its precursor, in particular IPP.
[10529] [0015.0.24.24] In one embodiment, the term "carotene, in
particular beta-carotene," or "the fine chemical" or "the
respective fine chemical" means at least one chemical compound with
a carotene-, in particular beta-carotene-like activity.
[10530] In a preferred embodiment, the term "carotene precursor, in
particular IPP" means a chemical compound, which expression
increases preferably the activity or amount of carotenes,
preferably of beta-carotenes, in an organism, alone or in
combination with other chemical compounds, e.g. other expression
tools, like genes conferring reactions for the conversion of IPP or
the production of IPP, e.g. genes being involved in the production
or conversion of mevalonic acid, the first specific precursor of
terpenoids, or acetyl-CoA via HMG-CoA
(3-hydroxy-3-methylglutaryl-CoA), that is itself converted to
isopentenyl pyrophosphate (IPP), or geranylgeranyl pyrophosphate
(GGPP) molecules which produce colorless phytoene, which is the
initial carotenoid, or of the existent alternative,
mevalonate-independent pathway for IPP formation that was
characterized initially in several species of eubacteria, i.e. in a
green alga, and in the plastids of higher plants, and comprising
the transketolase-type condensation reaction of pyruvate and
D-glyceraldehylde-3-phosphate to yield
1-deoxy-D-xylulose-5-phosphate (DXP), or one of the desaturases,
which confer the series of desaturation reactions to convert the
conversion product of IPP phytoene to phytofluenell, -tcarotene,
neurosporene and finally to lycopene, or the cyclization reaction
to .beta.-carotene that contains two .beta.-ionene rings.
[10531] In one embodiment, the term "the fine chemical" means a
"carotene, in particular beta-carotene, and its precursor, in
particular IPP". In one embodiment, the term "the fine chemical"
means beta-carotene or IPP depending on the context in which the
term is used. Throughout the specification the term "the fine
chemical" includes the free fine chemicals, its salts, ester,
thioester or bound to other compounds such sugars or sugarpolymers,
like glucoside, e.g. diglucoside. In one embodiment, the term "the
fine chemical" means beta-carotene or IPP, resp., in free form or
their salts or their ester or bound to a glucoside, e.g a
diglucoside, resp.
[10532] [0016.0.24.24] Accordingly, the present invention relates
to a process comprising [10533] (a) increasing or generating the
activity of one or more b1829, b2699, YBR089C-A, YDR316W, YDR513W,
and/or YLL013C, and/or b0730, b1926, b2211, b3172, b4129, and/or
YDR407C and/or b0481, b0970, b1736, b1738 and/or b3160 protein(s)
in a non-human organism in one or more parts thereof; and [10534]
(b) growing the organism under conditions which permit the
production of the fine chemical, thus, carotene, in particular
beta-carotene, or its precursor, in particular IPP, resp., in said
organism.
[10535] Accordingly, the present invention relates to a process
comprising [10536] (a) increasing or generating the activity of one
or more proteins having the activity of a protein indicated in
Table IIA or IIB, column 3, lines 279, 282, 285, 286, 288, and/or
289, or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637
to 641, resp., or having the sequence of a polypeptide encoded by a
nucleic acid molecule indicated in Table IA or IB, column 5 or 7,
lines 278 to 289 and/or lines 637 to 641, resp., in a non-human
organism in one or more parts thereof; and [10537] (b) growing the
organism under conditions which permit the production of the fine
chemical, thus, a carotene, in particular beta-carotene, or its
precursor, in particular IPP, resp., or fine chemicals comprising
carotene, in particular beta-carotene, or its precursor, in
particular IPP, resp., in said organism.
[10538] [0016.1.24.24] Accordingly, in one embodiment the term "the
fine chemical" means "beta-carotene" in relation to all sequences
listed in the Tables I to IV, lines 279, 282, 285, 286, 288, and/or
289 and/or lines 637 to 641 or homologs thereof and means "IPP" in
relation to all sequences listed in Table I to IV, lines 278, 280,
281, 283, 284, and/or 287 and/or lines 637 to 641, or homologs
thereof. Accordingly, the term "the fine chemical" can mean
"beta-carotene" or "IPP", owing to circumstances and the context.
In order to illustrate that the meaning of the term "the respective
fine chemical" means "beta-carotene", and/or "IPP" owing to the
sequences listed in the context the term "the respective fine
chemical" is also used.
[10539] [0017.0.0.24] to [0018.0.0.24]: see [0017.0.0.0] to
[0018.0.0.0]
[10540] [0019.0.24.24] Advantageously the process for the
production of the respective fine chemical leads to an enhanced
production of the a carotene, in particular beta-carotene, a
precursor thereof, e.g. IPP. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table IA or IB, column 3, lines 279, 282, 285, 286,
288, and/or 289, and/or lines 278, 280, 281, 283, 284, and/or 287
and/or lines 637 to 641 or encoded by nucleic acid molecule
indicated in Table IA or IB, columns 5 or 7, lines 279, 282, 285,
286, 288, and/or 289, and/or lines 278, 280, 281, 283, 284, and/or
287 and/or lines 637 to 641.
[10541] [0020.0.24.24] Surprisingly it was found, that the
transgenic expression of the Escherichia coli K12 or Saccharomyces
cerevisiae proteins b1829, b2699, YBR089C-A, YDR316W, YDR513W, and
YLL013C in Arabidopsis thaliana conferred an increase in the
beta-carotene ("the fine chemical" or "the fine respective
chemical" in respect to said proteins and their homologs as wells
as the encoding nucleic acid molecules, in particular as indicated
in Table I to IV, column 3, lines 279, 282, 285, 286, 288, and/or
289) content of the transformed plants.
[10542] Surprisingly it was found, that the transgenic expression
of the Escherichia coli K12 or Saccharomyces cerevisiae proteins
b0730, b1926, b2211, b3172, b4129, or YDR407C and/or b0481, b0970,
b1736, b1738 and/or b3160 in Arabidopsis thaliana conferred an
increase in the IPP ("the fine chemical" or "the fine respective
chemical" in respect to said proteins and their homologs as wells
as the encoding nucleic acid molecules, in particular as indicated
in Table I to IV, column 3, lines 278, 280, 281, 283, 284, or 287
or lines 637 to 641) content of the transformed plants.
[10543] [0021.0.0.24] see [0021.0.0.0]
[10544] [0022.0.24.24] The sequence of b0730 from Escherichia coli
K12 has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as a
transcriptional regulator of succinylCoA synthetase operon and a
fatty acyl response regulator. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein of
the superfamily of transcription regulator GntR, in particular
involved in regulation of C-compound and carbohydrate utilization,
transcriptional control, prokaryotic nucleoid, transcriptional
repressor, DNA binding, preferably being a transcriptional
regulator of succinylCoA synthetase operon and fatty acyl response
regulator, or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, meaning of the IPP, or of
carotenes, preferably in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a transcriptional regulator
of succinylCoA synthetase operon and a fatty acyl response
regulator, in particular of a lipoprotein of the superfamily of
transcription regulator GntR is increased or generated, e.g. from
E. coli or a homolog thereof.
[10545] The sequence of b1829 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a heat shock protein with
protease activity. Accordingly, in one embodiment, the process of
the present invention comprises the use of protein of the
superfamily of heat-shock protein htpX, in particular involved in
stress response, UNCLASSIFIED PROTEINS, pheromone response,
mating-type determination, sex-specific proteins, protein
modification, proteolytic degradation, preferably a heat shock
protein with protease activity or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of carotene, in particular beta-carotene, preferably in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention said activity,
e.g the activity of heat shock protein with protease activity is
increased or generated, e.g. from E. coli or a homolog thereof.
[10546] The sequence of b1926 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as flagellar protein fliT.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein of a flagellar protein
fliT from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of IPP, or of
carotenes, preferably in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention said activity, e.g. the activity of a
flagellar protein fliT is increased or generated, e.g. from E. coli
or a homolog thereof.
[10547] The sequence of b2699 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a DNA strand exchange and
recombination protein with protease and nuclease activity of the
superfamily of the recombination protein recA. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein of the superfamily of the recombination protein recA,
preferably with a DNA recombination and DNA repair activity, a
pheromone response activity, a mating-type determination activity,
a sex-specific protein activity, a nucleotide binding activity
and/or a protease and nuclease activity, in particular a DNA strand
exchange and recombination protein with protease and nuclease
activity from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of carotene, in
particular of beta-carotene, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention said activity, e.g. the activity
of a protease and nuclease activity, in particular a DNA strand
exchange and recombination protein with protease and nuclease
activity, in particular of the superfamily of the recombination
protein recA is increased or generated, e.g. from E. coli or a
homolog thereof.
[10548] The sequence of b2211 from Escherichia coli K12 has been
published in Blattner et al.,
[10549] Science 277(5331), 1453-1474, 1997, and its activity is
being defined as a ATP-binding transport proteins of the ABC
superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein of the superfamily
of unassigned ATP-binding cassette proteins, preferably with a
nucleotide binding, ABC transporters, CELLULAR TRANSPORT AND
TRANSPORT MECHANISMS activity, in particular a ATP-binding
transport proteins of the ABC superfamily protein activity from E.
coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of IPP, or of carotenes,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention said activity, e.g. the activity of a ATP-binding
transport proteins of the ABC superfamily is increased or
generated, e.g. from E. coli or a homolog thereof.
[10550] The sequence of b3172 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as argininosuccinate synthetase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein of the superfamily of
argininosuccinate synthase proteins, preferably involved in amino
acid biosynthesis, nitrogen and sulfur metabolism, biosynthesis of
the glutamate group (proline, hydroxyprolin, arginine, glutamine,
glutamate), degradation of amino acids of the glutamate group,
nitrogen and sulfur utilization, urea cycle, biosynthesis of
polyamines and creatine, biosynthesis of the aspartate family,
assimilation of ammonia, biosynthesis of the glutamate group,
preferably having a argininosuccinate synthetase activity from E.
coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of IPP, or of carotenes,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention said activity, e.g. the activity of a argininosuccinate
synthetase is increased or generated, e.g. from E. coli or a
homolog thereof.
[10551] The sequence of b4129 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as inducible lysine tRNA
synthetase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein of the superfamily
of lysine-tRNA ligase proteins, preferably involved in
aminoacyl-tRNA-synthetases, translation, preferably having a
inducible lysine tRNA synthetase activity from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of IPP, or of carotenes, preferably in free
or bound form in an organism or a part thereof, as mentioned. In
one embodiment, in the process of the present invention said
activity, e.g. the activity of a inducible lysine tRNA synthetase
is increased or generated, e.g. from E. coli or a homolog
thereof.
[10552] The sequence of YBR089C-A from Saccharomyces cerevisiae has
been published in Jacq et al., Nature 387 (6632 Suppl), 75-78,
1997, and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is being defined as nonhistone chromosomal protein.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein of the superfamily of
nonhistone chromosomal protein HMG-2, HMG box homology, unassigned
HMG box proteins, preferably being involved in transcriptional
control, nucleic acid binding, TRANSCRIPTION, preferably a
nonhistone chromosomal protein or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of carotene, in particular beta-carotene, in particular for
increasing the amount in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention said activity, e.g. the activity of a
nonhistone chromosomal protein. is increased or generated, e.g.
from Saccharomyces cerevisiae or a homolog thereof. The sequence of
YDR316W from Saccharomyces cerevisiae has been published in Jacq et
al., Nature 387 (6632 Suppl), 75-78, 1997, and Goffeau et al.,
Science 274 (5287), 546-547, 1996, and its activity is being
defined as putative S-adenosylmethionine-dependent
methyltransferase of the seven beta-strand family. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a protein of the superfamily of bioC homology, preferably
ofa putative S-adenosylmethionine-dependent methyltransferase of
the seven beta-strand family or its homolog, e.g. as shown herein,
for the production of the respective fine chemical, meaning of
carotene, in particular beta-carotene, in particular for increasing
the amount in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention said activity, e.g. the activity of a putative
S-adenosylmethionine-dependent methyltransferase of the seven
beta-strand family is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[10553] The sequence of YDR407C from Saccharomyces cerevisiae has
been published in Jacq et al., Nature 387 (6632 Suppl), 75-78,
1997, and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is being defined as targeting complex (TRAPP)
component involved in ER to Golgi membrane traffic. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a protein involved in the vesicular transport (Golgi
network, etc.), preferably being involved in targeting complex
(TRAPP) component involved in ER to Golgi membrane traffic, e.g. as
shown herein, for the production of the respective fine chemical,
meaning of IPP, or of carotenes, in particular for increasing the
amount in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention said activity, e.g. the activity of a protein. of the
vesicular transport (Golgi network, etc.) is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog thereof.
The sequence of YDR513W from Saccharomyces cerevisiae has been
published in Jacq et al., Nature 387 (6632 Suppl), 75-78, 1997, and
Goffeau et al., Science 274 (5287), 546-547, 1996, and its activity
is being defined as glutathione reductase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein of the superfamily of glutaredoxin, in particular
being involved in deoxyribonucleotide metabolism, cytoplasm, stress
response, detoxification, electron transport and
membrane-associated energy conservation, preferably a glutathione
reductase, or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, meaning of carotene, in particular
beta-carotene, in particular for increasing the amount in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention said activity,
e.g. the activity of a glutathione reductase is increased or
generated, e.g. from Saccharomyces cerevisiae or a homolog
thereof.
[10554] The sequence of YLL013C from Saccharomyces cerevisiae has
been published in Jacq et al., Nature 387 (6632 Suppl), 75-78,
1997, and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is being defined as a member of the PUF protein
family, which is named for the founding members, pumilio and Fbf.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein being involved in cell
differentiation, development (Systemic), nucleic acid binding,
transcriptional control, other transcription activities, preferably
a a member of the PUF protein family, which is named for the
founding members, pumilio and Fbf, or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of carotene, in particular beta-carotene, in particular for
increasing the amount in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention said activity, e.g. the activity of a a
member of the PUF protein family, which is named for the founding
members, pumilio and Fbf is increased or generated, e.g. from
Saccharomyces cerevisiae or a homolog thereof.
[10555] The sequence of b0481 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its protein is being defined as a protein with a YbaK-like
domain. Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein with a YbaK-like domain
from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of IPP,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention said protein with a YbaK-like domain is increased or
generated, e.g. from E. coli or a homolog thereof. The sequence of
b0970 from Escherichia coli K12 has been published in Blattner et
al., Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a glutamate receptor. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of Escherichia coli ybhL protein
superfamily, preferably a protein with the activity of a glutamate
receptor protein from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of glutamine
and/or isopentenylpyrophosphate, in particular for increasing the
amount of glutamine and/or isopentenylpyrophosphate, preferably
glutamine and/or isopentenylpyrophosphate in free or bound form in
an organism or a part thereof, as mentioned.
[10556] The sequence of b1736 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its protein is being defined as a protein with
[10557] PEP-dependent phosphotransferase activity. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a protein with PEP-dependent phosphotransferase activity
from E. coli or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of IPP,
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention the activity of the PEP-dependent phosphotransferase is
increased or generated, e.g. from E. coli or a homolog thereof.
[10558] The sequence of b1738 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its protein is being defined as a protein with PEP-dependent
phosphotransferase activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
with PEP-dependent phosphotransferase activity from E. coli or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of IPP, preferably in free or bound form in
an organism or a part thereof, as mentioned. In one embodiment, in
the process of the present invention the activity of a
PEP-dependent phosphotransferase is increased or generated, e.g.
from E. coli or a homolog thereof.
[10559] The sequence of b3160 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its protein is being defined as a monooxygenase protein with
luciferase-like ATPase activity. Accordingly, in one embodiment,
the process of the present invention comprises the use of a
monooxygenase protein with luciferase-like ATPase activity from E.
coli or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, meaning of IPP, preferably in free or
bound form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a monooxygenase protein with luciferase-like ATPase activity is
increased or generated, e.g. from E. coli or a homolog thereof.
[10560] [0023.0.24.24] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[10561] In one embodiment, the homolog of any one of the
polypeptides indicated in Table IIA or IIB, column 5 or 7, line 287
is a homolog of a polypeptide indicated in Table IIA or IIB, column
3, line 287 having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms preferably IPP, and in a
other preferred embodiment, also carotenes, in particular
beta-carotene. In one embodiment, the homologue is a homolog with a
sequence as indicated in Table I or II, column 7, line 287, resp.
In one embodiment, the homologue of one of the polypeptides
indicated in Table IIA or IIB, column 3, line 287 is derived from
an eukaryotic. In one embodiment, the homologue is derived from
Fungi. In one embodiment, the homologue of a polypeptide indicated
in Table IIA or IIB, column 3, line 287 is derived from Ascomyceta.
In one embodiment, the homologue of a polypeptide indicated in
Table IIA or IIB, column 3, line 287 is derived from
Saccharomycotina. In one embodiment, the homologue of a polypeptide
indicated in Table IIA or IIB, column 3, line 287 is derived from
Saccharomycetes. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 287 is a homologue
being derived from Saccharomycetales. In one embodiment, the
homologue of a polypeptide indicated in Table IIA or IIB, column 3,
line 287 is a homologue having the same or a similar activity being
derived from Saccharomycetaceae. In one embodiment, the homologue
of a polypeptide indicated in Table IIA or IIB, column 3, line 287
is a homologue having the same or a similar activity being derived
from Saccharomycetes.
[10562] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 5 or 7, line
278, 280, 281, 283, 284 or lines 637 to 641 is a homolog of the
polypeptide indicated in Table IIA or IIB, column 3, line 278, 280,
281, 283, 284 or lines 637 to 641, resp., having the same or a
similar activity. In particular an increase of activity confers an
increase in the content of the respective fine chemical in the
organisms preferably IPP, more preferably also carotenes, in
particular beta-carotene. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
line 278, 280, 281, 283, 284 or lines 637 to 641, resp. In one
embodiment, the homolog of one of the polypeptides indicated in
Table IIA or IIB, column 3, line 278, 280, 281, 283, 284 or lines
637 to 641 is derived from an bacteria. In one embodiment, the
homolog of a polypeptide indicated in Table IIA or IIB, column 3,
line 278, 280, 281, 283, 284 or lines 637 to 641 is derived from
Proteobacteria. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 278, 280, 281, 283,
284 or lines 637 to 641 is a homolog having the same or a similar
activity being derived from Gammaproteobacteria. In one embodiment,
the homolog of a polypeptide indicated in Table IIA or IIB, column
3, line 278, 280, 281, 283, 284 or lines 637 to 641 is derived from
Enterobacteriales. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 278, 280, 281, 283,
284 or lines 637 to 641 is a homolog being derived from
Enterobacteriaceae. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 278, 280, 281, 283,
284 or lines 637 to 641 is a homolog having the same or a similar
activity, in particular an increase of activity confers an increase
in the content of the IPP in the organisms or part thereof being
derived from Escherichia.
[10563] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 5 or 7, line
285, 286, 288 or 289 is a homolog of the polypeptide indicated in
Table IIA or IIB, column 3, line 285, 286, 288, or 289, resp.,
having the same or a similar activity. In particular an increase of
activity confers an increase in the content of the respective fine
chemical in the organisms, preferably beta-carotene. In one
embodiment, the homolog is a homolog with a sequence as indicated
in Table I or II, column 7, line 285, 286, 288 or 289, resp. In one
embodiment, the homolog of one of the polypeptides indicated in
Table IIA or IIB, column 3, line 285, 286, 288 or 289 is derived
from an eukaryotic. In one embodiment, the homolog is derived from
Fungi. In one embodiment, the homolog of a polypeptide indicated in
Table IIA or IIB, column 3, line 285, 286, 288 or 289 is derived
from Ascomyceta. In one embodiment, the homolog of a polypeptide
indicated in Table IIA or IIB, column 3, line 285, 286, 288 or 289
is derived from Saccharomycotina. In one embodiment, the homolog of
a polypeptide indicated in Table IIA or IIB, column 3, lines line
285, 286, 288 or 289 is derived from Saccharomycetes. In one
embodiment, the homolog of a polypeptide indicated in Table IIA or
IIB, column 3, line 285, 286, 288 or 289 is a homolog being derived
from Saccharomycetales. In one embodiment, the homolog of a
polypeptide indicated in Table IIA or IIB, column 3, line 285, 286,
288 or 289 is a homolog having the same or a similar activity being
derived from Saccharomycetaceae. In one embodiment, the homolog of
a polypeptide indicated in Table IIA or IIB, column 3, line 285,
286, 288 or 289 is a homolog having the same or a similar activity
being derived from Saccharomycetes.
[10564] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table IIA or IIB, column 5 or 7, line 279
or 282 is a homolog of the polypeptide indicated in Table IIA or
IIB, column 3, line 279 or 282, resp having the same or a similar
activity. In particular an increase of activity confers an increase
in the content of the respective fine chemical in the organisms,
preferably beta-carotene. In one embodiment, the homolog is a
homolog with a sequence as indicated in Table I or II, column 7,
line 279 or 282, resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table IIA or IIB, column 3, line 279 or
282 is derived from an bacteria. In one embodiment, the homolog of
a polypeptide indicated in Table IIA or IIB, column 3, line 279 or
282 is derived from Proteobacteria. In one embodiment, the homolog
of a polypeptide indicated in Table IIA or IIB, column 3, line 279
or 282 is a homolog having the same or a similar activity being
derived from Gammaproteobacteria. In one embodiment, the homolog of
a polypeptide indicated in Table IIA or IIB, column 3, line 279 or
282 is derived from Enterobacteriales. In one embodiment, the
homolog of a polypeptide indicated in Table IIA or IIB, column 3,
line 279 or 282 is a homolog being derived from Enterobacteriaceae.
In one embodiment, the homolog of a polypeptide indicated in Table
IIA or IIB, column 3, line 279 or 282 is a homolog having the same
or a similar activity, in particular an increase of activity
confers an increase in the content of the zeaxanthin in the
organisms or part thereof being derived from Escherichia.
[10565] [0023.1.24.24] Homologs of the polypeptides indicated in
Table IIA or IIB, column 3, lines 279, 282, 285, 286, 288, and/or
289, and/or lines 278, 280, 281, 283, 284, and/or 287 and/or lines
637 to 641, may be the polypeptides encoded by the nucleic acid
molecules indicated in Table IA or IB, column 7, lines 279, 282,
285, 286, 288, and/or 289, and/or lines 278, 280, 281, 283, 284,
and/or 287 and/or lines 637 to 641, or may be the polypeptides
indicated in Table IIA or IIB, column 7, lines 279, 282, 285, 286,
288 and/or 289, and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641.
[10566] Homologs of the polypeptides indicated in Table IIA or IIB,
column 3, lines 279, 282, 285, 286, 288, and/or 289, may be the
polypeptides encoded by the nucleic acid molecules indicated in
Table IA or IB, column 7, lines 279, 282, 285, 286, 288, and/or
289, respectively or may be the polypeptides indicated in Table IIA
or IIB, column 7, lines 279, 282, 285, 286, 288 and/or 289, having
a beta-carotene-content and/or -amount-increasing activity.
Homologs of the polypeptides indicated in Table IIA or IIB, column
3, lines 279, 282, 285, 286, 288 and/or 289 may be the polypeptides
encoded by the nucleic acid molecules indicated in Table IA or IB,
column 7, lines 279, 282, 285, 286, 288 and/or 289 or may be the
polypeptides indicated in Table IIA or IIB, column 7, lines 279,
282, 285, 286, 288 and/or 289 having a beta-carotene-content and/or
-amount-increasing activity.
[10567] Homologs of the polypeptides indicated in Table IIA or IIB,
column 3, lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637
to 641 may be the polypeptides encoded by the nucleic acid
molecules indicated in Table IA or IB, column 7, lines 278, 280,
281, 283, 284, and/or 287 and/or lines 637 to 641, respectively or
may be the polypeptides indicated in Table IIA or IIB, column 7,
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
having a IPP-content and/or -amount-increasing activity. Homologs
of the polypeptides indicated in Table IIA or IIB, column 3, lines
278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641 may be
the polypeptides encoded by the nucleic acid molecules indicated in
Table IA or IB, column 7, lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641 or may be the polypeptides indicated in
Table IIA or IIB, column 7, lines 278, 280, 281, 283, 284, and/or
287 and/or lines 637 to 641 having a IPP content- and/or
amount-increasing activity.
[10568] [0024.0.0.24] see [0024.0.0.0]
[10569] [0025.0.24.24] In accordance with the invention, a protein
or polypeptide has the "activity of an protein of the invention",
e.g. the activity of a protein indicated in Table IIA or IIB,
column 3, lines 279, 282, 285, 286, 288 and/or 289 if its de novo
activity, or its increased expression directly or indirectly leads
to an increased beta-carotene level in the organism or a part
thereof, preferably in a cell of said organism. In a preferred
embodiment, the protein or polypeptide has the above-mentioned
additional activities of a protein indicated in Table IIA or IIB,
column 3, lines 279, 282, 285, 286, 288, and/or 289. Throughout the
specification the activity or preferably the biological activity of
such a protein or polypeptide or an nucleic acid molecule or
sequence encoding such protein or polypeptide is identical or
similar if it still has the biological or enzymatic activity of any
one of the proteins indicated in Table IIA or IIB, column 3, lines
279, 282, 285, 286, 288, and/or 289, or which has at least 10% of
the original enzymatic activity, preferably 20%, particularly
preferably 30%, most particularly preferably 40% in comparison to
any one of the proteins indicated in Table IIA or IIB, column 3,
lines 285, 286, 288 or 289 of Saccharomyces cerevisiae and/or any
one of the proteins indicated in Table IIA or IIB, column 3, lines
279 or 282 of E. coli K12.
[10570] In accordance with the invention, a protein or polypeptide
has the "activity of an protein of the invention", e.g. the
activity of a protein indicated in Table IIA or IIB, column 3,
lines lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641 if its de novo activity, or its increased expression directly
or indirectly leads to an increased IPP level in the organism or a
part thereof, preferably in a cell of said organism. In a preferred
embodiment, the protein or polypeptide has the above-mentioned
additional activities of a protein indicated in Table IIA or IIB,
column 3, lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637
to 641. Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of any one of the proteins indicated in Table IIA or IIB,
column 3, lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637
to 641, or which has at least 10% of the original enzymatic
activity, preferably 20%, particularly preferably 30%, most
particularly preferably 40% in comparison to any one of the
proteins indicated in Table IIA or IIB, column 3, line 287 of
Saccharomyces cerevisiae and/or any one of the proteins indicated
in Table IIA or IIB, column 3, lines lines 278, 280, 281, 283
and/or 284 and/or lines 637 to 641 of E. coli K12.
[10571] In one embodiment, the polypeptide of the invention confers
said activity, e.g. the increase of the respective fine chemical in
an organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table I,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table I,
column 4 are derived from the same families, orders, classes or
phylums.
[10572] [0025.1.0.24] see [0025.1.0.0]
[10573] [0026.0.0.24] to [0033.0.0.24]: see [0026.0.0.0] to
[0033.0.0.0]
[10574] [0034.0.24.24] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of an protein having the activity of a
protein as indicated in Table IIA or IIB, column 3, lines 279, 282,
285, 286, 288, and/or 289, and/or lines 278, 280, 281, 283, 284,
and/or 287 and/or lines 637 to 641, or being encoded by a nucleic
acid molecule indicated in Table IA or IB, column 5, lines 279,
282, 285, 286, 288, and/or 289, and/or lines 278, 280, 281, 283,
284 and/or 287 and/or lines 637 to 641, or its homologs, e.g. as
indicated in Table IA or IB, column 7, lines 279, 282, 285, 286,
288, and/or 289, and/or lines 278, 280, 281, 283, 284, and/or 287
and/or lines 637 to 641, its biochemical or genetic causes. It
therefore shows the increased amount of the respective fine
chemical.
[10575] [0035.0.0.24] to [0038.0.0.24]: see [0035.0.0.0] to
[0038.0.0.0]
[10576] [0039.0.0.24] see [0039.0.0.0]
[10577] [0040.0.0.24] to [0044.0.0.24]: see [0040.0.0.0] to
[0044.0.0.0]
[10578] [0045.0.24.24] In case the activity of the Escherichia coli
K12 protein b0730 or its homologs e.g. a transcriptional regulator
of succinylCoA synthetase operon and fatty acyl response regulator,
e.g. as indicated in Table IIA or IIB, columns 5 or 7, 278, is
increased, preferably, in one embodiment the increase of the
respective fine chemical, preferably of IPP between 41% and 115% or
more is conferred.
[10579] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1829 or its homologs e.g. a heat shock protein
with protease activity, e.g. as indicated in Table IIA or IIB,
columns 5 or 7, line 279, is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of beta-carotene, between 33% and 74% or more is conferred.
[10580] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1926 or its homologs e.g. flagellar protein fliT,
e.g. as indicated in Table IIA or IIB, columns 5 or 7, line 280, is
increased, preferably, in one embodiment the increase of the
respective fine chemical, preferably of IPP, between 37% and 55% or
more is conferred.
[10581] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2211 or its homologs e.g. ATP-binding transport
proteins of the ABC superfamily, e.g. as indicated in Table IIA or
IIB, columns 5 or 7, line 281, is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of IPP, between 46% and 48% or more is conferred.
[10582] In one embodiment, in case the activity of the Escherichia
coli K12 protein b2699 or its homologs e.g. a DNA recombination and
DNA repair activity, a pheromone response activity, a mating-type
determination activity, a sex-specific protein activity, a
nucleotide binding activity and/or a protease and nuclease
activity, in particular a DNA strand exchange and recombination
protein with protease and nuclease activity, in particular of the
superfamily of the recombination protein recA, e.g. as indicated in
Table IIA or IIB, columns 5 or 7, line 282, is increased,
preferably, in one embodiment the increase of the respective fine
chemical, preferably of beta-carotene, between 43% and 76% or more
is conferred.
[10583] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3172 or its homologs e.g. argininosuccinate
synthetase, e.g. as indicated in Table IIA or IIB, columns 5 or 7,
line 283, is increased, preferably, in one embodiment the increase
of the respective fine chemical, preferably of IPP, between 35% and
74% or more is conferred.
[10584] In one embodiment, in case the activity of the Escherichia
coli K12 protein b4129 or its homologs e.g. an inducible lysine
tRNA synthetase, e.g. as indicated in Table IIA or IIB, columns 5
or 7, line 284, is increased, preferably, in one embodiment the
increase of the respective fine chemical, preferably of IPP,
between 34% and 63% or more is conferred.
[10585] In one embodiment, in case the activity of the
Saccharomyces cerevisiae protein YBR089C-A or its homologs, e.g. a
nonhistone chromosomal protein, e.g. as indicated in Table IIA or
IIB, columns 5 or 7, line 285, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of beta-carotene between 52% and 108% or more is conferred.
[10586] In one embodiment, in case the activity of the
Saccharomyces cerevisiae protein YDR316W or its homologs, e.g. a
putative S-adenosylmethionine-dependent methyltransferase of the
seven beta-strand family, e.g. as indicated in Table IIA or IIB,
columns 5 or 7, line 286, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of beta-carotene between 28% and 52% or more is conferred.
[10587] In one embodiment, in case the activity of the
Saccharomyces cerevisiae protein YDR407C or its homologs, e.g. a
targeting complex (TRAPP) component involved in ER to Golgi
membrane traffi or its homolog, e.g. as indicated in Table IIA or
IIB, columns 5 or 7, line 287, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of IPP between 54% and 214% or more is conferred.
[10588] In one embodiment, in case the activity of the
Saccharomyces cerevisiae protein YDR513W or its homologs, e.g. a
glutathione reductase, e.g. as indicated in Table IIA or IIB,
columns 5 or 7, line 288, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of beta-carotene between 39% and 43% or more is conferred.
[10589] In one embodiment, in case the activity of the
Saccharomyces cerevisiae protein YLL013C or its homologs, e.g. a
member of the PUF protein family, which is named for the founding
members, pumilio and Fbf, e.g. as indicated in Table IIA or IIB,
columns 5 or 7, line 289, is increased, preferably, in one
embodiment an increase of the respective fine chemical, preferably
of beta-carotene between 43% and 50% or more is conferred.
[10590] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0481 or its homologs e.g. a protein with a
YbaK-like domain, e.g. as indicated in Table IIA or IIB, columns 5
or 7, line 637, is increased, preferably, in one embodiment the
increase of the respective fine chemical, preferably of IPP,
between 35% and 39% or more is conferred.
[10591] In one embodiment, in case the activity of the Escherichia
coli K12 protein b0970 or its homologs e.g. a glutamate receptor,
e.g. as indicated in Table IIA or IIB, columns 5 or 7, line 638, is
increased, preferably, in one embodiment the increase of the
respective fine chemical, preferably of IPP, between 36% and 117%
or more is conferred.
[10592] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1736 or its homologs e.g. a PEP-dependent
phosphotransferase, e.g. as indicated in Table IIA or IIB, columns
5 or 7, line 639, is increased, preferably, in one embodiment the
increase of the respective fine chemical, preferably of IPP,
between 51% and 137% or more is conferred.
[10593] In one embodiment, in case the activity of the Escherichia
coli K12 protein b1738 or its homologs e.g a PEP-dependent
phosphotransferase, e.g. as indicated in Table IIA or IIB, columns
5 or 7, line 640, is increased, preferably, in one embodiment the
increase of the respective fine chemical, preferably of IPP,
between 63% and 160% or more is conferred.
[10594] In one embodiment, in case the activity of the Escherichia
coli K12 protein b3160 or its homologs e.g. a monooxygenase with
luciferase-like ATPase activity, e.g. as indicated in Table IIA or
IIB, columns 5 or 7, line 641, is increased, preferably, in one
embodiment the increase of the respective fine chemical, preferably
of IPP, between 35% and 47% or more is conferred.
[10595] [0046.0.24.24] In one embodiment, if the activity of the
Escherichia coli K12 protein b0730 or its homologs e.g.
transcriptional regulator of succinylCoA synthetase operon and
fatty acyl response regulator, e.g. as indicated in Table IIA or
IIB, columns 5 or 7, line 278, or of b1829 or its homologs, e.g.
heat shock protein with protease activity, e.g. as indicated in
Table IIA or IIB, columns 5 or 7, line 279, or of b1926 or its
homologs, e.g. flagellar protein fliT, e.g. as indicated in Table
IIA or IIB, columns 5 or 7, line 280, or of b2211 or its homologs,
e.g. ATP-binding transport proteins of the ABC superfamily, e.g. as
indicated in Table IIA or IIB, columns 5 or 7, line 281, or of
b2699 or its homologs, e.g. DNA strand exchange and recombination
protein with protease and nuclease activity, e.g. as indicated in
Table IIA or IIB, columns 5 or 7, line 282, or of b3172 or its
homologs, e.g. argininosuccinate synthetase, e.g. as indicated in
Table IIA or IIB, columns 5 or 7, line 283, or of b4129 or its
homologs, e.g. an inducible lysine tRNA synthetase, e.g. as
indicated in Table IIA or IIB, columns 5 or 7, line 284, is
increased; preferably an increase of the respective fine chemical
and of further carotenoids, preferably carotenes, is conferred.
[10596] In one embodiment, in case the activity of the
Saccharomyces cerevisiae protein YBR089C-A or its homologs, e.g. a
nonhistone chromosomal protein, e.g. as indicated in Table IIA or
IIB, columns 5 or 7, line 285 or YDR316W or its homologs, e.g. a
Putative S-adenosylmethionine-dependent methyltransferase of the
seven beta-strand family, e.g. as indicated in Table IIA or IIB,
columns 5 or 7, line 286 or YDR407C or its homologs, e.g. a
targeting complex (TRAPP) component involved in ER to Golgi
membrane traffic, e.g. as indicated in Table IIA or IIB, columns 5
or 7, line 287 or YDR513W or its homologs, e.g. a glutathione
reductase, e.g. as indicated in Table IIA or IIB, columns 5 or 7,
line 288 or YLL013C or its homologs, e.g. a member of the PUF
protein family, which is named for the founding members, pumilio
and Fbf, e.g. as indicated in Table IIA or IIB, columns 5 or 7,
line 289, e.g. as indicated in Table IIA or IIB, columns 5 or 7,
line 637 or b0481 or its homologs, e.g. a protein with a YbaK-like
domain, e.g. as indicated in Table IIA or IIB, columns 5 or 7, line
638 or b0970 or its homologs, e.g. a glutamate receptor, e.g. as
indicated in Table IIA or IIB, columns 5 or 7, line 639 or b1736 or
its homologs, e.g. a PEP-dependent phosphotransferase, e.g. as
indicated in Table IIA or IIB, columns 5 or 7, line 640 or b1738 or
its homologs, e.g. a PEP-dependent phosphotransferase, e.g. as
indicated in Table IIA or IIB, columns 5 or 7, line 641 or b3160 or
its homologs, e.g. a monooxygenase with luciferase-like ATPase
activity is increased, preferably an increase of the respective
fine chemical and of further carotenoids, preferably carotenes, is
conferred.
[10597] [0047.0.0.24] to [0048.0.0.24]: see [0047.0.0.0] to
[0048.0.0.0]
[10598] [0049.0.24.24] A protein having an activity conferring an
increase in the amount or level of the respective fine chemical
preferably has the structure of the polypeptide described herein.
In a particular embodiment, the polypeptides used in the process of
the present invention or the polypeptide of the present invention
comprises the sequence of a consensus sequence as indicated in
Table IV, columns 5 or 7, lines lines 279, 282, 285, 286, 288
and/or 289, or of a polypeptide as indicated in Table IA or IB,
columns 5 or 7, lines 279, 282, 285, 286, 288, and/or 289 or of a
functional homologue thereof as described herein, or of a
polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table IA or IB,
columns 5 or 7, lines 279, 282, 285, 286, 288, and/or 289 or its
herein described functional homologues and has the herein mentioned
activity conferring an increase in the beta-carotene level.
[10599] A protein having an activity conferring an increase in the
amount or level of the respective fine chemical preferably has the
structure of the polypeptide described herein. In a particular
embodiment, the polypeptides used in the process of the present
invention or the polypeptide of the present invention comprises the
sequence of a consensus sequence as indicated in Table IV, columns
5 or 7, lines 278, 280, 281, 283, 284 and/or 287 and/or 637 to 641,
or of a polypeptide as indicated in Table IIA or IIB, columns 5 or
7 lines 278, 280, 281, 283, 284 and/or 287 and/or 637 to 641 or of
a functional homologue thereof as described herein, or of a
polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table IA or IB,
columns 5 or 7, lines 278, 280, 281, 283, 284 and/or 287 and/or 637
to 641 or its herein described functional homologues and has the
herein mentioned activity conferring an increase in the IPP
level.
[10600] [0050.0.24.24] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of beta-carotene or IPP, resp., or their ester, or glucoside
thereof, e.g the diglucoside thereof.
[10601] [0051.0.24.24] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g compositions comprising IPP or
beta-carotene. Depending on the choice of the organism used for the
process according to the present invention, for example a
microorganism or a plant, compositions or mixtures of various IPP
or beta-carotenecan be produced.
[10602] [0052.0.0.24] see [0052.0.0.0]
[10603] [0053.0.24.24] In one embodiment, the process of the
present invention comprises one or more of the following steps
[10604] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptide of the invention, e.g. of a
polypeptide having an activity of a protein as indicated in Table
IIA or IIB, column 3, lines 279, 282, 285, 286, 288, and/or 289,
and/or lines 278, 280, 281, 283, 284, and/or 287 and/or 637 to 641,
or its homologs, e.g. as indicated in Table IIA or IIB, columns 5
or 7, lines 279, 282, 285, 286, 288, and/or 289, and/or lines 278,
280, 281, 283, 284, and/or 287 and/or 637 to 641, activity having
herein-mentioned the respective fine chemical increasing activity;
[10605] b) stabilizing a mRNA conferring the increased expression
of a protein encoded by the nucleic acid molecule of the invention,
e.g. of a polypeptide having an activity of a protein as indicated
in Table IIA or IIB, column 3, lines 279, 282, 285, 286, 288,
and/or 289 and/or 637 to 641, and/or lines 278, 280, 281, 283, 284,
and/or 287, or its homologs activity, e.g. as indicated in Table
IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288, and/or
289 and/or 637 to 641, and/or lines 278, 280, 281, 283, 284, and/or
287, or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned the respective fine chemical increasing
activity; [10606] c) increasing the specific activity of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
present invention having herein-mentioned the respective fine
chemical increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table IIA or IIB, column 3,
lines 279, 282, 285, 286, 288, and/or 289 and/or 637 to 641, and/or
lines 278, 280, 281, 283, 284, and/or 287, or its homologs
activity, e.g. as indicated in Table IIA or IIB, columns 5 or 7,
lines 279, 282, 285, 286, 288, and/or 289 and/or 637 to 641, and/or
lines 278, 280, 281, 283, 284, and/or 287, or decreasing the
inhibitory regulation of the polypeptide of the invention; [10607]
d) generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table IIA or IIB, column 3, lines 279, 282,
285, 286, 288, and/or 289 and/or 637 to 641, and/or lines 278, 280,
281, 283, 284, and/or 287, or its homologs activity, e.g. as
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288, and/or 289 and/or 637 to 641, and/or lines 278, 280, 281,
283, 284, and/or 287; [10608] e) stimulating activity of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the present invention or a polypeptide of
the present invention having herein-mentioned the respective fine
chemical increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table IIA or IIB, column 3,
lines 279, 282, 285, 286, 288, and/or 289 and/or 637 to 641, and/or
lines 278, 280, 281, 283, 284, and/or 287 or its homologs activity,
e.g. as indicated in Table IIA or IIB, columns 5 or 7, lines 279,
282, 285, 286, 288, and/or 289 and/or 637 to 641, and/or lines 278,
280, 281, 283, 284, and/or 287, by adding one or more exogenous
inducing factors to the organism or parts thereof; [10609] f)
expressing a transgenic gene encoding a protein conferring the
increased expression of a polypeptide encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention, having herein-mentioned the respective fine chemical
increasing activity, e.g. of a polypeptide having an activity of a
protein as indicated in Table IIA or IIB, column 3, lines 279, 282,
285, 286, 288, and/or 289 and/or 637 to 641, and/or lines 278, 280,
281, 283, 284, and/or 287 or its homologs activity, e.g. as
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288, and/or 289 and/or 637 to 641, and/or lines 278, 280, 281,
283, 284, and/or 287, and/or [10610] g) increasing the copy number
of a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned the respective fine chemical increasing
activity, e.g. of a polypeptide having an activity of a protein as
indicated in Table IIA or IIB, column 3, lines 279, 282, 285, 286,
288, and/or 289 and/or 637 to 641, and/or lines 278, 280, 281, 283,
284, and/or 287 or its homologs, e.g. as indicated in Table IIA or
IIB, columns 5 or 7, lines 279, 282, 285, 286, 288, and/or 289
and/or 637 to 641, and/or lines 278, 280, 281, 283, 284, and/or
287, activity. [10611] h) Increasing the expression of the
endogenous gene encoding the polypeptide of the invention, e.g. a
polypeptide having an activity of a protein as indicated in Table
IIA or IIB, column 3, lines 279, 282, 285, 286, 288, and/or 289
and/or 637 to 641, and/or lines 278, 280, 281, 283, 284, and/or 287
or its homologs activity, e.g. as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288, and/or 289 and/or
637 to 641 and/or lines 278, 280, 281, 283, 284, and/or 287, by
adding positive expression or removing negative expression
elements, e.g. homologous recombination can be used to either
introduce positive regulatory elements like for plants the 35S
enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; [10612]
and/or [10613] i) Modulating growth conditions of an organism in
such a manner, that the expression or activity of the gene encoding
the protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced respective fine chemical
production. [10614] j) selecting of organisms with especially high
activity of the proteins of the invention from natural or from
mutagenized resources and breeding them into the target organisms,
e.g. the elite crops.
[10615] [0054.0.24.24] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table IIA or IIB, columns 5
or 7, lines 279, 282, 285, 286, 288, and/or 289, and/or lines 278,
280, 281, 283, 284, and/or 287 and/or lines 637 to 641, resp., or
its homologs activity, e.g. as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288, and/or 289, and/or
lines 278, 280, 281, 283, 284, and/or 287 and/or lines 637 to 641
resp.
[10616] [0055.0.0.24] to [0064.0.0.24]: see [0055.0.0.0] to
[0064.0.0.0]
[10617] [0065.0.0.24]: see [0065.0.0.0]
[10618] [0066.0.0.24] to [0067.0.0.24]: see [0066.0.0.0] to
[0067.0.0.0]
[10619] [0068.0.24.24] The mutation is introduced in such a way
that the production of the respective fine chemical, in particular
beta-carotene or IPP is not adversely affected.
[10620] [0069.0.0.24] see [0069.0.0.0]
[10621] [0070.0.24.24] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolites composition in the organism, e.g. an advantageous
composition of carotenoids, in particular carotenes, in particular
beta-carotene, e.g. comprising a higher content of (from a
viewpoint of nutritional physiology limited) carotenoids, in
particular carotene, in particular beta-carotene or precursors
thereof, e.g. IPP.
[10622] [0071.0.0.24] see [0071.0.0.0]
[10623] [0072.0.24.24] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are
further carotenoids, e.g. carotenes or carotene, e.g.
ketocarentoids, or hydrocarotenoids, e.g. lutein, lycopene,
alpha-carotene, or beta-carentene, or other compounds for which IPP
is a precursor well known for the skilled.
[10624] [0073.0.24.24] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
(c) providing a non-human organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant; (d) increasing an activity of a polypeptide of the
invention or a homolog thereof, e.g. as indicated in Table IIA or
IIB, columns 5 or 7, lines 279, 282, 285, 286, 288, and/or 289,
and/or lines 278, 280, 281, 283, 284, and/or 287 and/or lines 637
to 641 or of a polypeptide being encoded by the nucleic acid
molecule of the present invention and described below, e.g.
conferring an increase of the respective fine chemical in an
organism, preferably in a microorganism, a non-human animal, a
plant or animal cell, a plant or animal tissue or a plant, (e)
growing an organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a plant
under conditions which permit the production of the respective fine
chemical in the organism, preferably the microorganism, the plant
cell, the plant tissue or the plant; and (f) if desired,
recovering, optionally isolating, the free and/or bound the
respective fine chemical and, optionally further free and/or bound
carotenoids, in particular carotene, in particular beta-carotene or
its precursor IPP, synthesized by the organism, the microorganism,
the non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[10625] [0074.0.24.24] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the respective fine chemical or the free and
bound the respective fine chemical but as option it is also
possible to produce, recover and, if desired isolate, other free
or/and bound carotenoids, in particular carotene, in particular
beta-carotene or the carotene precursor IPP.
[10626] [0075.0.0.24] to [0077.0.0.24]: see [0075.0.0.0] to
[0077.0.0.0]
[10627] [0078.0.24.24] The organism such as microorganisms or
plants or the recovered, and if desired isolated, the respective
fine chemical can then be processed further directly into
foodstuffs or animal feeds or for other applications, for example
according to the disclosures made in U.S. Pat. No. 6,399,115:
Method and composition for the treatment of benign prostate
hypertrophy (BPH) and prevention of prostate cancer
U.S. Pat. No. 6,399,114: Nutritional system for nervous system
disorders U.S. Pat. No. 6,399,060: Composition having nematicidal
activity U.S. Pat. No. 6,399,046: Use of a content of catechins or
a content of green tea extract in cosmetic preparations for tanning
the skin U.S. Pat. No. 6,395,782: Method of increasing longevity
and preventing body weight wasting in autoimmune disease by using
conjugated linoleic acid U.S. Pat. No. 6,395,508: Peptide mixture
and products thereof U.S. Pat. No. 6,395,315: Fermentation
composition, process for preparing the same, and use thereof U.S.
Pat. No. 6,395,311: Multicomponent biological vehicle U.S. Pat. No.
6,394,230: Sterol esters as food additives U.S. Pat. No. 6,391,640:
Methods and compositions for cellular and metabolic engineering
U.S. Pat. No. 6,391,332: Therapeutic micronutrient composition for
severe trauma, burns and critical illness U.S. Pat. No. 6,391,321:
Emulsifier-free finely disperse systems of the oil-in-water and
water-in-oil type U.S. Pat. No. 6,391,319: Cosmetic and
dermatological emulsions comprising alkyl glucosides and increased
electrolyte concentrations U.S. Pat. No. 6,391,289: Use of
sunscreen combinations comprising, as essential constituent,
4,4'-diarylbutadienes as photostable UV filters in cosmetic and
pharmaceutical preparations U.S. Pat. No. 6,387,961: Alkyl
2-acetamido-2-deoxyglucopyranoside and methods of emulsifying U.S.
Pat. No. 6,387,927: Epothilone derivatives and their use as
antitumor agents U.S. Pat. No. 6,387,883: Method for the prevention
and treatment of cachexia and anorexia U.S. Pat. No. 6,387,878:
Methods of treating intestinal ischemia using heparin-binding
epidermal growth factor U.S. Pat. No. 6,387,862: Bleach
compositions U.S. Pat. No. 6,387,418: Pomegranate extracts and
methods of using thereof U.S. Pat. No. 6,387,370: Compositions
containing extracts of Morinda citrifolia, red wine, prune,
blueberry, pomegranate, apple and enzyme mixture U.S. Pat. No.
6,387,355: Use of sunscreen combinations comprising, as essential
constituent, amino-substituted hydroxybenzophenones as photostable
UV filters in cosmetic and pharmaceutical preparations U.S. Pat.
No. 6,384,090: Preparation of active ingredient dispersions and
apparatus therefor U.S. Pat. No. 6,384,085: Material separated from
Ecklonia cava, method for extracting and purifying the same, and
use thereof as antioxidants U.S. Pat. No. 6,383,751: Assessing
lipid metabolism U.S. Pat. No. 6,383,543: Process for the
extraction of an organic salt from plants, the salt, and other
similar salts U.S. Pat. No. 6,383,524: Compositions and methods for
enhancing therapeutic effects U.S. Pat. No. 6,383,523:
Pharmaceutical compositions and methods for managing skin
conditions U.S. Pat. No. 6,383,503: PREPARATIONS OF THE W/O
EMULSION TYPE WITH AN INCREASED WATER CONTENT, ADDITIONALLY
COMPRISING ONE OR MORE ALKYLMETHICONE COPOLYOLS AND/OR
ALKYLDIMETHICONE COPOLYOLS, AND, IF DESIRED, CATIONIC POLYMERS U.S.
Pat. No. 6,383,474: Carotenoid preparation U.S. Pat. No. 6,383,473:
Solid composition for reducing tooth erosion U.S. Pat. No.
6,380,442: Process for the isolation of mixed carotenoids from
plants U.S. Pat. No. 6,380,232: Benzimidazole urea derivatives, and
pharmaceutical compositions and unit dosages thereof U.S. Pat. No.
6,380,227: Fermentative preparation process for and crystal forms
of cytostatics U.S. Pat. No. 6,379,697: Stabilization of
photosensitive materials U.S. Pat. No. 6,379,683: Nanocapsules
based on dendritic polymers U.S. Pat. No. 6,376,722: Lutein to
zeaxanthin isomerization process and product U.S. Pat. No.
6,376,717: Preparation of astaxanthin U.S. Pat. No. 6,376,544:
Nutritional product for a person having renal failure U.S. Pat. No.
6,376,498: Pharmaceutical, cosmetic and/or food composition with
antioxidant properties U.S. Pat. No. 6,376,455: Quaternary ammonium
compounds, compositions containing them, and uses thereof U.S. Pat.
No. 6,376,005: Antimicrobial composition for food and beverage
products U.S. Pat. No. 6,375,993: Pomegranate extracts and methods
of using thereof U.S. Pat. No. 6,375,992: Methods of hydrating
mammalian skin comprising oral administration of a defined
composition U.S. Pat. No. 6,375,963: Bioadhesive hot-melt extruded
film for topical and mucosal adhesion applications and drug
delivery and process for preparation thereof U.S. Pat. No.
6,375,956: Strip pack U.S. Pat. No. 6,375,873: Process and
apparatus for producing stably fine-particle powders U.S. Pat. No.
6,372,964: For higher basidiomycetes mushrooms grown as biomass in
submerged culture U.S. Pat. No. 6,372,946: Preparation of
4,4'-diketo-.beta.-carotene derivatives
[10628] The cited literature describe some preferred embodiments.
Said applications describe some advantageous embodiments without
meant to be limiting. The fermentation broth, fermentation
products, plants or plant products can be purified as described in
above mentioned applications and other methods known to the person
skilled in the art, e.g. as described in Methods in Enzymology:
Carotenoids, Part A:
[10629] Chemistry, Separation, Quantitation and Antioxidation, by
John N Abelson or Part B, Metabolism, Genetics, and Biochemistry,
or described herein below. Products of these different work-up
procedures are IPP and/or beta-carotenes and optional other
carotenoids, comprising compositions which still comprise
fermentation broth, plant particles and cell components in
different amounts, advantageously in the range of from 0 to 99% by
weight, preferably below 80% by weight, especially preferably
between below 50% by weight.
[10630] [0079.0.0.24] to [0084.0.0.24]: see [0079.0.0.0] to
[0084.0.0.0]
[10631] [0084.2.24.24] The invention also contemplates embodiments
in which the [3-carotene, or other carotenoid precursor compounds
in the production of the respective fine chemical like IPP, is
present in the flowers of the flowering plant chosen as the host
(for example, marigolds). The invention also contemplates
embodiments in which a host plant's flowers lack .beta.-carotene or
other carotenoid precursors, such as IPP. In a plant of the latter
type, the inserted DNA includes genes that code for carotenoid
precursors (compounds that can be converted biologically into
.beta.-carotene) and a ketolase, as well as a hydroxylase, if
otherwise absent.
[10632] In one embodiment, preferred flowering plants include, but
are not limited to: Amaryllidaceae (Allium, Narcissus); Apocynaceae
(Catharanthus); Asteraceae, alternatively Compositae (Aster,
Calendula, Callistephus, Cichorium, Coreopsis, Dahlia,
Dendranthema, Gazania, Gerbera, Helianthus, Helichrysum, Lactuca,
Rudbeckia, Tagetes, Zinnia); Balsaminaceae (Impatiens); Begoniaceae
(Begonia); Caryophyllaceae (Dianthus); Chenopodiaceae (Beta,
Spinacia); Cucurbitaceae (Citrullus, Curcurbita, Cucumis);
Cruciferae (Alyssum, Brassica, Erysimum, Matthiola, Raphanus);
Gentinaceae (Eustoma); Geraniaceae (Pelargonium); Graminae,
alternatively Poaceae, (Avena, Horedum, Oryza, Panicum, Pennisetum,
Poa, Saccharum, Secale, Sorghum, Triticum, Zea); Euphorbiaceae
(Poinsettia); Labiatae (Salvia); Leguminosae (Glycine, Lathyrus,
Medicago, Phaseolus, Pisum); Liliaceae (Cilium); Lobeliaceae
(Lobelia); Malvaceae (Abelmoschus, Gossypium, MaIva);
Plumbaginaceae (Limonium); Polemoniaceae (Phlox); Primulaceae
(Cyclamen); Ranunculaceae (Aconitum, Anemone, Aquilegia, Caltha,
Delphinium, Ranunculus); Rosaceae (Rosa); Rubiaceae (Pentas);
Scrophulariaceae (Angelonia, Antirrhinum, Torenia); Solanaceae
(Capsicum, Lycopersicon, Nicotiana, Petunia, Solanum); Umbelliferae
(Apium, Daucus, Pastinaca); Verbenaceae (Verbena, Lantana);
Violaceae (Viola).
[10633] [0085.0.24.24] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [10634] a) a nucleic acid sequence as
indicated in Table IA or IB, columns 5 or 7, lines 279, 282, 285,
286, 288, and/or 289, and/or lines 278, 280, 281, 283, 284, and/or
287 and/or lines 637 to 641, or a derivative thereof, or [10635] b)
a genetic regulatory element, for example a promoter, which is
functionally linked to the nucleic acid sequence as indicated in
Table IA or IB, columns 5 or 7, lines 279, 282, 285, 286, 288,
and/or 289, and/or lines 278, 280, 281, 283, 284, and/or 287 and/or
lines 637 to 641, or a derivative thereof, or [10636] c) (a) and
(b) is/are not present in its/their natural genetic environment or
has/have been modified by means of genetic manipulation methods, it
being possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[10637] [0086.0.0.24] to [0087.0.0.24]: see [0086.0.0.0] to
[0087.0.0.0]
[10638] [0088.0.24.24] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned
carotene, in particular beta-carotene and the general amount of
xanthohylls as energy source in feed. Further, this is also
important for the production of cosmetic compostions since, for
example, the antioxidant level of plant extracts is dependent on
the amount of th abovementioned carotene, in particular
beta-carotene and/or the amount of carotenoids as antioxidants.
[10639] [0088.1.0.24] see [0088.1.0.0]
[10640] [0089.0.0.24] to [0090.0.0.24]: see [0089.0.0.0] to
[0090.0.0.0]
[10641] [0091.0.24.24] Thus, the content of plant components and
preferably also further impurities is as low as possible, and the
abovementioned carotene, in particular beta-carotene, in particular
the fine chemical, is/are obtained in as pure form as possible. In
these applications, the content of plant components advantageously
amounts to less than 10%, preferably 1%, more preferably 0.1%, very
especially preferably 0.01% or less.
[10642] [0092.0.0.24] to [0094.0.0.24]: see [0092.0.0.0] to
[0094.0.0.0]
[10643] [0095.0.24.24] It may be advantageous to increase the pool
of said carotenoids, in particular carotene, in particular
beta-carotene in the transgenic organisms by the process according
to the invention in order to isolate high amounts of the pure
respective fine chemical.
[10644] [0096.0.24.24] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptide or a compound, which
functions as a sink for the desired fine chemical, for example
zeaxanthin or cryptoxanthin in the organism, is useful to increase
the production of the respective fine chemical. It has been
reported, that the inhibition of Lyopene production increases the
amount of other carotene, in particular beta-carotene in the cell.
Further it may be, that the inhibition of enzymes using zeaxanthin
or cryptoxanthin as substrate increases the amount of said
chemicals in a cell. For example, in one embodiment, it can be
advantageous to inhibit the production of astaxanthin, if a high
amount of cryptoxanthin or zeaxanthin is desired.
[10645] [0097.0.24.24] Glucosides, in particular, dicglucosides of
the zeaxanthin and beta-cryptoxanthin as well as other modification
of zeaxanthin and cryptoxanthin are known to a person skilled in
the art. In may also be advantageous to increase the content of the
bound respective fine chemical, e.g. of modification of zeaxanthin
and cryptoxanthin, in particular its glucosides, e.g.
diglucosides.
[10646] [0098.0.24.24] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated. The production of further carotenoids, e.g.
carotenes or carotene, in particular beta-carotene, in particular
ketocarentoids or hydrocarotenoids, e.g. lutein, lycopene,
alpha-carotene, or beta-carentene, or compounds for which the
respective fine chemical is a biosynthesis precursor compounds,
e.g. astaxanthin, or mixtures thereof or mixtures of other
carotenoids, in particular of carotene, in particular
beta-carotene, by the process according to the invention is
advantageous.
[10647] [0099.0.24.24] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[10648] [0100.0.24.24] Transgenic plants which comprise the
carotenoids such as said carotene, in particular beta-carotene,
e.g. cryptoxanthin or zeaxanthin (or astaxanthin as it is
synthesized from cryptoxanthin or zeaxanthin) synthesized in the
process according to the invention can advantageously be marketed
directly without there being any need for the carotenoids
synthesized to be isolated. Plants for the process according to the
invention are listed as meaning intact plants and all plant parts,
plant organs or plant parts such as leaf, stem, seeds, root,
tubers, anthers, fibers, root hairs, stalks, embryos, calli,
cotelydons, petioles, harvested material, plant tissue,
reproductive tissue and cell cultures which are derived from the
actual transgenic plant and/or can be used for bringing about the
transgenic plant. In this context, the seed comprises all parts of
the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue.
[10649] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, (in the form of their oils, fats,
lipids, as extracts, e.g. ether, alcohol, or other organic solvents
or water containing extract and/or free carotene, in particular
beta-carotene. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. E.g., oils,
fats, and/or lipids comprising carotene, in particular
beta-carotene can be obtained by what is known as cold beating or
cold pressing without applying heat. To allow for greater ease of
disruption of the plant parts, specifically the seeds, they can
previously be comminuted, steamed or roasted. Seeds, which have
been pretreated in this manner can subsequently be pressed or
extracted with solvents such as warm hexane. The solvent is
subsequently removed. In the case of microorganisms, the latter
are, after harvesting, for example extracted directly without
further processing steps or else, after disruption, extracted via
various methods with which the skilled worker is familiar.
Thereafter, the resulting products can be processed further, i.e.
degummed and/or refined. In this process, substances such as the
plant mucilages and suspended matter can be first removed. What is
known as desliming can be affected enzymatically or, for example,
chemico-physically by addition of acid such as phosphoric acid.
[10650] Because carotenoids in microorganisms are localized
intracellular, their recovery essentials comes down to the
isolation of the biomass. Well-established approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Of the residual
hydrocarbon, adsorbed on the cells, has to be removed. Solvent
extraction or treatment with surfactants have been suggested for
this purpose. However, it can be advantageous to avoid this
treatment as it can result in cells devoid of most carotenoids.
[10651] [0101.0.24.24] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michel, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[10652] [0102.0.24.24] Carotene, in particular beta-carotene, in
particular beta-cryptoxanthin or zeaxanthin can for example be
detected advantageously via HPLC, LC or GC separation methods. The
unambiguous detection for the presence of carotene, in particular
beta-carotene, in particular beta-cryptoxanthin or zeaxanthin
containing products can be obtained by analyzing recombinant
organisms using analytical standard methods: LC, LC-MS, MS or TLC).
The material to be analyzed can be disrupted by sonication,
grinding in a glass mill, liquid nitrogen and grinding, cooking, or
via other applicable methods.
[10653] [0103.0.24.24] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [10654] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide having a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
279, 282, 285, 286, 288, and/or 289, and/or lines 278, 280, 281,
283, 284, and/or 287 and/or lines 637 to 641 or a fragment thereof,
which confers an increase in the amount of the respective fine
chemical in an organism or a part thereof; [10655] b) nucleic acid
molecule comprising, preferably at least the mature form, of a
nucleic acid molecule having a sequence as indicated in Table IA or
IB, columns 5 or 7, lines 279, 282, 285, 286, 288, and/or 289,
and/or lines 278, 280, 281, 283, 284, and/or 287 and/or lines 637
to 641, [10656] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [10657] d) nucleic
acid molecule encoding a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [10658] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under under stringent
hybridization conditions and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[10659] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[10660] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [10661] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers pairs having a sequence as indicated in
Table III, columns 7, lines 279, 282, 285, 286, 288, and/or 289,
and/or lines 278, 280, 281, 283, 284, and/or 287 and/or lines 637
to 641, and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [10662] i) nucleic
acid molecule encoding a polypeptide which is isolated, e.g. from
an expression library, with the aid of monoclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (h), preferably to (a) to (c), and and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [10663] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence having a
sequences as indicated in Table IV, columns 7, lines 279, 282, 285,
286, 288, and/or 289, and/or lines 278, 280, 281, 283, 284, and/or
287 and/or lines 637 to 641 and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [10664] k) nucleic acid molecule comprising one or more of
the nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of the polypeptide indicated in Table
IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288, and/or
289, and/or lines 278, 280, 281, 283, 284, and/or 287 and/or lines
637 to 641, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[10665] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[10666] [0104.0.24.24] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence indicated in
Table IA or IB, columns 5 or 7, lines 279, 282, 285, 286, 288,
and/or 289, and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641 by one or more nucleotides. In one embodiment, the
nucleic acid molecule used in the process of the invention does not
consist of the sequence indicated in Table IA or IB, columns 5 or
7, lines 279, 282, 285, 286, 288, and/or 289, and/or lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641. In one
embodiment, the nucleic acid molecule of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to the
sequence indicated in Table IA or IB, columns 5 or 7, lines 279,
282, 285, 286, 288, and/or 289, and/or lines 278, 280, 281, 283,
284 and/or 287 and/or lines 637 to 641. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288, and/or 289, and/or lines 278, 280, 281, 283, 284 and/or
287 and/or lines 637 to 641.
[10667] [0105.0.0.24] to [0107.0.0.24]: see [0105.0.0.0] to
[0107.0.0.0]
[10668] [0108.0.24.24] Nucleic acid molecules with the sequence as
indicated in Table IA or IB, columns 5 or 7, lines 279, 282, 285,
286, 288, and/or 289, and/or lines 278, 280, 281, 283, 284 and/or
287 and/or lines 637 to 641, nucleic acid molecules which are
derived from an amino acid sequences as indicated in Table IIA or
IIB, columns 5 or 7, lines 279, 282, 285, 286, 288, and/or 289,
and/or lines 278, 280, 281, 283, 284, and/or 287 and/or lines 637
to 641 or from polypeptides comprising the consensus sequence as
indicated in Table IV, columns 7, lines 279, 282, 285, 286, 288,
and/or 289, and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641, or their derivatives or homologues encoding
polypeptides with the enzymatic or biological activity of an
activity of a polypeptide as indicated in Table IIA or IIB, column
3, 5 or 7, lines 279, 282, 285, 286, 288, and/or 289, and/or lines
278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641, e.g.
conferring the increase of the respective fine chemical, meaning
carotenoids, in particular carotene, in particular beta-carotene or
the carotene precursor IPP, resp., after increasing its expression
or activity, are advantageously increased in the process according
to the invention.
[10669] [0109.0.24.24] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular IPP or
beta-carotene, produced in the process according to the
invention.
[10670] [0110.0.0.24] see. [0110.0.0.0]
[10671] [0111.0.0.24] see [0111.0.0.0]
[10672] [0112.0.24.24] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table IIA or IIB, column 3, lines
279, 282, 285, 286, 28 and/or 289, and/or lines 278, 280, 281, 283,
284 and/or 287 and/or lines 637 to 641 or having the sequence of a
polypeptide as indicated in Table IIA or IIB, columns 5 and 7,
lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641 and conferring an
increase in the IPP and/or beta-carotene level, respectively.
[10673] [0113.0.0.24] to [0114.0.0.24]: see [0113.0.0.0] to
[0114.0.0.0]
[10674] [0115.0.0.24] see [0115.0.0.0]
[10675] [0116.0.0.24] to [0120.0.0.24] see [0116.0.0.0] to
[0120.0.0.0]
[10676] [0120.1.0.24] Production strains which are also
advantageously selected in the process according to the invention
are microorganisms selected from the group green algae, like
Spongioccoccum exentricum, Chlorella sorokiniana (pyrenoidosa,
7-11-05), or form the group of fungi like fungi belonging to the
Daccrymycetaceae family, or non-photosynthetic bacteria, like
methylotrophs, flavobacteria, actinomycetes, like streptomyces
chrestomyceticus, Mycobacteria like Mycobacterim phlei, or
Rhodobacter capsulatus.
[10677] [0121.0.24.24] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table IIA or
IIB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641 or the functional homologues thereof as described herein,
preferably conferring above-mentioned activity I.e. they confer a
beta-carotene increase after increasing the activity of the
polypeptide sequences indicated in Table IIA or IIB, columns 5 or
7, lines 279, 282, 285, 286, 288, and/or 289 or conferring a IPP
level increase after increasing the activity of the polypeptide
sequences indicated in Table IIA or IIB, columns 5 or 7, lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641.
[10678] [0122.0.0.24] to [0127.0.0.24]: see [0122.0.0.0] to
[0127.0.0.0]
[10679] [0128.0.24.24] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, columns 7,
lines 279, 282, 285, 286, 288 and/or 289, and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, by means of
polymerase chain reaction can be generated on the basis of a
sequence shown herein, for example the sequence as indicated in
Table IA or IB, columns 5 or 7, respective lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641 or the sequences derived from a sequences
as indicated in Table IIA or IIB, columns 5 or 7, respective lines
279, 282, 285, 286, 288 and/or 289, and/or lines 278, 280, 281,
283, 284 and/or 287 and/or lines 637 to 641, resp.
[10680] [0129.0.24.24] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence indicated in Table IV,
columns 7, lines 279, 282, 285, 286, 288 and/or 289, and/or lines
278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641 is
derived from said alignments.
[10681] [0130.0.24.24] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of the
respective fine chemical after increasing the expression or
activity the protein comprising said fragment.
[10682] [0131.0.0.24] to [0138.0.0.24]: see [0131.0.0.0] to
[0138.0.0.0]
[10683] [0139.0.24.24] Polypeptides having above-mentioned
activity, i.e. conferring the respective fine chemical level
increase, derived from other organisms, can be encoded by other DNA
sequences which hybridise to a sequence indicated in Table IA or
IB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 for
beta-carotene or indicated in Table IA or IB, columns 5 or 7, lines
278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641 for IPP
under relaxed hybridization conditions and which code on expression
for peptides having the respective fine chemical, i.e.
beta-carotene or IPP, resp., --increasing activity.
[10684] [0140.0.0.24] to [0146.0.0.24]: see [0140.0.0.0] to
[0146.0.0.0]
[10685] [0147.0.24.24] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
IA or IB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641 is one which is sufficiently complementary to one of said
nucleotide sequences such that it can hybridise to one of said
nucleotide sequences, thereby forming a stable duplex. Preferably,
the hybridisation is performed under stringent hybridization
conditions. However, a complement of one of the herein disclosed
sequences is preferably a sequence complement thereto according to
the base pairing of nucleic acid molecules well known to the
skilled person. For example, the bases A and G undergo base pairing
with the bases T and U or C, resp. and visa versa. Modifications of
the bases can influence the base-pairing partner.
[10686] [0148.0.24.24] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table IA or IB, columns 5 or 7,
lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, or a portion
thereof and preferably has above mentioned activity, in particular
having a IPP or beta-carotene-increasing activity after increasing
the activity or an activity of a product of a gene encoding said
sequences or their homologs.
[10687] [0149.0.24.24] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table IA or IB, columns 5 or
7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, or a portion
thereof and encodes a protein having above-mentioned activity, e.g.
conferring a IPP or beta-carotene increase, resp., and optionally,
the activity of protein indicated in Table IIA or IIB, column 5,
lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641.
[10688] [00149.1.24.24] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table IA or IB, columns 5 or 7, lines 279,
282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284,
and/or 287 and/or lines 637 to 641 has further one or more of the
activities annotated or known for the a protein as indicated in
Table IIA or IIB, column 3, lines 279, 282, 285, 286, 288 and/or
289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines
637 to 641.
[10689] [0150.0.24.24] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table IA or IB, columns 5 or 7, lines
279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283,
284 and/or 287 and/or lines 637 to 641, for example a fragment
which can be used as a probe or primer or a fragment encoding a
biologically active portion of the polypeptide of the present
invention or of a polypeptide used in the process of the present
invention, i.e. having above-mentioned activity, e.g. conferring an
increase of beta-carotene or IPP, resp., if its activity is
increased. The nucleotide sequences determined from the cloning of
the present protein-according-to-the-invention-encoding gene allows
for the generation of probes and primers designed for use in
identifying and/or cloning its homologues in other cell types and
organisms. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, 15 preferably about 20 or 25, more
preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one of the sequences set forth, e.g., as indicated in
Table IA or IB, columns 5 or 7, lines 279, 282, 285, 286, 288
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641, an anti-sense sequence of one of the sequences,
e.g., as indicated in Table IA or IB, columns 5 or 7, lines 279,
282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284,
and/or 287 and/or lines 637 to 641, or naturally occurring mutants
thereof. Primers based on a nucleotide of invention can be used in
PCR reactions to clone homologues of the polypeptide of the
invention or of the polypeptide used in the process of the
invention, e.g. as the primers described in the examples of the
present invention, e.g. as shown in the examples. A PCR with the
primer pairs indicated in Table III, column 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641 will result in a fragment of a
polynucleotide sequence as indicated in Table IA or IB, columns 5
or 7, lines 279, 282, 285, 286, 288 and/or 289, and/or lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641 or its gene
product.
[10690] [0151.0.0.24]: see [0151.0.0.0]
[10691] [0152.0.24.24] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283,
284 and/or 287 such that the protein or portion thereof maintains
the ability to participate in the respective fine chemical
production, in particular a beta-carotene (lines 279, 282, 285,
286, 288, and/or 289) or IPP (lines 278, 280, 281, 283, 284 and/or
287 and/or lines 637 to 641) increasing activity as mentioned above
or as described in the examples in plants or microorganisms is
comprised.
[10692] [0153.0.24.24] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641 such that the
protein or portion thereof is able to participate in the increase
of the respective fine chemical production. In one embodiment, a
protein or portion thereof as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641
has for example an activity of a polypeptide indicated in Table IIA
or IIB, column 3, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641.
[10693] [0154.0.24.24] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table
[10694] IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288,
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641 and has above-mentioned activity, e.g. conferring
preferably the increase of the respective fine chemical.
[10695] [0155.0.0.24] to [0156.0.0.24]: see [0155.0.0.0] to
[0156.0.0.0]
[10696] [0157.0.24.24] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences as
indicated in Table IA or IB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641 (and portions thereof) due to degeneracy of
the genetic code and thus encode a polypeptide of the present
invention, in particular a polypeptide having above mentioned
activity, e.g. conferring an increase in the respective fine
chemical in a organism, e.g. as polypeptides comprising the
sequence as indicated in Table IV, columns 5 or 7, lines 279, 282,
285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641 or as polypeptides depicted in
Table IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641 or the functional homologues. Advantageously, the
nucleic acid molecule of the invention comprises, or in an other
embodiment has, a nucleotide sequence encoding a protein
comprising, or in an other embodiment having, an amino acid
sequence of a consensus sequences as indicated in Table IV, columns
5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641 or of the
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283,
284 and/or 287 and/or lines 637 to 641, resp., or the functional
homologues. In a still further embodiment, the nucleic acid
molecule of the invention encodes a full length protein which is
substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table IV, columns 5 or 7, lines
279, 282, 285, 286, 288, and/or 289 and/or lines 278, 280, 281,
283, 284 and/or 287 and/or lines 637 to 641 or of a polypeptide as
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289, and/or lines 278, 280, 281, 283, 284 and/or
287 and/or lines 637 to 641 or the functional homologues. However,
in a preferred embodiment, the nucleic acid molecule of the present
invention does not consist of a sequence as indicated in Table IA
or IB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641, resp.
[10697] [0158.0.0.24] to [0160.0.0.24]: see [0158.0.0.0] to
[0160.0.0.0]
[10698] [0161.0.24.24] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table IA or IB, columns 5 or 7, lines 279,
282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641. The nucleic acid molecule is
preferably at least 20, 30, 50, 100, 250 or more nucleotides in
length.
[10699] [0162.0.0.24] see [0162.0.0.0]
[10700] [0163.0.24.24] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table IA or IB, columns 5 or 7, lines 279, 282,
285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641 corresponds to a
naturally-occurring nucleic acid molecule of the invention. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein). Preferably, the nucleic
acid molecule encodes a natural protein having above-mentioned
activity, e.g. conferring the increase of the amount of the
respective fine chemical in a organism or a part thereof, e.g. a
tissue, a cell, or a compartment of a cell, after increasing the
expression or activity thereof or the activity of a protein of the
invention or used in the process of the invention.
[10701] [0164.0.0.24] see [0164.0.0.0]
[10702] [0165.0.24.24] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table IA or IB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641 resp.
[10703] [0166.0.0.24] to [0167.0.0.24]: see [0166.0.0.0] to
[0167.0.0.0]
[10704] [0168.0.24.24] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641, resp., yet retain said activity described herein. The nucleic
acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641 resp., and is capable of participation in
the increase of production of the respective fine chemical after
increasing its activity, e.g.
[10705] its expression. Preferably, the protein encoded by the
nucleic acid molecule is at least about 60% identical to a sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282,
285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641 resp., more preferably at least
about 70% identical to one of the sequences as indicated in Table
IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or
289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines
637 to 641, resp., even more preferably at least about 80%, 90%,
95% homologous to a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., and most preferably at least about 96%, 97%, 98%, or 99%
identical to the sequence as indicated in Table IIA or IIB, columns
5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641.
[10706] [0169.0.0.24] to [0172.0.0.24]: see [0169.0.0.0] to
[0172.0.0.0]
[10707] [0173.0.24.24] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 34228 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 34228 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[10708] [0174.0.0.24]: see [0174.0.0.0]
[10709] [0175.0.24.24] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 34229 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 34229 by the above program algorithm with the
above parameter set, has a 80% homology.
[10710] [0176.0.24.24] Functional equivalents derived from one of
the polypeptides as indicated in Table IIA or IIB, columns 5 or 7,
lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, resp., according
to the invention by substitution, insertion or deletion have at
least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65%
or 70% by preference at least 80%, especially preferably at least
85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at
least 95%, 97%, 98% or 99% homology with one of the polypeptides as
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641, resp., according to the invention and are
distinguished by essentially the same properties as a polypeptide
as indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282,
285, 286, 288 and/or 289, and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641, resp.
[10711] [0177.0.24.24] Functional equivalents derived from a
nucleic acid sequence as indicated in Table IA or IB, columns 5 or
7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, resp., according
to the invention by substitution, insertion or deletion have at
least 30%, 35%, 40%, 45% or 50%, preferably at least 55%, 60%, 65%
or 70% by preference at least 80%, especially preferably at least
85% or 90%, 91%, 92%, 93% or 94%, very especially preferably at
least 95%, 97%, 98% or 99% homology with one of the polypeptides as
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641, resp., according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283,
284 and/or 287 and/or lines 637 to 641, resp.
[10712] [0178.0.0.24] see [0178.0.0.0]
[10713] [0179.0.24.24] A nucleic acid molecule encoding an
homologous to a protein sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289, and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., can be created by introducing one or more nucleotide
substitutions, additions or deletions into a nucleotide sequence of
the nucleic acid molecule of the present invention, in particular
as indicated in Table IA or IB, columns 5 or 7, lines 279, 282,
285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641, resp., such that one or more
amino acid substitutions, additions or deletions are introduced
into the encoded protein. Mutations can be introduced into the
encoding sequences of a sequences as indicated in Table IA or IB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., by standard techniques, such as site-directed mutagenesis
and PCR-mediated mutagenesis.
[10714] [0180.0.0.24] to [0183.0.0.24]: see [0180.0.0.0] to
[0183.0.0.0]
[10715] [0184.0.24.24] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table IA or IB, columns 5 or
7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, resp., or of the
nucleic acid sequences derived from a sequences as indicated in
Table IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641, resp., comprise also allelic variants with at
least approximately 30%, 35%, 40% or 45% homology, by preference at
least approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
IA or IB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641, resp., or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[10716] [0185.0.24.24] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table IA or IB, columns 5 or 7, lines 279, 282, 285, 286, 288
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641, resp., In one embodiment, it is preferred that
the nucleic acid molecule comprises as little as possible other
nucleotides not shown in any one of sequences as indicated in Table
IA or IB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284, and/or 287 and/or lines 637
to 641, resp., In one embodiment, the nucleic acid molecule
comprises less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or
40 further nucleotides. In a further embodiment, the nucleic acid
molecule comprises less than 30, 20 or 10 further nucleotides. In
one embodiment, a nucleic acid molecule used in the process of the
invention is identical to a sequences as indicated in Table IA or
IB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp.
[10717] [0186.0.24.24] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., In one embodiment, the nucleic acid molecule encodes less
than 150, 130, 100, 80, 60, 50, 40 or 30 further amino acids. In a
further embodiment, the encoded polypeptide comprises less than 20,
15, 10, 9, 8, 7, 6 or 5 further amino acids. In one embodiment, the
encoded polypeptide used in the process of the invention is
identical to the sequences as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp.,
[10718] [0187.0.24.24] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641, resp.,
comprises less than 100 further nucleotides. In a further
embodiment, said nucleic acid molecule comprises less than 30
further nucleotides. In one embodiment, the nucleic acid molecule
used in the process is identical to a coding sequence encoding a
sequences as indicated in Table IIA or IIB, columns 5 or 7, lines
279, 282, 285, 286, 288, and/or 289, and/or lines 278, 280, 281,
283, 284 and/or 287 and/or lines 637 to 641, resp.
[10719] [0188.0.24.24] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or
289 and/or lines 278, 280, 281, 283, 284, and/or 287 and/or lines
637 to 641, resp., and is expressed under identical conditions.
[10720] [0189.0.24.24] Homologues of a sequences as indicated in
Table IA or IB, columns 5 or 7, lines 279, 282, 285, 286, 288
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641, resp., or of a derived sequences as indicated in
Table IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641, resp., also mean truncated sequences, cDNA,
single-stranded DNA or RNA of the coding and noncoding DNA
sequence. Homologues of said sequences are also understood as
meaning derivatives, which comprise noncoding regions such as, for
example, UTRs, terminators, enhancers or promoter variants. The
promoters upstream of the nucleotide sequences stated can be
modified by one or more nucleotide substitution(s), insertion(s)
and/or deletion(s) without, however, interfering with the
functionality or activity either of the promoters, the open reading
frame (=ORF) or with the 3'-regulatory region such as terminators
or other 3' regulatory regions, which are far away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[10721] [0190.0.0.24]: see [0190.0.0.0]
[10722] [0191.0.0.24] The organisms or part thereof produce
according to the herein mentioned process of the invention an
increased level of free and/or bound the respective fine chemical
compared to said control or selected organisms or parts
thereof.
[10723] [0192.0.0.24] to [0203.0.0.24]: see [0192.0.0.0] to
[0203.0.0.0]
[10724] [0204.0.24.24] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule, which comprises a
nucleic acid molecule selected from the group consisting of:
[10725] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp.; or a fragment thereof conferring an increase in the amount
of the respective fine chemical, i.e. beta-carotene (lines lines
279, 282, 285, 286, 288, and/or 289) or IPP (lines 278, 280, 281,
283, 284 and/or 287 and/or lines 637 to 641), resp., in an organism
or a part thereof [10726] b) nucleic acid molecule comprising,
preferably at least the mature form, of a nucleic acid molecule as
indicated in Table IA or IB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641, resp., or a fragment thereof conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [10727] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [10728]
d) nucleic acid molecule encoding a polypeptide whose sequence has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [10729] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [10730] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [10731] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [10732]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
primers or primer pairs as indicated in Table III, columns 5 or 7,
lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641 and conferring an
increase in the amount of the respective fine chemical, i.e.
beta-carotene (lines lines 279, 282, 285, 286, 288 and/or 289) or
IPP (lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641), resp., in an organism or a part thereof; [10733] i) nucleic
acid molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [10734] j) nucleic acid molecule which encodes a
polypeptide comprising a consensus sequence as indicated in Table
IV, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289,
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641 and conferring an increase in the amount of the respective fine
chemical, i.e. beta-carotene (lines lines 279, 282, 285, 286, 288
and/or 289) or IPP (lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641), resp., in an organism or a part thereof; [10735]
k) nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of a polypeptide as indicated in
Table IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287, resp.,
and conferring an increase in the amount of the respective fine
chemical, i.e. beta-carotene (lines lines 279, 282, 285, 286, 288
and/or 289) or IPP (lines 278, 280, 281, 283, 284, and/or 287
and/or lines 637 to 641), resp., in an organism or a part thereof;
and [10736] l) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of a nucleic acid molecule as indicated in Table IA or
IB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., or a nucleic acid molecule encoding, preferably at least the
mature form of, a polypeptide as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; or which
encompasses a sequence which is complementary thereto; whereby,
preferably, the nucleic acid molecule according to (a) to (l)
distinguishes over a sequence as indicated in Table IA or IB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289, and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., by one or more nucleotides. In one embodiment, the nucleic
acid molecule of the invention does not consist of the sequence as
indicated in Table IA or IB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641, resp. In an other embodiment, the nucleic
acid molecule of the present invention is at least 30% identical
and less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence as indicated in Table IA or IB, columns 5 or 7, lines 279,
282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641, resp. In a further embodiment
the nucleic acid molecule does not encode a polypeptide sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641, resp. Accordingly, in one embodiment, the
nucleic acid molecule of the present invention encodes in one
embodiment a polypeptide which differs at least in one or more
amino acids from a polypeptide indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641
does not encode a protein of a sequence as indicated in Table IIA
or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641. Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table IIA or IIB, columns 5
or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641. In a further
embodiment, the protein of the present invention is at least 30%
identical to a protein sequence indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641
and less than 100%, preferably less than 99.999%, 99.99% or 99.9%,
more preferably less than 99%, 98%, 97%, 96% or 95% identical to a
sequence as indicated in Table IIA or IIB, columns 5 or 7, lines
279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283,
284 and/or 287 and/or lines 637 to 641.
[10737] [0205.0.0.24] to [0206.0.0.24]: see [0205.0.0.0] to
[0206.0.0.0]
[10738] [0207.0.24.24] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the carotenoid metabolism, the
carotene, in particular beta-carotene metabolism, the astaxanthin
metabolism, the amino acid metabolism, of glycolysis, of the
tricarboxylic acid metabolism or their combinations. As described
herein, regulator sequences or factors can have a positive effect
on preferably the gene expression of the genes introduced, thus
increasing it. Thus, an enhancement of the regulator elements may
advantageously take place at the transcriptional level by using
strong transcription signals such as promoters and/or enhancers. In
addition, however, an enhancement of translation is also possible,
for example by increasing mRNA stability or by inserting a
translation enhancer sequence.
[10739] [0208.0.0.24] to [0226.0.0.24]: see [0208.0.0.0] to
[0226.0.0.0]
[10740] [0227.0.24.24] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[10741] In addition to a sequence indicated in Table IA or IB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641 or
its derivatives, it is advantageous to express and/or mutate
further genes in the organisms. Especially advantageously,
additionally at least one further gene of the carotenoid, in
particular carotene biosynthetic pathway, e.g. one of the above
mentioned genes of this pathway, or e.g. for the synthesis of
astaxanthin or for another provitamin A or for another carotenoids
or carotene, is expressed in the organisms such as plants or
microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the amino acids desired since, for example, feedback
regulations no longer exist to the same extent or not at all. In
addition it might be advantageously to combine one or more of the
sequences indicated in Table IA or IB, columns 5 or 7, lines 279,
282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641, resp., with genes which
generally support or enhances to growth or yield of the target
organisms, for example genes which lead to faster growth rate of
microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[10742] [0228.0.24.24] In a further embodiment of the process of
the invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the carotenoid, in
particular carotenes metabolism, in particular in synthesis of
beta-cryptoxanthin, zeaxanthin, astaxanthin or lutein.
[10743] [0229.0.24.24] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the carotenoids biosynthetic
pathway, such as phytoene synthase (Psy), which is an important
control point for the regulation of the flux (Fraser et al., 2002),
phytoene desaturase (Pds), z-carotene desaturase, above mentioned
enzymes (s. introduction of the application), e.g. hydroxylases
such as beta-carotene hydroxylase (U.S. Pat. No. 6,214,575),
ketolases, or cyclases such as the beta-cyclase (U.S. Pat. No.
6,232,530) or oxygenases such as the beta-C4-oxygenase described in
U.S. Pat. No. 6,218,599 or homologs thereof, astaxanthin synthase
(U.S. Pat. No. 6,365,386), or other genes as described in U.S. Pat.
No. 6,150,130. These genes can lead to an increased synthesis of
the essential carotenoids, in particular carotene, in particular
beta-carotenes.
[10744] [0230.0.0.24] see [230.0.0.0].
[10745] [0231.0.24.24] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a beta-carotene or IPP degrading
protein is attenuated, in particular by reducing the rate of
expression of the corresponding gene.
[10746] [0232.0.0.24] to [0276.0.0.24]: see [0232.0.0.0] to
[0276.0.0.0]
[10747] [0277.0.24.24] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously. The fine respective chemical, i.e. e.g. beta-carotene
or IPP, and other carotenoids, in particular carotenes produced by
this process can be obtained by harvesting the organisms, either
from the crop in which they grow, or from the field. This can be
done via pressing or extraction of the plant parts
[10748] [0278.0.0.24] to [0282.0.0.24]: see [0278.0.0.0] to
[0282.0.0.0]
[10749] [0283.0.24.24] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table IIA
or IIB, column 3, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., or an antibody against a polypeptide as indicated in Table
IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or
289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines
637 to 641, resp., which can be produced by standard techniques
utilizing the polypeptid of the present invention or fragment
thereof, i.e., the polypeptide of this invention. Preferred are
monoclonal antibodies.
[10750] [0284.0.0.24] see [0284.0.0.0]
[10751] [0285.0.24.24] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or
289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines
637 to 641, resp., or as coded by a nucleic acid molecule as
indicated in Table IA or IB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641, resp., or functional homologues
thereof.
[10752] [0286.0.24.24] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table IV, columns 5 or 7, lines 279, 282, 285, 286,
288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641 and in one another embodiment, the present
invention relates to a polypeptide comprising or consisting of a
consensus sequence as indicated in Table IV, columns 5 or 7, lines
279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283,
284 and/or 287 and/or lines 637 to 641 whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table IV, column 7, lines 287 to 289 and/or 637 to 641, resp.
[10753] [0287.0.0.24] to [0289.0.0.24]: see [0287.0.0.0] to
[0289.0.0.0]
[10754] [00290.0.24.24] The alignment was performed with the
Software AlignX (Sep. 25, 2002) a component of Vector NTI Suite
8.0, InforMax.TM., Invitrogen.TM. life science software, U.S. Main
Office, 7305 Executive Way, Frederick, Md. 21704, USA with the
following settings: For pairwise alignments: gap opening penalty:
10.0; gap extension penalty 0.1. For multiple alignments: Gap
opening penalty: 10.0; Gap extension penalty: 0.1; Gap separation
penalty range: 8; Residue substitution matrix: blosum62;
Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0.5;
Consensus calculation options: Residue fraction for consensus: 0.9.
Presettings were selected to allow also for the alignment of
conserved amino acids
[10755] [0291.0.24.24] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[10756] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288, and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., by one or more amino acids. In one embodiment, polypeptide
distinguishes from a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., by more than 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, even more preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641, resp., by not more than 80% or 70% of the
amino acids, preferably not more than 60% or 50%, more preferred
not more than 40% or 30%, even more preferred not more than 20% or
10%. In an other embodiment, said polypeptide of the invention does
not consist of a sequence as indicated in Table IIA or IIB, columns
5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641.
[10757] [0292.0.0.24] see [0292.0.0.0]
[10758] [0293.0.24.24] In one embodiment, the invention relates to
polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or by a nucleic acid molecule used in the process of the
invention. In one embodiment, the polypeptide of the invention has
a sequence which distinguishes from a sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641, resp., by one or more amino acids. In an other
embodiment, said polypeptide of the invention does not consist of
the sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, resp. In a
further embodiment, said polypeptide of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical. In one
embodiment, said polypeptide does not consist of the sequence
encoded by a nucleic acid molecules as indicated in Table IA or IB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp.
[10759] [0294.0.24.24] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table IIA or IIB, column 3, lines 279, 282, 285, 286,
288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641 resp., which distinguishes over a sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282,
285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641, resp., by one or more amino
acids, preferably by more than 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, even more
preferred are more than 40, 50, or 60 amino acids but even more
preferred by less than 70% of the amino acids, more preferred by
less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[10760] [0295.0.0.24] to [0296.0.0.24]: see [0295.0.0.0] to
[0296.0.0.0]
[10761] [0297.0.0.24] see [0297.0.0.0]
[10762] [00297.1.24.24] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table IIA or IIB, columns 3, 5 or 7, lines 278 to 289
and/or lines 637 to 641, resp.
[10763] [0298.0.24.24] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282,
285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641, resp. The portion of the
protein is preferably a biologically active portion as described
herein. Preferably, the polypeptide used in the process of the
invention has an amino acid sequence identical to a sequence as
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284, and/or
287 and/or lines 637 to 641 resp.
[10764] [0299.0.24.24] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table IA or IB, columns 5 or 7,
lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, resp. The
preferred polypeptide of the present invention preferably possesses
at least one of the activities according to the invention and
described herein. A preferred polypeptide of the present invention
includes an amino acid sequence encoded by a nucleotide sequence
which hybridizes, preferably hybridizes under stringent conditions,
to a nucleotide sequence as indicated in Table IA or IB, columns 5
or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641, resp., or
which is homologous thereto, as defined above.
[10765] [0300.0.24.24] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table IIA or
IIB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641, resp., in amino acid sequence due to natural variation or
mutagenesis, as described in detail herein. Accordingly, the
polypeptide comprise an amino acid sequence which is at least about
35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about
75%, 80%, 85% or 90, and more preferably at least about 91%, 92%,
93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%,
99% or more homologous to an entire amino acid sequence of as
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641, resp.
[10766] [0301.0.0.24] see [0301.0.0.0]
[10767] [0302.0.24.24] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641 resp., or the amino acid sequence of a protein
homologous thereto, which include fewer amino acids than a full
length polypeptide of the present invention or used in the process
of the present invention or the full length protein which is
homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[10768] [0303.0.0.24] see [0303.0.0.0]
[10769] [0304.0.24.24] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
IIA or IIB, column 3, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641 but having differences in the sequence from said wild-type
protein. These proteins may be improved in efficiency or activity,
may be present in greater numbers in the cell than is usual, or may
be decreased in efficiency or activity in relation to the wild type
protein.
[10770] [0305.0.0.24] to [0308.0.0.24]: see [0305.0.0.0] to
[0308.0.0.0]
[10771] [0309.0.0.24] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table IIA or
IIB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641, resp., refers to a polypeptide having an amino acid sequence
corresponding to the polypeptide of the invention or used in the
process of the invention, whereas a "non-polypeptide of the
invention" or "other polypeptide" not being indicated in Table IIA
or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641, resp., refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous a
polypeptide of the invention, preferably which is not substantially
homologous to a polypeptide as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., e.g., a protein which does not confer the activity described
herein or annotated or known for as indicated in Table IIA or IIB,
column 3, lines 279, 282, 285, 286, 288 and/or 289 and/or lines
278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641, resp.,
and which is derived from the same or a different organism. In one
embodiment, a "non-polypeptide of the invention" or "other
polypeptide" not being indicated in Table IIA or IIB, columns 5 or
7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, resp., does not
confer an increase of the respective fine chemical in an organism
or part thereof.
[10772] [0310.0.0.24] to [0334.0.0.24]: see [0310.0.0.0] to
[0334.0.0.0]
[10773] [0335.0.24.24] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table IA or IB, columns
5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641, resp.,
and/or homologs thereof. As described inter alia in WO 99/32619,
dsRNAi approaches are clearly superior to traditional antisense
approaches. The invention therefore furthermore relates to
double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived there from), bring about
altered metabolic activity by the reduction in the expression of a
nucleic acid sequences as indicated in Table IA or IB, columns 5 or
7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, resp., and/or
homologs thereof. In a double-stranded RNA molecule for reducing
the expression of an protein encoded by a nucleic acid sequence
sequences as indicated in Table IA or IB, columns 5 or 7, lines
279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283,
284 and/or 287 and/or lines 637 to 641, resp., and/or homologs
thereof, one of the two RNA strands is essentially identical to at
least part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[10774] [0336.0.0.24] to [0342.0.0.24]: see [0336.0.0.0] to
[0342.0.0.0]
[10775] [0343.0.24.24] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table IA or IB, columns 5 or 7, lines 279,
282, 285, 286, 288, and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641, resp., or its homolog is not
necessarily required in order to bring about effective reduction in
the expression. The advantage is, accordingly, that the method is
tolerant with regard to sequence deviations as may be present as a
consequence of genetic mutations, polymorphisms or evolutionary
divergences. Thus, for example, using the dsRNA, which has been
generated starting from a sequence as indicated in Table IA or IB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641,
resp., or homologs thereof of the one organism, may be used to
suppress the corresponding expression in another organism.
[10776] [0344.0.0.24] to [0350.0.0.24]: see [0344.0.0.0] to
[0350.0.0.0]
[10777] [0351.0.0.24] to [0361.0.0.24]: see [0351.0.0.0] to
[0361.0.0.0]
[10778] [0362.0.24.24] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table IIA or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641, resp., e.g. encoding a polypeptide having protein
activity, as indicated in Table IIA or IIB, columns 3, lines 279,
282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641, resp. Due to the above
mentioned activity the respective fine chemical content in a cell
or an organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. Transgenic for a polypeptide having an activity of a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283,
284 and/or 287 and/or lines 637 to 641, resp., means herein that
due to modulation or manipulation of the genome, an activity as
annotated for a polypeptide as indicated in Table IIA or IIB,
column 3, lines 279, 282, 285, 286, 288 and/or 289 and/or lines
278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641, e.g.
having a sequence as indicated in Table IIA or IIB, columns 5 or 7,
lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, resp., is
increased in a cell or an organism or a part thereof. Examples are
described above in context with the process of the invention.
[10779] [0363.0.0.24] see [0363.0.0.0]
[10780] [0364.0.24.24] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table IIA or IIB, column 3, 5 or 7, lines 279, 282,
285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641, resp., with the corresponding
protein-encoding sequence as indicated in Table IA or IB, column 5
or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641, resp.,
--becomes a transgenic expression cassette when it is modified by
non-natural, synthetic "artificial" methods such as, for example,
mutagenization. Such methods have been described (U.S. Pat. No.
5,565,350; WO 00/15815; also see above).
[10781] [0365.0.0.24] to [0373.0.0.24]: see [0365.0.0.0] to
[0373.0.0.0]
[10782] [0374.0.24.24] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. The respective fine chemical
produced in the process according to the invention may, however,
also be isolated from the plant in the form of their free
beta-carotene or free IPP or bound in or to compounds or moieties,
like glucosides, e.g. diglucosides. The respective fine chemical
produced by this process can be harvested by harvesting the
organisms either from the culture in which they grow or from the
field. This can be done via expressing, grinding and/or extraction,
salt precipitation and/or ion-exchange chromatography or other
chromatographic methods of the plant parts, preferably the plant
seeds, plant fruits, plant tubers and the like.
[10783] [0375.0.0.24] to [0376.0.0.24]: see [0375.0.0.0] to
[0376.0.0.0]
[10784] [0377.0.24.24] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced carotenoids, e.g the fine chemical(s) comprising
composition is isolated. In one embodiment, the produced respective
fine chemical is isolated.
[10785] [0378.0.24.24] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the respective
fine chemical produced in the process can be isolated. The
resulting respective fine chemical can, if appropriate,
subsequently be further purified, if desired mixed with other
active ingredients such as vitamins, amino acids, carbohydrates,
antibiotics and the like, and, if appropriate, formulated.
[10786] [0379.0.24.24] In one embodiment, the product of the
process is a mixture of the fine chemicals. In one embodiment, the
product is a mixture of the respective fine chemicals with other
carotenoids.
[10787] [0380.0.24.24] The respective fine chemicals obtained in
the process are suitable as starting material for the synthesis of
further products of value. For example, they can be used in
combination with each other or alone for the production of
pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates to a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the respective fine chemical
containing composition produced by the process of the invention or
the respective fine chemical produced by the process of the
invention--if desired--and formulating the product with a
pharmaceutical or cosmetic acceptable carrier or formulating the
product in a form acceptable for an application in agriculture. A
further embodiment according to the invention is the use of the the
respective fine chemical produced in the process or of the
transgenic organisms in animal feeds, foodstuffs, medicines, food
supplements, cosmetics or pharmaceuticals or for the production of
other carotenoids, e.g. in after isolation of the respective fine
chemical or without, e.g. in situ, e.g within the organism used for
the process for the production of the respective fine chemical.
[10788] [0381.0.0.24] to [0382.0.0.24]: see [0381.0.0.0] to
[0382.0.0.0]
[10789] [0383.0.24.24] ./.
[10790] [0384.0.0.24] see [0384.0.0.0]
[10791] [0385.0.24.24] The fermentation broths obtained in this
way, containing in particular IPP or beta-carotene in mixtures with
other carotenoids, in particular with other carotenoids, in
particular carotenes, or containing microorganisms or parts of
microorganisms, like plastids, containing the respective fine
chemical or the carotenoids produced in mixtures with other
carotenoids, in particular with other carotene, normally have a dry
matter content of from 1 to 70% by weight, preferably 7.5 to 25% by
weight. Sugar-limited fermentation is additionally advantageous,
e.g. at the end, for example over at least 30% of the fermentation
time. This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 10 g/l,
preferably to 0 to 3 g/l during this time. The fermentation broth
is then processed further. Depending on requirements, the biomass
can be removed or isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth or left completely in it.
[10792] The fermentation broth can then be thickened or
concentrated by known methods, such as, for example, with the aid
of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[10793] As carotenoids are often localized in membranes or
plastids, in one embodiment it is advantageous to avoid a leaching
of the cells when the biomass is isolated entirely or partly by
separation methods, such as, for example, centrifugation,
filtration, decantation, coagulation/flocculation or a combination
of these methods, from the fermentation broth. The dry biomass can
directly be added to animal feed, provided the carotenoids
concentration is sufficiently high and no toxic compounds are
present. In view of the instability of carentoids, conditions for
drying, e.g. spray or flash-drying, can be mild and can be avoiding
oxidation and cis/trans isomerization. For example antioxidants,
e.g. BHT, ethoxyquin or other, can be added. In case the
carotenoids concentration in the biomass is to dilute, solvent
extraction can be used for their isolation, e.g. with alcohols,
ether or other organic solvents, e.g. with methanol, ethanol,
aceton, alcoholic potassium hydroxide, glycerol-phenol, liquefied
phenol or for example with acids or bases, like trichloroacetatic
acid or potassium hydroxide. A wide range of advantageous Methods
and techniques for the isolation of carotenoids, in particular of
the respective fine chemical, in particular of IPP or beta-carotine
can be found in the state of the art. In case phenol is used it can
for example be removed with ether and water extraction and the dry
eluate comprises a mixture of the carotenoids of the biomass.
[10794] [0386.0.24.24] Accordingly, it is possible to further
purify the respective fine chemicals or other carotenoids, in
particular the carotenes produced according to the invention. For
this purpose, the product-containing composition, e.g. a total or
partial lipid extraction fraction using organic solvents, e.g. as
described above, is subjected for example (without meaning to be
limited) to a saponification to remove triglycerides, partition
between e.g. hexane/methanol (separation of non-polar epiphase from
more polar hypophasic derivates) and separation via e.g. an open
column chromatography or HPLC in which case the desired product or
the impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use.
[10795] [0387.0.0.24] to [0392.0.0.24]: see [0387.0.0.0] to
[0392.0.0.0]
[10796] [0393.0.24.24] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [10797] (f) contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [10798] (g) identifying the nucleic acid molecules,
which hybridize under relaxed stringent conditions with the nucleic
acid molecule of the present invention in particular to the nucleic
acid molecule sequence as indicated in Table IA or IB, columns 5 or
7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641, resp., and,
optionally, isolating the full length cDNA clone or complete
genomic clone; [10799] (h) introducing the candidate nucleic acid
molecules in host cells, preferably in a plant cell or a
microorganism, appropriate for producing the respective fine
chemical; [10800] (i) expressing the identified nucleic acid
molecules in the host cells; [10801] (j) assaying the respective
fine chemical level in the host cells; and [10802] (k) identifying
the nucleic acid molecule and its gene product which expression
confers an increase in the respective fine chemical level in the
host cell after expression compared to the wild type.
[10803] [0394.0.0.24] to [0398.0.0.24]: see [0394.0.0.0] to
[0398.0.0.0]
[10804] [0399.0.24.24] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increased the respective fine chemical production in a
plant or microorganism, comprising the steps:
(d) culturing a cell or tissue or microorganism or maintaining a
plant expressing the polypeptide according to the invention or a
nucleic acid molecule encoding said polypeptide and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and the polypeptide of the present invention or used
in the process of the invention; and (e) identifying if the
compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout
system.
[10805] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in respective fine chemical
production.
[10806] [00399.1.24.24] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table IIA
or IIB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641 or a homolog thereof, e.g. comparing the phenotype of nearly
identical organisms with low and high activity of a protein as
indicated in Table IIA or IIB, columns 5 or 7, lines 279, 282, 285,
286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641 after incubation with the drug.
[10807] [0400.0.0.24] to [0415.0.0.24]: see [0400.0.0.0] to
[0415.0.0.0]
[10808] [0416.0.0.24] see [0416.0.0.0]
[10809] [0417.0.24.24] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the carotenoids, in particular carotene
production biosynthesis pathways. In particular, the overexpression
of the polypeptide of the present invention or the polypeptide
described for the process of the invention may protect an organism
such as a microorganism or a plant against inhibitors, which block
the carotenoid or carotene synthesis, in particular the respective
fine chemical synthesis in said organism. Examples of inhibitors or
herbicides blocking the synthesis of carotenoids in organism such
as microorganism or plants are for example classified in two
groups. The first group consists of inhibitors that cause the
accumulation of early intermediates in the pathway, particularly
the colorless phytene, e.g. diphenylamine. Other inhibitors
preferentially block late reactions in the pathway, notably the
cyclization of lycopene. Inhibitors are e.g. nicotine,
2-(4-chlorophenylthio)-triethylamine and other substituted amines
as well as nitrogenous heterocyclic bases, e.g. imidazole.
[10810] As carotene may protect organisms against damages of
oxidative stress, especially singlet oxygens, a increased level of
the respective respective fine chemical can protect plants against
herbicides which cause the toxic build-up of oxidative compounds,
e.g. singlet oxygen. For example, inhibition of the
protoporphorineogen oxidase (Protox), an enzyme important in the
synthesis of chlorophyll and heme biosynthesis results in the loss
of chlorophyll and carotenoids and in leaky membranes; the membrane
destruction is due to creation of free oxygen radicals (which is
also reported for other classic photosynthetic inhibitor
herbicides).
[10811] Accordingly, in one embodiment, the increase of the level
of the respective fine chemical is used to protect plants against
herbicides destroying membranes due to the creation of free oxygen
radicals.
[10812] Examples of inhibitors or herbicides building up oxidative
stress are aryl triazion, e.g. sulfentrazone, carfentrazone, or
diphenylethers, e.g. acifluorfen, lactofen, or oxyfluorfen, or
N-Phenylphthalimide, e.g. flumiclorac or flumioxazin, substituted
ureas, e.g. fluometuron, tebuthiuron, or diuron, linuron, or
triazines, e.g. atrazine, prometryn, ametryn, metributzin,
prometon, simazine, or hexazinone, or uracils, e.g. bromacil or
terbacil.
[10813] Carotenoid inhibitors are e.g. Pyridines and Pyridazinones,
e.g. norflurazon, fluridone or dithiopyr. Thus, in one embodiment,
the present invention relates to the use of an increase of the
respective fine chemical according to the present invention for the
protection of plants against carotenoids inhibitors as pyridines
and pyridazinones.
[10814] [0418.0.0.24] to [0423.0.0.24]: see [0418.0.0.0] to
[0423.0.0.0]
[10815] [0424.0.24.24] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other carotenoids, in
particular other carotene, e.g. astaxanthin, alpha or
gamma-carotene etc.
[10816] Accordingly, the nucleic acid of the invention, or the
nucleic acid molecule identified with the method of the present
invention or the complement sequences thereof, the polypeptide of
the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the respective fine chemical in a organism or part
thereof, e.g. in a cell.
[10817] [0424.1.0.24] In a further embodiment the present invention
relates to the use of the antagonist of the present invention, the
plant of the present invention or a part thereof, the microorganism
or the host cell of the present invention or a part thereof for the
production a cosmetic composition or a pharmaceutical composition.
Such a composition has antioxidative activity, photoprotective
activity, tanning activity, can be used for the treating of high
levels of cholesterol and/or lipids, can be used to protect, treat
or heal the above mentioned diseases, e.g. retinal disorders,
hyperholsterolemia, hyperlipidemia, and ahterosclerosis, or can be
used for the cleaning, conditioning, and/or treating of the skin,
e.g. if combined with a pharmaceutically or cosmetically acceptable
carrier.
[10818] The respective fine chemical, in particular beta-carotene
can be also used as stabilizer of other colours or oxygen sensitive
compounds.
[10819] [0425.0.0.24] to [0434.0.0.24]: see [0425.0.0.0] to
[0434.0.0.0]
[0435.0.24.24] Example 3
In-Vivo and In-Vitro Mutagenesis
[10820] [0436.0.24.24] An in vivo mutagenesis of organisms such as
green algae (e.g. Spongiococcum sp, e.g. Spongiococcum exentricum,
Chlorella sp.), Saccharomyces, Mortierella, Escherichia and others
mentioned above, which are beneficial for the production of
carotenoid, e.g. carotene, in particular beta-carotene, or its
precursour IPP, can be carried out by passing a plasmid DNA (or
another vector DNA) containing the desired nucleic acid sequence or
nucleic acid sequences, e.g. the nucleic acid molecule of the
invention or the vector of the invention, through E. coli and other
microorganisms (for example Bacillus spp. or yeasts such as
Saccharomyces cerevisiae) which are not capable of maintaining the
integrity of its genetic information.
[10821] Usual mutator strains have mutations in the genes for the
DNA repair system [for example mutHLS, mutD, mutT and the like; for
comparison, see Rupp, W. D. (1996) DNA repair mechanisms in
Escherichia coli and Salmonella, pp. 2277-2294, ASM: Washington].
The skilled worker knows these strains. The use of these strains is
illustrated for example in Greener, A. and Callahan, M. (1994)
Strategies 7; 32-34.
[10822] In-vitro mutation methods such as increasing the
spontaneous mutation rates by chemical or physical treatment are
well known to the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[10823] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[10824] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.24.24] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpine
[10825] [0438.0.24.24] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology, VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[10826] [0439.0.24.24] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255f) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[10827] [0440.0.24.24] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002) teaches a homolog recombination system dependent on
rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this system
for homologous recombination are vectors derived from the Ylp
series. Plasmid vectors derived for example from the 2.mu.-Vector
are known by the skilled worker and used for the expression in
yeasts. Other preferred vectors are for example pART1, pCHY21 or
pEVP11 as they have been described by McLeod et al. (EMBO J. 1987,
6:729-736) and Hoffman et al. (Genes Dev. 5, 1991: 561-571.) or
Russell et al. (J. Biol. Chem. 258, 1983: 143-149.). Other
beneficial yeast vectors are plasmids of the REP, REP-X, pYZ or RIP
series.
[10828] [0441.0.0.24] see [0441.0.0.0]
[10829] [0442.0.0.24] see [0442.0.0.0]
[10830] [0443.0.0.24] see [0443.0.0.0]
[0444.0.24.24] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[10831] [0445.0.24.24] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[10832] [0446.0.0.24] to [0450.0.0.24]: see [0446.0.0.0] to
[0450.0.0.0]
[10833] [0451.0.0.24] see [0451.0.5.5]
[10834] [0452.0.0.24] to [0454.0.0.24]: see [0452.0.0.0] to
[0454.0.0.0]
[10835] [0455.0.24.24] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a fatty acid) can be determined by growing the
modified microorganisms or the modified plant under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the elevated production of
desired product (i.e. of the lipids or a fatty acid). These
analytical techniques are known to the skilled worker and comprise
spectroscopy, thin-layer chromatography, various types of staining
methods, enzymatic and microbiological methods and analytical
chromatography such as high-performance liquid chromatography (see,
for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2,
p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.,
(1987) "Applications of HPLC in Biochemistry" in: Laboratory
Techniques in Biochemistry and
[10836] Molecular Biology, Vol. 17; Rehm et al. (1993)
Biotechnology, Vol. 3, Chapter III: "Product recovery and
purification", p. 469-714, VCH: Weinheim; Better, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[10837] In addition to the abovementioned processes, plant lipids
are extracted from plant material as described by Cahoon et al.
(1999) Proc. Natl. Acad. Sci. USA 96 (22): 12935-12940 and Browse
et al. (1986) Analytic Biochemistry 152:141-145. The qualitative
and quantitative analysis of lipids is described by Christie,
William W., Advances in Lipid Methodology, Ayr/Scotland: Oily Press
(Oily Press Lipid Library; 2); Christie, William W., Gas
Chromatography and Lipids. A Practical Guide--Ayr, Scotland: Oily
Press, 1989, Repr. 1992, IX, 307 pp. (Oily Press Lipid Library; 1);
"Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952)--16
(1977) under the title: Progress in the Chemistry of Fats and Other
Lipids CODEN.
[10838] [0456.0.0.24]: see [0456.0.0.0]
[0457.0.24.24] Example 9
Purification of the Carotene and Determination of the Content
[10839] [0458.0.24.24] Abbreviations: GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[10840] The unambiguous detection for the presence of beta-carotene
or IPP can be obtained by analyzing recombinant organisms using
analytical standard methods: LC, LC-MSMS or TLC, as described The
total carotene produced in the organism for example in yeasts used
in the inventive process can be analysed for example according to
the following procedure:
[10841] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods.
[10842] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[10843] A typical sample pretreatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography. E.g.:
[10844] For analysis, solvent delivery and aliquot removal can be
accomplished with a robotic system comprising a single injector
valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000
W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of
50% potassium hydroxide hydro-ethanolic solution (4 water:1
ethanol) can be added to each vial, followed by the addition of 3
ml of octanol. The saponification treatment can be conducted at
room temperature with vials maintained on an IKA HS 501 horizontal
shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours
at 250 movements/minute, followed by a stationary phase of
approximately one hour.
[10845] Following saponification, the supernatant can be diluted
with 0.24 ml of methanol. The addition of methanol cqan be
conducted under pressure to ensure sample homogeneity. Using a 0.25
ml syringe, a 0.1 ml aliquot can be removed and transferred to HPLC
vials for analysis.
[10846] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete
with a quaternary pump, vacuum degassing system, six-way injection
valve, temperature regulated autosampler, column oven and
Photodiode Array detector can be used [Agilent Technologies
available through Ultra Scientific Inc., 250 Smith Street, North
Kingstown,
[10847] RI]. The column can be a Waters YMC30, 5-micron,
4.6.times.250 mm with a guard column of the same material [Waters,
34 Maple Street, Milford, Mass.]. The solvents for the mobile phase
can be 81 methanol: 4 water: 15 tetrahydrofuran (THF) stabilized
with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injections were
20 l. Separation can be isocratic at 30.degree. C. with a flow rate
of 1.7 ml/minute. The peak responses can be measured by absorbance
at 447 nm.
[10848] Carotenoid compositions can be determined for wild-type and
mutant samples selected from those identified in a screening
procedure. Petal samples can be stored in a -80.degree. C. freezer
until mutants were identified. Samples can be lyophilized, and the
dried tissue can be stored under argon at -80.degree. C. until
ready for analysis.
[10849] Extraction procedures can be performed under red light.
Dried petals can be ground to pass through a No. 40 sieve mesh
size. A ground sample can be accurately weighed and transferred
into a 100 ml red volumetric flask. To the sample, 500 microliters
I) of
[10850] H.sub.2O can be added, and the mixture can be swirled for 1
minute. Thirty ml of extractant solvent (10 ml hexane+7 ml
acetone+6 ml absolute alcohol+7 ml toluene) can be added, and the
flask can be shaken at 160 rpm for 10 minutes.
[10851] For saponification, 2 ml of 40% methanolic KOH can be added
into the flask, which can be then swirled for one minute. The flask
can be placed in a 56.degree. C. H.sub.2O bath for 20 minutes. An
air condenser can be attached to prevent loss of solvent. The
sample can be cooled in the dark for one hour with the condenser
attached. After cooling, 30 ml of hexane can be added, and the
flask can be shaken at 160 rpm for 10 minutes.
[10852] The shaken sample can be diluted to volume (100 ml) with
10% sodium sulfate solution and shaken vigorously for one minute.
The sample can be remained in the dark for at least 30 minutes. A
35 ml aliquot can be removed from the approximately 50 ml upper
phase, and transferred to a sample cup. An additional 30 ml of
hexane can be added into the flask that can be then shaken at 160
rpm for 10 minutes. After approximately one hour, the upper phases
can be combined. For HPLC analysis, 10 ml aliquots can be dried
under nitrogen and stored under argon at -80.degree. C.
[10853] HPLC equipment comprised an Alliance 2690 equipped with a
refrigerated autosampler, column heater and a Waters Photodiode
Array 996 detector (Waters Corp., 34 Maple Street Milford, Mass.
01757). Separation can be obtained with a YMC30 column, 3 m,
2.0.times.150 mm with a guard column of the same material.
Standards can be obtained from ICC Indorespective fine chemicals
Somerville, N.J. 088876 and from DHI-Water & Environment,
DK-2970 Horsholm, Denmark.
[10854] The dried mutant samples can be resuspended in
tetrahydrofuran and methanol to a total volume of 200 l and
filtered, whereas the control can be not additionally concentrated.
Carotenoids can be separated using a gradient method. Initial
gradient conditions can be 90% methanol: 5% water: 5% methyl
tert-butyl ether at a flow rate of 0.4 milliliters per minute
(ml/min). From zero to 15 minutes, the mobile phase can be changed
from the initial conditions to 80 methanol: 5 water: 15 methyl
tert-butyl ether, and from 15 to 60 minutes to 20 methanol: 5
water: 75 methyl tert-butyl ether. For the following 10 minutes,
the mobile phase can be returned to the initial conditions and the
column equilibrated for an additional 10 minutes. The column
temperature can be maintained at 27.degree. C. and the flow rate
was 0.4 ml/minute. Injections were 10 l. The majority of peak
responses can be measured at 450 nm and additional areas added from
286, 348, 400 and 472 nm extracted channels.
[10855] [0459.0.24.24] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the carotene can be further purified with a
so-called RPHPLC. As eluent acetonitrile/water or
chloroform/acetonitrile mixtures can be used. If necessary, these
chromatography steps may be repeated, using identical or other
chromatography resins. The skilled worker is familiar with the
selection of suitable chromatography resin and the most effective
use for a particular molecule to be purified.
[10856] [0460.0.0.24] see [0460.0.0.0]
[0461.0.24.24] Example 10
Cloning SEQ ID NO: 34228 for the Expression in Plants
[10857] [0462.0.0.24] see [0462.0.0.0]
[10858] [0463.0.24.24] SEQ ID NO: 34228 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[10859] [0464.0.0.24] to [0466.0.0.24]: see [0464.0.0.0] to
[0466.0.0.0]
[10860] [0466.1.0.24] In case the Herculase enzyme can be used for
the amplification, the PCR amplification cycles were as follows: 1
cycle of 2-3 minutes at 94.degree. C., followed by 25-30 cycles of
in each case 30 seconds at 94.degree. C., 30 seconds at
55-60.degree. C. and 5-10 minutes at 72.degree. C., followed by 1
cycle of 10 minutes at 72.degree. C., then 4.degree. C.
[10861] [0467.0.24.24] The following primer sequences were selected
for the gene SEQ ID NO: 34228: [10862] i) forward primer (SEQ ID
NO: 34320) [10863] ii) reverse primer (SEQ ID NO: 34321) or were
selected for the genes described in Table III, column 5, lines 278
to 289 or lines 637 to 641 as described in Table III, column 7,
lines 278 to 289 or lines 637 to 641.
[10864] [0468.0.0.24] to [0479.0.0.24]: see [0468.0.0.0] to
[0479.0.0.0]
[0480.0.24.24] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 34228
[10865] [0481.0.0.24] to [0513.0.0.24]: see [0481.0.0.0] to
[0513.0.0.0]
[10866] [0514.0.24.24] As an alternative, carotenes can be detected
as described in Deli, J. & Molnar, P. Paprika carotenoids:
Analysis, isolation, structure eucidation. Curr. Org. Chem. 6,
1197-1219 (2004) or Fraser, P. D., Pinto, M. E., Holloway, D. E.
& Bramley, P. M. Technical advance: application of
high-performance liquid chromatography with photodiode array
detection to the metabolic profiling of plant isoprenoids. Plant J.
24, 551-558 (2000).
[10867] The results of the different plant analyses can be seen
from the table 1 which follows:
TABLE-US-00141 TABLE 1 ORF Metabolite Method Min Max b0730
Isopentenyl Pyrophosphate LC 1.41 2.15 b1829 beta-Carotene LC 1.33
1.74 b1926 Isopentenyl Pyrophosphate LC 1.37 1.55 b2211 Isopentenyl
Pyrophosphate LC 1.46 1.48 b2699 beta-Carotene LC 1.43 1.76 b3172
Isopentenyl Pyrophosphate LC 1.35 1.74 b4129 Isopentenyl
Pyrophosphate LC 1.34 1.63 YBR089C-A beta-Carotene LC 1.52 2.08
YDR316W beta-Carotene LC 1.28 1.52 YDR407C Isopentenyl
Pyrophosphate LC 1.54 3.14 YDR513W beta-Carotene LC 1.39 1.43
YLL013C beta-Carotene LC 1.43 1.50 b0970 Isopentenyl Pyrophosphate
LC 1.36 2.17 b0481 Isopentenyl Pyrophosphate LC 1.35 1.39 b1736
Isopentenyl Pyrophosphate LC 1.51 2.37 b1738 Isopentenyl
Pyrophosphate LC 1.63 2.60 b3160 Isopentenyl Pyrophosphate LC 1.35
1.47
[10868] [0515.0.24.24] to [0552.0.0.24]: see [0515.0.0.0] to
[0552.0.0.0]
[0552.1.24.24]: Example 15
Metabolite Profiling Info from Zea mays
[10869] Zea mays plants were engineered, grown and analyzed as
described in Example 14c.
[10870] The results of the different Zea mays plants analysed can
be seen from Table 2 which follows:
TABLE-US-00142 TABLE 2 ORF Metabolite Min Max b1829 beta-Carotene
1.40 1.76
[10871] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in beta-Carotene in
genetically modified corn plants expressing the Escherichia coli
nucleic acid sequence b1829 resp.
[10872] In case the activity of the Escherichia coli protein b1829
or a heat shock protein with protease activity or its homolog, is
increased in corn plants, preferably, an increase of the fine
chemical beta-Carotene between 40% and 76% is conferred.
[10873] [00552.2.0.24]: see [00552.2.0.0]
[10874] [0553.0.24.24] [10875] 1. A process for the production of
carotene and/or IPP, which comprises (a) increasing or generating
the activity of a protein as indicated in Table IIA or IIB, columns
5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278,
280, 281, 283, 284 and/or 287 and/or lines 637 to 641 or a
functional equivalent thereof in a non-human organism, or in one or
more parts thereof; and (b) growing the organism under conditions
which permit the production of carotene and/or IPP in said
organism. [10876] 2. A process for the production of carotene
and/or IPP, comprising the increasing or generating in an organism
or a part thereof the expression of at least one nucleic acid
molecule comprising a nucleic acid molecule selected from the group
consisting of: [10877] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table IIA or IIB, columns 5 or 7, lines
279, 282, 285, 286, 288, and/or 289, and/or lines 278, 280, 281,
283, 284 and/or 287 and/or lines 637 to 641 or a fragment thereof,
which confers an increase in the amount of carotene and/or IPP in
an organism or a part thereof; [10878] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table IA or
IB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641;
[10879] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of carotene and/or IPP in an
organism or a part thereof; [10880] d) nucleic acid molecule which
encodes a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
carotene and/or IPP in an organism or a part thereof; [10881] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to [10882] (c) under under stringent hybridisation
conditions and conferring an increase in the amount of carotene
and/or IPP in an organism or a part thereof; [10883] f) nucleic
acid molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table III, columns 5 or 7, lines lines 279, 282, 285, 286, 288
and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287 and/or
lines 637 to 641 and conferring an increase in the amount of
carotene and/or IPP in an organism or a part thereof; [10884] g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of carotene and/or IPP in an organism or a
part thereof; [10885] h) nucleic acid molecule encoding a
polypeptide comprising a consensus as indicated in Table IV,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641
and conferring an increase in the amount of carotene and/or IPP in
an organism or a part thereof; and [10886] i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of carotene and/or IPP in an organism or a part thereof.
[10887] or comprising a sequence which is complementary thereto.
[10888] 3. The process of claim 1 or 2, comprising recovering of
the free or bound caroteneand/or IPP. [10889] 4. The process of any
one of claims 1 to 3, comprising the following steps: [10890] (a)
selecting an organism or a part thereof expressing a polypeptide
encoded by the nucleic acid molecule characterized in claim 2;
[10891] (b) mutagenizing the selected organism or the part thereof;
[10892] (c) comparing the activity or the expression level of said
polypeptide in the mutagenized organism or the part thereof with
the activity or the expression of said polypeptide of the selected
organisms or the part thereof; [10893] (d) selecting the mutated
organisms or parts thereof, which comprise an increased activity or
expression level of said polypeptide compared to the selected
organism or the part thereof; [10894] (e) optionally, growing and
cultivating the organisms or the parts thereof; and [10895] (f)
recovering, and optionally isolating, the free or bound carotene
and/or IPP produced by the selected mutated organisms or parts
thereof. [10896] 5. The process of any one of claims 1 to 4,
wherein the activity of said protein or the expression of said
nucleic acid molecule is increased or generated transiently or
stably. [10897] 6. An isolated nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[10898] a) nucleic acid molecule encoding of a polypeptide as
indicated in Table IIA or IIB, columns 5 or 7, lines lines 279,
282, 285, 286, 288 and/or 289 and/or lines 278, 280, 281, 283, 284
and/or 287 and/or lines 637 to 641 or a fragment thereof, which
confers an increase in the amount of carotene in an organism or a
part thereof; [10899] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table IA or IB, columns 5 or
7, lines 279, 282, 285, 286, 288 and/or 289 and/or lines 278, 280,
281, 283, 284 and/or 287 and/or lines 637 to 641; [10900] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of carotene and/or IPP in an
organism or a part thereof; [10901] d) nucleic acid molecule which
encodes a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
carotene and/or IPP in an organism or a part thereof; [10902] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of carotene and/or IPP in an
organism or a part thereof; [10903] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table III,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641
and conferring an increase in the amount of carotene and/or IPP in
an organism or a part thereof; [10904] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
carotene and/or IPP in an organism or a part thereof; [10905] h)
nucleic acid molecule encoding a polypeptide comprising a consensus
as indicated in Table IV, columns 5 or 7, lines 279, 282, 285, 286,
288 and/or 289 and/or lines 278, 280, 281, 283, 284 and/or 287
and/or lines 637 to 641 and conferring an increase in the amount of
carotene and/or IPP in an organism or a part thereof; and [10906]
i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of carotene and/or IPP
in an organism or a part thereof. [10907] whereby the nucleic acid
molecule distinguishes over the sequence as indicated in Table IA
or IB, columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289
and/or lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to
641 by one or more nucleotides. [10908] 7. A nucleic acid construct
which confers the expression of the nucleic acid molecule of claim
6, comprising one or more regulatory elements. [10909] 8. A vector
comprising the nucleic acid molecule as claimed in claim 6 or the
nucleic acid construct of claim 7. [10910] 9. The vector as claimed
in claim 8, wherein the nucleic acid molecule is in operable
linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [10911] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [10912] 11. The host cell of claim 10, which is a
transgenic host cell. [10913] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [10914] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [10915] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table IIA or IIB,
columns 5 or 7, lines 279, 282, 285, 286, 288 and/or 289 and/or
lines 278, 280, 281, 283, 284 and/or 287 and/or lines 637 to 641 by
one or more amino acids 15. An antibody, which binds specifically
to the polypeptide as claimed in claim 14. [10916] 16. A plant
tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [10917] 17. A method for screening for
agonists and antagonists of the activity of a polypeptide encoded
by the nucleic acid molecule of claim 6 conferring an increase in
the amount of carotene and/or IPP in an organism or a part thereof
comprising: [10918] (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of carotene and/or IPP in an organism or a part thereof with
a candidate compound or a sample comprising a plurality of
compounds under conditions which permit the expression the
polypeptide; [10919] (b) assaying the linoleic acid level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [10920] (c)
identifying a agonist or antagonist by comparing the measured
carotene and/or IPP level or polypeptide expression level with a
standard linoleic acid or polypeptide expression level measured in
the absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. [10921] 18. A process for
the identification of a compound conferring increased carotene
and/or IPP production in a plant or microorganism, comprising the
steps: [10922] (a) culturing a plant cell or tissue or
microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of carotene and/or IPP in an organism or a
part thereof and a readout system capable of interacting with the
polypeptide under suitable conditions which permit the interaction
of the polypeptide with said readout system in the presence of a
compound or a sample comprising a plurality of compounds and
capable of providing a detectable signal in response to the binding
of a compound to said polypeptide under conditions which permit the
expression of said readout system and of the polypeptide encoded by
the nucleic acid molecule of claim 6 conferring an increase in the
amount of carotene and/or IPP in an organism or a part thereof;
[10923] (b) identifying if the compound is an effective agonist by
detecting the presence or absence or increase of a signal produced
by said readout system. [10924] 19. A method for the identification
of a gene product conferring an increase in carotene and/or IPP
production in a cell, comprising the following steps: [10925] (a)
contacting the nucleic acid molecules of a sample, which can
contain a candidate gene encoding a gene product conferring an
increase in carotene and/or IPP after expression with the nucleic
acid molecule of claim 6; [10926] (b) identifying the nucleic acid
molecules, which hybridise under relaxed stringent conditions with
the nucleic acid molecule of claim 6; [10927] (c) introducing the
candidate nucleic acid molecules in host cells appropriate for
producing carotene and/or IPP; [10928] (d) expressing the
identified nucleic acid molecules in the host cells; [10929] (e)
assaying the carotene and/or IPP level in the host cells; and
[10930] (f) identifying nucleic acid molecule and its gene product
which expression confers an increase in the carotene and/or IPP
level in the host cell in the host cell after expression compared
to the wild type. [10931] 20. A method for the identification of a
gene product conferring an increase in carotene and/or IPP
production in a cell, comprising the following steps: a)
identifying in a data bank nucleic acid molecules of an organism;
which can contain a candidate gene encoding a gene product
conferring an increase in the carotene and/or IPP amount or level
in an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing carotene and/or IPP; c) expressing the
identified nucleic acid molecules in the host cells; d) assaying
the carotene and/or IPP level in the host cells; and e) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the carotene and/or IPP level in the host cell after
expression compared to the wild type. [10932] 21. A method for the
production of an agricultural composition comprising the steps of
the method of any one of claims 17 to 20 and formulating the
compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture.
[10933] 22. A composition comprising the nucleic acid molecule of
claim 6, the polypeptide of claim 14, the nucleic acid construct of
claim 7, the vector of any one of claim 8 or 9, an antagonist or
agonist identified according to claim 17, the compound of claim 18,
the gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [10934] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
carotene and/or IPP after expression. [10935] 24. Use of the
polypeptide of claim 14 or the nucleic acid construct claim 7 or
the gene product identified according to the method of claim 19 or
20 for identifying compounds capable of conferring a modulation of
carotene and/or IPP levels in an organism.
[10936] 25. Cosmetic, pharmaceutical, food or feed composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20. [10937] 26. The method of any one of
claims 1 to 5, the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20, wherein the carotene is beta-carotene
[10938] 27. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the protection of a plant against
a oxidative stress. [10939] 28. Use of the nucleic acid molecule of
claim 6, the polypeptide of claim 14, the nucleic acid construct of
claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the harvested material of claim
16, the host cell of claim 10 to 12 or the gene product identified
according to the method of claim 19 or 20 for the protection of a
plant against a oxidative stress causing or a carotenoid synthesis
inhibiting herbicide. [10940] 29. Use of the agonist identified
according to claim 17, the plant or plant tissue of claim 16, the
harvested material of claim 16, or the host cell of claim 10 to 12
for the production of a cosmetic composition.
[10941] [0554.0.0.24] Abstract: see [0554.0.0.0]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[10942] [0000.0.0.25] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[10943] [0001.0.0.25] see [0001.0.0.0]
[10944] [0002.0.25.25] Carbohydrates are aldehyde or ketone
compounds with multiple hydroxyl groups. Many carbohydrates have
the empirical formula (CH2O)n; some also contain nitrogen,
phosphorus, or sulfur.
[10945] Carbohydrates are classsfied in monosaccharides,
oligosaccharides, and polysaccharides.
[10946] Monosaccharides, or simple sugars, consist of a single
polyhydroxy aldehyde or ketone unit. Monosaccharides of more than
four carbons tend to have cyclic structures.
[10947] Oligosaccharides consist of short chains of monosaccharide
units, or residues, usually 2 to 19 units, joined by glycosidic
bonds.
[10948] The polysaccharides are sugar polymers containing more than
20 or so monosaccharide units, and some have hundreds or thousands
of units. Some polysaccharides are linear chains; others are
branched.
[10949] Carbohydrates are called saccharides or, if they are
relatively small, sugars.
[10950] In the present invention, saccharides means all of the
aforementioned carbohydrate, e.g. monosaccharides, preferably
fructose, glucose, inositol, galactose, arabinose xylose or other
pentoses or hexoses; oligosaccharides, preferably disaccharides
like sucrose, lactose or trisaccharides like raffinose; or
polysaccharides like starch or cellulose.
[10951] [0003.0.25.25] Carbohydrates are the most abundant class of
organic compounds found in living organisms.
[10952] They are a major source of metabolic energy, both for
plants and for animals. Aside from the sugars and starches that
meet this vital nutritional role, carbohydrates function in energy
storage (for example starch or glycogen), in signaling (by
glycoproteins and glycolipids, e.g. blood group determinants), fuel
the nervous system, muscle and virtually all cells, are parts of
nucleic acids (in genes, mRNA, tRNA, ribosomes), and as cell
surface markers as recognition sites on cell surfaces and signaling
in glycolipids and glycoproteins and also serve as a structural
material for example as cell wall components (cellulose).
[10953] [0004.0.25.25] Glucose, also called dextrose, is the most
widely distributed sugar in the plant and animal kingdoms and it is
the sugar present in blood as "blood sugar. It occupies a central
position in the metabolism of plants, animals, and many
microorganisms. Glucose is rich in potential energy, and thus a
good fuel; in the body is catabolised to produce ATP. It is stored
as a high molecular weight polymer such as starch or glycogen or is
converted to fatty acids. It is also a remarkably versatile
precursor, capable of supplying a huge array of metabolic
intermediates for biosynthetic reactions.
[10954] Based on its manifold features, glucose is used in
nutrition and medicine.
[10955] Fructose, also called levulose or "fruit sugar, is the most
important ketose sugar.
[10956] Fructose is a hexose and is a reducing sugar. Fructose is
used a a sweetener by diabetics because it does not rise the blood
sugar level, even in large amounts.
[10957] Fructose and glucose are the main carbohydrate constituents
of honey. Those hexoses are further the main components of many
oligo- and polysaccharides, like sucrose, raffinose, stachyose,
trehalose, starch, cellulose or dextran.
[10958] The most frequent disaccharide is sucrose (saccharose,
b-D-fructofuranosyl-a-D-glucopyranosid, cane sugar, beet sugar,
sugar in a narrow sense of a name for commercially available
sucrose meaning sucrose is the sugar that is commonly called
"sugar) which consists of the six-carbon sugars D-glucose and
D-fructose. It is formed by plants but not by animals. Sucrose is a
major intermediate product of photosynthesis; in many plants it is
the principal form in which sugar is transported from the leaves to
other parts of the plant body. In mammalians sucrose is an
obligatory component of blood and its content in blood is kept at
the stable level. It is strongly necessary for brain cells as well
as for normal functioning of the central nervous system. Sugar is
widely-known as a source of glycogen--a substance, feeding liver,
heart and muscles. It is one of the most widely-used food products
and is the major disaccharide in most diets. It is present in
honey, maple sugar, fruits, berries, and vegetables. It may be
added to food products as liquid or crystalline sucrose or as
invert sugar. It is commercially prepared from sugar cane or sugar
beets. Sucrose can provide a number of desirable functional
qualities to food products including sweetness, mouth-feel, and the
ability to transform between amorphous and crystalline states.
High-concentrated sucrose is a natural preserving agent, it
determines gel-formation processes, gives necessary viscosity to
the products. Sucrose is a raw material for caramel, colour
etc.
[10959] Sucrose is further an excellent fermentation feedstock,
which is of specific interest for fermentation industry (including
a number of non-food industries--pharmaceutical industries). The
presence of eight hydroxyl groups in the sucrose molecule provides
a theoretical possibility of a very large number of sucrose
derivatives. Sucrose derivatives are used by industries in
production of detergents, emulsifiers (sucrose+fatty acids) and
adhesives (sucrose octa acetate).
[10960] Sucrose is a precursor to a group of carbohydrates in
plants known as the raffinose family of oligosaccharides found in
many plant seeds especially legumes. This family contains the
trisaccharide raffinose, the tetrasaccharide stachyose and the
pentasaccharide verbascose. Oligosaccharides of the
raffinose-series are major components in many food legumes
(Shallenberger et al., J. Agric. Food Chem., 9,1372; 1961).
Raffinose
(beta-D-fructofuranosyl-6-O-alpha-D-galactopyranosyl-alpha-D-gl-
ucopyranosid, melitriose, gossypose, melitose), which consists of
sucrose with .alpha.-galactose attached through its C-4 atom to the
1 position on the fructose residue and is thought to be second only
to sucrose among the nonstructural carbohydrates with respect to
abundance in the plant kingdom. It may be ubiquitous, at least
among higher plants. Raffinose accumulate in significant quantities
in the edible portion of many economically significant crop
species. Examples include soybean, sugar beet, cotton, canola and
all of the major edible leguminous crops including beans, peas,
lentil and lupine.
[10961] An important key intermediates in the formation of
raffinose and stachyose is myo-inositol
(cyclohexan-1,2,3,4,5,6-hexaole), the most common cyclitol.
Myo-inositol is fundamental to many different aspects of plant
growth and development. In addition to its role as the precursor
for phytic acid biosynthesis, myo-inositol is also used for uronide
and pentose biosynthesis, it is also present in phosphoinositides
of plant cell membranes, as well as other complex plant lipids
including glycophosphoceramides.
[10962] Furthermore, it is also a precursor of other naturally
occurring inositol isomers, and many of these as well as
myo-inositol are distributed as methyl ethers in a species specific
pattern throughout the plant kingdom. Myo-inositol is an important
growth factor.
[10963] The most carbohydrates found in nature occur as
polysaccharides which are polymers of medium to high molecular
weight. Polysaccharides, also called glycans, differ from each
other in the identity of their recurring monosaccharide units, in
the length of their chains, in the types of bonds linking the
units, and in the degree of branching.
[10964] Starch is the most valuable polysaccharide. Normal native
starches consist of a mixture of 15-30% amylose and 70-85%
amylopectin. Amylose structurally is a linear polymer of
anhydroglucose units, of molecular weight approximately between 40
000 and 340 000, the chains containing 250 to 2000 anhydroglucose
units. Amylopectin is considered to be composed of anhydroglucose
chains with many branch points; the molecular weight may reach as
high as 80 000 000.
[10965] Starch is the most important, abundant, digestible food
polysaccharide. It occurs as the reserve polysaccharide in the
leaf, stem, root, seed, fruit and pollen of many higher plants. It
occurs as discrete, partially-crystalline granules whose size,
shape, and gelatinization temperature depend on the botanical
source of the starch. Common food starches are derived from seed
(wheat, maize, rice, barley) and root (potato, cassava/tapioca)
sources. Starches have been modified to improve desired functional
characteristics and are added in relatively small amounts to foods
as food additives. Another important polysaccharide is cellulose.
Cellulose is the most commonly seen polysaccharide and scientist
estimate that over one trillion tons of cellulose are synthesized
by plants each year. Cellulose forms the cell wall of plants. It is
yet a third polymer of the monosaccharide glucose. Cellulose
differs from starch and glycogen because the glucose units form a
two-dimensional structure, with hydrogen bonds holding together
nearby polymers, thus giving the molecule added stability. A single
"cellulose fiber" can consist of up to 10000 individual
anhydroglucose units. In cellulose, the individual fiber molecules
are arranged in bundles and thus form so called micro fibrils which
ultimately result in a "densely woven" net like structure of
cellulose molecules. The strong cohesion between the individual
cellulose fibers is due to the huge number of strong hydrogen
bonds.
[10966] Cellulose is the major polysaccharide of grass, leaves and
trees and is said to include around 50% of all biological carbon
found on our planet. It is the basic material of natural substances
such as wood, flax or cotton and consists of long, unbranched fiber
molecules. Cellulose, as plant fiber, cannot be digested by human
beings therefore cellulose passes through the digestive tract
without being absorbed into the body. Some animals, such as cows
and termites, contain bacteria in their digestive tract that help
them to digest cellulose. Nevertheless, cellulose is of importance
in human nutrition in that fiber is an essential part of the diet,
giving bulk to food and promoting intestinal motility.
[10967] [0005.0.25.25] The polysaccharides starch and cellulose are
the most important raw material in the industrial and commercial
production of glucose. In the common procedure starch or cellulose
are acidly or enzymatically hydrolysed to glucose.
[10968] Fructose is usually also produced from starch by
enzymatically transforming it into glucose syrup and subsequently
treating with isomerase, leading to a conversion of glucose to
fructose.
[10969] Succrose is obtained commercially from the expressed juice
of sugar cane or of sugar beet.
[10970] Myo-inositol exists in nature either in its free form
(found, for example, in sugarcane, beet molasses, and almond hulls)
or as a hexaphosphate called phytin (found, for example, in corn
steep liquor). Industrial purification of phytin from corn steep
liquor involves precipitation with calcium, followed by hydrolysis
with a strong acid.
[10971] Separation of free form inositols from plant extracts
involves treatment with acid and separation of myo-inositol by
column (U.S. Pat. No. 5,482,631) or the use of ion-exchange (U.S.
Pat. No. 4,482,761).
[10972] Cellulose is a very important industrial product. As
disclosed above, it serves as row material for monosaccharides. It
is further used in the manufacture of paper, textiles, plastics,
explosives, packaging material (Cellophane.RTM.), feed, food and
fermentation products. Cellulose is obtained primarily by acid or
alkaline hydrolize.
[10973] Starch occurs intracellularly as large clusters or
granules. These granular starch consists of microscopic granules,
which differ in size and shape, depending on the plant source. The
granules are insoluble in water at room temperature. There is a
quite number of methods known for the extraction of starch. For
example a slurry of grinded starch containing plant material is
heated, whereby the granules swell and eventually burst, dispersing
the starch molecules into the solution. During the liquefaction
step, the long-chained starch is further degraded into smaller
branched and linear units (maltodextrins) by an alpha-amylase. A
large number of processes have been described for converting starch
to starch hydrolysates, such as maltose, glucose or specialty
syrups, either for use as sweeteners or as precursors for other
saccharides such as fructose. A process for enzymatic hydrolysis of
granular starch into a soluble starch hydrolysate is disclosed in
US 20050042737.
[10974] [0006.0.25.25] Carbohydrates play a major role in human and
animal diets, comprising some 40-75% of energy intake. Their most
important nutritional property is digestibility. Some of them are
hydrolyzed by enzymes of the human gastrointestinal system to
monosaccharides that are absorbed in the small intestine and enter
the pathways of carbohydrate metabolism. Others can be digested by
certain animals. Carbohydrates, fat and protein are the energy
yielding nutrients in animal feed. In the average diet for farm
animals, carbohydrates are included at levels of 70-80%. For
example pig diets are mainly based on cereals which contain the
main part of the energy providing nutrients that are essential for
pigs.
[10975] With view to the increasing global demand for food because
of the growing world population and at the same time the shrinking
availability of arable land, it is important to increase the food
and feed quality, particulary the availability of certain essential
nutrients, preferably carbohydrates, preferably polysaccharides
like starch or cellulose and/or monosaccharides like fructose,
glucose and/or myo-inositol and/or trisaccharides like raffinose
and/or disaccharides like sucrose. Nutritional improvements in
foods and feeds could help to meet these demands for improved
quality. Modern agricultural biotechnology, which involves the
application of cellular and molecular techniques to transfer DNA
that encodes a desired trait to food and feed crops, is a powerful
complement to traditional methods to meet global food and feed
requirements.
[10976] [0007.0.25.25] Furthermore the physicochemical properties
such as viscosity and the capacity to bind water and ions, vary
between different cereals. Consequently, different cereal
properties affect digestion and fermentation as well as microbial
populations in the gastro-intestinal tract in various ways.
Gastro-intestinal disturbances comprise a major problem for health
of humans and animals.
[10977] There is a need for suitable dietary composition and food
or feed ingredients, preferably cereals, legumes or fruits which
promotes a beneficial gut environment and thereby preventing
gastro-intestinal disorders.
[10978] Therefore improving the quality of foodstuffs and animal
feeds is an important task of the food-and-feed industry. This is
necessary since, for example, carbohydrates, which occur in plants
and some microorganisms are limited with regard to the supply of
mammals. Especially advantageous for the quality of foodstuffs and
animal feeds is as balanced as possible a carbohydrate profile in
the diet since a great excess of some sugars above a specific
concentration in the food has only some or little or no positive
effect.
[10979] [0008.0.25.25] Genetically modified plants having improved
nutritional profiles are known in the state of art. US 20030070192
discloses a DNA expression cassette which alters the sugar alcohol
of tranformed plants. U.S. Pat. No. 5,908,975 concerns methods for
synthesis and accumulation of fructose polymers in transgenic
plants by selective expression of bacterial fructosyltransferase
genes using tissue specific promoters and a vacuole targeting
sequence.
[10980] WO89/12386 describes a method for the production of glucose
and fructose polymers in transgenic tomato plants.
[10981] A stress tolerance sequences including proteins like
galactinol synthase (GOLS) and raffinose synthase (RAFS), which are
up regulated in response to stress and lead to the production of
raffinose is disclosed in US 20050055748.
[10982] U.S. Pat. No. 6,887,708 provides nucleotide sequences
encoding polypeptides having the function of GIGANTEA gene of
Arabidopsis thaliana which allows the manipulation of the starch
accumulation process in plants.
[10983] Grain having an embryo with a genotype heterozygous for two
or more wild type genes (for example, Aa/Bb) and an endosperm
having a genotype heterozygous for such genes and leading to plants
with altered the normal starch synthesis pathway is disclosed in US
20050091716.
[10984] [0009.0.25.25] Nevertheles, there is a constant need for
providing novel enzyme activities or direct or indirect regulators
and thus alternative methods with advantageous properties for
producing carbohydrates, preferably polysaccharides like starch or
cellulose and/or monosaccharides like fructose, glucose and/or
myo-inositol and/or trisaccharides like raffinose and/or
disaccharides like sucrose or its precursor in organisms, e.g. in
transgenic organisms.
[10985] [0010.0.25.25] Another problem is the seasonal change in
carbohydrate composition of plants and optimum harvest periods for
are complicated by issues of timing.
[10986] [0011.0.25.25] To ensure constantly a high quality of foods
and animal feeds, it is necessary to add one or a plurality of
carbohydrates, preferably polysaccharides like starch or cellulose
and/or monosaccharides like fructose, glucose and/or myo-inositol
and/or trisaccharides like raffinose and/or disaccharides like
sucrose in a balanced manner to suit the organism.
[10987] [0012.0.25.25] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes which
participate in the biosynthesis of carbohydrates, preferably
polysaccharides like starch or cellulose and/or monosaccharides
like fructose, glucose and/or myo-inositol and/or trisaccharides
like raffinose and/or disaccharides like sucrose and make it
possible to produce them specifically on an industrial scale
without unwanted byproducts forming. In the selection of genes for
or regulators of biosynthesis two characteristics above all are
particularly important. On the one hand, there is as ever a need
for improved processes for obtaining the highest possible contents
of carbohydrates, preferably polysaccharides like starch or
cellulose and/or monosaccharides like fructose, glucose and/or
myo-inositol and/or trisaccharides like raffinose and/or
disaccharides like sucrose; on the other hand as less as possible
byproducts should be produced in the production process.
[10988] The added carbohydrates further beneficially affects the
microflora by selectively stimulating the growth and/or activity of
beneficial bacteria.
[10989] Another aspect is the significant reduction of cost of
production and manufacturing not only to the nutrition, in
particular sweetener industry, but also agriculture and cosmetic
and health industry.
[10990] [0013.0.0.25] see [0013.0.0.0]
[10991] [0014.0.25.25] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose. Accordingly, in the
present invention, the term "the fine chemical" as used herein
relates to (a) "carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose". Further, the term "the fine chemicals" as
used herein also relates to compositions of fine chemicals
comprising carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose.
[10992] [0015.0.25.25] In one embodiment, the term "carbohydrate"
or "the fine chemical" or "the respective fine chemical" means at
least one chemical compound with carbohydrate activity selected
from the group of preferably polysaccharides, more preferably
starch and/or cellulose and/or monosaccharides, more preferably
fructose, glucose and/or myo-inositol and/or trisaccharides, more
preferably raffinose and/or disaccharides, more preferably sucrose.
In an preferred embodiment, the term "the fine chemical" or the
term "carbohydrate" or the term "the respective fine chemical"
means at least one chemical compound with carbohydrate activity
selected from the group "carbohydrates, preferably polysaccharides,
more preferably starch and/or cellulose and/or monosaccharides,
more preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose".
[10993] An increased carbohydrate content normally means an
increased total carbohydrate content. However, an increased
carbohydrate content also means, in particular, a modified content
of the above-described compounds with carbohydrate activity,
without the need for an inevitable increase in the total
carbohydrate content. In a preferred embodiment, the term "the fine
chemical" means carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose in free form or its salts or its ester or
its ether or bound to acylglycerol.
[10994] [0015.1.22.22] A measure for the content of the
polysaccharides, preferably starch and cellulose, of the invention
can be the content of anhydroglucose. This compound is the analyte
which indicates the presence of the polysaccharides, preferably
starch and cellulose, of the invention if the samples are prepared
and measured as described in the examples.
[10995] [0016.0.25.25] Accordingly, the present invention relates
to a process comprising [10996] (a) increasing or generating the
activity of one or more [10997] b0252, b1430, b1693, b3231,
YER174C, b0050-b1539, b3919 and/or b4232-protein(s) or of a protein
having the sequence of a polypeptide encoded by a nucleic acid
molecule indicated in Table II, columns 5 or 7, lines 290 to 294
and/or 604 to 607; [10998] in a non-human organism in one or more
parts thereof and [10999] (b) growing the organism under conditions
which permit the production of the fine chemical, meaning of starch
and/or cellulose or fine chemicals comprising starch and/or
cellulose in said organism; [11000] or [11001] (a) increasing or
generating the activity of one or more b0161, b0695, b1708, b1926,
b2597, b2664, b4239, b4327, b0124, b0149, b1318, b1463, b2491,
b3260, b3578, b3619 and/or b4122-protein(s) or of a protein having
the sequence of a polypeptide encoded by a nucleic acid molecule
indicated in Table II, columns 5 or 7, lines 295 to 302 and/or 608
to 616; [11002] in a non-human organism in one or more parts
thereof and [11003] (b) growing the organism under conditions which
permit the production of the fine chemical, meaning of fructose or
fine chemicals comprising fructose in said organism; [11004] or
[11005] (a) increasing or generating the activity of one or more
b0161, b1708, b1926, b2599, b4239, YJL072C, b1463, b1736, b2491,
b3578 and/or b3619-protein(s) or of a protein having the sequence
of a polypeptide encoded by a nucleic acid molecule indicated in
Table II, columns 5 or 7, lines 303 to 308 and/or 617 to 621;
[11006] in a non-human organism in one or more parts thereof and
[11007] (b) growing the organism under conditions which permit the
production of the fine chemical, meaning of glucose or fine
chemicals comprising glucose in said organism; [11008] or [11009]
(a) increasing or generating the activity of one or more b0138,
b0290, b2023, b2699, b3172, b3430, b4129, YBR204C, YDR112W,
YGR261C, YIL150C, YJL099W, YOR044W, YOR350C, b1463, b1961, b4074
and/or YHR072W-A-protein(s) or of a protein having the sequence of
a polypeptide encoded by a nucleic acid molecule indicated in Table
II, columns 5 or 7, lines 309 to 322 and/or 622 to 625; [11010] in
a non-human organism in one or more parts thereof and [11011] (b)
growing the organism under conditions which permit the production
of the fine chemical, meaning of myo-inositol or fine chemicals
comprising myo-inositol in said organism; [11012] or [11013] (a)
increasing or generating the activity of one or more b0161, b0730,
b1701, b1886, b2664, b2699, b3601, YBR184W, YGR261C and/or
b3578-protein(s) or of a protein having the sequence of a
polypeptide encoded by a nucleic acid molecule indicated in Table
II, columns 5 or 7, lines 323 to 331 and/or 626;
[11014] in a non-human organism in one or more parts thereof and
[11015] (b) growing the organism under conditions which permit the
production of the fine chemical, meaning of raffinose or fine
chemicals comprising raffinose in said organism; [11016] or [11017]
(a) increasing or generating the activity of one or more b0019
and/or b4239-protein(s) or of a protein having the sequence of a
polypeptide encoded by a nucleic acid molecule indicated in Table
II, columns 5 or 7, lines 332 to 333; [11018] in a non-human
organism in one or more parts thereof and [11019] (b) growing the
organism under conditions which permit the production of the fine
chemical, meaning of sucrose or fine chemicals comprising sucrose
in said organism.
[11020] Accordingly, the present invention relates to a process for
the production of carbohydrates comprising [11021] (a) increasing
or generating the activity of one or more proteins having the
activity of a protein indicated in Table II, column 3, lines 290 to
333 and/or lines 604 to 626 or having the sequence of a polypeptide
encoded by a nucleic acid molecule indicated in Table I, column 5
or 7, lines 290 to 333 and/or lines 604 to 626, in a non-human
organism in one or more parts thereof and [11022] (b) growing the
organism under conditions which permit the production of the fine
chemical, thus, carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose resp.
[11023] [0016.1.25.25] Accordingly, the term "the fine chemical"
means in one embodiment "polysaccharides, preferably starch and/or
cellulose" in relation to all sequences listed in Table I to IV,
lines 290 to 294 and/or and/or 604 to 607 or homologs thereof
and
means in one embodiment "monosaccharides, preferably fructose" in
relation to all sequences listed in Table I to IV, lines 295 to 302
and/or 608 to 616 or homologs thereof, and means in one embodiment
"monosaccharides, preferably glucose" in relation to all sequences
listed in Tables I to IV, lines 303 to 308 and/or 617 to 621 or
homologs thereof and means in one embodiment "monosaccharides,
preferably myo-inositol" in relation to all sequences listed in
Table I to IV, lines 309 to 322 and/or 622 to 625, and means in one
embodiment "trisaccharides, preferably raffinose" in relation to
all sequences listed in Table I to IV, lines 323 to 331 and/or 626,
and means in one embodiment "disaccharides, preferably sucrose" in
relation to all sequences listed in Table I to IV, lines 332 to
333.
[11024] Accordingly, in one embodiment the term "the fine chemical"
means "fructose", "glucose" and "raffinose" in relation to all
sequences listed in Table I to IV, lines 295, 303 and 323 and/or
lines 614 and 620 and 626;
[11025] in one embodiment the term "the fine chemical" means
"fructose", "glucose" and "myo-inositol" in relation to all
sequences listed in Table I to IV, lines 611, 617 and 622 and/or
lines 611 and 617 and 622;
[11026] in one embodiment the term "the fine chemical" means
"fructose" and "glucose" in relation to all sequences listed in
Table I to IV, lines 297 and 304 and/or lines 612 and 619 and/or
lines 614 and 620 and/or lines 615 and 621 and or lines 611 and
617;
[11027] in one embodiment the term "the fine chemical" means
"fructose" and "glucose" in relation to all sequences listed in
Table I to IV, lines 298 and 305,
[11028] in one embodiment the term "the fine chemical" means
"fructose" and "raffinose" in relation to all sequences listed in
Table I to IV, lines 300 and 327;
[11029] in one embodiment the term "the fine chemical" means
"fructose", "glucose" and "sucrose" in relation to all sequences
listed in Table I to IV, lines 301,307 and 333;
[11030] in one embodiment the term "the fine chemical" means
"myo-inositol" and "raffinose" in relation to all sequences listed
in Table I to IV, lines 312 and 328;
[11031] in one embodiment the term "the fine chemical" means
"myo-inositol" and "raffinose" in relation to all sequences listed
in Table I to IV, lines 318 and 331;
[11032] in one embodiment the term "the fine chemical" means
"myo-inositol" and "fructose" in relation to all sequences listed
in Table I to IV, lines 611 and 622;
[11033] in one embodiment the term "the fine chemical" means
"myo-inositol" and "glucose" in relation to all sequences listed in
Table I to IV, lines 617 and 622;
[11034] in one embodiment the term "the fine chemical" means
"fructose" and "raffinose" in relation to all sequences listed in
Table I to IV, lines 614 and 626;
[11035] in one embodiment the term "the fine chemical" means
"glucose" and "raffinose" in relation to all sequences listed in
Table I to IV, lines 620 and 626;
[11036] Accordingly, the term "the fine chemical" can mean "starch
and/or cellulose", "fructose", "glucose", "myo-inositol",
"raffinose" and/or "sucrose", owing to circumstances and the
context. In order to illustrate that the meaning of the term "the
fine chemical" means "starch and/or cellulose", "fructose",
"glucose", "myo-inositol", "raffinose" and/or "sucrosel" the term
"the respective fine chemical" is also used.
[11037] [0017.0.25.25] to [0018.0.25.25]: see [0017.0.0.0] to
[0018.0.0.0]
[11038] [0019.0.25.25] Advantageously the process for the
production of the respective fine chemical leads to an enhanced
production of the respective fine chemical. The terms "enhanced" or
"increase" mean at least a 10%, 20%, 30%, 40% or 50%, preferably at
least 60%, 70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%,
400% or 500% higher production of the respective fine chemical in
comparison to the reference as defined below, e.g. that means in
comparison to an organism without the aforementioned modification
of the activity of a protein having the activity of a protein
indicated in Table II, column 3, lines 290 to 333 and/or lines 604
to 626 or encoded by nucleic acid molecule indicated in Table I,
columns 5 or 7, lines 290 to 333 and/or lines 604 to 626.
[11039] [0020.0.25.25] Surprisingly it was found, that the
transgenic expression of at least one of the Saccaromyces
cerevisiae protein(s) indicated in Table II, Column 3, line 294 for
starch and/or cellulose;
[11040] line 308 for glucose;
lines 316 to 322 and/or 625 for myo-inositol; lines 330 to 331 for
raffinose; in Arabidopsis thaliana conferred an increase in the
respective fine chemical content of the transformed plants and/or
at least one of the Escherichia coli K12 proteins indicated in
Table II, Column 3, lines 290 to 293 and/or 604 to 607 for starch
and/or cellulose; lines 295 to 302 and/or 608 to 616 for fructose;
lines 303 to 307 and/or 617 to 621 for glucose; lines 309 to 315
and/or 622 to 624 for myo-inositol; lines 323 to 329 and/or 626 for
raffinose; lines 332 to 333 for sucrose; in Arabidopsis thaliana
conferred an increase in the respective fine chemical content of
the transformed plants.
[11041] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein as indicated in
Table II, column 5, lines 295, 303 and 323 and/or lines 614 and 620
and 626 in Arabidopsis thaliana conferred an increase in fructose,
glucose or raffinose (or the respective fine chemical) content of
the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of fructose; in one
embodiment, said protein or its homologs are used for the
production of glucose; in one embodiment, said protein or its
homologs are used for the production of raffinose; in one
embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of: fructose, glucose and raffinose.
[11042] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein as indicated in
Table II, column 5, lines 611, 617 and 622 in Arabidopsis thaliana
conferred an increase in fructose, glucose or myo-inositol (or the
respective fine chemical) content of the transformed plants. Thus,
in one embodiment, said protein or its homologs are used for the
production of fructose; in one embodiment, said protein or its
homologs are used for the production of glucose; in one embodiment,
said protein or its homologs are used for the production of
myo-inositol; in one embodiment, said protein or its homologs are
used for the production of one or more fine chemical selected from
the group consisting of: fructose, glucose and myo-inositol.
[11043] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein as indicated in
Table II, column 5, lines 297 and 304 and/of lines 298 and 305
and/or and/or lines 612 and 619 and/or lines 614 and 620 in
Arabidopsis thaliana conferred an increase in fructose or glucose
(or the respective fine chemical) content of the transformed
plants. Thus, in one embodiment, said protein or its homologs are
used for the production of fructose; in one embodiment, said
protein or its homologs are used for the production of glucose; in
one embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of: fructose and glucose.
[11044] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein as indicated in
table II, line 300 and 327 in Arabidopsis thaliana conferred an
increase in fructose or raffinose (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of frutose; in
one embodiment, said protein or its homologs are used for the
production of raffinose; in one embodiment, said protein or its
homologs are used for the production of fructose and raffinose.
[11045] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein as indicated in
table II, lines 301, 307 and 333 in Arabidopsis thaliana conferred
an increase in fructose, glucose or sucrose (or the respective fine
chemical) content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of fructose; in one embodiment, said protein or its
homologs are used for the production of glucose; in one embodiment,
said protein or its homologs are used for the production of
sucrose; in one embodiment, said protein or its homologs are used
for the production of fructose and glucose; in one embodiment, said
protein or its homologs are used for the production of fructose and
sucrose; in one embodiment, said protein or its homologs are used
for the production of glucose and sucrose; in one embodiment, said
protein or its homologs are used for the production of fructose,
glucose and sucrose.
[11046] Accordingly, it was surprisingly found, that the transgenic
expression of the Escherichia coli K12 protein as indicated in
table II, line 312 and 328, or the Saccaromyces cerevisiae proteins
as indicated in Table II, column 3, lines 318 and 331 in
Arabidopsis thaliana conferred an increase in myo-inositol or
raffinose (or the respective fine chemical) content of the
transformed plants. Thus, in one embodiment, one of said proteins
or its homologs are used for the production of myo-inositol; in one
embodiment, said protein or its homologs are used for the
production of raffinose; in one embodiment, said protein or its
homologs are used for the production of myo-inositol and
raffinose.
[11047] [0021.0.25.25] see [0021.0.0.0]
[11048] [0022.0.25.25] The sequence of b0019 from Escherichia coli
K12 has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as a Na+/H+
antiporter. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the Na+/H+-exchanging protein nhaA superfamily,
preferably a protein with a Na+/H+ antiporter protein activity or
its homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably sucrose in free or
bound form in an organism or a part thereof, as mentioned.
[11049] The sequence of b0138 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative fimbrial-like
adhesin protein. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the Escherichia coli K12 fimbrial-like adhesin protein
or its homolog, e.g. as shown herein, from Escherichia coli K12 or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably myo-inositol in free
or bound form in an organism or a part thereof, as mentioned.
[11050] The sequence of b0161 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a periplasmic serine protease
(heat shock protein). Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of the helicobacter serine proteinase superfamily,
preferably a protein with a periplasmic serine protease (heat shock
protein) activity or its homolog, e.g. as shown herein, from
Escherichia coli K12 or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of carbohydrate,
preferably fructose, glucose and/or raffinose in free or bound form
in an organism or a part thereof, as mentioned.
[11051] The sequence of b0252 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is not yet defined but it is a conserved protein
of unknown function, similarity with helicase and ligase proteins.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the helicase or ligase protein, preferably a protein with activity
of b0252 protein from Escherichia coli K12 or its homolog, e.g. as
shown herein, from Escherichia coli K12 or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
carbohydrate, preferably starch and/or cellulose in free or bound
form in an organism or a part thereof, as mentioned.
[11052] The sequence of b0290 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a receptor protein.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Escherichia coli yagW protein superfamily, preferably a protein
with a receptor protein activity or its homolog, e.g. as shown
herein, from Escherichia coli K12 or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
carbohydrate, preferably myo-inositol in free or bound form in an
organism or a part thereof, as mentioned.
[11053] The sequence of b0695 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a sensory histidine kinase in
two-component regulatory system. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of the sensor histidine kinase
superfamily, preferably a protein with a sensory histidine kinase
in two-component regulatory system activity or its homolog, e.g. as
shown herein, from Escherichia coli K12 or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
carbohydrate, preferably fructose in free or bound form in an
organism or a part thereof, as mentioned.
[11054] The sequence of b0730 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a transcriptional regulator of
succinylCoA synthetase operon and fatty acyl response regulator.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the transcription regulator GntR superfamily, preferably a protein
with a transcriptional regulator of succinylCoA synthetase operon
and fatty acyl response regulator activity or its homolog, e.g. as
shown herein, from Escherichia coli K12 or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
carbohydrate, preferably raffinose in free or bound form in an
organism or a part thereof, as mentioned.
[11055] The sequence of b1430 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a
S-adenosyl-L-methionine-dependent methyltransferase conferring
tellurite resistance. Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of the hemagglutinin hag1 superfamily, preferably a
protein with a S-adenosyl-L-methionine-dependent methyltransferase
activity conferring tellurite resistance or its homolog, e.g. as
shown herein, from Escherichia coli K12 or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
carbohydrate, preferably starch and/or cellulose in free or bound
form in an organism or a part thereof, as mentioned.
[11056] The sequence of b1693 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a 3-dehydroquinate
dehydratase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the 3-dehydroquinate dehydratase superfamily,
preferably a protein with a 3-dehydroquinate dehydratase activity
or its homolog, e.g. as shown herein, from Escherichia coli K12 or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably starch and/or
cellulose in free or bound form in an organism or a part thereof,
as mentioned.
[11057] The sequence of b1701 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a CoenzymeA-dependent ligase
with firefly luciferase-like ATPase Domain. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the 4-coumarate-CoA ligase or
acetate-CoA ligase superfamily, preferably a protein with a
CoenzymeA-dependent ligase activity or its homolog, e.g. as shown
herein, from Escherichia coli K12 or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
carbohydrate, preferably raffinose in free or bound form in an
organism or a part thereof, as mentioned.
[11058] The sequence of b1708 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a lipoprotein. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of the protein H11314
superfamily, preferably a protein with a lipoprotein activity or
its homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably fructose and/or
glucose in free or bound form in an organism or a part thereof, as
mentioned.
[11059] The sequence of b1886 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a methyl-accepting chemotaxis
protein II or aspartate sensor receptor. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the methyl-accepting
chemotaxis protein superfamily, preferably a protein with a
methyl-accepting chemotaxis protein II or aspartate sensor receptor
activity or its homolog, e.g. as shown herein, from Escherichia
coli K12 or its homolog, e.g. as shown herein, for the production
of the fine chemical, meaning of carbohydrate, preferably raffinose
in free or bound form in an organism or a part thereof, as
mentioned.
[11060] The sequence of b1926 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a flagellar protein fliT.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
flagellar protein fliT or its homolog, e.g. as shown herein, from
Escherichia coli K12 or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of carbohydrate,
preferably fructose and/or glucose in free or bound form in an
organism or a part thereof, as mentioned.
[11061] The sequence of b2023 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a glutamine amidotransferase
subunit of imidazole glycerol phosphate synthase heterodimer.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the amidotransferase hisH superfamily with trpG homology,
preferably a protein with a glutamine amidotransferase subunit of
imidazole glycerol phosphate synthase heterodimer activity or its
homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably myo-inositol in free
or bound form in an organism or a part thereof, as mentioned.
[11062] The sequence of b2597 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a ribosome associated factor.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Pseudomonas putida protein rpoX superfamily, preferably a
protein with a ribosome associated factor activity or its homolog,
e.g. as shown herein, from Escherichia coli
[11063] K12 or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of carbohydrate,
preferably fructose in free or bound form in an organism or a part
thereof, as mentioned.
[11064] The sequence of b2599 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a bifunctional enzyme:
chorismate mutase P (N-terminal) and prephenate dehydratase
(C-terminal). Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the pheA bifunctional enzyme superfamily with
prephenate dehydratase homology, preferably a protein with a
bifunctional enzyme: chorismate mutase P (N-terminal) and
prephenate dehydratase (C-terminal) activity or its homolog, e.g.
as shown herein, from Escherichia coli K12 or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
carbohydrate, preferably glucose in free or bound form in an
organism or a part thereof, as mentioned.
[11065] The sequence of b2664 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a transcriptional repressor
with DNA-binding Winged helix domain (GntR familiy). Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product with an activity of the transcription
regulator gabP superfamily, preferably a protein with a putative
transcriptional repressor with DNA-binding Winged helix domain
(GntR familiy) activity or its homolog, e.g. as shown herein, from
Escherichia coli K12 or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of carbohydrate,
preferably fructose and/or raffinose in free or bound form in an
organism or a part thereof, as mentioned.
[11066] The sequence of b2699 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a DNA strand exchange and
recombination protein with protease and nuclease activity.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the recombination protein recA superfamily, preferably a protein
with a DNA strand exchange and recombination protein with protease
and nuclease activity or its homolog, e.g. as shown herein, from
Escherichia coli K12 or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of carbohydrate,
preferably myo-inositol and/or raffinose in free or bound form in
an organism or a part thereof, as mentioned.
[11067] The sequence of b3172 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474,1997,
and its activity is being defined as a argininosuccinate
synthetase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the argininosuccinate synthetase superfamily,
preferably a protein with a argininosuccinate synthetase activity
or its homolog, e.g. as shown herein, from Escherichia coli K12 or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably myo-inositol in free
or bound form in an organism or a part thereof, as mentioned.
[11068] The sequence of b3231 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a 50S ribosomal subunit
protein L13. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the Escherichia coli ribosomal protein L13 superfamily,
preferably a protein with a 50S ribosomal subunit protein L13
activity or its homolog, e.g. as shown herein, from Escherichia
coli K12 or its homolog, e.g. as shown herein, for the production
of the fine chemical, meaning of carbohydrate, preferably starch
and/or cellulose in free or bound form in an organism or a part
thereof, as mentioned.
[11069] The sequence of b3430 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a glucose-1-phosphate
adenylyltransferase. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of the glucose-1-phosphate adenylyltransferase
superfamily, preferably a protein with a glucose-1-phosphate
adenylyltransferase activity or its homolog, e.g. as shown herein,
from Escherichia coli K12 or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of carbohydrate,
preferably myo-inositol in free or bound form in an organism or a
part thereof, as mentioned.
[11070] The sequence of b3601 from Escherichia coli K12 has been
published in Blattner et al.,
[11071] Science 277(5331), 1453-1474, 1997, and its activity is
being defined as a transcriptional repressor for mannitol
utilization. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein with a
transcriptional repressor activity or its homolog, e.g. as shown
herein, from Escherichia coli K12 or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
carbohydrate, preferably raffinose in free or bound form in an
organism or a part thereof, as mentioned.
[11072] The sequence of b4129 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a (inducible) lysine tRNA
synthetase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the lysine-tRNA ligase superfamily, preferably a
protein with a lysine tRNA synthetase activity or its homolog, e.g.
as shown herein, from Escherichia coli K12 or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
carbohydrate, preferably myo-inositol in free or bound form in an
organism or a part thereof, as mentioned.
[11073] The sequence of b4239 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a trehalose-6-P hydrolase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the alpha-glucosidase superfamily with alpha-amylase core homology,
preferably a protein with a trehalose-6-P hydrolase activity or its
homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably fructose, glucose
and/or sucrose in free or bound form in an organism or a part
thereof, as mentioned.
[11074] The sequence of b4327 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a HTH-type transcriptional
regulator with periplasmic binding protein domain (LysR family).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the protein b2409 superfamily with alpha-amylase core homology,
preferably a protein with a HTH-type transcriptional regulator with
periplasmic binding protein domain (LysR family) activity or its
homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably fructose in free or
bound form in an organism or a part thereof, as mentioned.
[11075] The sequence of b4327 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a HTH-type transcriptional
regulator with periplasmic binding protein domain (LysR family).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the protein b2409 superfamily with alpha-amylase core homology,
preferably a protein with a HTH-type transcriptional regulator with
periplasmic binding protein domain (LysR family) activity or its
homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably fructose in free or
bound form in an organism or a part thereof, as mentioned.
[11076] The sequence of YBR184w from Saccharomyces cerevisiae has
been published in Goffeau, Science 274 (5287), 546-547, 1996, and
in Feldmann, EMBO J., 13, 5795-5809, 1994, and its activity is is
not yet characterized. Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of the protein YBR184w superfamily, preferably a
protein with a protein YBR184w activity or its homolog, e.g. as
shown herein, from Saccaromyces cerevisiae or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
carbohydrate, preferably raffinose in free or bound form in an
organism or a part thereof, as mentioned.
[11077] The sequence of YBR204C from Saccharomyces cerevisiae has
been published in Goffeau, Science 274 (5287), 546-547, 1996, and
in Feldmann, EMBO J., 13, 5795-5809, 1994, and its activity is
being defined as a peroxisomal lipase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein with a peroxisomal lipase activity or its homolog,
e.g. as shown herein, from Saccaromyces cerevisiae or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of carbohydrate, preferably myo-inositol in free or bound
form in an organism or a part thereof, as mentioned.
[11078] The sequence of YDR112w from Saccharomyces cerevisiae
(accession number S69752), and its activity is not yet defined.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein with a YDR112w activity or
its homolog, e.g. as shown herein, from Saccaromyces cerevisiae or
its homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably myo-inositol in free
or bound form in an organism or a part thereof, as mentioned.
[11079] The sequence of YER174C from Saccharomyces cerevisiae has
been published in Dietrich, Nature 387 (6632 Suppl), 78-81, 1997,
and Goffeau, Science 274 (5287), 546-547, 1996, and its activity is
being defined as a hydroperoxide and superoxide-radical responsive
glutathione-dependent oxidoreductase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the thioredoxin superfamily,
preferably a protein with a hydroperoxide and superoxide-radical
responsive glutathione-dependent oxidoreductase activity or its
homolog, e.g. as shown herein, from Saccaromyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably starch and/or
cellulose in free or bound form in an organism or a part thereof,
as mentioned.
[11080] The sequence of YGR261C from Saccharomyces cerevisiae has
been published in Tettelin, Nature 387 (6632 Suppl), 81-84, 1997,
and Goffeau, Science 274 (5287), 546-547, 1996, and its activity is
being defined as a clathrin assembly complex beta adaptin
component. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the beta-adaptin superfamily, preferably a protein with
a clathrin assembly complex beta adaptin component activity or its
homolog, e.g. as shown herein, from Saccaromyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably myo-inositol and/or
raffinose in free or bound form in an organism or a part thereof,
as mentioned.
[11081] The sequence of YIL150C from Saccharomyces cerevisiae has
been published in Goffeau st al., Science 274 (5287), 546-547, 1996
and Churcher et al., Nature 387 (6632 Suppl), 84-87, 1997, and its
activity is being defined as a chromatin binding protein, required
for S-phase (DNA synthesis) initiation or completion. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a protein with a chromatin binding protein, required for
S-phase (DNA synthesis) initiation or completion activity or its
homolog, e.g. as shown herein, from Saccaromyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably myo-inositol in free
or bound form in an organism or a part thereof, as mentioned.
[11082] The sequence of YJL072C from Saccharomyces cerevisiae has
been published in Goffeau, A., Science 274 (5287), 546-547, 1996
and Galibert, F., EMBO J. 15 (9), 2031-2049, 1996, and its activity
is being defined as a subunit of the GINS complex required for
chromosomal DNA replication. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of the Saccharomyces cerevisiae membrane
protein YJL072c superfamily, preferably a protein with a subunit of
the GINS complex required for chromosomal DNA replication activity
or its homolog, e.g. as shown herein, from Saccaromyces cerevisiae
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of carbohydrate, preferably glucose in free
or bound form in an organism or a part thereof, as mentioned.
[11083] The sequence of YJL099W from Saccharomyces cerevisiae has
been published in Goffeau, A., Science 274 (5287), 546-547, 1996
and Galibert, F., EMBO J. 15 (9), 2031-2049, 1996, and its activity
is being defined as a protein involved in chitin biosynthesis
and/or its regulation. Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of a protein involved in chitin biosynthesis and/or its
regulation or its homolog, e.g. as shown herein, from
[11084] Saccaromyces cerevisiae or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
carbohydrate, preferably myo-inositol in free or bound form in an
organism or a part thereof, as mentioned.
[11085] The sequence of YOR044W from Saccharomyces cerevisiae has
been published in Dujon, Nature 387 (6632 Suppl), 98-102, 1997, and
Goffeau, Science 274 (5287), 546-547, 1996, and its activity is not
yet characterized. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of the Saccharomyces cerevisiae membrane protein YOR044w
superfamily, preferably a protein with a YOR044W activity or its
homolog, e.g. as shown herein, from Saccaromyces cerevisiae or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of carbohydrate, preferably myo-inositol in free
or bound form in an organism or a part thereof, as mentioned.
[11086] The sequence of YOR350C from Saccharomyces cerevisiae has
been published in Dujon, Nature 387 (6632 Suppl), 98-102, 1997, and
Goffeau, Science 274 (5287), 546-547, 1996, and its activity is not
yet characterized. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of the Saccharomyces cerevisiae MNE1 protein superfamily,
preferably a protein with a YOR350C activity or its homolog, e.g.
as shown herein, from Saccaromyces cerevisiae or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
of carbohydrate, preferably myo-inositol in free or bound form in
an organism or a part thereof, as mentioned.
[11087] The sequence of b0050 (Accession number NP.sub.--414592)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a conserved protein potentially involved in protein
protein interaction. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of apaG protein superfamily, preferably a protein with the
activity of a conserved protein potentially involved in
protein-protein interaction from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
starch and/or cellulose, particular for increasing the amount of
starch and/or cellulose, preferably starch and/or cellulose in free
or bound form in an organism or a part thereof, as mentioned. The
sequence of b0124 (Accession number NP.sub.--414666) from
Escherichia coli K12 has been published in Blattner et al., Science
277 (5331), 1453-1474, 1997, and its activity is being defined as a
glucose dehydrogenase. Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of glucose dehydrogenase (pyrroloquinoline-quinone)
superfamily, preferably a protein with the activity of a glucose
dehydrogenase from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of fructose,
particular for increasing the amount of fructose, preferably
fructose in free or bound form in an organism or a part thereof, as
mentioned.
[11088] The sequence of b0149 (Accession number NP.sub.--414691)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a bifunctional penicillin-binding protein 1b: glycosyl
transferase (N-terminal); transpeptidase (C-terminal). Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product with an activity of penicillin-binding
protein 1B superfamily, preferably a protein with the activity of a
bifunctional penicillin-binding protein 1b: glycosyl transferase
(N-terminal); transpeptidase (C-terminal) from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of fructose, in particular for increasing the
amount of fructose, preferably fructose in free or bound form in an
organism or a part thereof, as mentioned.
[11089] The sequence of b1318 from Escherichia coli K12 (Accession
number NP.sub.--415834) has been published in Blattner, Science
277(5331), 1453-1474, 1997, and its activity is beeing defined as a
sugar transport protein (of the ABC superfamily). Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with the activity of the inner membrane
protein malK (with ATP-binding cassette homology) superfamily,
preferably a protein with a sugar transport protein (of the ABC
superfamily) activity from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
fructose, in particular for increasing the amount of fructose in
free or bound form in an organism or a part thereof, as
mentioned.
[11090] The sequence of b1463 from Escherichia coli K12 (Accession
number NP.sub.--415980) has been published in Blattner, Science
277(5331), 1453-1474, 1997, and its activity is beeing defined as a
N-hydroxyarylamine 0-acetyltransferase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with the activity of the arylamine
acetyltransferase superfamily, preferably a protein with a
N-hydroxyarylamine 0-acetyltransferase activity from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of fructose and/of glucose and/or myo-inositol,
in particular for increasing the amount of fructose, in particular
for increasing the amount of glucose, in particular for increasing
the amount of myo-inositol, in particular for increasing the amount
of fructose and glucose, in particular for increasing the amount of
fructose and myo-inositol, in particular for increasing the amount
of glucose and myo-inositol, in particular for increasing the
amount of fructose and glucose and myo-inositol, preferably of
fructose and/or glucose and/or myo-inositol in free or bound form
in an organism or a part thereof, as mentioned.
[11091] The sequence of b1539 (Accession number NP.sub.--416057)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a NADP-dependent L-serine/L-allo-threonine
dehydrogenase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of ribitol dehydrogenase, short-chain alcohol
dehydrogenase homology superfamily, preferably a protein with the
activity of a NADP-dependent L-serine/L-allo-threonine
dehydrogenase from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of starch and/or
cellulose, particular for increasing the amount of starch and/or
cellulose, preferably starch and/or cellulose in free or bound form
in an organism or a part thereof, as mentioned.
[11092] The sequence of b1736 (Accession number NP.sub.--416250)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a PEP-dependent phosphotransferase enzyme,
cellobiose/arbutin/salicin sugar-specific protein. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of phosphotransferase system
lactose-specific enzyme II, factor III superfamily, preferably a
protein with the activity of a PEP-dependent phosphotransferase
enzyme, cellobiose/arbutin/salicin sugar-specific protein from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of glucose, in particular for increasing
the amount of glucose, preferably glucose in free or bound form in
an organism or a part thereof, as mentioned.
[11093] The sequence of b1961 (Accession number NP.sub.--416470)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a DNA cytosine methylase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of site-specific
methyltransferase (cytosine-specific) EcoRII superfamily,
preferably a protein with the activity of a DNA cytosine methylase
from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of myo-inositol, in
particular for increasing the amount of myo-inositol, preferably
myo-inositol in free or bound form in an organism or a part
thereof, as mentioned.
[11094] The sequence of b2491 (Accession number NP.sub.--416986)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a transcriptional activator for expression of
hydrogenase 4 genes, interacts with sigma 54 (EBP family).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
RNA polymerase sigma factor interaction domain homology,
transcription activator fhlA, nif-specific regulatory protein,
response regulator homology superfamily, preferably a protein with
the activity of a transcriptional activator for expression of
hydrogenase 4 genes, interacts with sigma 54 (EBP family) from E.
coli or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of fructose and/or glucose, in
particular for increasing the amount of fructose, in particular for
increasing the amount of glucose, in particular for increasing the
amount of fructose and glucose, preferably fructose and/or glucose
in free or bound form in an organism or a part thereof, as
mentioned.
[11095] The sequence of b3260 (Accession number NP.sub.--417726)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a tRNA-dihydrouridine synthase B. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of transcription regulator yacF
superfamily, preferably a protein with the activity of a
tRNA-dihydrouridine synthase B from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
fructose, in particular for increasing the amount of fructose,
preferably fructose in free or bound form in an organism or a part
thereof, as mentioned.
[11096] The sequence of b3578 (Accession number YP 026232) from
Escherichia coli K12 has been published in Blattner et al., Science
277(5331), 1453-1474, 1997, and its activity is being defined as
putative component of transport system. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a putative component of transport system from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of fructose and/or glucose and/or raffinose, in
particular for increasing the amount of fructose, in particular for
increasing the amount of glucose, in particular for increasing the
amount of raffinose, in particular for increasing the amount of
fructose and glucose, in particular for increasing the amount of
fructose and raffinose, in particular for increasing the amount of
glucose and raffinose, in particular for increasing the amount of
fructose, glucose and raffinose, preferably fructose and/or glucose
and/of raffinose in free or bound form in an organism or a part
thereof, as mentioned.
[11097] The sequence of b3619 (Accession number NP.sub.--418076)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a ADP-L-glycero-D-mannoheptose-6-epimerase,
NAD(P)-binding. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of ADPglyceromanno-heptose 6-epimerase, UDPglucose
4-epimerase homology superfamily, preferably a protein with the
activity of a ADP-L-glycero-D-mannoheptose-6-epimerase,
NAD(P)-binding from E. coli or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of fructose and/or
glucose, in particular for increasing the amount of fructose, in
particular for increasing the amount of glucose, in particular for
increasing the amount of fructose and glucose, preferably fructose
and/or glucose in free or bound form in an organism or a part
thereof, as mentioned.
[11098] The sequence of b3919 (Accession number NP.sub.--418354)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a triosephosphate isomerase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of triose-phosphate isomerase
superfamily, preferably a protein with the activity of a
triosephosphate isomerase from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
starch and/or cellulose, in particular for increasing the amount of
starch and/or cellulose, preferably starch and/or cellulose in free
or bound form in an organism or a part thereof, as mentioned.
[11099] The sequence of b4074 (Accession number NP.sub.--418498)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a cytochrome c-type biogenesis protein. Accordingly, in
one embodiment, the process of the present invention comprises the
use of a gene product with an activity of nrfE protein superfamily,
preferably a protein with the activity of a Cytochrome c-type
biogenesis protein from E. coli or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
myo-inositol, in particular for increasing the amount of
myo-inositol, preferably myo-inositol in free or bound form in an
organism or a part thereof, as mentioned.
[11100] The sequence of b4122 (Accession number NP.sub.--418546)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a fumarase B (fumarate hydratase Class I). Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product with an activity of iron-dependent
fumarate hydratase and/or iron-dependent tartrate dehydratase alpha
chain homology superfamily, preferably a protein with the activity
of a fumarase B (fumarate hydratase Class I) from E. coli or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of fructose, in particular for increasing the
amount of fructose, preferably fructose in free or bound form in an
organism or a part thereof, as mentioned.
[11101] The sequence of b4232 from Escherichia coli K12 (ACCESSION
No. NP.sub.--416986) has been published in Blattner, Science
277(5331), 1453-1474, 1997, and its activity is beeing defined as a
fructose-1,6-bisphosphatase. Accordingly, in one embodiment, the
process of the present invention comprises the use of a
"fructose-1,6-bisphosphatase" from E. coli or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
methionin, in particular for increasing the amount of methionine in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of a protein of the superfamily "fructose-bisphosphatase",
preferably having an activity in C-compound and carbohydrate
metabolism, C-compound and carbohydrate utilization, energy,
glycolysis and gluconeogenesis, plastid, photosynthesis, more
preferred having an "fructose-1,6-bisphosphatase"-activity, is
increased or generated, e.g. from E. coli or a homolog thereof.
Accordingly, in one embodiment, in the process of the present
invention the activity of a "fructose-1,6-bisphosphatase" or its
homolog is increased for the production of the fine chemical,
meaning of starch and/or cellulose, in particular for increasing
the amount of starch and/or cellulose in free or bound form in an
organism or a part thereof, as mentioned.
[11102] The sequence of YHR072W-A from Saccharomyces cerevisiae
(ACCESSION NP.sub.--058135) has been published in Goffeau et al.,
Science 274 (5287), 546-547, 1996 and its activity is beeing
defined as that of a constituent of small nucleolar
ribonucleoprotein particles containing H/ACA-type snoRNAs, which
are required for pseudouridylation and processing of pre-18S rRNA.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a constituent of small nucleolar
ribonucleoprotein particles containing H/ACA-type snoRNAs, which
are required for pseudouridylation and processing of pre-18S rRNA
from Saccaromyces cerevisiae or its homolog, e.g. as shown herein,
for the production of the fine chemical, meaning of myo-inositol,
in particular for increasing the amount of myo-inositol, preferably
myo-inositol in free or bound form in an organism or a part
thereof, as mentioned.
[11103] [0023.0.25.25] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[11104] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, line 294 for starch
and/or cellulose; line 308 for glucose; lines 316 to 322 and/or 625
for myo-inositol; lines 330 to 331 and/or 626 for raffinose resp.,
is a homolog having the same or a similar activity, resp. In
particular an increase of activity confers an increase in the
content of the respective fine chemical in the organisms. In one
embodiment, the homolog is a homolog with a sequence as indicated
in Table I or II, column 7, line 294 for starch and/or cellulose;
line 308 for glucose; lines 316 to 322 and/or 625 for myo-inositol;
lines 330 to 331 and/or 626 for raffinose resp. In one embodiment,
the homolog of one of the polypeptides indicated in Table II,
column 3, line 294 for starch and/or cellulose; line 308 for
glucose; lines 316 to 322 and/or 625 for myo-inositol; lines 330 to
331 and/or 626 for raffinose resp., is derived from an eukaryotic.
In one embodiment, the homolog is derived from Fungi. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 294, is derived from Ascomyceta. In one embodiment,
the homolog of a polypeptide indicated in Table II, column 3, line
294, is derived from Saccharomycotina. In one embodiment, the
homolog of a polypeptide indicated in Table II, column 3, line 294
for starch and/or cellulose; line 308 for glucose; lines 316 to 322
and/or 625 for myo-inositol; lines 330 to 331 and/or 626 for
raffinose resp., is derived from Saccharomycetes. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, line 294 for starch and/or cellulose; line 308 for
glucose; lines 316 to 322 and/or 625 for myo-inositol; lines 330 to
331 and/or 626 for raffinose resp., is a homolog being derived from
Saccharomycetales. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 294 for starch and/or
cellulose; line 308 for glucose; lines 316 to 322 and/or 625 for
myo-inositol; lines 330 to 331 and/or 626 for raffinose resp. is a
homolog having the same or a similar activity being derived from
Saccharomycetaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, line 294 for starch and/or
cellulose; line 308 for glucose; lines 316 to 322 and/or 625 for
myo-inositol; lines 330 to 331 and/or 626 for raffinose resp., is a
homolog having the same or a similar activity being derived from
Saccharomycetes.
[11105] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 290 to 293 is a
homolog having the same or a similar activity. In particular an
increase of activity confers an increase in the content of the
respective fine chemical in the organisms preferably of
carbohydrate, preferably starch and/or cellulose. In one
embodiment, the homolog is a homolog with a sequence as indicated
in Table I or II, column 7, lines 290 to 293, resp. In one
embodiment, the homolog of one of the polypeptides indicated in
Table II, column 3,290 to 293 is derived from an bacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 290 to 293 is derived from Proteobacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 290 to 293 is a homolog having the same or a
similar activity being derived from Gammaproteobacteria. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 290 to 293 is derived from Enterobacteriales. In
one embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 290 to 293 is a homolog being derived from
Enterobacteriaceae. In one embodiment, the homolog of a polypeptide
indicated in Table II, column 3, lines 290 to 293 is a homolog
having the same or a similar activity and being derived from
Escherichia.
[11106] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table II, column 3, lines 290 to 293
and/or 604 to 607 for starch and/or cellulose; lines 295 to 302
and/or 608 to 616 for fructose; lines 303 to 307 and/or 617 to 621
for glucose; lines 309 to 315 and/or 622 to 624 for myo-inositol;
lines 323 to 329 for raffinose; lines 332 to 333 for sucrose, resp.
is a homolog having the same or a similar activity. In particular
an increase of activity confers an increase in the content of the
respective fine chemical in the organisms preferably of
carbohydrate, preferably fructose. In one embodiment, the homolog
is a homolog with a sequence as indicated in Table I or II, column
7, lines 290 to 293 and/or 604 to 607 for starch and/or cellulose;
lines 295 to 302 and/or 608 to 616 for fructose; lines 303 to 307
and/or 617 to 621 for glucose; lines 309 to 315 and/or 622 to 624
for myo-inositol; lines 323 to 329 for raffinose; lines 332 to 333
for sucrose, resp. In one embodiment, the homolog of one of the
polypeptides indicated in Table II, column 3, lines 290 to 293
and/or 604 to 607 for starch and/or cellulose; lines 295 to 302
and/or 608 to 616 for fructose; lines 303 to 307 and/or 617 to 621
for glucose; lines 309 to 315 and/or 622 to 624 for myo-inositol;
lines 323 to 329 for raffinose; lines 332 to 333 for sucrose, resp.
is derived from an bacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 290 to 293
and/or 604 to 607 for starch and/or cellulose; lines 295 to 302
and/or 608 to 616 for fructose; lines 303 to 307 and/or 617 to 621
for glucose; lines 309 to 315 and/or 622 to 624 for myo-inositol;
lines 323 to 329 for raffinose; lines 332 to 333 for sucrose, resp.
is derived from Proteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 290 to 293
and/or 604 to 607 for starch and/or cellulose; lines 295 to 302
and/or 608 to 616 for fructose; lines 303 to 307 and/or 617 to 621
for glucose; lines 309 to 315 and/or 622 to 624 for myo-inositol;
lines 323 to 329 for raffinose; lines 332 to 333 for sucrose, resp.
is a homolog having the same or a similar activity being derived
from Gammaproteobacteria. In one embodiment, the homolog of a
polypeptide indicated in Table II, column 3, lines 290 to 293
and/or 604 to 607 for starch and/or cellulose; lines 295 to 302
and/or 608 to 616 for fructose; lines 303 to 307 and/or 617 to 621
for glucose; lines 309 to 315 and/or 622 to 624 for myo-inositol;
lines 323 to 329 for raffinose; lines 332 to 333 for sucrose, resp.
is derived from Enterobacteriales. In one embodiment, the homolog
of a polypeptide indicated in Table II, column 3, lines 290 to 293
and/or 604 to 607 for starch and/or cellulose; lines 295 to 302
and/or 608 to 616 for fructose; lines 303 to 307 and/or 617 to 621
for glucose; lines 309 to 315 and/or 622 to 624 for myo-inositol;
lines 323 to 329 for raffinose; lines 332 to 333 for sucrose, resp.
is a homolog being derived from Enterobacteriaceae. In one
embodiment, the homolog of a polypeptide indicated in Table II,
column 3, lines 290 to 293 and/or 604 to 607 for starch and/or
cellulose; lines 295 to 302 and/or 608 to 616 for fructose; lines
303 to 307 and/or 617 to 621 for glucose; lines 309 to 315 and/or
622 to 624 for myo-inositol; lines 323 to 329 for raffinose; lines
332 to 333 for sucrose, resp. is a homolog having the same or a
similar activity and being derived from Escherichia.
[11107] [0023.1.25.25] Homologs of the polypeptide indicated in
Table II, column 3, lines 290 to 333 and/or lines 604 to 626 may be
the polypeptides encoded by the nucleic acid molecules indicated in
Table I, column 7, lines 290 to 333 and/or lines 604 to 626, resp.,
or may be the polypeptides indicated in Table II, column 7, lines
290 to 333 and/or lines 604 to 626, resp.
[11108] [0024.0.0.9] see [0024.0.0.0]
[11109] [0025.0.25.25] In accordance with the invention, a protein
or polypeptide has the "activity of an protein of the invention",
e.g. the activity of a protein indicated in Table II, column 3,
lines 290 to 294 and/or 604 to 607 for starch and/or cellulose;
lines 295 to 302 and/or 608 to 616 for fructose; lines 303 to 308
and/or 617 to 621 for glucose; lines 309 to 322 and/or 622 to 625
for myo-inositol; lines 323 to 331 and/or 626 for raffinose; lines
332 to 333 for sucrose; , resp., if its de novo activity, or its
increased expression directly or indirectly leads to increased
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose, resp., in
the organism or a part thereof, preferably in a cell of said
organism. In a preferred embodiment, the protein or polypeptide has
the above-mentioned additional activities of a protein indicated in
Table II, column 3, lines 290 to 294 and/or 604 to 607 for starch
and/or cellulose; lines 295 to 302 and/or 608 to 616 for fructose;
lines 303 to 308 and/or 617 to 621 for glucose; lines 309 to 322
and/or 622 to 625 for myo-inositol; lines 323 to 331 and/or 626 for
raffinose; lines 332 to 333 for sucrose, resp. Throughout the
specification the activity or preferably the biological activity of
such a protein or polypeptide or an nucleic acid molecule or
sequence encoding such protein or polypeptide is identical or
similar if it still has the biological or enzymatic activity of any
one of the proteins indicated in Table II, column 3, lines 290 to
294 and/or 604 to 607 for starch and/or cellulose; lines 295 to 302
and/or 608 to 616 for fructose; lines 303 to 308 and/or 617 to 621
for glucose; lines 309 to 322 and/or 622 to 625 for myo-inositol;
lines 323 to 331 and/or 626 for raffinose; lines 332 to 333 for
sucrose, resp., or which has at least 10% of the original enzymatic
activity, preferably 20%, particularly preferably 30%, most
particularly preferably 40% in comparison to any one of the
proteins indicated in Table II, column 3, line 294 for starch
and/or cellulose; line 308 for glucose; lines 316 to 322 and/or 625
for myo-inositol; lines 330 to 331 and/or 626 for raffinose resp.
of Saccharomyces cerevisiae and/or any one of the proteins
indicated in Table II, column 3, lines 290 to 293 and/or 604 to 607
for starch and/or cellulose; lines 295 to 302 and/or 608 to 616 for
fructose; lines 303 to 307 for glucose; lines 309 to 315 and/or 622
to 624 for myo-inositol; lines 323 to 329 for raffinose; lines 332
to 333 for sucrose, resp. of E. coli K12.
[11110] [0025.1.0.25]: see [0025.1.0.0]
[11111] [0026.0.0.25] to [0033.0.0.25]: see [0026.0.0.0] to
[0033.0.0.0]
[11112] [0034.0.25.25] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity of the polypeptide of the invention, e.g. as
result of an increase in the level of the nucleic acid molecule of
the present invention or an increase of the specific activity of
the polypeptide of the invention. E.g., it differs by or in the
expression level or activity of an protein having the activity of a
protein as indicated in Table II, column 3, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp.;, or being encoded by a
nucleic acid molecule indicated in Table I, column 5, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp.;, or its
homologs, e.g. as indicated in Table I, column 7, lines 290 to 294
and/or 604 to 607;
[11113] lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp.; its biochemical or genetical
causes and therefore shows the increased amount of the respective
fine chemical.
[11114] [0035.0.0.25] to [0044.0.0.25]: see [0035.0.0.0] to
[0044.0.0.0]
[11115] [0045.0.25.25] In one embodiment the activity of the
Escherichia coli K12 protein b0019 or its homologs, e.g. the
activity of a protein of the Na+/H+-exchanging protein nhaA
superfamily is increased, preferably, of a protein having a Na+/H+
antiporter activity, e.g. as indicated in Table I, columns 5 or 7,
line 332, is increased conferring an increase of the respective
fine chemical, preferably of sucrose, between 22% and 55% or
more.
[11116] In one embodiment the activity of the Escherichia coli K12
protein b0138 or its homologs, e.g. having a fimbrial-like adhesin
protein activity, e.g. as indicated in Table I, columns 5 or 7,
line 309, is increased conferring an increase of the respective
fine chemical, preferably of myo-inositol, between 32% and 46% or
more.
[11117] In one embodiment the activity of the Escherichia coli K12
protein b0161 or its homologs, e.g. the activity of a protein of
the Helicobacter serine proteinase superfamily is increased,
preferably, of a protein having a periplasmic serine protease (heat
shock protein) activity, e.g. as indicated in Table I, columns 5 or
7, lines 295,303 and 323, is increased conferring an increase of
the respective fine chemical, preferably of fructose (82% to
1155%), glucose (86% to 338%) and/or raffinose (128% to 197%)
resp., between 82% and 1155% or more.
[11118] In one embodiment the activity of the Escherichia coli K12
protein b0252 or its homologs, e.g. having a b0252 protein activity
and similarity with helicase and ligase proteins, e.g. as indicated
in Table I, columns 5 or 7, line 290 is increased conferring an
increase of the respective fine chemical, preferably of starch
and/or cellulose, between 37% and 53% or more.
[11119] In one embodiment the activity of the Escherichia coli K12
protein b0290 or its homologs, e.g. the activity of a protein of
the Escherichia coli yagW protein superfamily is increased,
preferably, of a protein having a receptor protein activity, e.g.
as indicated in Table I, columns 5 or 7, line 310, is increased
conferring an increase of the respective fine chemical, preferably
of myo-inositol, between 23% and 50% or more.
[11120] In one embodiment the activity of the Escherichia coli K12
protein b0695 or its homologs, e.g. the activity of a protein
having homology with the sensor histidine kinase superfamily is
increased, preferably, of a protein having a activity of sensory
histidine kinase in two-component regulatory system, e.g. as
indicated in Table I, columns 5 or 7, line 296, is increased
conferring an increase of the respective fine chemical, preferably
of fructose, between 69% and 1046% or more.
[11121] In one embodiment the activity of the Escherichia coli K12
protein b0730 or its homologs, e.g. the activity of a protein of
the transcription regulator GntR superfamily is increased,
preferably, of a protein having a activity of a transcriptional
regulator of succinylCoA synthetase operon and fatty acyl response
regulator, e.g. as indicated in Table I, columns 5 or 7, line 324,
is increased conferring an increase of the respective fine
chemical, preferably of raffinose, between 93% and 616% or
more.
[11122] In one embodiment the activity of the Escherichia coli K12
protein b1430 or its homologs, e.g. the activity of a protein of
the hemagglutinin hag1 superfamily is increased, preferably, of a
protein having a activity of putative
S-adenosyl-L-methionine-dependent methyltransferase conferring
tellurite resistance, e.g. as indicated in Table I, columns 5 or 7,
line 291, is increased conferring an increase of the respective
fine chemical, preferably of starch and/or cellulose, between 31%
and 58% or more.
[11123] In one embodiment the activity of the Escherichia coli K12
protein b1693 or its homologs, e.g. the activity of a protein of
the 3-dehydroquinate dehydratase superfamily is increased,
preferably, of a protein having a 3-dehydroquinate dehydratase
activity, e.g. as indicated in Table I, columns 5 or 7, line 292,
is increased conferring an increase of the respective fine
chemical, preferably of starch and/or cellulose, between 34% and
116% or more.
[11124] In one embodiment the activity of the Escherichia coli K12
protein b1701 or its homologs, e.g. the activity of a protein with
homology to the 4-coumarate-CoA ligase and/or acetate-CoA ligase
superfamily is increased, preferably, of a protein having a
activity of a CoenzymeA-dependent ligase with firefly
luciferase-like ATPase Domain, e.g. as indicated in Table I,
columns 5 or 7, line 325, is increased conferring an increase of
the respective fine chemical, preferably of raffinose, between 57%
and 958% or more.
[11125] In one embodiment the activity of the Escherichia coli K12
protein b1708 or its homologs, e.g. the activity of a protein of
the protein H11314 superfamily is increased, preferably, of a
protein having a lipoprotein activity, e.g. as indicated in Table
I, columns 5 or 7, lines 297 and 304, is increased conferring an
increase of the respective fine chemical, preferably of fructose
(77% to 2664%) and/or glucose (62% to 942%), between 62% and 2664%
or more.
[11126] In one embodiment the activity of the Escherichia coli K12
protein b1886 or its homologs, e.g. the activity of a protein of
the methyl-accepting chemotaxis protein superfamily is increased,
preferably, of a protein having a methyl-accepting chemotaxis
protein II and/or aspartate sensor receptor activity, e.g. as
indicated in Table I, columns 5 or 7, line 326, is increased
conferring an increase of the respective fine chemical, preferably
of raffinose, between 64% and 391% or more.
[11127] In one embodiment the activity of the Escherichia coli K12
protein b1926 or its homologs, e.g. having a flagellar protein fliT
activity, e.g. as indicated in Table I, columns 5 or 7, lines 298
and 305, is increased conferring an increase of the respective fine
chemical, preferably of fructose (86% to 193%) and/or glucose (63%
to 88%), between 63% and 193% or more.
[11128] In one embodiment the activity of the Escherichia coli K12
protein b2023 or its homologs, e.g. the activity of a protein of
the amidotransferase hisH superfamily with trpG homology is
increased, preferably, of a protein having a activity of a
glutamine amidotransferase subunit of imidazole glycerol phosphate
synthase heterodimer, e.g. as indicated in Table I, columns 5 or 7,
line 311, is increased conferring an increase of the respective
fine chemical, preferably of myo-inositol, between 31% and 110% or
more.
[11129] In one embodiment the activity of the Escherichia coli K12
protein b2597 or its homologs, e.g. the activity of a protein of
the Pseudomonas putida hypothetical protein rpoX superfamily is
increased, preferably, of a protein having a activity of a ribosome
associated factor, e.g. as indicated in Table I, columns 5 or 7,
line 299, is increased conferring an increase of the respective
fine chemical, preferably of fructose, between 90% and 105% or
more.
[11130] In one embodiment the activity of the Escherichia coli K12
protein b2599 or its homologs, e.g. the activity of a protein of
the pheA bifunctional enzyme superfamily with prephenate
dehydratase homology is increased, preferably, of a proteinhaving a
activity of a bifunctional enzyme: chorismate mutase P (N-terminal)
and prephenate dehydratase (C-terminal), e.g. as indicated in Table
I, columns 5 or 7, line 306, is increased conferring an increase of
the respective fine chemical, preferably of glucose, between 65%
and 171% or more.
[11131] In one embodiment the activity of the Escherichia coli K12
protein b2664 or its homologs, e.g. the activity of a protein of
the transcription regulator gabP superfamily is increased,
preferably, of a protein having a transcriptional repressor with
DNA-binding Winged helix domain (GntR familiy) activity, e.g. as
indicated in Table I, columns 5 or 7, lines 300 and 327, is
increased conferring an increase of the respective fine chemical,
preferably of fructose (77% to 4086%) and/or raffinose (72% to
1267%), between 72% and 4086% or more.
[11132] In one embodiment the activity of the Escherichia coli K12
protein b2699 or its homologs, e.g. the activity of a protein of
the recombination protein recA superfamily is increased,
preferably, of a protein having a activity of a DNA strand exchange
and recombination protein with protease and nuclease activity, e.g.
as indicated in Table I, columns 5 or 7, lines 312 and 328, is
increased conferring an increase of the respective fine chemical,
preferably of myo-inositol (86% to 690%) and/or raffinose (61% to
2408%), between 61% and 2408% or more.
[11133] In one embodiment the activity of the Escherichia coli K12
protein b3172 or its homologs, e.g. the activity of a protein of
the argininosuccinate synthase superfamily is increased,
preferably, of a protein having a argininosuccinate synthetase
activity, e.g. as indicated in Table I, columns 5 or 7, line 313,
is increased conferring an increase of the respective fine
chemical, preferably of myo-inositol, between 26% and 144% or
more.
[11134] In one embodiment the activity of the Escherichia coli K12
protein b3231 or its homologs, e.g. the activity of a protein of
the Escherichia coli ribosomal protein L13 superfamily is
increased, preferably, of a protein having a 50S ribosomal subunit
protein L13 activity, e.g. as indicated in Table I, columns 5 or 7,
line 293, is increased conferring an increase of the respective
fine chemical, preferably of starch and/or cellulose, between 31%
and 74% or more.
[11135] In one embodiment the activity of the Escherichia coli K12
protein b3430 or its homologs, e.g. the activity of a protein of
the glucose-1-phosphate adenylyltransferase superfamily is
increased, preferably, of a protein having a glucose-1-phosphate
adenylyltransferase activity, e.g. as indicated in Table I, columns
5 or 7, line 314, is increased conferring an increase of the
respective fine chemical, preferably of myo-inositol, between 34%
and 116% or more.
[11136] In one embodiment the activity of the Escherichia coli K12
protein b3601 or its homologs, e.g. having a transcriptional
repressor for mannitol utilization activity, e.g. as indicated in
Table I, columns 5 or 7, line 329, is increased conferring an
increase of the respective fine chemical, preferably of raffinose,
between 84% and 135% or more.
[11137] In one embodiment the activity of the Escherichia coli K12
protein b4129 or its homologs, e.g. the activity of a protein of
the lysine-tRNA ligase superfamily is increased, preferably, of a
protein having a (inducible) lysine tRNA synthetase activity, e.g.
as indicated in Table I, columns 5 or 7, line 315, is increased
conferring an increase of the respective fine chemical, preferably
of myo-inositol, between 29% and 48% or more.
[11138] In one embodiment the activity of the Escherichia coli K12
protein b4239 or its homologs, e.g. the activity of a protein of
the alpha-glucosidase superfamily with alpha-amylase core homology
is increased, preferably, of a protein having a trehalose-6-P
hydrolase activity, e.g. as indicated in Table I, columns 5 or 7,
lines 301, 307 and 333, is increased conferring an increase of the
respective fine chemical, preferably of fructose (321% to 768%),
glucose (100% to 385%) and/or sucrose (64% to 169%) resp., between
64% and 768% or more.
[11139] In one embodiment the activity of the Escherichia coli K12
protein b4327 or its homologs, e.g. the activity of a protein of
the protein b2409 superfamily is increased, preferably, of a
protein having a HTH-type transcriptional regulator with
periplasmic binding protein domain (LysR family) activity, e.g. as
indicated in Table I, columns 5 or 7, line 302, is increased
conferring an increase of the respective fine chemical, preferably
of fructose, between 97% and 275% or more.
[11140] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YBR184W or its homologs, e.g. the activity of a
YBR184W protein superfamily-protein is increased, preferably, an
activity of YBR184W protein, e.g. as indicated in Table I, columns
5 or 7, line 330 is increased conferring an increase of the
respective fine chemical, preferably of raffinose between 87% and
478% or more.
[11141] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YBR204C or its homologs, e.g. an activity of
peroxisomal lipase protein, e.g. as indicated in Table I, columns 5
or 7, line 316, is increased conferring an increase of the
respective fine chemical, preferably of myo-inositol between 27%
and 192% or more.
[11142] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YDR112W or its homologs, e.g. an activity of
YDR112W protein, e.g. as indicated in Table I, columns 5 or 7, line
317, is increased conferring an increase of the respective fine
chemical, preferably of myo-inositol between 41% and 90% or
more.
[11143] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER174C or its homologs, e.g. the activity of a
protein of the thioredoxin homology superfamily is increased,
preferably, an activity of hydroperoxide and superoxide-radical
responsive glutathione-dependent oxidoreductase protein, e.g. as
indicated in Table I, columns 5 or 7, line 294 is increased
conferring an increase of the respective fine chemical, preferably
of starch and/or cellulose between 34% and 84% or more.
[11144] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YGR261C or its homologs, e.g. the activity of a
protein of the beta-adaptin superfamily is increased, preferably,
an activity of clathrin assembly complex beta adaptin component
protein, e.g. as indicated in Table I, columns 5 or 7, lines 318
and 331, is increased conferring an increase of the respective fine
chemical, preferably of myo-inositol (124% to 698%) and/or
raffinose (95% to 2956%) resp., between 95% and 2956% or more.
[11145] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YIL150C or its homologs, e.g. the activity of a
chromatin binding protein, required for S-phase (DNA synthesis)
initiation or completion, e.g. as indicated in Table I, columns 5
or 7, line 319 is increased conferring an increase of the
respective fine chemical, preferably of myo-inositol between 400%
and 480% or more.
[11146] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YJL072C or its homologs, e.g. the activity of a
protein of the Saccharomyces cerevisiae membrane protein YJL072c
superfamily is increased, preferably, an activity of subunit of the
GINS complex required for chromosomal DNA replication protein, e.g.
as indicated in Table I, columns 5 or 7, line 308 is increased
conferring an increase of the respective fine chemical, preferably
of glucose between 58% and 293% or more.
[11147] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YJL099W or its homologs, e.g. the activity of a
protein involved in chitin biosynthesis and/or its regulation e.g.
as indicated in Table I, columns 5 or 7, line 320 is increased
conferring an increase of the respective fine chemical, preferably
of myo-inositol between 26% and 472% or more.
[11148] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR044W or its homologs, e.g. the activity of a
protein of the Saccharomyces cerevisiae membrane protein YOR044w
superfamily is increased, e.g. as indicated in Table I, columns 5
or 7, line 321 is increased conferring an increase of the
respective fine chemical, preferably of myo-inositol between 44%
and 160% or more.
[11149] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR350C or its homologs, e.g. the activity of a
protein of the Saccharomyces cerevisiae MNE1 protein superfamily is
increased, e.g. as indicated in Table I, columns 5 or 7, line 322
is increased conferring an increase of the respective fine
chemical, preferably of myo-inositol between 52% and 182% or
more.
[11150] In case the activity of the Escherichia coli K12 protein
b0050 or its homologs, as indicated in Table I, columns 5 or 7,
line 604, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of starch and/or
cellulose between 38% and 89% or more is conferred.
[11151] In case the activity of the Escherichia coli K12 protein
b0124 or its homologs, as indicated in Table I, columns 5 or 7,
line 608, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose between 100%
and 2926% or more is conferred.
[11152] In case the activity of the Escherichia coli K12 protein
b0149 or its homologs, as indicated in Table I, columns 5 or 7,
line 609, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose between 68%
and 95% or more is conferred.
[11153] In case the activity of the Escherichia coli K12 protein
b1318 or its homologs, as indicated in Table I, columns 5 or 7,
line 610, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose between 103%
and 199% or more is conferred.
[11154] In case the activity of the Escherichia coli K12 protein
b1463 or its homologs, as indicated in Table I, columns 5 or 7,
lines 611 and 617 and 622, e.g. protein with an activity as defined
in [0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose between 92%
and 453%, preferably of glucose between 57% and 618%, preferably of
myo-inositol between 36% and 96%, preferably of fructose and
glucose between 57% and 618%, preferably of fructose and
myo-inositol between 36% and 453%, preferably of glucose and
myo-inositol between 36% and 618%, preferably of fructose and
glucose and myo-inositol between 36% and 618%, or more is
conferred.
[11155] In case the activity of the Escherichia coli K12 protein
b1539 or its homologs, as indicated in Table I, columns 5 or 7,
line 605, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of starch and/or
cellulose between 39% and 92% or more is conferred.
[11156] In case the activity of the Escherichia coli K12 protein
b1736 or its homologs, as indicated in Table I, columns 5 or 7,
line 618, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glucose between 61%
and 110% or more is conferred.
[11157] In case the activity of the Escherichia coli K12 protein
b1961 or its homologs, as indicated in Table I, columns 5 or 7,
line 623, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of myo-inositol between
25% and 58% or more is conferred.
[11158] In case the activity of the Escherichia coli K12 protein
b2491 or its homologs, as indicated in Table I, columns 5 or 7,
lines 612 and 619, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose between 123%
and 278%, preferably of glucose between 81% and 289%, in particular
for increasing the amount of fructose and glucose between 81% and
289%, or more is conferred.
[11159] In case the activity of the Escherichia coli K12 protein
b3260 or its homologs, as indicated in Table I, columns 5 or 7,
line 613, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose between 87%
and 472% or more is conferred.
[11160] In case the activity of the Escherichia coli K12 protein
b3578 or its homologs, as indicated in Table I, columns 5 or 7,
lines 614 and 620 and 626, e.g. protein with an activity as defined
in [0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose between 89%
and 106%, preferably of glucose between 65% and 110%, preferably of
raffinose between 55% and 84%, preferably of fructose and glucose
between 65% and 110%, preferably of glucose and raffinose between
55% and 110%, preferably of fructose and raffinose between, 55% and
106%, preferably of fructose and glucose and raffinose between 55%
and 110%, or more is conferred.
[11161] In case the activity of the Escherichia coli K12 protein
b3619 or its homologs, as indicated in Table I, columns 5 or 7,
line 615 and 621, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose between 61%
and 111%, preferably of glucose between 60% and 119%, preferably of
fructose and glucose between 60% and 119% or more is conferred.
[11162] In case the activity of the Escherichia coli K12 protein
b3919 or its homologs, as indicated in Table I, columns 5 or 7,
line 606, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of starch and/or
cellulose between 33% and 52% or more is conferred.
[11163] In case the activity of the Escherichia coli K12 protein
b4074 or its homologs, as indicated in Table I, columns 5 or 7,
line 624, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of myo-inositol between
25% and 50% or more is conferred.
[11164] In case the activity of the Escherichia coli K12 protein
b4122 or its homologs, as indicated in Table I, columns 5 or 7,
line 616, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose between 53%
and 502% or more is conferred.
[11165] In case the activity of the Escherichia coli K12 protein
b4232 or its homologs, as indicated in Table I, columns 5 or 7,
line 607, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of starch and/or
cellulose between 35% and 61% or more is conferred.
[11166] In case the activity of the Saccharomyces cerevisiae
protein YHR072W-A or its homologs, as indicated in Table I, columns
5 or 7, line 625, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of myo-inositol between
27% and 53% or more is conferred.
[11167] [0046.0.25.25] In one embodiment the activity of the
Escherichia coli K12 protein b0019 or its homologs, e.g. the
activity of a protein of the Na+/H+-exchanging protein nhaA
superfamily is increased, preferably, of a protein having a Na+/H+
antiporter activity, e.g. as indicated in Table I, columns 5 or 7,
line 332, is increased conferring an increase of the respective
fine chemical, preferably of sucrose and of further
carbohydrate(s).
[11168] In one embodiment the activity of the Escherichia coli K12
protein b0138 or its homologs, e.g. having a fimbrial-like adhesin
protein activity, e.g. as indicated in Table I, columns 5 or 7,
line 309, is increased conferring an increase of the respective
fine chemical, preferably of myo-inositol and of further
carbohydrate(s).
[11169] In one embodiment the activity of the Escherichia coli K12
protein b0161 or its homologs, e.g. the activity of a protein of
the Helicobacter serine proteinase superfamily is increased,
preferably, of a protein having a periplasmic serine protease (heat
shock protein) activity, e.g. as indicated in Table I, columns 5 or
7, lines 295, 303 and 323, is increased conferring an increase of
the respective fine chemical, preferably of fructose, glucose
and/or raffinose resp. and of further carbohydrate(s).
[11170] In one embodiment the activity of the Escherichia coli K12
protein b0252 or its homologs, e.g. having a b0252 protein activity
and similarity with helicase and ligase proteins, e.g. as indicated
in Table I, columns 5 or 7, line 290 is increased conferring an
increase of the respective fine chemical, preferably of starch
and/or cellulose and of further carbohydrate(s).
[11171] In one embodiment the activity of the Escherichia coli K12
protein b0290 or its homologs, e.g. the activity of a protein of
the Escherichia coli yagW protein superfamily is increased,
preferably, of a protein having a receptor protein activity, e.g.
as indicated in Table I, columns 5 or 7, line 310, is increased
conferring an increase of the respective fine chemical, preferably
of myo-inositol and of further carbohydrate(s).
[11172] In one embodiment the activity of the Escherichia coli K12
protein b0695 or its homologs, e.g. the activity of a protein
having homology with the sensor histidine kinase superfamily is
increased, preferably, of a protein having a activity of sensory
histidine kinase in two-component regulatory system, e.g. as
indicated in Table I, columns 5 or 7, line 296, is increased
conferring an increase of the respective fine chemical, preferably
of fructose and of further carbohydrate(s).
[11173] In one embodiment the activity of the Escherichia coli K12
protein b0730 or its homologs, e.g. the activity of a protein of
the transcription regulator GntR superfamily is increased,
preferably, of a protein having a activity of a transcriptional
regulator of succinylCoA synthetase operon and fatty acyl response
regulator, e.g. as indicated in
[11174] Table I, columns 5 or 7, line 324, is increased conferring
an increase of the respective fine chemical, preferably of
raffinose and of further carbohydrate(s).
[11175] In one embodiment the activity of the Escherichia coli K12
protein b1430 or its homologs, e.g. the activity of a protein of
the hemagglutinin hag1 superfamily is increased, preferably, of a
protein having a activity of putative
S-adenosyl-L-methionine-dependent methyltransferase conferring
tellurite resistance, e.g. as indicated in Table I, columns 5 or 7,
line 291, is increased conferring an increase of the respective
fine chemical, preferably of starch and/or cellulose and of further
carbohydrate(s).
[11176] In one embodiment the activity of the Escherichia coli K12
protein b1693 or its homologs, e.g. the activity of a protein of
the 3-dehydroquinate dehydratase superfamily is increased,
preferably, of a protein having a 3-dehydroquinate dehydratase
activity, e.g. as indicated in Table I, columns 5 or 7, line 292,
is increased conferring an increase of the respective fine
chemical, preferably of starch and/or cellulose and of further
carbohydrate(s).
[11177] In one embodiment the activity of the Escherichia coli K12
protein b1701 or its homologs, e.g. the activity of a protein with
homology to the 4-coumarate-CoA ligase and/or acetate-CoA ligase
superfamily is increased, preferably, of a protein having a
activity of a CoenzymeA-dependent ligase with firefly
luciferase-like ATPase Domain, e.g. as indicated in Table I,
columns 5 or 7, line 325, is increased conferring an increase of
the respective fine chemical, preferably of raffinose and of
further carbohydrate(s).
[11178] In one embodiment the activity of the Escherichia coli K12
protein b1708 or its homologs, e.g. the activity of a protein of
the protein H11314 superfamily is increased, preferably, of a
protein having a lipoprotein activity, e.g. as indicated in Table
I, columns 5 or 7, lines 297 and 304, is increased conferring an
increase of the respective fine chemical, preferably of fructose
and/or glucose and of further carbohydrate(s).
[11179] In one embodiment the activity of the Escherichia coli K12
protein b1886 or its homologs, e.g. the activity of a protein of
the methyl-accepting chemotaxis protein superfamily is increased,
preferably, of a protein having a methyl-accepting chemotaxis
protein II and/or aspartate sensor receptor activity, e.g. as
indicated in Table I, columns 5 or 7, line 326, is increased
conferring an increase of the respective fine chemical, preferably
of raffinose and of further carbohydrate(s).
[11180] In one embodiment the activity of the Escherichia coli K12
protein b1926 or its homologs, e.g. having a flagellar protein fliT
activity, e.g. as indicated in Table I, columns 5 or 7, lines 298
and 305, is increased conferring an increase of the respective fine
chemical, preferably of fructose and/or glucose and of further
carbohydrate(s).
[11181] In one embodiment the activity of the Escherichia coli K12
protein b2023 or its homologs, e.g. the activity of a protein of
the amidotransferase hisH superfamily with trpG homology is
increased, preferably, of a protein having a activity of a
glutamine amidotransferase subunit of imidazole glycerol phosphate
synthase heterodimer, e.g. as indicated in Table I, columns 5 or 7,
line 311, is increased conferring an increase of the respective
fine chemical, preferably of myo-inositol and of further
carbohydrate(s).
[11182] In one embodiment the activity of the Escherichia coli K12
protein b2597 or its homologs, e.g. the activity of a protein of
the Pseudomonas putida hypothetical protein rpoX superfamily is
increased, preferably, of a protein having a activity of a ribosome
associated factor, e.g. as indicated in Table I, columns 5 or 7,
line 299, is increased conferring an increase of the respective
fine chemical, preferably of fructose and of further
carbohydrate(s).
[11183] In one embodiment the activity of the Escherichia coli K12
protein b2599 or its homologs, e.g. the activity of a protein of
the pheA bifunctional enzyme superfamily with prephenate
dehydratase homology is increased, preferably, of a proteinhaving a
activity of a bifunctional enzyme: chorismate mutase P (N-terminal)
and prephenate dehydratase (C-terminal), e.g. as indicated in Table
I, columns 5 or 7, line 306, is increased conferring an increase of
the respective fine chemical, preferably of glucose and of further
carbohydrate(s).
[11184] In one embodiment the activity of the Escherichia coli K12
protein b2664 or its homologs, e.g. the activity of a protein of
the transcription regulator gabP superfamily is increased,
preferably, of a protein having a transcriptional repressor with
DNA-binding
[11185] Winged helix domain (GntR familiy) activity, e.g. as
indicated in Table I, columns 5 or 7, lines 300 and 327, is
increased conferring an increase of the respective fine chemical,
preferably of fructose and/or raffinose and of further
carbohydrate(s).
[11186] In one embodiment the activity of the Escherichia coli K12
protein b2699 or its homologs, e.g. the activity of a protein of
the recombination protein recA superfamily is increased,
preferably, of a protein having a activity of a DNA strand exchange
and recombination protein with protease and nuclease activity, e.g.
as indicated in Table I, columns 5 or 7, lines 312 and 328, is
increased conferring an increase of the respective fine chemical,
preferably of myo-inositol and/or raffinose and of further
carbohydrate(s).
[11187] In one embodiment the activity of the Escherichia coli K12
protein b3172 or its homologs, e.g. the activity of a protein of
the argininosuccinate synthase superfamily is increased,
preferably, of a protein having a argininosuccinate synthetase
activity, e.g. as indicated in Table I, columns 5 or 7, line 313,
is increased conferring an increase of the respective fine
chemical, preferably of myo-inositol and of further
carbohydrate(s).
[11188] In one embodiment the activity of the Escherichia coli K12
protein b3231 or its homologs, e.g. the activity of a protein of
the Escherichia coli ribosomal protein L13 superfamily is
increased, preferably, of a protein having a 50S ribosomal subunit
protein L13 activity, e.g. as indicated in Table I, columns 5 or 7,
line 293, is increased conferring an increase of the respective
fine chemical, preferably of starch and/or cellulose and of further
carbohydrate(s).
[11189] In one embodiment the activity of the Escherichia coli K12
protein b3430 or its homologs, e.g. the activity of a protein of
the glucose-1-phosphate adenylyltransferase superfamily is
increased, preferably, of a protein having a glucose-1-phosphate
adenylyltransferase activity, e.g. as indicated in Table I, columns
5 or 7, line 314, is increased conferring an increase of the
respective fine chemical, preferably of myo-inositol and of further
carbohydrate(s).
[11190] In one embodiment the activity of the Escherichia coli K12
protein b3601 or its homologs, e.g. having a transcriptional
repressor for mannitol utilization activity, e.g. as indicated in
Table I, columns 5 or 7, line 329, is increased conferring an
increase of the respective fine chemical, preferably of raffinose
and of further carbohydrate(s).
[11191] In one embodiment the activity of the Escherichia coli K12
protein b4129 or its homologs, e.g. the activity of a protein of
the lysine-tRNA ligase superfamily is increased, preferably, of a
protein having a (inducible) lysine tRNA synthetase activity, e.g.
as indicated in Table I, columns 5 or 7, line 315, is increased
conferring an increase of the respective fine chemical, preferably
of myo-inositol and of further carbohydrate(s).
[11192] In one embodiment the activity of the Escherichia coli K12
protein b4239 or its homologs, e.g. the activity of a protein of
the alpha-glucosidase superfamily with alpha-amylase core homology
is increased, preferably, of a protein having a trehalose-6-P
hydrolase activity, e.g. as indicated in Table I, columns 5 or 7,
lines 301, 307 and 333, is increased conferring an increase of the
respective fine chemical, preferably of fructose, glucose and/or
sucrose resp., and of further carbohydrate(s).
[11193] In one embodiment the activity of the Escherichia coli K12
protein b4327 or its homologs, e.g. the activity of a protein of
the protein b2409 superfamily is increased, preferably, of a
protein having a HTH-type transcriptional regulator with
periplasmic binding protein domain (LysR family) activity, e.g. as
indicated in Table I, columns 5 or 7, line 302, is increased
conferring an increase of the respective fine chemical, preferably
of fructose and of further carbohydrate(s).
[11194] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YBR184W or its homologs, e.g. the activity of a
YBR184W protein superfamily-protein is increased, preferably, an
activity of YBR184W protein, e.g. as indicated in Table I, columns
5 or 7, line 330 is increased conferring an increase of the
respective fine chemical, preferably of raffinose and of further
carbohydrate(s).
[11195] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YBR204C or its homologs, e.g. an activity of
peroxisomal lipase protein, e.g. as indicated in Table I, columns 5
or 7, line 316, is increased conferring an increase of the
respective fine chemical, preferably of myo-inositol and of further
carbohydrate(s).
[11196] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YDR112W or its homologs, e.g. an activity of
YDR112W protein, e.g. as indicated in Table I, columns 5 or 7, line
317, is increased conferring an increase of the respective fine
chemical, preferably of myo-inositol and of further
carbohydrate(s).
[11197] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YER174C or its homologs, e.g. the activity of a
protein of the thioredoxin homology superfamily is increased,
preferably, an activity of hydroperoxide and superoxide-radical
responsive glutathione-dependent oxidoreductase protein, e.g. as
indicated in Table I, columns 5 or 7, line 294 is increased
conferring an increase of the respective fine chemical, preferably
of starch and/or cellulose and of further carbohydrate(s).
[11198] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YGR261C or its homologs, e.g. the activity of a
protein of the beta-adaptin superfamily is increased, preferably,
an activity of clathrin assembly complex beta adaptin component
protein, e.g. as indicated in Table I, columns 5 or 7, lines 318
and 331, is increased conferring an increase of the respective fine
chemical, preferably of myo-inositol and/or raffinose resp., and of
further carbohydrate(s).
[11199] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YIL150C or its homologs, e.g. the activity of a
chromatin binding protein, required for S-phase (DNA synthesis)
initiation or completion, e.g. as indicated in Table I, columns 5
or 7, line 319 is increased conferring an increase of the
respective fine chemical, preferably of myo-inositol and of further
carbohydrate(s).
[11200] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YJL072C or its homologs, e.g. the activity of a
protein of the Saccharomyces cerevisiae membrane protein YJL072c
superfamily is increased, preferably, an activity of subunit of
the
[11201] GINS complex required for chromosomal DNA replication
protein, e.g. as indicated in Table I, columns 5 or 7, line 308 is
increased conferring an increase of the respective fine chemical,
preferably of glucose and of further carbohydrate(s).
[11202] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YJL099W or its homologs, e.g. the activity of a
protein involved in chitin biosynthesis and/or its regulation e.g.
as indicated in Table I, columns 5 or 7, line 320 is increased
conferring an increase of the respective fine chemical, preferably
of myo-inositol and of further carbohydrate(s).
[11203] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR044W or its homologs, e.g. the activity of a
protein of the Saccharomyces cerevisiae membrane protein YOR044w
superfamily is increased, e.g. as indicated in Table I, columns 5
or 7, line 321 is increased conferring an increase of the
respective fine chemical, preferably of myo-inositol and of further
carbohydrate(s).
[11204] In one embodiment, the activity of the Saccharomyces
cerevisiae protein YOR350C or its homologs, e.g. the activity of a
protein of the Saccharomyces cerevisiae MNE1 protein superfamily is
increased, e.g. as indicated in Table I, columns 5 or 7, line 322
is increased conferring an increase of the respective fine
chemical, preferably of myo-inositol and of further
carbohydrate(s).
[11205] In case the activity of the Escherichia coli K12 protein
b0050 or its homologs, as indicated in Table I, columns 5 or 7,
line 604, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of starch and/or
cellulose and of one or more other carbohydrate(s) is
conferred.
[11206] In case the activity of the Escherichia coli K12 protein
b0124 or its homologs, as indicated in Table I, columns 5 or 7,
line 608, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose and of one or
more other carbohydrate(s) is conferred.
[11207] In case the activity of the Escherichia coli K12 protein
b0149 or its homologs, as indicated in Table I, columns 5 or 7,
line 609, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose and of one or
more other carbohydrate(s) is conferred.
[11208] In case the activity of the Escherichia coli K12 protein
b1318 or its homologs, as indicated in Table I, columns 5 or 7,
line 610, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose and of one or
more other carbohydrate(s) is conferred.
[11209] In case the activity of the Escherichia coli K12 protein
b1463 or its homologs, as indicated in Table I, columns 5 or 7,
lines 611 and 617 and 622, e.g. protein with an activity as defined
in [0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose and/or
glucose and/or myo-inositol and of one or more other
carbohydrate(s) conferred.
[11210] In case the activity of the Escherichia coli K12 protein
b1539 or its homologs, as indicated in Table I, columns 5 or 7,
line 605, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of starch and/or
cellulose and of one or more other carbohydrate(s) is
conferred.
[11211] In case the activity of the Escherichia coli K12 protein
b1736 or its homologs, as indicated in Table I, columns 5 or 7,
line 618, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of glucose and of one or
more other carbohydrate(s) conferred.
[11212] In case the activity of the Escherichia coli K12 protein
b1961 or its homologs, as indicated in Table I, columns 5 or 7,
line 623, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of myo-inositol and of
one or more other carbohydrate(s) is conferred.
[11213] In case the activity of the Escherichia coli K12 protein
b2491 or its homologs, as indicated in Table I, columns 5 or 7,
lines 612 and 619, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose and/orglucose
and of one or more other carbohydrate(s) is conferred.
[11214] In case the activity of the Escherichia coli K12 protein
b3260 or its homologs, as indicated in Table I, columns 5 or 7,
line 613, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose and of one or
more other carbohydrate(s) is conferred.
[11215] In case the activity of the Escherichia coli K12 protein
b3578 or its homologs, as indicated in Table I, columns 5 or 7,
lines 614 and 620 and 626, e.g. protein with an activity as defined
in [0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose and/or of
glucose and/or raffinose and of one or more other carbohydrate(s)
is conferred.
[11216] In case the activity of the Escherichia coli K12 protein
b3619 or its homologs, as indicated in Table I, columns 5 or 7,
line 615 and 621, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose and/or of
glucose and of one or more other carbohydrate(s) is conferred.
[11217] In case the activity of the Escherichia coli K12 protein
b3919 or its homologs, as indicated in Table I, columns 5 or 7,
line 606, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of starch and/or
cellulose and of one or more other carbohydrate(s) is
conferred.
[11218] In case the activity of the Escherichia coli K12 protein
b4074 or its homologs, as indicated in Table I, columns 5 or 7,
line 624, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of myo-inositol and of
one or more other carbohydrate(s) is conferred.
[11219] In case the activity of the Escherichia coli K12 protein
b4122 or its homologs, as indicated in Table I, columns 5 or 7,
line 616, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of fructose and of one or
more other carbohydrate(s) is conferred.
[11220] In case the activity of the Escherichia coli K12 protein
b4232 or its homologs, as indicated in Table I, columns 5 or 7,
line 607, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of starch and/or
cellulose and of one or more other carbohydrate(s) is
conferred.
[11221] In case the activity of the Saccharomyces cerevisiae
protein YHR072W-A or its homologs, as indicated in Table I, columns
5 or 7, line 625, e.g. protein with an activity as defined in
[0022.0.25.25], is increased, preferably, in one embodiment the
increase of the fine chemical, preferably of myo-inositol and of
one or more other carbohydrate(s) is conferred.
[11222] [0047.0.0.25] to [0048.0.0.25]: see [0047.0.0.0] to
[0048.0.0.0]
[11223] [0049.0.25.25] A protein having an activity conferring an
increase in the amount or level of the starch and/or cellulose
preferably has the structure of the polypeptide described herein.
In a particular embodiment, the polypeptides used in the process of
the present invention or the polypeptide of the present invention
comprises the sequence of a consensus sequence a as indicated in
Table IV, column 7, lines 290 to 294 and/or 604 to 607 and/or the
sequence of a polypeptide as indicated in Table II, columns 5 or 7,
lines 290 to 294 and/or 604 to 607, or of a functional homologue
thereof as described herein, or of a polypeptide encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 290 to 294
and/or 604 to 607 or its herein described functional homologues and
has the herein mentioned activity conferring an increase in the
starch and/or cellulose level.
[11224] A protein having an activity conferring an increase in the
amount or level of the fructose preferably has the structure of the
polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence a as indicated in Table IV, column 7, lines 295
to 302 and/or 608 to 616 and/or the sequence of a polypeptide as
indicated in Table II, columns 5 or 7, lines 295 to 302 and/or 608
to 616, or of a functional homologue thereof as described herein,
or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 295 to 302 and/or 608 to 616 or its
herein described functional homologues and has the herein mentioned
activity conferring an increase in the fructose level.
[11225] A protein having an activity conferring an increase in the
amount or level of the glucose preferably has the structure of the
polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence a as indicated in Table IV, column 7, lines 303
to 308 and/or 617 to 621 and/or the sequence of a polypeptide as
indicated in Table II, columns 5 or 7, lines 303 to 308 and/or 617
to 621, or of a functional homologue thereof as described herein,
or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 303 to 308 and/or 617 to 621 or its
herein described functional homologues and has the herein mentioned
activity conferring an increase in the glucose level.
[11226] A protein having an activity conferring an increase in the
amount or level of the myo-inositol preferably has the structure of
the polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence a as indicated in Table IV, column 7, lines 309
to 322 and/or 622 to 625 and/or the sequence of a polypeptide as
indicated in Table II, columns 5 or 7, lines 309 to 322 and/or 622
to 625, or of a functional homologue thereof as described herein,
or of a polypeptide encoded by the nucleic acid molecule
characterized herein or the nucleic acid molecule according to the
invention, for example by a nucleic acid molecule as indicated in
Table I, columns 5 or 7, lines 309 to 322 and/or 622 to 625 or its
herein described functional homologues and has the herein mentioned
activity conferring an increase in the myo-inositol level.
[11227] A protein having an activity conferring an increase in the
amount or level of the raffinose preferably has the structure of
the polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence a as indicated in Table IV, column 7, lines 323
to 331 and/or 626 and/or the sequence of a polypeptide as indicated
in Table II, columns 5 or 7, lines 323 to 331 and/or 626, or of a
functional homologue thereof as described herein, or of a
polypeptide encoded by the nucleic acid molecule characterized
herein or the nucleic acid molecule according to the invention, for
example by a nucleic acid molecule as indicated in Table I, columns
5 or 7, lines 323 to 331 and/or 626 or its herein described
functional homologues and has the herein mentioned activity
conferring an increase in the raffinose level.
[11228] A protein having an activity conferring an increase in the
amount or level of the sucrose preferably has the structure of the
polypeptide described herein. In a particular embodiment, the
polypeptides used in the process of the present invention or the
polypeptide of the present invention comprises the sequence of a
consensus sequence a as indicated in Table IV, column 7, lines 332
to 333 and/or the sequence of a polypeptide as indicated in Table
II, columns 5 or 7, lines 332 to 333, or of a functional homologue
thereof as described herein, or of a polypeptide encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by a nucleic acid
molecule as indicated in Table I, columns 5 or 7, lines 332 to 333
or its herein described functional homologues and has the herein
mentioned activity conferring an increase in the sucrose level.
[11229] [0050.0.25.25] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, ester and ethers, preferably etherificated
with other mono-, di-oligo- or polysaccharides, with alkyl, alkenyl
or alkinyl alcohols.
[11230] [0051.0.25.25] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g compositions comprising carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose resp., Depending on
the choice of the organism used for the process according to the
present invention, for example a microorganism or a plant,
compositions or mixtures of various respective fine chemicals can
be produced.
[11231] [0052.0.0.25] see [0052.0.0.0]
[11232] [0053.0.25.25] In one embodiment, the process of the
present invention comprises one or more of the following steps
[11233] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having an activity of a protein as indicated in Table
II, column 3, lines 290 to 294 and/or 604 to 607 for starch and/or
cellulose; lines 295 to 302 and/or 608 to 616 for fructose; lines
303 to 308 and/or 617 to 621 for glucose; lines 309 to 322 and/or
622 to 625 for myo-inositol; lines 323 to 331 and/or 626 for
raffinose; lines 332 to 333 for sucrose; resp., or its homologs,
e.g. as indicated in Table II, columns 5 or 7, lines 290 to 294
and/or 604 to 607 for starch and/or cellulose; lines 295 to 302
and/or 608 to 616 for fructose; lines 303 to 308 and/or 617 to 621
for glucose; lines 309 to 322 and/or 622 to 625 for myo-inositol;
lines 323 to 331 and/or 626 for raffinose; lines 332 to 333 for
sucrose, resp., activity having herein-mentioned the respective
fine chemical-increasing activity; [11234] b) stabilizing a mRNA
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 290 to 294 and/or 604 to 607 for starch and/or cellulose;
lines 295 to 302 and/or 608 to 616 for fructose; lines 303 to 308
and/or 617 to 621 for glucose; lines 309 to 322 and/or 622 to 625
for myo-inositol; lines 323 to 331 and/or 626 for raffinose; lines
332 to 333 for sucrose, resp., or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 290 to 294 and/or 604
to 607 for starch and/or cellulose; lines 295 to 302 and/or 608 to
616 for fructose; lines 303 to 308 and/or 617 to 621 for glucose;
lines 309 to 322 and/or 622 to 625 for myo-inositol; lines 323 to
331 and/or 626 for raffinose; lines 332 to 333 for sucrose; resp.,
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned the respective fine chemical-increasing
activity; [11235] c) increasing the specific activity of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
present invention having herein-mentioned the respective fine
chemical-increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 290
to 294 and/or 604 to 607 for starch and/or cellulose; lines 295 to
302 and/or 608 to 616 for fructose; lines 303 to 308 and/or 617 to
621 for glucose; lines 309 to 322 and/or 622 to 625 for
myo-inositol; lines 323 to 331 and/or 626 for raffinose; lines 332
to 333 for sucrose; resp., or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 290 to 294 and/or 604
to 607 for starch and/or cellulose; lines 295 to 302 and/or 608 to
616 for fructose; lines 303 to 308 and/or 617 to 621 for glucose;
lines 309 to 322 and/or 622 to 625 for myo-inositol; lines 323 to
331 and/or 626 for raffinose; lines 332 to 333 for sucrose; resp.,
or decreasing the inhibiitory regulation of the polypeptide of the
invention; [11236] d) generating or increasing the expression of an
endogenous or artificial transcription factor mediating the
expression of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention having herein-mentioned the
respective fine chemical-increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 290 to 294 and/or 604 to 607 for starch and/or cellulose;
lines 295 to 302 and/or 608 to 616 for fructose; lines 303 to 308
and/or 617 to 621 for glucose; lines 309 to 322 and/or 622 to 625
for myo-inositol; lines 323 to 331 and/or 626 for raffinose; lines
332 to 333 for sucrose; resp., or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 290 to 294 and/or 604
to 607 for starch and/or cellulose; lines 295 to 302 and/or 608 to
616 for fructose; lines 303 to 308 and/or 617 to 621 for glucose;
lines 309 to 322 and/or 622 to 625 for myo-inositol; lines 323 to
331 and/or 626 for raffinose; lines 332 to 333 for sucrose, resp.;
[11237] e) stimulating activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention having herein-mentioned the respective fine
chemical-increasing activity, e.g. of a polypeptide having an
activity of a protein as indicated in Table II, column 3, lines 290
to 294 and/or 604 to 607 for starch and/or cellulose; lines 295 to
302 and/or 608 to 616 for fructose; lines 303 to 308 and/or 617 to
621 for glucose; lines 309 to 322 and/or 622 to 625 for
myo-inositol; lines 323 to 331 and/or 626 for raffinose; lines 332
to 333 for sucrose, resp., or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 290 to 294 and/or 604
to 607 for starch and/or cellulose; lines 295 to 302 and/or 608 to
616 for fructose; lines 303 to 308 and/or 617 to 621 for glucose;
lines 309 to 322 and/or 622 to 625 for myo-inositol; lines 323 to
331 and/or 626 for raffinose; lines 332 to 333 for sucrose, resp.,
by adding one or more exogenous inducing factors to the organisms
or parts thereof; [11238] f) expressing a transgenic gene encoding
a protein conferring the increased expression of a polypeptide
encoded by the nucleic acid molecule of the present invention or a
polypeptide of the present invention, having herein-mentioned the
respective fine chemical-increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 290 to 294 and/or 604 to 607 for starch and/or cellulose;
lines 295 to 302 and/or 608 to 616 for fructose; lines 303 to 308
and/or 617 to 621 for glucose; lines 309 to 322 and/or 622 to 625
for myo-inositol; lines 323 to 331 and/or 626 for raffinose; lines
332 to 333 for sucrose, resp., or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 290 to 294 and/or 604
to 607 for starch and/or cellulose; lines 295 to 302 and/or 608 to
616 for fructose; lines 303 to 308 and/or 617 to 621 for glucose;
lines 309 to 322 and/or 622 to 625 for myo-inositol; lines 323 to
331 and/or 626 for raffinose; lines 332 to 333 for sucrose, resp.,
and/or [11239] g) increasing the copy number of a gene conferring
the increased expression of a nucleic acid molecule encoding a
polypeptide encoded by the nucleic acid molecule of the invention
or the polypeptide of the invention having herein-mentioned the
respective fine chemical-increasing activity, e.g. of a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 290 to 294 and/or 604 to 607 for starch and/or cellulose;
lines 295 to 302 and/or 608 to 616 for fructose; lines 303 to 308
and/or 617 to 621 for glucose; lines 309 to 322 and/or 622 to 625
for myo-inositol; lines 323 to 331 and/or 626 for raffinose; lines
332 to 333 for sucrose, resp., or its homologs, e.g. as indicated
in Table II, columns 5 or 7, lines 290 to 294 and/or 604 to 607 for
starch and/or cellulose; lines 295 to 302 and/or 608 to 616 for
fructose; lines 303 to 308 and/or 617 to 621 for glucose; lines 309
to 322 and/or 622 to 625 for myo-inositol; lines 323 to 331 and/or
626 for raffinose; lines 332 to 333 for sucrose; resp., activity.
[11240] h) Increasing the expression of the endogenous gene
encoding the polypeptide of the invention, e.g. a polypeptide
having an activity of a protein as indicated in Table II, column 3,
lines 290 to 294 and/or 604 to 607 for starch and/or cellulose;
lines 295 to 302 and/or 608 to 616 for fructose; lines 303 to 308
and/or 617 to 621 for glucose; lines 309 to 322 and/or 622 to 625
for myo-inositol; lines 323 to 331 and/or 626 for raffinose; lines
332 to 333 for sucrose, resp., or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, lines 290 to 294 and/or 604
to 607 for starch and/or cellulose; lines 295 to 302 and/or 608 to
616 for fructose; lines 303 to 308 and/or 617 to 621 for glucose;
lines 309 to 322 and/or 622 to 625 for myo-inositol; lines 323 to
331 and/or 626 for raffinose; lines 332 to 333 for sucrose; resp.,
by adding positive expression or removing negative expression
elements, e.g. homologous recombination can be used to either
introduce positive regulatory elements like for plants the 35S
enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; [11241]
i) Modulating growth conditions of an organism in such a manner,
that the expression or activity of the gene encoding the protein of
the invention or the protein itself is enhanced for example
microorganisms or plants can be grown for example under a higher
temperature regime leading to an enhanced expression of heat shock
proteins, which can lead an enhanced fine chemical production
and/or [11242] j) selecting of organisms with especially high
activity of the proteins of the invention from natural or from
mutagenized resources and breeding them into the target organisms,
eg the elite crops.
[11243] [0054.0.25.25] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table II, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
or its homologs activity, e.g. as indicated in Table II, columns 5
or 7 lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or
608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322
and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp.
[11244] [0055.0.0.25] to [0064.0.0.25]: see [0055.0.0.0] to
[0064.0.0.0]
[11245] [0065.0.0.25]: see [0065.0.0.0]
[11246] [0066.0.0.25] to [0067.0.0.25]: see [0066.0.0.0] to
[0067.0.0.0]
[11247] [0068.0.25.25] The mutation is introduced in such a way
that the production of the carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucroseis not adversely
affected.
[11248] [0069.0.0.25] see [0069.0.0.0]
[11249] [0070.0.25.25] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolites composition in the organism, e.g. an advantageous
composition carbohydrates comprising a higher content of (from a
viewpoint of nutritional physiology limited) preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose.
[11250] [0071.0.25.25] Preferably the composition further comprises
higher amounts of metabolites positively affecting or lower amounts
of metabolites negatively affecting the nutrition or health of
animals or humans provided with said compositions or organisms of
the invention or parts thereof. Likewise, the number or activity of
further genes which are required for the import or export of
nutrients or metabolites, including carbohydrates or its
precursors, required for the cell's biosynthesis of
carbohydratesmay be increased so that the concentration of
necessary or relevant precursors, cofactors or intermediates within
the cell(s) or within the corresponding storage compartments is
increased. Owing to the increased or novel generated activity of
the polypeptide of the invention or the polypeptide used in the
method of the invention or owing to the increased number of nucleic
acid sequences of the invention and/or to the modulation of further
genes which are involved in the biosynthesis of the carbohydrates,
e.g. by increasing the activity of enzymes synthesizing precursors
or by destroying the activity of one or more genes which are
involved in the breakdown of the carbohydrates, it is possible to
increase the yield, production and/or production efficiency of
carbohydratesin the host organism, such as the plants or the
microorganims.
[11251] [0072.0.25.25] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to polysaccharides, more preferably starch and/or
cellulose and/or monosaccharides, more preferably fructose, glucose
and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose, further
carbohydrates, preferably saccharides.
[11252] [0073.0.25.25] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
a) providing a non-human organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant; b) increasing an activity of a polypeptide of the
invention or a homolog thereof, e.g. as indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., or of a polypeptide being encoded by the nucleic
acid molecule of the present invention and described below, e.g.
conferring an increase of the respective fine chemical in an
organism, preferably in a microorganism, a non-human animal, a
plant or animal cell, a plant or animal tissue or a plant, c)
growing an organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a plant
under conditions which permit the production of the respective fine
chemical in the organism, preferably the microorganism, the plant
cell, the plant tissue or the plant; and d) if desired, revovering,
optionally isolating, the free and/or bound the respective fine
chemical and, optionally further free and/or bound carbohydrate(s)
synthetized by the organism, the microorganism, the non-human
animal, the plant or animal cell, the plant or animal tissue or the
plant.
[11253] [0074.0.25.25] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the respective fine chemical or the free and
bound the respective fine chemical but as option it is also
possible to produce, recover and, if desired isolate, other free
or/and bound carbohydrate(s).
[11254] [0075.0.0.25] to [0077.0.0.25]: see [0075.0.0.0] to
[0077.0.0.0]
[11255] [0078.0.25.25] The organism such as microorganisms or
plants or the recovered, and if desired isolated, respective fine
chemical can then be processed further directly into foodstuffs or
animal feeds or for other applications in nutrition or medicine or
cosmetics, for example according to the disclosures made in U.S.
Pat. No. 6,669,962: Starch microcapsules for delivery of active
agents; US 20050042737: Starch process; US 20050054071: Enzymes for
starch processing; US 20050091716: Novel plants and processes for
obtaining them; U.S. Pat. Nos. 5,096,594 and 5,482,631 discloses a
method of purifying cyclitols; U.S. Pat. No. 4,997,489 discloses
soaking almond hulls in water to obtain a syrup containing
fructose, glucose, inositol, and sorbitol; U.S. Pat. No. 5,296,364
discloses a microbial method for producing inositol; U.S. Pat. No.
4,734,402; U.S. Pat. No. 4,788,065; U.S. Pat. No. 6,465,037 and
U.S. Pat. No. 6,355,295: relates to soy food ingredient based on
carbohydrates, U.S. Pat. No. 6,653,451; US 20040128713: pertains to
soybean plants having in their seeds significantly lower contents
of raffinose, stachyose and phytic acid and significantly higher
contents of sucrose and inorganic phosphate; US 20050008713
discloses compositions of plant carbohydrates for dietary
supplements and nutritional support; which are expressly
incorporated herein by reference. The fermentation broth,
fermentation products, plants or plant products can be treated and
processed as described in above mentioned applications or by other
methods known to the person skilled in the art and described herein
below.
[11256] In the method for producing carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose according to the
invention, the cultivation step of the genetically modified
organisms, also referred to as transgenic organisms hereinbelow, is
preferably followed by harvesting said organisms and isolating the
respective carbohydrate(s) from said organisms.
[11257] The organisms are harvested in a manner known per se and
appropriate for the particular organism. Microorganisms such as
bacteria, mosses, yeasts and fungi or plant cells which are
cultured in liquid media by fermentation may be removed, for
example, by centrifugation, decanting or filtration. Plants are
grown on solid media in a manner known per se and harvested
accordingly.
[11258] Carbohydrates, preferably polysaccharides, more preferably
starch and/or cellulose and/or monosaccharides, more preferably
fructose, glucose and/or myo-inositol and/or trisaccharides, more
preferably raffinose and/or disaccharides, more preferably sucrose
are isolated from the harvested biomass in a manner known per se,
for example by extraction and, where appropriate, further chemical
or physical purification processes such as, for example, chemical
and/or enzymatical degradation, precipitation methods,
crystallography, thermal separation methods such as rectification
methods or physical separation methods such as, for example,
chromatography.
[11259] Products of these different work-up procedures are
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose comprising
compositions, e.g. compostions comprising carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose which still comprise
fermentation broth, plant particles and/or cell components in
different amounts, advantageously in the range of from 0 to 99% by
weight, preferably below 80% by weight, especially preferably
between 50%, 40%, 30%, 20%, 20%, 10%, 5%, 3%, 2%, 1%, 05%, 0.1%,
0.01% and 0% by weight resp.
[11260] In one embodiment, preferred plants include, but are not
limited to: sugar beet, sugar cane, soybeans and/or potato (Solanum
tuberosum).
[11261] [0079.0.0.25] to [0084.0.0.25]: see [0079.0.0.0] to
[0084.0.0.0]
[11262] [0085.0.25.25] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [11263] a) a nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., or a derivative thereof, or
[11264] b) a genetic regulatory element, for example a promoter,
which is functionally linked to the nucleic acid sequence as
indicated in Table I, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., or a derivative thereof, or
[11265] c) (a) and (b) is/are not present in its/their natural
genetic environment or has/have been modified by means of genetic
manipulation methods, it being possible for the modification to be,
by way of example, a substitution, addition, deletion, inversion or
insertion of one or more nucleotide. "Natural genetic environment"
means the natural chromosomal locus in the organism of origin or
the presence in a genomic library. In the case of a genomic
library, the natural, genetic environment of the nucleic acid
sequence is preferably at least partially still preserved. The
environment flanks the nucleic acid sequence at least on one side
and has a sequence length of at least 50 bp, preferably at least
500 bp, particularly preferably at least 1000 bp, very particularly
preferably at least 5000 bp.
[11266] [0086.0.0.25] to [0087.0.0.25]: see [0086.0.0.0] to
[0087.0.0.0]
[11267] [0088.0.25.25] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content the
respective fine chemical is modified advantageously owing to the
nucleic acid molecule of the present invention expressed. This is
important for humans and animals since, for example, the
nutritional value of plants for nutrition is dependent on the
abovementioned carbohydrates and the general amount of saccharides
as energy source in feed.
[11268] [0088.1.0.25] to [0095.0.0.25]: see [0088.1.0.0] to
[0095.0.0.0]
[11269] [0096.0.25.25] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptide or a compound, which
functions as a sink for the desired fine chemical, for example
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose in the
organism, is useful to increase the production of the respective
fine chemical.
[11270] [0097.0.0.25] see [0097.0.0.0]
[11271] [0098.0.25.25] In a preferred embodiment, the respective
fine chemical (carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose) is produced in accordance with the
invention and, if desired, is isolated. The production of
carbohydrates, or mixtures thereof or mixtures with other compounds
by the process according to the invention is advantageous.
[11272] [0099.0.25.25] In the case of the fermentation of
microorganisms, the abovementioned carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose may accumulate in the
medium and/or the cells. If microorganisms are used in the process
according to the invention, the fermentation broth can be processed
after the cultivation. Depending on the requirement, all or some of
the biomass can be removed from the fermentation broth by
separation methods such as, for example, centrifugation,
filtration, decanting or a combination of these methods, or else
the biomass can be left in the fermentation broth. The fermentation
broth can subsequently be reduced, or concentrated, with the aid of
known methods such as, for example, rotary evaporator, thin-layer
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. Afterwards advantageously further compounds for
formulation can be added such as corn starch or silicates. This
concentrated fermentation broth advantageously together with
compounds for the formulation can subsequently be processed by
lyophilization, spray drying, and spray granulation or by other
methods. Preferably the respective fine chemical or the
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose
compositions are isolated from the organisms, such as the
microorganisms or plants or the culture medium in or on which the
organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[11273] [0100.0.25.25] Transgenic plants which comprise the
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose synthesized
in the process according to the invention can advantageously be
marketed directly without there being any need for the oils, lipids
or fatty acids synthesized to be isolated. Plants for the process
according to the invention are listed as meaning intact plants and
all plant parts, plant organs or plant parts such as leaf, stem,
seeds, root, tubers, anthers, fibers, fruits, root hairs, stalks,
embryos, calli, cotelydons, petioles, harvested material, plant
tissue, reproductive tissue and cell cultures which are derived
from the actual transgenic plant and/or can be used for bringing
about the transgenic plant. In this context, the seed comprises all
parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue.
[11274] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, in the form of their extracts,
e.g. water containing extract, or as fibre in case of starch and/or
cellulose, as degradation products (chemical ar enzymatical
degradation) or crystallisation product or as their ethers and/or
esters and/or free carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose. The respective fine chemical produced by
this process can be obtained by harvesting the organisms, either
from the crop in which they grow, or from the field. This can be
done via pressing or extraction of the plant parts, e.g. in the
plant stem, seeds, root, tubers, anthers, fibers, fruits. To
increase the efficiency of extraction it is beneficial to clean, to
temper and if necessary to hull and to flake the plant material
especially the stem, seeds, root, tubers, anthers, fibers, fruits.
extracts and/carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose can be obtained by what is known as cold
beating or cold pressing without applying heat. To allow for
greater ease of disruption of the plant parts, specifically the
stem, seeds, root, tubers, anthers, fibers, fruits, they can
previously be comminuted, steamed or roasted. Plant parts, which
have been pretreated in this manner can subsequently be pressed or
extracted with solvents or water. The solvent is subsequently
removed. In the case of microorganisms, the latter are, after
harvesting, for example extracted directly without further
processing steps or else, after disruption, extracted via various
methods with which the skilled worker is familiar. Thereafter, the
resulting products can be processed further, i.e. degradiated,
crystallized and/or refined.
[11275] [0101.0.25.25] see [0101.0.0.0]
[11276] [0102.0.25.25] Carbohydrates, preferably polysaccharides,
more preferably starch and/or cellulose and/or monosaccharides,
more preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose can for example be analyzed advantageously
via HPLC or GC separation methods and detected by MS oder MSMS
methods. The unambiguous detection for the presence of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrosecontaining
products can be obtained by analyzing recombinant organisms using
analytical standard methods: GC, GC-MS, LC,
[11277] LC-MSMS or TLC, as described on several occasions. The
carbohydrates can be analized further in plant extracts by
anion-exchange chromatography with pulsed amperometric detection
(Cataldi et al., Anal Chem.; 72(16):3902-7, 2000), by enzymatic
"BioAnalysis" usind test kits from R-Biopharm and Roche or from
Megazyme, Ireland.
[11278] [0103.0.25.25] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [11279] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide having a
sequence as indicated in Table II, columns 5 or 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp., or a fragment
thereof, which confers an increase in the amount of the respective
fine chemical in an organism or a part thereof; [11280] b) nucleic
acid molecule comprising, preferably at least the mature form, of a
nucleic acid molecule having a sequence as indicated in Table I,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., [11281] c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as result of the degeneracy of the
genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [11282]
d) nucleic acid molecule encoding a polypeptide which has at least
50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [11283] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [11284] f) nucleic acid molecule encoding a polypeptide,
the polypeptide being derived by substituting, deleting and/or
adding one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[11285] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [11286] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers pairs having a sequence as indicated in
Table III, column 7, lines 290 to 294 and/or 604 to 607; lines 295
to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [11287]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [11288] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequences as indicated in Table IV, column 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [11289] k) nucleic acid
molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of the polypeptide indicated in Table II, columns 5 or 7, lines 290
to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; and [11290] l) nucleic
acid molecule which is obtainable by screening a suitable library
under stringent conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k), preferably to
(a) to (c), or with a fragment of at least 15 nt, preferably 20 nt,
30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k), preferably to (a) to (c), and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which comprises a
sequence which is complementary thereto.
[11291] [00103.1.0.4.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table IA, columns 5 or 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp., by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of the sequence shown
in indicated in Table I A, columns 5 or 7, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp. In one embodiment, the
nucleic acid molecule used in the process of the invention is less
than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table I A, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II A, columns 5 or 7, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp.
[11292] [00103.2.0.4.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table I B, columns 5 or 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp., by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of the sequence shown
in indicated in Table I B, columns 5 or 7, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp.: In one embodiment, the
nucleic acid molecule used in the process of the invention is less
than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table I B, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table II B, columns 5 or 7, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp.
[11293] [0104.0.25.25] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence indicated in
Table I, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines
295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621;
lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626;
lines 332 to 333, resp., by one or more nucleotides. In one
embodiment, the nucleic acid molecule used in the process of the
invention does not consist of the sequence indicated in Table I,
columns 5 or 7 lines 290 to 294 and/or 604 to 607; lines 295 to 302
and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to
322 and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to
333, resp. In one embodiment, the nucleic acid molecule of the
present invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence indicated in Table I, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp. In
another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp.
[11294] [0105.0.0.25] to [0107.0.0.25]: see [0105.0.0.0] to
[0107.0.0.0]
[11295] [0108.0.25.25] Nucleic acid molecules with the sequence as
indicated in Table I, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., nucleic acid molecules which
are derived from an amino acid sequences as indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., or from polypeptides comprising the consensus
sequence as indicated in Table IV, column 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp. or their derivatives
or homologues encoding polypeptides with the enzymatic or
biological activity of an activity of a polypeptide as indicated in
Table II, column 3, 5 or 7, lines 290 to 294 and/or 604 to 607;
lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to
621; lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or
626; lines 332 to 333, resp., or conferring an increase of the
respective fine chemical, meaning carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose after increasing its
expression or activity are advantageously increased in the process
according to the invention.
[11296] [0109.0.0.25] see [0109.0.0.0]
[11297] [0110.0.25.25] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
used in the process of the invention, e.g. of a protein as
indicated in Table II, column 5, lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., or being encoded by a nucleic
acid molecule indicated in Table I, column 5, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp., or of its homologs,
e.g. as indicated in Table II, column 7, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp., can be determined from
generally accessible databases.
[11298] [0111.0.0.25] see [0111.0.0.0]
[11299] [0112.0.25.25] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table II, column 3, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., or having the
sequence of a polypeptide as indicated in Table II, columns 5 and
7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608
to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
and conferring an increase in the level of carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose.
[11300] [0113.0.0.25] to [0120.0.0.25]: see [0113.0.0.0] to
[0120.0.0.0]
[11301] [0121.0.25.25] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., or the functional homologues thereof as
described herein, preferably conferring above-mentioned activity,
i.e. conferring an increase in the level of the fine chemical of
the invention after increasing the activity of the polypeptide
sequences indicated in Table II, columns 5 or 7, lines 290 to 294
and/or 604 to 607 for starch and/or cellulose; lines 295 to 302
and/or 608 to 616 for fructose; lines 303 to 308 and/or 617 to 621
for glucose; lines 309 to 322 and/or 622 to 625 for myo-inositol;
lines 323 to 331 and/or 626 for raffinose; lines 332 to 333 for
sucrose resp.
[11302] [0122.0.0.25] to [0127.0.0.259]: see [0122.0.0.0] to
[0127.0.0.0]
[11303] [0128.0.9.9] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table III, columns 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
by means of polymerase chain reaction can be generated on the basis
of a sequence shown herein, for example the sequence as indicated
in Table I, columns 5 or 7, lines 290 to 294 and/or 604 to 607;
lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to
621; lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or
626; lines 332 to 333, resp., or the sequences derived from a
sequence as indicated in Table II, columns 5 or 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp.
[11304] [0129.0.25.25] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved regions are those, which show a
very little variation in the amino acid in one particular position
of several homologs from different origin. The consensus sequences
indicated in Table IV, column 7, lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., are derived from said
alignments.
[11305] [0130.0.25.25] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose after
increasing the expression or activity of the protein comprising
said fragment.
[11306] [0131.0.0.25] to [0138.0.0.25]: see [0131.0.0.0] to
[0138.0.0.0]
[11307] [0139.0.25.25] Polypeptides having above-mentioned
activity, i.e. conferring the increase of the respective fine
chemical level, derived from other organisms, can be encoded by
other DNA sequences which hybridize to a sequences indicated in
Table I, columns 5 or 7, preferably table I B, lines 290 to 294
and/or 604 to 607 for starch and/or cellulose; lines 295 to 302
and/or 608 to 616 for fructose; lines 303 to 308 and/or 617 to 621
for glucose; lines 309 to 322 and/or 622 to 625 for myo-inositol;
lines 323 to 331 and/or 626 for raffinose; lines 332 to 333 for
sucrose resp., under relaxed hybridization conditions and which
code on expression for peptides having the respective fine
chemical, in particular, of starch and/or cellulose and/fructose
and/or glucose and/or myo-inositol and/or raffinose and/or sucrose,
resp., increasing activity.
[11308] [0140.0.0.25] to [0146.0.0.25]: see [0140.0.0.0] to
[0146.0.0.0]
[11309] [0147.0.25.25]: Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
I, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., is one which is sufficiently complementary to
one of said nucleotide sequences such that it can hybridize to one
of said nucleotide sequences, thereby forming a stable duplex.
Preferably, the hybridisation is performed under stringent
hybridization conditions. However, a complement of one of the
herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[11310] [0148.0.25.25] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table I, preferably table I B,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., or a portion thereof and preferably has above
mentioned activity, in particular having a carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose increasing activity
after increasing the activity or an activity of a product of a gene
encoding said sequences or their homologs.
[11311] [0149.0.25.25] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table I, preferably table I
B, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., or a portion thereof and encodes a protein
having above-mentioned activity, e.g. conferring increase of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose resp., and
optionally, the activity of a protein indicated in Table II, column
5, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608
to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp.
[11312] [00149.1.25.25] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table I,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., preferably of Table I B, columns 5 or 7, lines
290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616;
lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to
625; lines 323 to 331 and/or 626; lines 332 to 333, resp. has
further one or more of the activities annotated or known for the a
protein as indicated in Table II, column 3 lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp.
[11313] [0150.0.25.25] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table I, preferably Table I B,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., for example a fragment which can be used as a
probe or primer or a fragment encoding a biologically active
portion of the polypeptide of the present invention or of a
polypeptide used in the process of the present invention, i.e.
having above-mentioned activity, e.g. conferring an increase of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose resp., if
its activity is increased. The nucleotide sequences determined from
the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table I, columns
5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or
608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322
and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp., an anti-sense sequence of one of the sequences, e.g., as
indicated in Table I, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., or naturally occurring mutants
thereof. Primers based on a nucleotide of invention can be used in
PCR reactions to clone homologues of the polypeptide of the
invention or of the polypeptide used in the process of the
invention, e.g. as the primers described in the examples of the
present invention, e.g. as shown in the examples. A
[11314] PCR with the primer pairs indicated in Table III, column 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
will result in a fragment of a polynucleotide sequence as indicated
in Table I, columns 5 or 7, lines 290 to 294 and/or 604 to 607;
lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to
621; lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or
626; lines 332 to 333, resp., or its gene product.
[11315] [0151.0.0.25]: see [0151.0.0.0]
[11316] [0152.0.25.25] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table II, columns 5 or 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp., such that the
protein or portion thereof maintains the ability to participate in
the respective fine chemical production, in particular an activity
increasing the level of carbohydrates, preferably polysaccharides,
more preferably starch and/or cellulose and/or monosaccharides,
more preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose resp., as mentioned above or as described
in the examples in plants or microorganisms is comprised.
[11317] [0153.0.25.25] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table II, columns 5 or 7, lines 290
to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., such that the
protein or portion thereof is able to participate in the increase
of the respective fine chemical production. In one embodiment, a
protein or portion thereof as indicated in Table II, columns 5 or
7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608
to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
has for example an activity of a polypeptide as indicated in Table
II, column 3, lines 290 to 294 and/or 604 to 607; lines 295 to 302
and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to
322 and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to
333, resp.
[11318] [0154.0.25.25] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table II, columns 5 or 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp., and has
above-mentioned activity, e.g. conferring preferably the increase
of the respective fine chemical.
[11319] [0155.0.0.25] to [0156.0.0.25]: see [0155.0.0.0] to
[0156.0.0.0]
[11320] [0157.0.25] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences
indicated in Table I, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp. (and portions thereof) due to
degeneracy of the genetic code and thus encode a polypeptide of the
present invention, in particular a polypeptide having above
mentioned activity, e.g. conferring an increase in the respective
fine chemical in a organism, e.g. as that polypeptides comprising a
consensus sequence as indicated in Table IV, column 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp. or of the
polypeptide as indicated in Table II, columns 5 or 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp. or their
functional homologues. Advantageously, the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, a consensus sequences as indicated in Table IV,
column 7, lines 290 to 294 and/or 604 to 607; lines 295 to 302
and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to
322 and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to
333, resp. or of the polypeptide as indicated in Table II, column
7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608
to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp. or
the functional homologues. In a still further embodiment, the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention encodes a full length protein
which is substantially homologous to an amino acid sequence
comprising a consensus sequence as indicated in Table IV, column 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp. or
of a polypeptide as indicated in Table II, columns 5 or 7, lines
290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616;
lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to
625; lines 323 to 331 and/or 626; lines 332 to 333, resp. or the
functional homologues thereof. However, in a preferred embodiment,
the nucleic acid molecule of the present invention does not consist
of a sequence as indicated in Table I, columns 5 or 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., preferably as
indicated in Table I A, columns 5 or 7 lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp. Preferably the nucleic acid
molecule of the invention is a functional homologue or identical to
a nucleic acid molecule indicated in Table I B, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp.
[11321] [0158.0.0.25] to [0160.0.0.25]: see [0158.0.0.0] to
[0160.0.0.0]
[11322] [0161.0.25.25]: Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table I, columns 5 or 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp. The nucleic acid
molecule is preferably at least 20, 30, 50, 100, 250 or more
nucleotides in length.
[11323] [0162.0.0.25]: see [0162.0.0.0]
[11324] [0163.0.25.25]: Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table I, columns 5 or 7, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp., corresponds to a
naturally-occurring nucleic acid molecule of the invention. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein). Preferably, the nucleic
acid molecule encodes a natural protein having above-mentioned
activity, e.g. conferring the respective fine chemical increase
after increasing the expression or activity thereof or the activity
of a protein of the invention or used in the process of the
invention.
[11325] [0164.0.0.25]: see [0164.0.0.0]
[11326] [0165.0.25.25] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table I, columns 5 or 7 lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp.
[11327] [0166.0.0.25] to [0167.0.0.25]: see [0166.0.0.0] to
[0167.0.0.0]
[11328] [0168.0.25.25]: Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., preferably of Table II B, column 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp. yet retain
said activity described herein. The nucleic acid molecule can
comprise a nucleotide sequence encoding a polypeptide, wherein the
polypeptide comprises an amino acid sequence at least about 50%
identical to an amino acid sequence as indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., preferably of Table II B, column 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp. and is capable
of participation in the increase of production of the respective
fine chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., preferably of Table II B, column 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., more
preferably at least about 70% identical to one of the sequences as
indicated in Table II, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., preferably of Table II B,
column 7, lines 290 to 294 and/or 604 to 607; lines 295 to 302
and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to
322 and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to
333, resp., even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table II, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
preferably of Table II B, column 7, lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., and most preferably at least
about 96%, 97%, 98%, or 99% identical to the sequence as indicated
in Table II, columns 5 or 7, lines 290 to 294 and/or 604 to 607;
lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to
621; lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or
626; lines 332 to 333, resp., preferably of Table II B, column 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp.
[11329] [0169.0.0.25] to [0172.0.0.25]: see [0169.0.0.0] to
[0172.0.0.0]
[11330] [0173.0.25.25]: For example a sequence, which has 80%
homology with sequence SEQ ID NO: 28606 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 28606 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[11331] [0174.0.0.25]: see [0174.0.0.0]
[11332] [0175.0.25.25]: For example a sequence which has a 80%
homology with sequence SEQ ID NO: 28607 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 28607 by the above program algorithm with the
above parameter set, has a 80% homology.
[11333] [0176.0.25.25]: Functional equivalents derived from one of
the polypeptides as indicated in Table II, columns 5 or 7, lines
290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616;
lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to
625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
according to the invention by substitution, insertion or deletion
have at least 30%, 35%, 40%, 45% or 50%, preferably at least 55%,
60%, 65% or 70% by preference at least 80%, especially preferably
at least 85% or 90%, 91%, 92%, 93% or 94%, very especially
preferably at least 95%, 97%, 98% or 99% homology with one of the
polypeptides as indicated in Table II, columns 5 or 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., according to
the invention and are distinguished by essentially the same
properties as a polypeptide as indicated in Table II, columns 5 or
7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608
to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp.
[11334] [0177.0.25.25]: Functional equivalents derived from a
nucleic acid sequence as indicated in Table I, preferably in Table
I B, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295
to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table II,
columns 5 or 7 lines 290 to 294 and/or 604 to 607; lines 295 to 302
and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to
322 and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to
333, resp., according to the invention and encode polypeptides
having essentially the same properties as a polypeptide as
indicated in Table II, preferably in Table I B, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp.
[11335] [0178.0.0.25]: see [0178.0.0.0]
[11336] [0179.0.25.25]: A nucleic acid molecule encoding an
homologous to a protein sequence as indicated in Table II, columns
5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or
608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322
and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp., preferably in Table I B, column 7, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp., can be created by
introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of the nucleic acid molecule
of the present invention, in particular as indicated in Table I,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences of a
sequences as indicated in Table I, columns 5 or 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp., by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
[11337] [0180.0.0.25] to [0183.0.0.25]: see [0180.0.0.0] to
[0183.0.0.0]
[11338] [0184.0.25.25] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table I, columns 5 or 7,
preferably in Table I B, column 7, lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., or of the nucleic acid
sequences derived from a sequences as indicated in Table II,
columns 5 or 7, preferably in Table II B, column 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., comprise also
allelic variants with at least approximately 30%, 35%, 40% or 45%
homology, by preference at least approximately 50%, 60% or 70%,
more preferably at least approximately 90%, 91%, 92%, 93%, 94% or
95% and even more preferably at least approximately 96%, 97%, 98%,
99% or more homology with one of the nucleotide sequences shown or
the abovementioned derived nucleic acid sequences or their
homologues, derivatives or analogues or parts of these. Allelic
variants encompass in particular functional variants which can be
obtained by deletion, insertion or substitution of nucleotides from
the sequences shown, preferably from a sequence as indicated in
Table I, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines
295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621;
lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626;
lines 332 to 333, resp., or from the derived nucleic acid
sequences, the intention being, however, that the enzyme activity
or the biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[11339] [0185.0.25.25] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table I, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines
295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621;
lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626;
lines 332 to 333, resp., preferably in Table I B, column 7, lines
290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616;
lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to
625; lines 323 to 331 and/or 626; lines 332 to 333, resp. In one
embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotides not shown in any
one of sequences as indicated in Table I, columns 5 or 7, lines 290
to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., preferably in
Table I B, column 7, lines 290 to 294 and/or 604 to 607; lines 295
to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp. In one embodiment, the nucleic acid molecule
comprises less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or
40 further nucleotides. In a further embodiment, the nucleic acid
molecule comprises less than 30, 20 or 10 further nucleotides. In
one embodiment, a nucleic acid molecule used in the process of the
invention is identical to a sequences as indicated in Table I,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., preferably in Table I B, column 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp.
[11340] [0186.0.25.25] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table II, columns
5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or
608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322
and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp., preferably in Table II B, column 7, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp. In one embodiment, the
nucleic acid molecule encodes less than 150, 130, 100, 80, 60, 50,
40 or 30 further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table II, columns 5 or 7, lines 290 to 294 and/or 604 to 607;
lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to
621; lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or
626; lines 332 to 333, resp., preferably in Table II B, lines 290
to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp.
[11341] [0187.0.25.25] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table II, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
preferably in Table II B, column 7, lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., and comprises less than 100
further nucleotides. In a further embodiment, said nucleic acid
molecule comprises less than 30 further nucleotides. In one
embodiment, the nucleic acid molecule used in the process is
identical to a coding sequence encoding a polypeptide as indicated
in Table II, columns 5 or 7, lines 290 to 294 and/or 604 to 607;
lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to
621; lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or
626; lines 332 to 333, resp., preferably in Table II B, lines 290
to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp. [0188.0.25.25]
Polypeptides (=proteins), which still have the essential biological
or enzymatic activity of the polypeptide of the present invention
conferring an increase of the respective fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide as indicated in Table II, columns 5 or 7, lines
290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616;
lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to
625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
preferably compared to a sequence as indicated in Table II, column
3 and 5, lines 290 to 294 and/or 604 to 607; lines 295 to 302
and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to
322 and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to
333, resp., and expressed under identical conditions.
[11342] In one embodiment, the polypeptide of the invention is a
homolog consisting of or comprising the sequence as indicated in
Table II B, columns 7, lines 290 to 294 and/or 604 to 607; lines
295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621;
lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626;
lines 332 to 333, resp.
[11343] [0189.0.25.25] Homologues of a sequences as indicated in
Table I, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines
295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621;
lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626;
lines 332 to 333, resp., or of a sequence derived from a sequence
as indicated in Table II, columns 5 or 7, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp., also mean truncated
sequences, cDNA, single-stranded DNA or RNA of the coding and
noncoding DNA sequence. Homologues of said sequences are also
understood as meaning derivatives, which comprise noncoding regions
such as, for example, UTRs, terminators, enhancers or promoter
variants. The promoters upstream of the nucleotide sequences stated
can be modified by one or more nucleotide substitution(s),
insertion(s) and/or deletion(s) without, however, interfering with
the functionality or activity either of the promoters, the open
reading frame (=ORF) or with the 3'-regulatory region such as
terminators or other 3' regulatory regions, which are far away from
the ORF. It is furthermore possible that the activity of the
promoters is increased by modification of their sequence, or that
they are replaced completely by more active promoters, even
promoters from heterologous organisms. Appropriate promoters are
known to the person skilled in the art and are mentioned herein
below.
[11344] [0190.0.0.25] see [0190.0.0.0]
[11345] [0191.0.0.25] see [0191.0.0.0]:
[11346] [0192.0.0.25] to [0203.0.0.25] see [0192.0.0.0] to
[0203.0.0.0]
[11347] [0204.0.25.25] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule, which comprises a
nucleic acid molecule selected from the group consisting of:
[11348] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table II, columns 5
or 7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or
608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322
and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp., preferably in Table II B, column 7, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp., or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, i.e. carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose resp., in an organism or a part thereof
[11349] b) nucleic acid molecule comprising, preferably at least
the mature form, of a nucleic acid molecule as indicated in Table
I, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., preferably in Table I B, column 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., or a fragment
thereof conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [11350] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [11351] d) nucleic acid molecule encoding a polypeptide
whose sequence has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [11352]
e) nucleic acid molecule which hybridizes with a nucleic acid
molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [11353]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c),
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [11354] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[11355] h) nucleic acid molecule comprising a nucleic acid molecule
which is obtained by amplifying a cDNA library or a genomic library
using primers or primer pairs as indicated in Table III, column or
7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608
to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
and conferring an increase in the amount of the respective fine
chemical, i.e. carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose resp. in an organism or a part thereof;
[11356] i) nucleic acid molecule encoding a polypeptide which is
isolated, e.g. from a expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (g), preferably to (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [11357] j) nucleic acid
molecule which encodes a polypeptide comprising a consensus
sequence as indicated in Table IV, column 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp., and conferring an
increase in the amount of the respective fine chemical, i.e.
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose resp., in
an organism or a part thereof; [11358] k) nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a
domaine of a polypeptide as indicated in Table II, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
preferably in Table II B, column 7, lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., and conferring an increase in
the amount of the respective fine chemical, i.e. carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose resp., in an organism
or a part thereof; and [11359] l) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of a nucleic acid molecule as indicated in Table I,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., preferably in Table I B, column 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., or a nucleic
acid molecule encoding, preferably at least the mature form of, a
polypeptide as indicated in Table II, columns 5 or 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., preferably in
Table II B, column 7, lines 290 to 294 and/or 604 to 607; lines 295
to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
encompasses a sequence which is complementary thereto; whereby,
preferably, the nucleic acid molecule according to (a) to (l)
distinguishes over the sequence indicated in Table IA or, columns 5
or 7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or
608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322
and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp., by one or more nucleotides. In one embodiment, the nucleic
acid molecule does not consist of the sequence shown and indicated
in Table I A or I B, columns 5 or 7 lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp.: In one embodiment, the nucleic
acid molecule is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table I A or I B, columns 5 or
7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608
to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp. In
another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table II A or II B, columns
5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or
608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322
and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp. In an other embodiment, the nucleic acid molecule of the
present invention is at least 30%, 40%, 50%, or 60% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table I A or I B, columns 5 or 7, lines 290
to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp. In a further
embodiment the nucleic acid molecule does not encode a polypeptide
sequence as indicated in Table II A or II B, columns 5 or 7, lines
290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616;
lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to
625; lines 323 to 331 and/or 626; lines 332 to 333, resp.
Accordingly, in one embodiment, the nucleic acid molecule of the
differs at least in one or more residues from a nucleic acid
molecule indicated in Table I A or I B, columns 5 or 7, lines 290
to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp. Accordingly,
in one embodiment, the nucleic acid molecule of the present
invention encodes a polypeptide, which differs at least in one or
more amino acids from a polypeptide indicated in Table II A or II
B, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp. In another embodiment, a nucleic acid molecule
indicated in Table I A or I B, columns 5 or 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp. does not encode a
protein of a sequence indicated in Table II A or II B, columns 5 or
7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608
to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.
Accordingly, in one embodiment, the protein encoded by a sequences
of a nucleic acid according to (a) to (l) does not consist of a
sequence as indicated in Table II A or II B, columns 5 or 7, lines
290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616;
lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to
625; lines 323 to 331 and/or 626; lines 332 to 333, resp. In a
further embodiment, the protein of the present invention is at
least 30%, 40%, 50%, or 60% identical to a protein sequence
indicated in Table II A or II B, columns 5 or 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp. and less than 100%,
preferably less than 99.999%, 99.99% or 99.9%, more preferably less
than 99%, 985, 97%, 96% or 95% identical to a sequence as indicated
in Table I A or II B, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp.
[11360] [0205.0.0.25] to [0206.0.0.25] see [0205.0.0.0] to
[0206.0.0.0]
[11361] [0207.0.25.25] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the carbohydrate or starch
metabolism, the glucose metabolism, the saccharide metabolism, the
metabolism of glycolysis, or their combinations. As described
herein, regulator sequences or factors can have a positive effect
on preferably the gene expression of the genes introduced, thus
increasing it. Thus, an enhancement of the regulator elements may
advantageously take place at the transcriptional level by using
strong transcription signals such as promoters and/or enhancers. In
addition, however, an enhancement of translation is also possible,
for example by increasing mRNA stability or by inserting a
translation enhancer sequence.
[11362] [0208.0.0.25] to [0226.0.0.25]: see [0208.0.0.0] to
[0226.0.0.0]
[11363] [0227.0.25.25]: The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[11364] In addition to a sequence indicated in Table I, columns 5
or 7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or
608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322
and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp., or its derivatives, it is advantageous additionally to
express and/or mutate further genes in the organisms. Especially
advantageously, additionally at least one further gene of the
carbohydrate biosynthetic pathway is expressed in the organisms
such as plants or microorganisms. It is also possible that the
regulation of the natural genes has been modified advantageously so
that the gene and/or its gene product is no longer subject to the
regulatory mechanisms which exist in the organisms. This leads to
an increased synthesis of the carbohydrate desired since, for
example, feedback regulations no longer exist to the same extent or
not at all. In addition it might be advantageously to combine one
or more of the sequences indicated in Table I, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
with genes which generally support or enhances to growth or yield
of the target organisms, for example genes which lead to faster
growth rate of microorganisms or genes which produces stress-,
pathogen, or herbicide resistant plants.
[11365] [0228.0.25.25: In a further embodiment of the process of
the invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the carbohydrate
metabolism, in particular in synthesis of polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose.
[11366] [0229.0.25.25]: Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the carbohydrate biosynthetic
pathway. These genes can lead to an increased synthesis of the
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose resp.
[11367] [0230.0.0.25] see [0230.0.0.0]
[11368] [0231.0.25.25] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a carbohydrate degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[11369] [0232.0.0.25] to [0276.0.0.25]: see [0232.0.0.0] to
[0276.0.0.0]
[11370] [0277.0.25.25] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
is familiar. For example, via extraction, chemical or enzymatical
deg radiation, crystallization, salt precipitation, and/or
different chromatography methods. The process according to the
invention can be conducted batchwise, semibatchwise or
continuously. The respective fine chemcical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts
[11371] [0278.0.0.25] to [0282.0.0.25]: see [0278.0.0.0] to
[0282.0.0.0]
[11372] [0283.0.25.25]: Moreover, a native polypeptide conferring
the increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table II,
column 3, lines 290 to 294 and/or 604 to 607; lines 295 to 302
and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to
322 and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to
333, resp., or an antibody against a polypeptide as indicated in
Table II, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines
295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621;
lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626;
lines 332 to 333, resp., which can be produced by standard
techniques utilizing the polypeptid of the present invention or
fragment thereof, i.e., the polypeptide of this invention.
Preferred are monoclonal antibodies.
[11373] [0284.0.0.25]: see [0284.0.0.0]
[11374] [0285.0.25.25]: In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
II, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295
to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., or as encoded by a nucleic acid molecule as
indicated in Table I, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., or functional homologues
thereof.
[11375] [0286.0.25.25]: In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table IV, column 7, lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp. In another embodiment, the
present invention relates to a polypeptide comprising or consisting
of a consensus sequence as indicated in Table IV, column 7, lines
290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616;
lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to
625; lines 323 to 331 and/or 626; lines 332 to 333, resp., whereby
20 or less, preferably 15 or 10, preferably 9, 8, 7, or 6, more
preferred 5 or 4, even more preferred 3, even more preferred 2,
even more preferred 1, most preferred 0 of the amino acids
positions indicated can be replaced by any amino acid and/or be
absent.
[11376] In one embodiment, the present invention relates to the
method of the present invention comprising a polypeptide or to a
polypeptide comprising more than one consensus sequences (of an
individual line) as indicated in Table IV, column 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp.
[11377] [0287.0.0.25] to [0290.0.0.25]: see [0287.0.0.0] to
[0290.0.0.0]
[11378] [0291.0.25.25]: In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of
plant or microorganism specific consensus sequences.
[11379] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table II A or II B,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp. by one or more amino acids. In one embodiment,
polypeptide distinguishes form a sequence as indicated in Table II
A or IIB, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines
295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621;
lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626;
lines 332 to 333, resp. by more than 1, 2, 3, 4, 5, 6, 7, 8 or 9
amino acids, preferably by more than 10, 15, 20, 25 or 30 amino
acids, evenmore preferred are more than 40, 50, or 60 amino acids
and, preferably, the sequence of the polypeptide of the invention
distinguishes from a sequence as indicated in Table II A or II B,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp. by not more than 80% or 70% of the amino acids,
preferably not more than 60% or 50%, more preferred not more than
40% or 30%, even more preferred not more than 20% or 10%. In an
other embodiment, said polypeptide of the invention does not
consist of a sequence as indicated in Table II A or II B, columns 5
or 7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or
608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322
and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp.
[11380] [0292.0.0.25]: see [0292.0.0.0]
[11381] [0293.0.25.25]: In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the process
of the invention.
[11382] In one embodiment, the polypeptide of the invention has a
sequence which distinguishes from a sequence as indicated in Table
II A or II B, columns 5 or 7, lines 290 to 294 and/or 604 to 607;
lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to
621; lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or
626; lines 332 to 333, resp. by one or more amino acids. In an
other embodiment, said polypeptide of the invention does not
consist of the sequence as indicated in Table II A or II B, columns
5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or
608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322
and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to 333,
resp. In a further embodiment, said polypeptide of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by a nucleic acid molecules as indicated in
Table I A or I B, columns 5 or 7, lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp.
[11383] [0294.0.25.25] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table II, column 3, lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., which distinguishes over a
sequence as indicated in Table II A or II B, columns 5 or 7, lines
290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616;
lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to
625; lines 323 to 331 and/or 626; lines 332 to 333, resp. by one or
more amino acids, preferably by more than 5, 6, 7, 8 or 9 amino
acids, preferably by more than 10, 15, 20, 25 or 30 amino acids,
evenmore preferred are more than 40, 50, or 60 amino acids but even
more preferred by less than 70% of the amino acids, more preferred
by less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[11384] [0295.0.0.25] to [0297.0.0.25]: see [0295.0.0.0] to
[0297.0.0.0]
[11385] [0297.1.25.25] Non polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table II, columns 3, 5 or 7, lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp.
[11386] [0298.0.25.25]: A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table II, columns 5 or 7 lines 290 to 294 and/or
604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308
and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to
331 and/or 626; lines 332 to 333, resp. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table II, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines
295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621;
lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626;
lines 332 to 333, resp.
[11387] [0299.0.25.25]: Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table I, columns 5 or 7, lines
290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616;
lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to
625; lines 323 to 331 and/or 626; lines 332 to 333, resp. The
preferred polypeptide of the present invention preferably possesses
at least one of the activities according to the invention and
described herein. A preferred polypeptide of the present invention
includes an amino acid sequence encoded by a nucleotide sequence
which hybridizes, preferably hybridizes under stringent conditions,
to a nucleotide sequence as indicated in Table I, columns 5 or 7
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
or which is homologous thereto, as defined above.
[11388] [0300.0.25.25]: Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., in amino acid sequence due to natural variation
or mutagenesis, as described in detail herein. Accordingly, the
polypeptide comprise an amino acid sequence which is at least about
35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%, preferably at least about
75%, 80%, 85% or 90, and more preferably at least about 91%, 92%,
93%, 94% or 95%, and most preferably at least about 96%, 97%, 98%,
99% or more homologous to an entire amino acid sequence of a
sequence as indicated in Table II A or II B, columns 5 or 7, lines
290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616;
lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to
625; lines 323 to 331 and/or 626; lines 332 to 333, resp.
[11389] [0301.0.0.25] see [0301.0.0.0]
[11390] [0302.0.25.25] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table II, columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines
295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621;
lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or
626;
[11391] lines 332 to 333, resp., or the amino acid sequence of a
protein homologous thereto, which include fewer amino acids than a
full length polypeptide of the present invention or used in the
process of the present invention or the full length protein which
is homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[11392] [0303.0.0.25]: see [0303.0.0.0]
[11393] [0304.0.25.25]: Manipulation of the nucleic acid molecule
of the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
II, column 3, lines 290 to 294 and/or 604 to 607; lines 295 to 302
and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to
322 and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to
333, resp., but having differences in the sequence from said
wild-type protein. These proteins may be improved in efficiency or
activity, may be present in greater numbers in the cell than is
usual, or may be decreased in efficiency or activity in relation to
the wild type protein.
[11394] [0305.0.0.25] to [0308.0.0.25]: see [0305.0.0.0] to
[0308.0.0.0]
[11395] [0309.0.25.25]: In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., refers to a polypeptide having an amino acid
sequence corresponding to the polypeptide of the invention or used
in the process of the invention, whereas a "non-polypeptide of the
invention" or "other polypeptide" not being indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., refers to a polypeptide having an amino acid
sequence corresponding to a protein which is not substantially
homologous a polypeptide of the invention, preferably which is not
substantially homologous to a polypeptide as indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., e.g., a protein which does not confer the
activity described herein or annotated or known for as indicated in
Table II, column 3, lines 290 to 294 and/or 604 to 607; lines 295
to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., and which is derived from the same or a
different organism. In one embodiment, a "non-polypeptide of the
invention" or "other polypeptide" not being indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., does not confer an increase of the respective
fine chemical in an organism or part therof.
[11396] [0310.0.0.25] to [0334.0.0.25]: see [0310.0.0.0] to
[0334.0.0.0]
[11397] [0335.0.25.25]: The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table I, columns 5 or
7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608
to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
and/or homologs thereof. As described inter alia in WO 99/32619,
dsRNAi approaches are clearly superior to traditional antisense
approaches. The invention therefore furthermore relates to
double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived therefrom), bring about altered
metabolic activity by the reduction in the expression of a nucleic
acid sequences as indicated in Table I, columns 5 or 7, lines 290
to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., and/or
homologs thereof. In a double-stranded RNA molecule for reducing
the expression of an protein encoded by a nucleic acid sequence
sequences as indicated in Table I, columns 5 or 7, lines 290 to 294
and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines 303 to
308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625; lines
323 to 331 and/or 626; lines 332 to 333, resp., and/or homologs
thereof, one of the two RNA strands is essentially identical to at
least part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[11398] [0336.0.0.25] to [0342.0.0.25]: see [0336.0.0.0] to
[0342.0.0.0]
[11399] [0343.0.25.25]: As has already been described, 100%
sequence identity between the dsRNA and a gene transcript of a
nucleic acid sequence as indicated in Table I, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
or its homolog is not necessarily required in order to bring about
effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table I, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., or homologs thereof of the one
organism, may be used to suppress the corresponding expression in
another organism.
[11400] [0344.0.0.25] to [0361.0.0.25]: see [0344.0.0.0] to
[0361.0.0.0]
[11401] [0362.0.25.25]: Accordingly the present invention relates
to any cell transgenic for any nucleic acid characterized as part
of the invention, e.g. conferring the increase of the respective
fine chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table II, lines 290 to 294 and/or 604 to 607; lines 295 to 302
and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines 309 to
322 and/or 622 to 625; lines 323 to 331 and/or 626; lines 332 to
333, resp., e.g. encoding a polypeptide having protein activity, as
indicated in Table II, columns 3, lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp. Due to the above mentioned
activity the respective fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. Transgenic for a polypeptide having an activity of a
polypeptide as indicated in Table II, columns 5 or 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., means herein
that due to modulation or manipulation of the genome, an activity
as annotated for a polypeptide as indicated in Table II, column 3,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
e.g. having a sequence as indicated in Table II, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
is increased in a cell or an organism or a part thereof. Examples
are described above in context with the process of the
invention
[11402] [0363.0.0.25]: see [0363.0.0.0]
[11403] [0364.0.0.25]: see [0364.0.0.0]
[11404] [0365.0.0.25] to [0373.0.0.25]: see [0365.0.0.0] to
[0373.0.0.0]
[11405] [0374.0.25.25]: Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. Carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose resp., produced in
the process according to the invention may, however, also be
isolated from the plant in the form of their free form or bound in
or to compounds or moieties. Carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose resp., produced by
this process can be harvested by harvesting the organisms either
from the culture in which they grow or from the field. This can be
done via expressing, grinding and/or extraction, salt precipitation
and/or ion-exchange chromatography or other chromatographic methods
of the plant parts, preferably the plant seeds, plant fruits, plant
tubers and the like.
[11406] [0375.0.0.25] to [0376.0.0.25]: see [0375.0.0.0] to
[0376.0.0.0]
[11407] [0377.0.25.25]: Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced carbohydrates, preferably polysaccharides, more preferably
starch and/or cellulose and/or monosaccharides, more preferably
fructose, glucose and/or myo-inositol and/or trisaccharides, more
preferably raffinose and/or disaccharides, more preferably sucrose
comprising composition or the produced the respective fine chemical
is isolated.
[11408] [0378.0.25.25]: In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose resp.,
produced in the process can be isolated. The resulting
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose resp., can,
if appropriate, subsequently be further purified, if desired mixed
with other active ingredients such as vitamins, amino acids,
carbohydrates, antibiotics and the like, and, if appropriate,
formulated.
[11409] [0379.0.25.25]: In one embodiment, the carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose resp., is a mixture
comprising of one or more the respective fine chemicals. In one
embodiment, the respective fine chemical means here carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose. In one embodiment,
carbohydrate means here a mixture of the respective fine
chemicals.
[11410] [0380.0.25.25]: The carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose resp., obtained in
the process are suitable as starting material for the synthesis of
further products of value. For example, they can be used in
combination with each other or alone for the production of
pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose comprising
composition produced or the respective fine chemical produced if
desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose resp., produced in
the process or of the transgenic organisms in animal feeds,
foodstuffs, medicines, food supplements, cosmetics or
pharmaceuticals.
[11411] [0381.0.0.25] to [0382.0.0.25]: see [0381.0.0.0] to
[0382.0.0.0]
[11412] [0383.0.9.25]: ./.
[11413] [0384.0.0.25]: see [0384.0.0.0]
[11414] [0385.0.25.25]: The fermentation broths obtained in this
way, containing in particular carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose, resp., in mixtures
with other compounds, in particular with other carbohydrates, or
fatty acids building lipids or containing microorganisms or parts
of microorganisms, like plastids, containing carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose resp., in mixtures
with other compounds, e.g. carbohydrates, normally have a dry
matter content of from 7.5 to 25% by weight.
[11415] The fermentation broth can then be thickened or
concentrated by known methods, such as, for example, with the aid
of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[11416] [0386.0.0.25] -/-
[11417] [0387.0.0.25] to [0392.0.0.25]: see [0387.0.0.0] to
[0392.0.0.0]
[11418] [0393.0.25.25]: In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [11419] (a) contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [11420] (b) identifying the nucleic acid molecules,
which hybridize under relaxed stringent conditions with the nucleic
acid molecule of the present invention in particular to the nucleic
acid molecule sequence as indicated in Table I, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
preferably in Table I B, lines 290 to 294 and/or 604 to 607; lines
295 to 302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621;
lines 309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626;
lines 332 to 333, resp. and, optionally, isolating the full length
cDNA clone or complete genomic clone; [11421] (c) introducing the
candidate nucleic acid molecules in host cells, preferably in a
plant cell or a microorganism, appropriate for producing the
respective fine chemical; [11422] (d) expressing the identified
nucleic acid molecules in the host cells; [11423] (e) assaying the
the respective fine chemical level in the host cells; and [11424]
(f) identifying the nucleic acid molecule and its gene product
which expression confers an increase in the respective the
respective fine chemical level in the host cell after expression
compared to the wild type.
[11425] [0394.0.0.25] to [0399.0.0.25]: see [0394.0.0.0] to
[0399.0.0.0]
[11426] [00399.1.9.9]: One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table II,
columns 5 or 7, lines 290 to 294 and/or 604 to 607; lines 295 to
302 and/or 608 to 616; lines 303 to 308 and/or 617 to 621; lines
309 to 322 and/or 622 to 625; lines 323 to 331 and/or 626; lines
332 to 333, resp., or a homolog thereof, e.g. comparing the
phenotyp of nearly identical organisms with low and high activity
of a protein as indicated in Table II, columns 5 or 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., after
incubation with the drug.
[11427] [0400.0.0.25] to [0416.0.0.25]: see [0400.0.0.0] to
[0416.0.0.0]
[11428] [0417.0.9.9]: The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose, resp., production
biosynthesis pathways. In particular, the overexpression of the
polypeptide of the present invention may protect an organism such
as a microorganism or a plant against inhibitors, which block the
carbohydrate, in particular the respective fine chemical synthesis
in said organism. Examples of inhibitors or herbicides blocking the
synthesis in organism such as microorganism or plants are for
example the cellulose synthesis inhibitors 2,6-dichlorobenzonitrile
(DCB),
N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-
-diamine, called AE F150944 or isoxaben.
[11429] [0418.0.0.25] to [0423.0.0.25]: see [0418.0.0.0] to
[0423.0.0.0]
[11430] [0424.0.25.25]: Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other
carbohydrate.
[11431] Accordingly, the nucleic acid of the invention, or the
nucleic acid molecule identified with the method of the present
invention or the complement sequences thereof, the polypeptide of
the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the respective fine chemical in a organism or part
thereof, e.g. in a cell.
[11432] [0425.0.0.25] to [0454.0.0.25]: see [0425.0.0.0] to
[0453.0.0.0]
[0454.0.25.25] Example 8
Analysis of the Effect of the Nucleic Acid Molecule on the
Production of the Fine Chemical Resp
[11433] [0455.0.25.25] The effect of the genetic modification in C.
glutamicum or other microbial especially bacterial strains for
carbohydrate production on the production of an carbohydrate can be
determined by growing the modified microorganisms under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the increased production of the
amino acid. Such analytical techniques are well known to the
skilled worker and encompass spectroscopy, thin-layer
chromatography, various types of staining methods, enzymatic and
microbiological methods and analytical chromatography such as
high-performance liquid chromatography (see, for example, Ullman,
Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp.
443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987)
"Applications of HPLC in Biochemistry" in: Laboratory Techniques in
Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993)
Biotechnology, Vol. 3, Chapter III: "Product recovery and
purification", pp. 469-714, VCH: Weinheim; Better, P. A. et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F. and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A. and Henry, J. D. (1988) Biochemical Separations,
in Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3; chapter
11, pp. 1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation
and purification techniques in biotechnology, Noyes
Publications).
[11434] [0456.0.0.25]: see [0456.0.0.0]
[0457.0.25.25]: Example 9
Purification of the Carbohydrates, Preferably Polysaccharides, More
Preferably Starch and/or Cellulose and/or Monosaccharides, More
Preferably Fructose, Glucose and/or Myo-Inositol and/or
Trisaccharides, More Preferably Raffinose and/or Disaccharides,
More Preferably Sucrose
[11435] [0458.0.25.25]: Abbreviations: GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[11436] The unambiguous detection for the presence of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrosecan be
obtained by analyzing recombinant organisms using analytical
standard methods: GC, GC-MS or TLC, as described (1997, in:
Advances on Lipid Methodology, Fourth Edition: Christie, Oily
Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). The
total carbohydrate produced in the organism for example in yeasts
used in the inventive process can be analysed for example according
to the following procedure: The material such as yeasts, E. coli or
plants to be analyzed can be disrupted by sonication, grinding in a
glass mill, liquid nitrogen and grinding or via other applicable
methods.
[11437] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[11438] -[0459.0.25.25]: -/-
[11439] [0460.0.0.25] see [0460.0.0.0]
[0461.0.25.25] Example 10
Cloning SEQ ID NO: 28606 for the Expression in Plants
[11440] [0462.0.0.25]: see [0462.0.0.0]
[11441] [0463.025.25] SEQ ID NO: 28606 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[11442] [0464.0.0.25] to [0466.0.0.25]: see [0464.0.0.0] to
[0466.0.0.0]
[11443] [0467.025.25] The following primer sequences were selected
for the gene SEQ ID NO: 28606:
TABLE-US-00143 i) forward primer (SEQ ID NO: 28696) atgaaacatc
tgcatcgatt ctttag, ii) reverse primer (SEQ ID NO: 28697) ttaaactgat
ggacgcaaac gaacg
[11444] [0468.0.0.25] to [0479.0.0.25]: see [0468.0.0.0] to
[0479.0.0.0]
[0480.025.25]: Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 28606
[11445] [0481.0.0.25] to [0513.0.0.25]: see [0481.0.0.0] to
[0513.0.0.0]
[11446] [0514.0.9.9]: As an alternative, the carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose can be detected
advantageously as for example described by Sonnebald et al., (Nat
Biotechnol. 1997 August; 15(8):794-7), or Panikulangara et al.,
Plant Physiol. 2004 October; 136(2):3148-58.
[11447] The results of the different plant analyses can be seen
from the table 1, which follows:
TABLE-US-00144 TABLE 1 ORF Metabolite analyte Method Min Max b0019
Sucrose Sucrose GC 1.22 1.55 b0138 myo-Inositol myo-Inositol GC
1.32 1.46 b0161 Fructose Fructose GC 1.82 12.55 b0161 Glucose
Glucose GC 1.86 4.38 b0161 Raffinose Raffinose LC 2.28 2.97 b0252
starch/cellulose Anhydroglucose GC 1.37 1.53 b0290 myo-Inositol
myo-Inositol GC 1.23 1.50 b0695 Fructose Fructose GC 1.69 11.46
b0730 Raffinose Raffinose LC 1.93 7.16 b1430 starch/cellulose
Anhydroglucose GC 1.31 1.58 b1693 starch/cellulose Anhydroglucose
GC 1.34 2.16 b1701 Raffinose Raffinose LC 1.57 10.58 b1708 Fructose
Fructose GC 1.77 27.64 b1708 Glucose Glucose GC 1.62 10.42 b1886
Raffinose Raffinose LC 1.64 4.91 b1926 Fructose Fructose GC 1.86
2.93 b1926 Glucose Glucose GC 1.63 1.88 b2023 myo-Inositol
myo-Inositol GC 1.31 2.10 b2597 Fructose Fructose GC 1.90 2.05
b2599 Glucose Glucose GC 1.65 2.71 b2664 Fructose Fructose GC 1.77
41.86 b2664 Raffinose Raffinose LC 1.72 13.67 b2699 myo-Inositol
myo-Inositol GC 1.86 7.90 b2699 Raffinose Raffinose LC 1.61 25.08
b3172 myo-Inositol myo-Inositol GC 1.26 2.44 b3231 starch/cellulose
Anhydroglucose GC 1.31 1.74 b3430 myo-Inositol myo-Inositol GC 1.34
2.16 b3601 Raffinose Raffinose LC 1.84 2.35 b4129 myo-Inositol
myo-Inositol GC 1.29 1.48 b4239 Fructose Fructose GC 4.21 8.68
b4239 Glucose Glucose GC 2.00 4.85 b4239 Sucrose Sucrose GC 1.64
2.69 b4327 Fructose Fructose GC 1.97 3.75 YBR184W Raffinose
Raffinose LC 1.87 5.78 YBR204C myo-Inositol myo-Inositol GC 1.27
2.92 YDR112W myo-Inositol myo-Inositol GC 1.41 1.90 YER174C
starch/cellulose Anhydroglucose GC 1.34 1.84 YGR261C myo-Inositol
myo-Inositol GC 2.24 7.98 YGR261C Raffinose Raffinose LC 1.95 30.56
YIL150C myo-Inositol myo-Inositol GC 4.80 4.80 YJL072C Glucose
Glucose GC 1.58 3.93 YJL099W myo-Inositol myo-Inositol GC 1.26 5.72
YOR044W myo-Inositol myo-Inositol GC 1.44 2.60 YOR350C myo-Inositol
myo-Inositol GC 1.52 2.82 b0050 starch and/or Anhydroglucose GC
1.38 1.89 cellulose b0124 Fructose Fructose GC 2.00 30.26 b0149
Fructose Fructose GC 1.68 1.95 b1318 Fructose Fructose GC 2.03 2.99
b1463 Fructose Fructose GC 1.92 5.53 b1463 Glucose Glucose GC 1.57
7.18 b1463 myo-Inositol myo-Inositol GC 1.36 1.96 b1539 starch
and/or Anhydroglucose GC 1.39 1.92 cellulose b1736 Glucose Glucose
GC 1.61 2.10 b1961 myo-Inositol myo-Inositol GC 1.25 1.58 b2491
Fructose Fructose GC 2.23 3.78 b2491 Glucose Glucose GC 1.81 3.89
b3260 Fructose Fructose GC 1.87 5.72 b3578 Fructose Fructose GC
1.89 2.06 b3578 Glucose Glucose GC 1.65 2.10 b3578 Raffinose
Raffinose LC 1.55 1.84 b3619 Fructose Fructose GC 1.61 2.11 b3619
Glucose Glucose GC 1.60 2.19 b3919 starch and/or Anhydroglucose GC
1.33 1.52 cellulose b4074 myo-Inositol myo-Inositol GC 1.25 1.50
b4122 Fructose Fructose GC 1.53 6.02 b4232 starch and/or
Anhydroglucose GC 1.35 1.61 cellulose YHR072W-A myo-Inositol
myo-Inositol GC 1.27 1.53
[11448] [0515.0.0.25] to [0552.0.0.25]: see [0515.0.0.0] to
[0552.0.0.0]
[0552.1.25.25]: Example 15
Metabolite Profiling Info from Zea mays
[11449] Zea mays plants were engineered as described in Example
14c.
[11450] The results of the different Zea mays plants analysed can
be seen from Table 2 which follows:
TABLE-US-00145 TABLE 2 ORF_NAME Metabolite Min Max b2699 Raffinose
2.06 5.58 YIL150C myo-Inositol 2.03 2.91 b4239 Fructose 1.83 1.95
b4239 Glucose 2.18 2.29
[11451] Table 2 exhibits the metabolic data from maize, shown in
either TO or T1, describing the increase in raffinose and/or
myo-Inositol and/or fructose and/or glucose in genetically modified
corn plants expressing the Saccharomyces cerevisiae nucleic acid
sequence YIL150C and/or E. coli nucleic acid sequence b2699 or
b4239 resp.
[11452] In one embodiment, in case the activity of the Saccaromyces
cerevisiae protein YIL150C or its homologs, e.g. "a chromatin
binding protein, required for S-phase (DNA synthesis) initiation or
completion", is increased in corn plants, preferably, an increase
of the fine chemical myo-inositol between 103% and 191% is
conferred.
[11453] In case the activity of the E. coli protein b2699 or a DNA
strand exchange and recombination protein with protease and
nuclease activity, preferably a gene product with an activity of
recombination protein recA superfamily or its homolog, is increased
in corn plants, preferably, an increase of the fine chemical
raffinose between 106% and 458% is conferred.
[11454] In one embodiment, in case the activity of the E. coli
protein b4239 or its homologs, e.g. "the activity of a protein of
the alpha-glucosidase superfamily with alpha-amylase core homology
is increased, preferably, of a protein having a trehalose-6-P
hydrolase activity", is increased in corn plants, preferably, an
increase of the fine chemical fructose between 83% and 95% is
conferred. and/or an increase of the fine chemical glucose between
118% and 129% is conferred.
[11455] [0552.2.0.25]: see [0552.2.0.0]
[11456] [0553.0.25.25] [11457] 1. A process for the production of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose, which
comprises [11458] (a) increasing or generating the activity of a
protein as indicated in Table II, columns 5 or 7, lines 290 to 294
and/or 604 to 607 for starch and/or cellulose; [11459] lines 295 to
302 and/or 608 to 616 for fructose; [11460] lines 303 to 308 and/or
617 to 621 for glucose; [11461] lines 309 to 322 and/or 622 to 625
for myo-inositol; [11462] lines 323 to 331 and/or 626 for
raffinose; [11463] lines 332 to 333 for sucrose; [11464] resp., or
a functional equivalent thereof in a non-human organism, or in one
or more parts thereof; and [11465] (b) growing the organism under
conditions which permit the production of carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose resp. in said
organism. [11466] 2. A process for the production of carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose, comprising the
increasing or generating in an organism or a part thereof the
expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[11467] a) nucleic acid molecule encoding of a polypeptide as
indicated in Table II, columns 5 or 7, lines 290 to 294 and/or 604
to 607 for starch and/or cellulose; [11468] lines 295 to 302 and/or
608 to 616 for fructose; [11469] lines 303 to 308 and/or 617 to 621
for glucose; [11470] lines 309 to 322 and/or 622 to 625 for
myo-inositol; [11471] lines 323 to 331 and/or 626 for raffinose;
[11472] lines 332 to 333 for sucrose; [11473] resp., or a fragment
thereof, which confers an increase in the amount of carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrosein an organism or a
part thereof; [11474] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 290 to 294 and/or 604 to 607 for starch and/or cellulose;
[11475] lines 295 to 302 and/or 608 to 616 for fructose; [11476]
lines 303 to 308 and/or 617 to 621 for glucose; [11477] lines 309
to 322 and/or 622 to 625 for myo-inositol; [11478] lines 323 to 331
and/or 626 for raffinose; [11479] lines 332 to 333 for sucrose
resp.; [11480] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose in an organism or a
part thereof; [11481] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose in an
organism or a part thereof; [11482] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose in an organism or a part thereof; [11483]
f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table III, column 7, lines 290 to 294 and/or 604 to
607 for starch and/or cellulose; [11484] lines 295 to 302 and/or
608 to 616 for fructose; [11485] lines 303 to 308 and/or 617 to 621
for glucose; [11486] lines 309 to 322 and/or 622 to 625 for
myo-inositol; [11487] lines 323 to 331 and/or 626 for raffinose;
[11488] lines 332 to 333 for sucrose resp., and conferring an
increase in the amount of carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrosein an organism or a
part thereof; [11489] g) nucleic acid molecule encoding a
polypeptide which is isolated with the aid of monoclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (f) and conferring an increase in the amount of yin an
organism or a part thereof; [11490] h) nucleic acid molecule
encoding a polypeptide comprising a consensus as indicated in Table
IV, column 7, lines 290 to 294 and/or 604 to 607 for starch and/or
cellulose; [11491] lines 295 to 302 and/or 608 to 616 for fructose;
[11492] lines 303 to 308 and/or 617 to 621 for glucose; [11493]
lines 309 to 322 and/or 622 to 625 for myo-inositol; [11494] lines
323 to 331 and/or 626 for raffinose; [11495] lines 332 to 333 for
sucroseresp., and conferring an increase in the amount of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose in an
organism or a part thereof; and [11496] i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof. [11497] or comprising a sequence which is complementary
thereto. [11498] 3. The process of claim 1 or 2, wherein the
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose is
isolated. [11499] 4. The process of any one of claims 1 to 3,
comprising the following steps: [11500] (a) selecting an organism
or a part thereof expressing a polypeptide encoded by the nucleic
acid molecule characterized in claim 2; [11501] (b) mutagenizing
the selected organism or the part thereof; [11502] (c) comparing
the activity or the expression level of said polypeptide in the
mutagenized organism or the part thereof with the activity or the
expression of said polypeptide of the selected organisms or the
part thereof; [11503] (d) selecting the mutated organisms or parts
thereof, which comprise an increased activity or expression level
of said polypeptide compared to the selected organism or the part
thereof; [11504] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [11505] (f) recovering, and
optionally isolating, the free or bound carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose produced by the
selected mutated organisms or parts thereof. [11506] 5. The process
of any one of claims 1 to 4, wherein the activity of said protein
or the expression of said nucleic acid molecule is increased or
generated transiently or stably. [11507] 6. An isolated nucleic
acid molecule comprising a nucleic acid molecule selected from the
group consisting of: [11508] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table II, columns 5 or 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., or a fragment
thereof, which confers an increase in the amount of carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrosein an organism or a
part thereof; [11509] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table I, columns 5 or 7,
lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608 to
616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.;
[11510] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose in an organism or a
part thereof; [11511] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose in an
organism or a part thereof; [11512] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose in an organism or a part thereof; [11513]
f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table III, column 7, lines 290 to 294 and/or 604 to
607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., and conferring an increase in
the amount of carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose in an organism or a part thereof; [11514]
g) nucleic acid molecule encoding a polypeptide which is isolated
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (f) and conferring
an increase in the amount of carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose in an organism or a
part thereof; [11515] h) nucleic acid molecule encoding a
polypeptide comprising a consensus as indicated in Table IV, column
7, lines 290 to 294 and/or 604 to 607; lines 295 to 302 and/or 608
to 616; lines 303 to 308 and/or 617 to 621; lines 309 to 322 and/or
622 to 625; lines 323 to 331 and/or 626; lines 332 to 333, resp.,
and conferring an increase in the amount of carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose in an organism or a
part thereof; and [11516] i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrosein an organism or a part thereof. whereby
the nucleic acid molecule distinguishes over the sequence as
indicated in Table I A, columns 5 or 7, lines 290 to 294 and/or 604
to 607; lines 295 to 302 and/or 608 to 616; lines 303 to 308 and/or
617 to 621; lines 309 to 322 and/or 622 to 625; lines 323 to 331
and/or 626; lines 332 to 333, resp., by one or more nucleotides.
[11517] 7. A nucleic acid construct which confers the expression of
the nucleic acid molecule of claim 6, comprising one or more
regulatory elements. [11518] 8. A vector comprising the nucleic
acid molecule as claimed in claim 6 or the nucleic acid construct
of claim 7. [11519] 9. The vector as claimed in claim 8, wherein
the nucleic acid molecule is in operable linkage with regulatory
sequences for the expression in a prokaryotic or eukaryotic, or in
a prokaryotic and eukaryotic, host. [11520] 10. A host cell, which
has been transformed stably or transiently with the vector as
claimed in claim 8 or 9 or the nucleic acid molecule as claimed in
claim 6 or the nucleic acid construct of claim 7 or produced as
described in claim any one of claims 2 to 5.
[11521] 11. The host cell of claim 10, which is a transgenic host
cell. [11522] 12. The host cell of claim 10 or 11, which is a plant
cell, an animal cell, a microorganism, or a yeast cell, a fungus
cell, a prokaryotic cell, an eukaryotic cell or an archaebacterium.
[11523] 13. A process for producing a polypeptide, wherein the
polypeptide is expressed in a host cell as claimed in any one of
claims 10 to 12. [11524] 14. A polypeptide produced by the process
as claimed in claim 13 or encoded by the nucleic acid molecule as
claimed in claim 6 whereby the polypeptide distinguishes over a
sequence as indicated in Table II A, columns 5 or 7, lines 290 to
294 and/or 604 to 607; lines 295 to 302 and/or 608 to 616; lines
303 to 308 and/or 617 to 621; lines 309 to 322 and/or 622 to 625;
lines 323 to 331 and/or 626; lines 332 to 333, resp., by one or
more amino acids [11525] 15. An antibody, which binds specifically
to the polypeptide as claimed in claim 14. [11526] 16. A plant
tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [11527] 17. A method for screening for
agonists and antagonists of the activity of a polypeptide encoded
by the nucleic acid molecule of claim 6 conferring an increase in
the amount of carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose in an organism or a part thereof
comprising: [11528] (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose in an organism or a part thereof with a
candidate compound or a sample comprising a plurality of compounds
under conditions which permit the expression the polypeptide;
[11529] (b) assaying the carbohydrates, preferably polysaccharides,
more preferably starch and/or cellulose and/or monosaccharides,
more preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucroselevel or the polypeptide expression level in
the cell, tissue, plant or microorganism or the media the cell,
tissue, plant or microorganisms is cultured or maintained in; and
[11530] (c) identifying a agonist or antagonist by comparing the
measured carbohydrates, preferably polysaccharides, more preferably
starch and/or cellulose and/or monosaccharides, more preferably
fructose, glucose and/or myo-inositol and/or trisaccharides, more
preferably raffinose and/or disaccharides, more preferably sucrose
level or polypeptide expression level with a standard
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucroseor
polypeptide expression level measured in the absence of said
candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. [11531] 18. A process for the
identification of a compound conferring increased carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose production in a plant
or microorganism, comprising the steps: [11532] (a) culturing a
plant cell or tissue or microorganism or maintaining a plant
expressing the polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose in an organism or a
part thereof and a readout system capable of interacting with the
polypeptide under suitable conditions which permit the interaction
of the polypeptide with said readout system in the presence of a
compound or a sample comprising a plurality of compounds and
capable of providing a detectable signal in response to the binding
of a compound to said polypeptide under conditions which permit the
expression of said readout system and of the polypeptide encoded by
the nucleic acid molecule of claim 6 conferring an increase in the
amount of carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose in an organism or a part thereof; [11533]
(b) identifying if the compound is an effective agonist by
detecting the presence or absence or increase of a signal produced
by said readout system. [11534] 19. A method for the identification
of a gene product conferring an increase in carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucroseproduction in a cell,
comprising the following steps: [11535] (a) contacting the nucleic
acid molecules of a sample, which can contain a candidate gene
encoding a gene product conferring an increase in carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose after expression with
the nucleic acid molecule of claim 6; [11536] (b) identifying the
nucleic acid molecules, which hybridise under relaxed stringent
conditions with the nucleic acid molecule of claim 6; [11537] (c)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose; [11538] (d)
expressing the identified nucleic acid molecules in the host cells;
[11539] (e) assaying the carbohydrates, preferably polysaccharides,
more preferably starch and/or cellulose and/or monosaccharides,
more preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucroselevel in the host cells; and [11540] (f)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucroselevel in the host cell
in the host cell after expression compared to the wild type.
[11541] 20. A method for the identification of a gene product
conferring an increase in carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose production in a cell,
comprising the following steps: [11542] (a) identifying in a data
bank nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the carbohydrates, preferably polysaccharides, more preferably
starch and/or cellulose and/or monosaccharides, more preferably
fructose, glucose and/or myo-inositol and/or trisaccharides, more
preferably raffinose and/or disaccharides, more preferably sucrose
amount or level in an organism or a part thereof after expression,
and which are at least 20% homologous to the nucleic acid molecule
of claim 6; [11543] (b) introducing the candidate nucleic acid
molecules in host cells appropriate for producing carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose; [11544] (c)
expressing the identified nucleic acid molecules in the host cells;
[11545] (d) assaying the carbohydrates, preferably polysaccharides,
more preferably starch and/or cellulose and/or monosaccharides,
more preferably fructose, glucose and/or myo-inositol and/or
trisaccharides, more preferably raffinose and/or disaccharides,
more preferably sucrose level in the host cells; and [11546] (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucroselevel in the host cell
after expression compared to the wild type. [11547] 21. A method
for the production of an agricultural composition comprising the
steps of the method of any one of claims 17 to 20 and formulating
the compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. [11548] 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [11549] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably fructose,
glucose and/or myo-inositol and/or trisaccharides, more preferably
raffinose and/or disaccharides, more preferably sucrose after
expression. [11550] 24. Use of the polypeptide of claim 14 or the
nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose levels in an
organism. [11551] 25. Cosmetic, pharmaceutical, food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. [11552] 26. Use of the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the host cell of claim 10 to
12 or the gene product identified according to the method of claim
19 or 20 for the protection of a plant against a carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably fructose, glucose and/or
myo-inositol and/or trisaccharides, more preferably raffinose
and/or disaccharides, more preferably sucrose synthesis inhibiting
herbicide.
[11553] [0554.0.0.25] Abstract: see [0554.0.0.0]
PROCESS FOR THE CONTROL OF THE PRODUCTION OF FINE CHEMICALS
[11554] [0000.0.0.26] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and corresponding embodiments as
described herein as follows.
[11555] [0001.0.0.26] The present invention relates further to a
process for the control of the production of fine chemical in a
microorganism, a plant cell, a plant, a plant tissue or in one or
more parts thereof. The invention furthermore relates to nucleic
acid molecules, polypeptides, nucleic acid constructs, vectors,
antisense molecules, antibodies, host cells, plant tissue,
propagation material, harvested material, plants, microorganisms as
well as agricultural compositions and to their use.
[11556] [0002.0.0.26] Certain products and by-products of
naturally-occurring metabolic processes in cells have utility in a
wide array of industries, including, but not limited to, the food,
feed, cosmetics, health care, and pharmaceutical industries and
agriculture. These molecules, collectively termed `fine chemicals`
include molecules such as vitamins for example vitamin A, D, E, K,
B.sub.1, B.sub.2, B.sub.6, B.sub.12, C, pantothenic acid, biotin or
folic acid; substances with vitamin-like character for example
vitamin F, lipoic acid, ubiquinones, choline, myoinsositiol,
vitamin U (S-methylmethionine), flavours for example vanillin,
coumarin, isoeugenol, eugenol, (R)-carvone, (S)-carvone, menthol,
jasmone or farnesol; nutraceuticals for example phytosterols,
flavonoids, anthocyanidins, isoflavons or isoprenoids; detergents;
fatty acids such as saturated fatty acids, mono unsaturated fatty
acids (singular MUFA, plural MUFAS), poly unsaturated fatty acids
(singular PUFA, plural PUFAS), waxes or lipids containing said
fatty acids; carbohydrates for example cellulose, starch, dextrin,
pectin, xanthangum, carrageenan or alginate; sugars for example
monosaccharides such as glucose, fructose, manose, sorbose, ribose,
ribulose, xylose, xylulose or galactose, disaccharides such as
lactose, sucrose, saccharose, maltose, isomaltose or cellobiose,
trisaccharides such as raffinose or maltotriose; carboxylic acids
for example citric acid, .alpha.-ketoglutaric acid, ferulic acid,
sinapic acid or lactic acid; carotinoids for example
.alpha.-carotene, .beta.-carotene, zeaxanthine, lutein,
astaxanthine, lycopene, phyotoene or phytofluene, amino acids for
example lysine, threonine, methionine, tryptophane, phenylalanine,
argenine, valine or tyrosine, cofactors for example heme or
quinines, enzymes for example lipases, esterases, proteases,
amylases, glucosidases etc. and other compounds [as described e.g.
in Kuninaka, A. (1996) Nucleotides and related compounds, p.
561-612, in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim,
in Industial Microbiology and Biotechnology, Demain et al., second
edition, ASM Press Washington, D.C. 1999, in Ullmann's Encyclopedia
of Industrial Chemistry, vol. A27, Vitamins, p. 443-613 (1996) VCH:
Weinheim and Ong, A. S., Niki, E. & Packer, L. (1995)
Nutrition, Lipids, Health, and Disease Proceedings of the
UNESCO/Confederation of Scientific and Technological Associations
in Malaysia, and the Society for Free Radical Research, Asia, held
Sep. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes,
and all other chemicals described in Gutcho (1983) Chemicals by
Fermentation, Noyes Data Corporation, ISBN: 0818805086 and
references contained therein].
[11557] [0003.0.0.26] Compounds with health promoting properties
that can be considered for inclusion into a nutraceutical or a
functional food or which are used in food, feed, cosmetics, and
pharmaceutical industries and agriculture are, for example, amino
acids, carotenoids, saturated and unsaturated fatty acids,
carbohydrates, oligosaccharides, fibres, vitamins and precursors,
minerals and others. Some of these compounds for example can block
or delay the development of cancer and arteriosclerosis.
[11558] Carotenoids can scavenge toxic oxygen radicals and function
as provitamins. Multiple unsaturated fatty acids may prevent heart
and vascular diseases. Oligosaccharides and fibres can bind toxic
compounds and may serve as food for, and this way improve the
quantity and quality of, the intestinal flora. Oligosaccharides and
fibres are poorly digestible and are therefore helpful in keeping
the dietary energy low.
[11559] [0004.0.0.26] Further advantageous properties of the fine
chemical of the invention are described above, preferably in the
respective paragraphs [0002.0.m.n] to [0011.0.m.n] whereby m and n
can be one or more numbers between zero to twenty-five, as
disclosed afore.
[11560] [0005.0.0.26] During the last decade, many millions of
hectares have been planted worldwide with transgenic crops. Over
90% of these crops provide transgenically the agronomic properties
of herbicide and pest tolerance.
[11561] Today genetic engineering of plants and microorganisms
intends to make new or improved products. This development enables
industry and farmers to produce higher-value products, for food and
feed, for medical and for industrial objectives. Further, it is
intended and expected to have a high economic impact.
[11562] Fine chemicals for nutraceuticals and pharmaceuticals can
be produced chemically or biotechnologically by micro-organisms,
animal cell cultures, and plants. Plants are one of the new hosts
that can serve for the production of recombinant
pharmaceuticals.
[11563] [0006.0.0.26] Microorganisms, plant cells, plants, plant
tissues or one or more parts thereof may serve as new hosts for the
production of fine chemicals, recombinant nutraceuticals and/or
pharmaceuticals. Nutraceuticals and pharmaceuticals can be
distinguished at best on the basis of their features, their
physiological activity, their effect on the metabolism animals and
human beings and their aim.
[11564] [0007.0.0.26] Nutraceuticals on the one hand aim to
maintain or to meliorate the health situation of principally
healthy humans or animals. They are compounds that are naturally
present in food or are added to foods for daily consumption. Such
foods are called `functional foods` and in the case of animal
application: `functional feed`. They can be supplied with a health
claim.
[11565] Synonymous to nutraceuticals and belonging to the same
field of terminology of are `functional foods`, `designer foods`,
`positive nutrition`, `foods with dietary supplements`, `foods with
functional ingredients`, `health food`, `dietary food`, `fuctional
food ingredient`, etc. Nutraceuticals are understood as a product
that can be a single well-defined food-compound with health
promoting characteristics, but also as complex foods with such
beneficial characteristics. Nutraceuticals may be briefly and
meaningless defined as nutritionally or medicinally enhanced foods
that provide physiological, medical and/or health benefits,
including the prevention and treatment of disease beyond basic
nutritional functions. The definition for a functional food
formulated by the EU is "foods that have been satisfactorily
demonstrated to affect beneficially one or more target functions of
the body, beyond adequate nutritional effects, in a way which is
relevant to either an improved state of health and well-being, or
reduction of the risk to diseases". The terms "functional feed" and
"functional crop" are used with similar meanings.
[11566] [0008.0.0.26] Pharmaceuticals on the other hand aim to cure
(human, animal) patients, to mitigate, or to serve in diagnostics.
They usually are purified, well defined medicinal and/or
therapeutic preparations that have passed the clinical tests and
that are supplied with a medicinal claim.
[11567] [0009.0.0.26] According to the Concerted Action on
Functional Food Sciences in Europe (FUFOSE), funded by the EU, a
food can be made a functional food by using different
approaches:
[11568] to eliminate a component known to cause deleterious effects
to the consumer (e.g. an allergenic protein), or to increase the
concentration of a natural component in food, or to add a component
which is not normally present in most foods, but for which
beneficial effects have been demonstrated, or to replace a
component, usually a macronutrient, the intake of which is usually
excessive by a component which has beneficial effects or to improve
the bioavailability of, or to modify, food components for which
beneficial effects have been demonstrated.
[11569] [0010.0.0.26] It would be advantageous to have cells,
microorganisms or plants which put a combination of metabolites at
disposal, whereby the combination of the metabolites may be used
for inclusion into a nutraceutical or a functional food or
feed.
[11570] [0011.0.0.26] One object of the present invention is to put
cells, microorganisms or plants at disposal, which deliver fine
chemicals, in proportions to be used as compounds with health
promoting properties that can be considered for inclusion into a
nutraceutical or a functional food or feed, preferably without
disproportional costs and efforts.
[11571] It is further object to present process for the control of
the production of the fine chemical in a microorganism, a plant
cell, a plant, a plant tissue which modifies the content of two or
more metabolites simultaneously.
[11572] [0012.0.0.26] It is generally accepted that a diet
consisting of an adequate number of calories and having sufficient
levels of vitamins and minerals allows for proper function of the
various systems, and is required to maintain a state of good
health. In addition, it is well-established that many diseases and
undesirable conditions can be prevented, slowed, or even reversed
by modifying the subjects dietary intake.
[11573] Improving the quality of foodstuffs and animal feeds is an
important task of the food-and-feed industry.
[11574] This is necessary since certain fine chemicals, for example
like some mentioned above amino acids, fatty acids, glycerides,
lipids, vitamins, carotenoids, phytosterols, organic compounds,
preferably organic acids as disclosed in [0002.0.16.16],
[0002.0.17.17], [0002.0.19.19], [0002.0.20.20] and/or
[0002.0.23.23], glycerol and derivates, coenzymes, galactolipids
and/or carbohydrates, saccharides and/or sugars, which occur in
plants are limited with regard to the supply of mammals. Especially
advantageous for the quality of foodstuffs and animal feeds is as
balanced as possible a metabolic profile since a great excess of
one fine chemical above a specific concentration in the food or
feed has no further positive effect on the utilization of the
nutrition since other fine chemicals suddenly become limiting. A
further increase in quality is only possible via addition of
further fine chemicals, which are limiting under these
conditions.
[11575] [0013.0.0.26] Currently, the production of recombinant fine
chemicals in plants or microorganismes is usually based on the
triggered production or increased production of one selected fine
chemical.
[11576] [0014.0.0.26] On the other hand it is known that one and
the same gene may have different characteristics and effects,
sometimes additional "side effects", the so called pleiotropic
effects.
[11577] The pleiotropic effect means that one gene may be
responsible for the development of several features and
characteristics, often unforeseen change of several characteristics
in transgene organisms. Therefore, pleiotropic effects may cause
various phenomena and processes in organisms, which could lead to
phenotypic changes in the organism.
[11578] [0015.0.0.26] An other object of the present invention is a
process for the production of fine chemicals which avoids
undesirable side effect as described above and/or which uses these
side effects for the production of combinations of fine chemicals
in defined ratios.
[11579] [0016.0.0.26] To ensure a high quality of foods and animal
feeds, it is often necessary to add a plurality of fine chemicals
in a balanced manner to suit the organism.
[11580] [0017.0.0.26] Accordingly, there is still a great demand
for suitable genes which encode enzymes or proteins which directly
or indirectly participate in the biosynthesis of the fine chemicals
and make it possible to produce certain said fine chemicals
specifically on an industrial scale without unwanted byproducts
forming.
[11581] There is also a demand to reduce the concentration or
availability of some undesired metabolites or compounds in plants.
McElroy (U.S. Pat. No. 6,750,379) for example discloses plants with
minor nutritional quality of the host plant to insect pests. The
insecticidal activity is confered by genes that code for enzymes
that facilitate the production of compounds that reduce the
nutritional quality, for instance gene encoding for lipoxygenases,
which have been shown to exhibit anti-nutritional effects on
insects and to reduce the nutritional quality of their diet.
[11582] [0018.0.0.26] In order to reduce byproducts or undesired
metabolites methods of recombinant DNA technology are known, which
induce a decresase in gene expression, like knock-out, antisenseRNA
or post-transcriptional gene silencing (PTGS) used to describe RNAi
(RNA interference), co-suppression and quelling, technologies.
These techniques are based on the downregulation or inactivation of
an endogenous gene. The inactivation takes place through knockout
methods by homologous recombination, for instance by the insertion
of sequences within a endogenous gene to disrupt it, rendering its
protein non-functional, or removing the gene entirely and for the
other methods by introducing a homologous transgene in the
cells.
[11583] Little is known to date on controlling the production of
single and/or certain fine chemicals by other methods of
recombinant DNA technology, like the overexpression of exogenous
genes.
[11584] Schomburg et al. (Plant Cell. 2003 January; 15 (1):
151-163) describes the decrease of endogenous Gibberellin levels in
tobacco caused by the the increased expression of the AtGA2ox7 or
AtGA2ox8 gens from Arabidopsis thaliana encoding for Gibberellin
2-oxidases.
[11585] [0019.0.0.26] From a practical standpoint it would be of
great advantage to control the production of fine chemicals and
preferably certain combination of fine chemicals in an organism
such as a microorganism or a plant in order to produce the fine
chemicals and preferably certain combination of fine chemicals in
an amount which provides optimal growth and health benefit to
animals or humans.
[11586] [0020.0.0.26] It is an object of the present invention to
develop an inexpensive process for the synthesis of fine chemicals
or specific combination of fine chemicals.
[11587] [0021.0.0.26] It is a further object of the present
invention to provide a process for the control of the production of
fine chemicals in a microorganism, a plant cell, a plant, a plant
tissue or in one or more parts thereof without the above mentioned
disadvantages.
[11588] [0022.0.0.26] It was now found that this object is achieved
by providing the process according to the invention and the
embodiments described herein and characterized in the claims.
[11589] [0023.0.0.26] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical can be one or more fine
chemical selected from the group
of the fine chemicals as described above, preferably in the
respective paragraphs [0014.0.m.n] to [0015.0.m.n] whereby m and n
can be one or more numbers between zero to twenty-five, as
disclosed afore; preferably selected from the group consisting of
Methionine, Threonine, Tryptophane, Isoleucine, Leucine, Valine,
Arginine, Glutamate, Glutamine, Proline, 5-Oxoproline, Alanine,
Aspartic acid, Citrulline, Glycine, Homoserine, Phenylalanine,
Serine, Tyrosine, gamma-Aminobutyric acid (GABA), Putrescine,
Shikimic Acid, Palmitic acid (C16:0), Linoleic acid
(C18:cis[9,12]2), Linolenic acid (C18:cis[9,12,15]3), Stearic acid
(C18:0), C20:1 fatty acid (Gadoleic acid), 2-Hydroxy-palmitic acid,
Heptadecanoic acid (C17:0), Hexadecadienoic acid (C16:2),
Hexadecatrienoic acid (C16:3), C24:1 fatty acid
(2-Hydroxy-tetracosenoic acid (2-OH--C24:1)), Behenic acid (C22:0),
Cerotic Acid (C26:0), Lignoceric acid (C24:0), Melissic Acid
(C30:0), Glycerol, lipid fraction, Glycerol, polar fraction,
Glycerol-3-phosphate, 2,3-Dimethyl-5-phytylquinol,
alpha-Tocopherol, beta/gamma-Tocopherol, Cryptoxanthin, Zeaxanthin,
Lutein, beta-Sitosterol, Campesterol, Anhydroglucose
(Starch/Celllulose), Fructose, Glucose, iso-Maltose, myo-Inositol,
Raffinose, Sucrose, UDPGlucose, Ferulic acid, Sinapic Acid,
Coenzyme Q10, Coenzyme Q9, beta-apo-8 Carotenal, beta-Carotene,
Isopentenyl Pyrophosphate, Citramalate, Fumarate, Glyceric acid,
Malate, Malate, Lacton of Trihydroxybutyric Acid, Pyruvate,
Succinate, Trihydroxybutanoic acid, Salicylic acid, Phosphate
(inorganic and from organic phosphates), Methylgalactopyranoside,
preferably as shown in table X.
[11590] [0024.0.0.26] Accordingly, the present invention relates to
a process as described above, preferably in the respective
paragraph [0016.0.m.n] whereby m and n can be one or more numbers
between zero to twenty-five, as disclosed afore, and conferring a
defined metabolic profile.
[11591] [0025.0.0.26] In one embodiment the present invention
relates to a process comprising
(a) increasing or generating the activity of one or more b0019,
b0050, b0057, b0112, b0124, b0138, b0149, b0161, b0175, b0196,
b0251, b0252, b0255, b0376, b0462, b0464, b0486, b0577, b0651,
b0695, b0730, b0828, b0847, b0849, b0880, b0970, b0986, b1097,
b1284, b1318, b1343, b1360, b1463, b1693, b1708, b1736, b1738,
b1829, b1886, b1896, b1926, b1961, b2023, b2078, b2095, b2211,
b2239, b2307, b2414, b2426, b2478, b2489, b2491, b2507, b2553,
b2576, b2597, b2599, b2664, b2699, b2703, b2710, b2753, b2796,
b2822, b3064, b3074, b3116, b3129, b3160, b3166, b3169, b3172,
b3231, b3256, b3260, b3430, b3457, b3462, b3578, b3619, b3644,
b3684, b3767, b3791, b3919, b3926, b3936, b3938, b3966, b3983,
b4004, b4054, b4063, b4074, b4122, b4129, b4139, b4232, b4239,
b4327, b4346, b4401, YAL049C, YBL015W, YBR084W, YBR089C-A, YBR184W,
YBR204C, YCL038C, YCR012W, YCR059C, YDL127W, YDR271C, YDR316W,
YDR447C, YDR513W, YEL045C, YEL046C, YER152C, YER156C, YER173W,
YER174C, YFL019C, YFL050C, YFL053W, YFR007W, YFR042W, YGL205W,
YGL237C, YGR101W, YGR104C, YGR261C, YHR072W, YHR072W-A, YHR130C,
YHR189W, YHR201C, YIL150C, YJL055W, YJL072C, YJL099W, YKL132C,
YKR057W, YLL013C, YLR082C, YLR089C, YLR224W, YLR255C, YLR375W,
YOR024W, YOR044W, YOR084W, YOR245C, YOR317W, YOR344C, YOR350C,
YPL099C, YPL268W, YPRO24W, YPR138C and/or YPR172W protein(s) or of
a protein having the sequence of a polypeptide encoded by a
corresponding nucleic acid molecule indicated in Table I, columns 5
or 7 or indicated in table V columns 5 or 7, in a non-human
organism or in one or more parts thereof and (b) growing the
organism under conditions which permit the production of the fine
chemical in said organism and/or conferring a defined metabolic
profile.
[11592] [0026.0.0.26] Accordingly, the present invention relates to
a process for the production of fine chemicals comprising
(a) increasing or generating the activity of one or more proteins
having the activity of a protein indicated in Table II or Table VI,
column 3, preferably as indicated in table X or having the sequence
of a polypeptide encoded by a corresponding nucleic acid molecule
indicated in Table I or table V, column 5 or 7, in a non-human
organism in one or more parts thereof and (b) growing the organism
under conditions which permit the production of the fine chemicals
and/or conferring a defined metabolic profile.
[11593] [0027.0.0.26] The corresponding nucleic acid molecule of a
polypeptide as indicated in table X can be find by searching Table
I, column 3, whereby the corresponding nucleic acid molecule is
indicated in table I, column 5 and the homologues in column 7.
[11594] [0028.0.0.26] For the purposes of the invention, as a rule
the term "fine chemical" is intended to encompass the term
"metabolite" and vice versa.
[11595] [0029.0.0.26] The "combination" of fine chemicals according
to the invention is defined as a metabolic profile. Metabolic
profile means a combination of different fine chemicals in certain
ratio, e.g. as disclosed in Table X.
[11596] [0030.0.0.26] The metabolic profile of a cell of the
invention is characterized by "increase of a metabolite content" or
"decrease of a metabolite content", which relates to the relative
increase or decrease of that metabolite content in cell, a
microorganism, a plant cell, a plant, a plant tissue or in one or
more parts thereof compared to the wild type cell, microorganism,
plant cell, plant, plant tissue or one or more parts thereof.
[11597] [0031.0.0.26] According to the invention, the metabolic
profile is expressed by the changes in the metabolite content, e.g.
the matabolic profile as indicated in table X and/or by the ratio
of the fine chemicals as indicated in table X.
[11598] In other words, the metabolic profile of a cell a
microorganism, a plant cell, a plant, a plant tissue or in one or
more parts thereof, e.g. transgenic, which has an increased or
generated activity of one protein selected from the group
consisting of b0019, b0050, b0057, b0112, b0124, b0138, b0149,
b0161, b0175, b0196, b0251, b0252, b0255, b0376, b0462, b0464,
b0486, b0577, b0651, b0695, b0730, b0828, b0847, b0849, b0880,
b0970, b0986, b1097, b1284, b1318, b1343, b1360, b1463, b1693,
b1708, b1736, b1738, b1829, b1886, b1896, b1926, b1961, b2023,
b2078, b2095, b2211, b2239, b2307, b2414, b2426, b2478, b2489,
b2491, b2507, b2553, b2576, b2597, b2599, b2664, b2699, b2703,
b2710, b2753, b2796, b2822, b3064, b3074, b3116, b3129, b3160,
b3166, b3169, b3172, b3231, b3256, b3260, b3430, b3457, b3462,
b3578, b3619, b3644, b3684, b3767, b3791, b3919, b3926, b3936,
b3938, b3966, b3983, b4004, b4054, b4063, b4074, b4122, b4129,
b4139, b4232, b4239, b4327, b4346, b4401, YAL049C, YBL015W,
YBR084W, YBR089C-A, YBR184W, YBR204C, YCL038C, YCR012W, YCR059C,
YDL127W, YDR271C, YDR316W, YDR447C, YDR513W, YEL045C, YEL046C,
YER152C, YER156C, YER173W, YER174C, YFL019C, YFL050C, YFL053W,
YFR007W, YFR042W, YGL205W, YGL237C, YGR101W, YGR104C, YGR261C,
YHR072W, YHR072W-A, YHR130C, YHR189W, YHR201C, YIL150C, YJL055W,
YJL072C, YJL099W, YKL132C, YKR057W, YLL013C, YLR082C, YLR089C,
YLR224W, YLR255C, YLR375W, YOR024W, YOR044W, YOR084W, YOR245C,
YOR317W, YOR344C, YOR350C, YPL099C, YPL268W, YPRO24W, YPR138C
and/or YPR172W, is defined by the content of the fine chemicals
and/or the ratio of the fine chemicals as disclosed in the column
beneath the respective protein.
[11599] [0032.0.0.26] The metabolic profile is characterized by the
metabolic content of the fine chemicals of the invention,
preferably a combination of
[11600] 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or
86 of the metabolites as shown in table X, preferably selected from
the group consisting of Methionine, Threonine, Tryptophane,
Isoleucine, Leucine, Valine, Arginine, Glutamate, Glutamine,
Proline, 5-Oxoproline, Alanine, Aspartic acid, Citrulline, Glycine,
Homoserine, Phenylalanine, Serine, Tyrosine, gamma-Aminobutyric
acid (GABA), Putrescine, Shikimic Acid, Palmitic acid (C16:0),
Linoleic acid (C18:cis[9,12]2), Linolenic acid (C18:cis[9,12,15]3),
Stearic acid (C18:0), C20:1 fatty acid (Gadoleic acid),
2-Hydroxy-palmitic acid, Heptadecanoic acid (C17:0),
Hexadecadienoic acid (C16:2), Hexadecatrienoic acid (C16:3), C24:1
fatty acid (2-Hydroxy-tetracosenoic acid (2-OH--C24:1)), Behenic
acid (C22:0), Cerotic Acid (C26:0), Lignoceric acid (C24:0),
Melissic Acid (C30:0), Glycerol, lipid fraction, Glycerol, polar
fraction, Glycerol-3-phosphate, 2,3-Dimethyl-5-phytylquinol,
alpha-Tocopherol, beta/gamma-Tocopherol, Cryptoxanthin, Zeaxanthin,
Lutein, beta-Sitosterol, Campesterol, Anhydroglucose
(Starch/Celllulose), Fructose, Glucose, iso-Maltose, myo-lnositol,
Raffinose, Sucrose, UDPGlucose, Ferulic acid, Sinapic Acid,
Coenzyme Q10, Coenzyme Q9, beta-apo-8 Carotenal, beta-Carotene,
Isopentenyl Pyrophosphate, Citramalate, Fumarate, Glyceric acid,
Malate, Malate, Lacton of Trihydroxybutyric Acid, Pyruvate,
Succinate, Trihydroxybutanoic acid, Salicylic acid, Phosphate
(inorganic and from organic phosphates) and
Methylgalactopyranoside.
[11601] A metabolic profile according to the present invention is
defined preferably by the ratio of concentrations of the fine
chemicals of the invention.
[11602] [0033.0.0.26] According to table X the increase of a
metabolite content, meaning of the concentration of a fine
chemical, is expressed by a numerical value greater than "1". A
numerical value of "2" means a duplication of the content of the
respective fine chemical compared to the relative metabolic profile
of the wild type cell, microorganism, plant cell, plant, plant
tissue or one or more parts thereof.
[11603] According to table X the decrease of a metabolite content,
meaning of the concentration of a fine chemical, is expressed by a
numerical value less than "1". A numerical value of "0.5" means a
halving of the content of the respective fine chemical compared to
the relative metabolic profile of the wild type cell,
microorganism, plant cell, plant, plant tissue or one or more parts
thereof.
[11604] No number in table X generally means a numerical value of
"1" concerning the metabolite profile, which is esentially
identical to the relative metabolic profile of the wild type cell,
microorganism, plant cell, plant, plant tissue or one or more parts
thereof.
[11605] Different from this general rule, for those metabolites
which were listed for two different methods (methods LC and GC in
column D) only the numerical value for one of the two methods were
listed.
[11606] Relative metabolic profile means the ratio of the
metabolites, preferably directed to the increase and/or decrease,
and not to the numerical value, of the metabolite content as
defined above.
[11607] In a preferred embodiment the relative metabolic profile is
50%, more preferred, 60%, even more preferred 70%, even more
preferred 80% or even more preferred 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% similar to a profile as depicted in any one of
the columns of table X and expressing the protein displayed in the
respective column of table X or a homolog thereof.
[11608] Preferably the relative metabolic profile is identical to
the corresponding metabolic profile a indicated in the column of
table X.
[11609] In other words, the numerical value indicated in table X
expresses the factor, by which the respective metabolite content is
changed comparing with the content of the wild type.
[11610] [0034.0.0.26] Preferably the metabolite content and/or the
ratio of the metabolites implies ranges of concentration of every
single fine chemical.
[11611] This means an increase of a metabolite content expressed by
a numerical value greater than one implies an increase of a
metabolite content by factor 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7;
1.8; 1.9; 2; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8; 2.9; 3; 3.1;
3.2; 3.3; 3.4; 3.5; 3.6; 3.7; 3.8; 3.9; 4; 4.1; 4.2; 4.3; 4.4; 4.5;
4.6; 4.7; 4.8; 4.9; 5; 5.1; 5.2; 5.3; 5.4; 5.5; 5.6; 5.7; 5.8; 5.9;
6; 6.1; 6.2; 6.3; 6.4; 6.5; 6.6; 6.7; 6.8; 6.9; 7; 7.1; 7.2; 7.3;
7.4; 7.5; 7.6; 7.7; 7.8; 7.9; 8; 8.1; 8.2; 8.3; 8.4; 8.5; 8.6; 8.7;
8.8; 8.9; 9; 9.1; 9.2; 9.3; 9.4; 9,5; 9,6; 9,7; 9,8; 9,9; 10; 10.1;
10.2; 10.3; 10.4; 10.5; 10.6; 10.7; 10.8; 10.9; 11; 11.1; 11.2;
11.3; 11.4; 11.5; 11.6; 11.7; 11.8; 11.9; 12; 12.1; 12.2; 12.3;
12.4; 12.5; 12.6; 12.7; 12.8; 12.9; 13; 13.1; 13.2; 13.3; 13.4;
13.5; 13.6; 13.7; 13.8; 13.9; 14; 14.1; 14.2; 14.3; 14.4; 14.5;
14.6; 14.7; 14.8; 14.9; 15; 15.1; 15.2; 15.3; 15.4; 15.5; 15.6;
15.7; 15.8; 15.9; 16; 16.1; 16.2; 16.3; 16.4; 16.5; 16.6; 16.7;
16.8; 16.9; 17; 17.1; 17.2; 17.3;
[11612] 17.4; 17.5; 17.6; 17.7; 17.8; 17.9; 18; 18.1; 18.2; 18.3;
18.4; 18.5; 18.6; 18.7; 18.8; 18.9; 19; 19.1; 19.2; 19.3; 19.4;
19.5; 19.6; 19.7; 19.8; 19.9; 20; 20.1; 20.2; 20.3; 20.4; 20.5;
20.6; 20.7; 20.8; 20.9; 21; 21.1; 21.2; 21.3; 21.4; 21.5; 21.6;
21.7; 21.8; 21.9; 22; 22.1; 22.2; 22.3; 22.4; 22.5; 22.6; 22.7;
22.8; 22.9; 23; 23.1; 23.2; 23.3; 23.4; 23.5; 23.6; 23.7; 23.8;
23.9; 24; 24.1; 24.2; 24.3; 24.4; 24.5; 24.6; 24.7; 24.8; 24.9; 25;
25.1;
[11613] 25.2; 25.3; 25.4; 25.5; 25.6; 25.7; 25.8; 25.9; 26; 26.1;
26.2; 26.3; 26.4; 26.5; 26.6; 26.7; 26.8; 26.9; 27; 27.1; 27.2;
27.3; 27.4; 27.5; 27.6; 27.7; 27.8; 27.9; 28; 28.1; 28.2; 28.3;
28.4; 28.5; 28.6; 28.7; 28.8; 28.9; 29; 29.1; 29.2; 29.3; 29.4;
29.5; 29.6; 29.7; 29.8; 29.9; 30; 31; 32; 33; 34; 35; 36; 37; 38;
39; 40; 41; 42; 43; 44; 45; 46; 47; 48; 49; 50; 55; 60; 65; 70; 75;
80; 85; 90; 95; 100 or more
preferably by the respective factor as indicated in table X or
more.
[11614] This means a decrease of a metabolite content, meaning of
the concentration of a fine chemical, expressed by a numerical
value less than one implies a decrease of a metabolite content by
factor 0,99; 0,98; 0,97; 0,96; 0,95; 0,94; 0,93; 0,92; 0,91; 0,9;
0,89; 0,88; 0,87; 0,86; 0,85; 0,84; 0,83; 0,82; 0,81; 0,8; 0,79;
0,78; 0,77; 0,76; 0,75; 0,74; 0,73; 0,72; 0,71; 0,7; 0,69; 0,68;
0,67; 0,66; 0,65; 0,64; 0,63; 0,62; 0,61; 0,6; 0,59; 0,58; 0,57;
0,56; 0,55; 0,54; 0,53; 0,52; 0,51; 0,5; 0,49; 0,48; 0,47; 0,46;
0,45; 0,44; 0,43; 0,42; 0,41; 0,4; 0,39; 0,38; 0,37; 0,36; 0,35;
0,34; 0,33; 0,32; 0,31; 0,3; 0,29; 0,28; 0,27; 0,26; 0,25; 0,24;
0,23; 0,22; 0,21; 0,2; 0,19; 0,18; 0,17; 0,16; 0,15; 0,14; 0,13;
0,12; 0,11; 0,1; 0,09; 0,08; 0,07; 0,06; 0,05; 0,04; 0,03; 0,02;
0,01 or less;
[11615] preferably by the respective factor as indicated in table X
or less.
[11616] [0035.0.0.26] In a further embodiment, the invention
relates to a process for the control of the production of fine
chemicals, whereby the fine chemical is one or more selected from
the group
of fine chemicals as described above, preferably in the respective
paragraphs [0014.0.m.n] to [0015.0.m.n] whereby m and n can be one
or more numbers between zero to twenty-five, as disclosed afore;
preferably selected from the group consisting of Methionine,
Threonine, Tryptophane, Isoleucine, Leucine, Valine, Arginine,
Glutamate, Glutamine, Proline, 5-Oxoproline, Alanine, Aspartic
acid, Citrulline, Glycine, Homoserine, Phenylalanine, Serine,
Tyrosine, gamma-Aminobutyric acid (GABA), Putrescine, Shikimic
Acid, Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2),
Linolenic acid (C18:cis[9,12,15]3), Stearic acid
[11617] (C18:0), C20:1 fatty acid (Gadoleic acid),
2-Hydroxy-palmitic acid, Heptadecanoic acid (C17:0),
Hexadecadienoic acid (C16:2), Hexadecatrienoic acid (C16:3), C24:1
fatty acid (2-Hydroxy-tetracosenoic acid (2-OH--C24:1)), Behenic
acid (C22:0), Cerotic Acid (C26:0), Lignoceric acid (C24:0),
Melissic Acid (C30:0), Glycerol, lipid fraction, Glycerol, polar
fraction, Glycerol-3-phosphate, 2,3-Dimethyl-5-phytylquinol,
alpha-Tocopherol, beta/gamma-Tocopherol, Cryptoxanthin, Zeaxanthin,
Lutein, beta-Sitosterol, Campesterol, Anhydroglucose
(Starch/Celllulose), Fructose, Glucose, iso-Maltose, myo-Inositol,
Raffinose, Sucrose, UDPGlucose, Ferulic acid, Sinapic Acid,
Coenzyme Q10, Coenzyme Q9, beta-apo-8 Carotenal, beta-Carotene,
Isopentenyl Pyrophosphate, Citramalate, Fumarate, Glyceric acid,
Malate, Malate, Lacton of Trihydroxybutyric Acid, Pyruvate,
Succinate, Trihydroxybutanoic acid, Salicylic acid, Phosphate
(inorganic and from organic phosphates), Methylgalactopyranoside,
preferably as shown in table X.
[11618] [0036.0.0.26] Accordingly, the present invention relates to
a process for the control of the production of fine chemicals as
described above, preferably in the respective paragraph
[0016.0.m.n] whereby m and n can be one or more numbers between
zero to twenty-five, as disclosed afore, and conferring a defined
metabolic profile.
[11619] [0037.0.0.26] Preferably the present invention relates to a
process comprising
(a) increasing or generating the activity of one or more b0019,
b0050, b0057, b0112, b0124, b0138, b0149, b0161, b0175, b0196,
b0251, b0252, b0255, b0376, b0462, b0464, b0486, b0577, b0651,
b0695, b0730, b0828, b0847, b0849, b0880, b0970, b0986, b1097,
b1284, b1318, b1343, b1360, b1463, b1693, b1708, b1736, b1738,
b1829, b1886, b1896, b1926, b1961, b2023, b2078, b2095, b2211,
b2239, b2307, b2414, b2426, b2478, b2489, b2491, b2507, b2553,
b2576, b2597, b2599, b2664, b2699, b2703, b2710, b2753, b2796,
b2822, b3064, b3074, b3116, b3129, b3160, b3166, b3169, b3172,
b3231, b3256, b3260, b3430, b3457, b3462, b3578, b3619, b3644,
b3684, b3767, b3791, b3919, b3926, b3936, b3938, b3966, b3983,
b4004, b4054, b4063, b4074, b4122, b4129, b4139, b4232, b4239,
b4327, b4346, b4401, YAL049C, YBL015W, YBR084W, YBR089C-A, YBR184W,
YBR204C, YCL038C, YCR012W, YCR059C, YDL127W, YDR271C, YDR316W,
YDR447C, YDR513W, YEL045C, YEL046C, YER152C, YER156C, YER173W,
YER174C, YFL019C, YFL050C, YFL053W, YFR007W, YFR042W, YGL205W,
YGL237C, YGR101W, YGR104C, YGR261C, YHR072W, YHR072W-A, YHR130C,
YHR189W, YHR201C, YIL150C, YJL055W, YJL072C, YJL099W, YKL132C,
YKR057W, YLL013C, YLR082C, YLR089C, YLR224W, YLR255C, YLR375W,
YOR024W, YOR044W, YOR084W, YOR245C, YOR317W, YOR344C, YOR350C,
YPL099C, YPL268W, YPRO24W, YPR138C and/or YPR172W and/or b0021,
b0043, b0134, b0186, b0186, b0328, b0677, b0734, b0763, b0895,
b0895, b1054, b1183, b1217, b1249, b1292, b1874, b2110, b2696,
b2901, b3025, b3091, b3335, b3709, b3825, b3924, b4101, b4113,
b4242, b4359, YGL005C and/or YML005W protein(s) or of a protein
having the sequence of a polypeptide encoded by a corresponding
nucleic acid molecule indicated in Table I, and/or V columns 5 or
7, in a non-human organism or in one or more parts thereof and (b)
growing the organism under conditions which permit the production
fine chemicals in defined ratios in said organism resulting in a
defined metabolic profile.
[11620] [0038.0.0.26] Accordingly, the present invention relates to
a process for the control of the production of fine chemicals
comprising
(a) increasing or generating the activity of one or more proteins
having the activity of a protein indicated in Table II, column 3,
preferably as indicated in table X and/or IX or having the sequence
of a polypeptide encoded by a corresponding nucleic acid molecule
indicated in Table I, column 5 or 7, in a non-human organism in one
or more parts thereof and (b) growing the organism under conditions
which permit the control of the production of fine chemicals in
defined ratios in said organism resulting in a defined metabolic
profile.
[11621] [0038.1.0.26] The present invention relates further to a
process for the the production of a biological composition of fine
chemicals in a defined ratio, preferably in a relative metabolic
profile as indicated in table X and/or IX. In a biological
composition according to the present invention, the fine chemicals
are biologically synthesized, meaning they were synthesized in a
cell, a microorganism, a plant cell, a plant, a plant tissue or in
one or more parts thereof.
[11622] [0038.2.0.26] The present invention relates further to a
biological composition of fine chemicals in a defined ratio,
preferably in a relative metabolic profile as indicated in table X
and/or IX, produced by the process of the invention.
[11623] This biological composition according to the invention can
be one or more cells of the invention, e.g of crude microorganism,
plant cell, plant, plant tissue or one or more parts thereof of the
invention, preferably a raw extract or a purified extract of the
cells of the invention, e.g microorganism, plant cell, plant, plant
tissue or one or more parts thereof of the invention, which all
comprise ne or more fine chemicals in a relative metabolic profile
as indicated in table X and/or IX.
[11624] [0038.3.0.26] According to the invention an extract of fine
chemicals is disclosed in paragraphs [0078.0.m.n], [0089.0.m.n],
[0102.0.m.n], [0277.0.m.n], [0458.0.m.n], [0489.0. m.n] to [0507.0.
m.n], [0514.0. m.n] or [0530.6. m.n], whereby m and n can be one or
more numbers between zero to twenty-five, as disclosed afore. An
extraction is further desdcribed in Jander et al., Plant Journal
(2004), 39, 465-475 or Summer et al., BMC Plant Biology 2005,
5:8.
[11625] [0038.4.0.26] According to the invention, the biological
composition at least one, or two or three or more, relative
metabolic profile as depicted in table X due to the overexpression
of at least one of the nucleic acid molecules or its homologues
coding for a protein as depicted in table X and/or IX.
[11626] [0039.0.0.26]
[11627] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0019 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, cerotic acid
(C26:0), lignoceric acid (C24:0), beta-sitosterol, and/or sucrose,
or mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11628] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0050 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamate, linoleic acid
(C18:cis[9,12]2), linolenic acid (C18:cis[9,12,15]3),
cryptoxanthin, beta-sitosterol, starch, and/or cellulose, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11629] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0057 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamate, citrulline,
glycine, serine, C24:1 fatty acid (2-Hydroxy-tetracosenoic acid
(2-OH--C24:1)) and/or glyceric acid, or mixtures thereof containing
at least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11630] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0112 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of alpha-tocopherol and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
shikimic acid, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11631] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0124 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of isoleucine, leucine, valine
and/or fructose and/or conferring a decrease of a fine chemical,
whereby the fine chemical is at least one compound selected from
the group consisting of homoserine, or mixtures thereof containing
at least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11632] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0138 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of proline and/or myo-inositol,
or mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11633] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0149 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of leucine, proline and/or
fructose and/or conferring a decrease of a fine chemical, whereby
the fine chemical is at least one compound selected from the group
consisting of glutamate, citrulline and/or stearic acid (C18:0), or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11634] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0161 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of tryptophane, isoleucine,
leucine, valine, arginine, glutamate, glutamine, 5-oxoproline,
aspartic acid, phenylalanine, stearic acid (C18:0),
2-hydroxy-palmitic acid, alpha-tocopherol, beta-sitosterol,
campesterol, fructose, glucose, raffinose, fumarate, malate and/or
salicylic acid, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11635] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0175 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of beta-tocopherol,
gamma-tocopherol and/or coenzyme Q9, or mixtures thereof containing
at least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11636] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0196 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of ferulic acid and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
glycerol (lipid fraction), or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11637] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0251 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of linoleic acid
(C18:cis[9,12]2) and/or linolenic acid (C18:cis[9,12,15]3), or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X. In one embodiment in
the process of the invention the activity of the Escherichia coli
K12 protein b0252 or its homologs, preferably as indicated in the
respective aforementioned paragraphs [23.1.m.n] (where m and n can
be one or more numbers between 0 to 25), e.g. the activity as
defined in the respective aforementioned paragraphs [22.0.m.n]
(where m and n can be one or more numbers between 0 to 25), is
increased, conferring an increase of a fine chemical, whereby the
fine chemical is at least one compound selected from the group
consisting of starch and/or cellulose and/or conferring a decrease
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of beta-apo-8 carotenal
and/or methylgalactopyranoside, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11638] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0255 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of linoleic acid
(C18:cis[9,12]2) and/or lignoceric acid (C24:0), or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11639] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0376 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of 5-oxoproline and/or linolenic
acid (C18:cis[9,12,15]3), or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11640] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0462 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of citrulline and/or conferring
a decrease of a fine chemical, whereby the fine chemical is at
least one compound selected from the group consisting of shikimic
acid and/or glycerol-3-phosphate, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11641] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0464 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of methionine, threonine and/or
campesterol and/or conferring a decrease of a fine chemical,
whereby the fine chemical is at least one compound selected from
the group consisting of sucrose and/or trihydroxybutanoic acid, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11642] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0486 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of tryptophane, valine,
glutamine, alanine and/or serine, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11643] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0577 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of aspartic acid, glycine,
linoleic acid (C18:cis[9,12]2) and/or linolenic acid
(C18:cis[9,12,15]3), or mixtures thereof containing at least two,
three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11644] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0651 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of gamma-aminobutyric acid
(GABA) and/or conferring a decrease of a fine chemical, whereby the
fine chemical is at least one compound selected from the group
consisting of trihydroxybutanoic acid, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11645] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0695 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of arginine, phenylalanine,
fructose and/or pyruvate, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11646] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0730 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamate, proline, aspartic
acid, linoleic acid (C18:cis[9,12]2), raffinose, ferulic acid,
coenzyme Q9, isopentenyl pyrophosphate, fumarate, malate and/or
succinate, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11647] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0828 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of isoleucine and/or valine, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11648] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0847 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of gamma-aminobutyric acid
[11649] (GABA) and/or conferring a decrease of a fine chemical,
whereby the fine chemical is at least one compound selected from
the group consisting of glycine and/or homoserine, or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11650] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0849 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamine, palmitic acid
(C16:0), linoleic acid (C18:cis[9,12]2) and/or linolenic acid
(C18:cis[9,12,15]3), or mixtures thereof containing at least two,
three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11651] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0880 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of cerotic acid (C26:0) and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
raffinose, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11652] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0970 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamine, 5-oxoproline,
tyrosine, alpha-tocopherol, isopentenyl pyrophosphate and/or
trihydroxybutanoic acid, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11653] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b0986 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of beta-tocopherol,
gamma-tocopherol and/or cryptoxanthin and/or conferring a decrease
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of tryptophane,
glycerol-3-phosphate, ferulic acid, fumarate and/or succinate, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11654] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1097 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of linoleic acid
(C18:cis[9,12]2) and/or C24:1 fatty acid (2-Hydroxy-tetracosenoic
acid (2-OH--C24:1)), or mixtures thereof containing at least two,
three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11655] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1284 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of arginine and/or conferring a
decrease of a fine chemical, whereby the fine chemical is at least
one compound selected from the group consisting of fumarate, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11656] In one embodiment in the process of the invention the
activity of the Escherichia coli
[11657] K12 protein b1318 or its homologs, preferably as indicated
in the respective aforementioned paragraphs [23.1.m.n] (where m and
n can be one or more numbers between 0 to 25), e.g. the activity as
defined in the respective aforementioned paragraphs [22.0.m.n]
(where m and n can be one or more numbers between 0 to 25), is
increased, conferring an increase of a fine chemical, whereby the
fine chemical is at least one compound selected from the group
consisting of tryptophane and/or fructose and/or conferring a
decrease of a fine chemical, whereby the fine chemical is at least
one compound selected from the group consisting of
glycerol-3-phosphate, or mixtures thereof containing at least two,
three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11658] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1343 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of methionine, valine,
glutamate, glutamine, 5-oxoproline, alanine, aspartic acid and/or
trihydroxybutanoic acid, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11659] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1360 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, proline and/or
citrulline, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11660] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1463 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of isoleucine, leucine,
fructose, glucose and/or myo-inositol, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11661] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1693 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamate, shikimic acid,
linoleic acid (C18:cis[9,12]2), starch, cellulose, citramalate
and/or glyceric acid and/or conferring a decrease of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of sucrose, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11662] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1708 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of isoleucine, leucine, valine,
phenylalanine, fructose and/or glucose, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11663] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1736 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamate, glucose and/or
isopentenyl pyrophosphate, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11664] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1738 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, glutamate,
isopentenyl pyrophosphate, fumarate and/or succinate, or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11665] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1829 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, isoleucine,
leucine, arginine, glutamine, serine, tyrosine, heptadecanoic acid
(C17:0), 2,3-dimethyl-5-phytylquinol, alpha-tocopherol,
beta-tocopherol, gamma-tocopherol, coenzyme Q10 and/or
beta-carotene, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11666] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1886 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamine, phenylalanine,
serine, cerotic acid (C26:0) and/or raffinose, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11667] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1896 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, glutamate,
5-oxoproline, aspartic acid, stearic acid (C18:0), cerotic acid
(C26:0), campesterol, sinapic acid, citramalate, fumarate, malate,
lacton of trihydroxybutyric acid, succinate and/or
trihydroxybutanoic acid and/or conferring a decrease of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of fructose and/or sucrose, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11668] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1926 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamine, fructose, glucose
and/or isopentenyl pyrophosphate and/or conferring a decrease of a
fine chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glycine, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11669] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b1961 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of myo-inositol, fumarate and/or
succinate, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11670] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2023 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of aspartic acid and/or
myo-inositol, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11671] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2078 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of phenylalanine and/or C24:1
fatty acid (2-Hydroxy-tetracosenoic acid (2-OH--C24:1)) and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
beta-apo-8 carotenal, or mixtures thereof containing at least two,
three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11672] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2095 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of arginine, alanine and/or
stearic acid (C18:0), or mixtures thereof containing at least two,
three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11673] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2211 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of cryptoxanthin and/or
isopentenyl pyrophosphate, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11674] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2239 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of citrulline and/or conferring
a decrease of a fine chemical, whereby the fine chemical is at
least one compound selected from the group consisting of
glycerol-3-phosphate and/or isopentenyl pyrophosphate, or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11675] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2307 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of arginine and/or glutamate, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11676] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2414 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of methionine, threonine,
valine, glutamine, citrulline, glycine, phenylalanine, serine
and/or ferulic acid, or mixtures thereof containing at least two,
three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11677] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2426 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamine, citrulline and/or
coenzyme Q9, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11678] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2478 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of campesterol and/or malate, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11679] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2489 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamine, alanine,
citrulline, glycine and/or serine, or mixtures thereof containing
at least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11680] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2491 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of tyrosine, fructose and/or
glucose and/or conferring a decrease of a fine chemical, whereby
the fine chemical is at least one compound selected from the group
consisting of hexadecatrienoic acid (C16:3), or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11681] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2507 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of aspartic acid and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
sucrose, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11682] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2553 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamine, proline and/or
glycine, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11683] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2576 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of alanine, aspartic acid,
glycine and/or phenylalanine, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11684] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2597 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of fructose and/or conferring a
decrease of a fine chemical, whereby the fine chemical is at least
one compound selected from the group consisting of citramalate, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11685] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2599 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glucose and/or succinate, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11686] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2664 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, isoleucine,
leucine, proline, serine, fructose, raffinose and/or salicylic
acid, or mixtures thereof containing at least two, three, four or
five compounds selected from the aforementioned groups, preferably
6, 7, 8 or 9 compounds selected from the aforementioned groups,
more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11687] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2699 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of proline, linolenic acid
(C18:cis[9,12,15]3), stearic acid (C18:0), alpha-tocopherol,
zeaxanthin, lutein, beta-sitosterol, campesterol, myo-inositol,
raffinose, coenzyme Q10, coenzyme Q9, beta-carotene, fumarate,
lacton of trihydroxybutyric acid, trihydroxybutanoic acid and/or
methylgalactopyranoside and/or conferring a decrease of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of arginine, citrulline and/or
glycine, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11688] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2703 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of coenzyme Q9 and/or conferring
a decrease of a fine chemical, whereby the fine chemical is at
least one compound selected from the group consisting of alanine,
or mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11689] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2710 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamate, linoleic acid
(C18:cis[9,12]2) and/or glycerol-3-phosphate, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11690] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2753 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of aspartic acid and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
beta-apo-8 carotenal or mixtures thereof containing at least two,
three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11691] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2796 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of phenylalanine and/or
salicylic acid, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11692] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b2822 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of linoleic acid
[11693] (C18:cis[9,12]2), linolenic acid (C18:cis[9,12,15]3) and/or
campesterol, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11694] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3064 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamine, serine and/or
linoleic acid (C18:cis[9,12]2), or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11695] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3074 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, tryptophane,
glutamate and/or ferulic acid, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11696] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3116 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamate, aspartic acid,
serine, fumarate, malate, succinate and/or salicylic acid, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11697] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3129 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of linolenic acid
(C18:cis[9,12,15]3), glyceric acid and/or methylgalactopyranoside,
or mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11698] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3160 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, glutamine,
citrulline, serine, alpha-tocopherol, isopentenyl pyrophosphate
and/or succinate, or mixtures thereof containing at least two,
three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11699] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3166 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamine and/or linoleic
acid (C18:cis[9,12]2), or mixtures thereof containing at least two,
three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11700] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3169 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamate, glutamine,
aspartic acid, malate and/or trihydroxybutanoic acid, or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11701] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3172 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of 5-oxoproline, aspartic acid,
myo-inositol, sinapic acid, isopentenyl pyrophosphate, fumarate
and/or malate, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11702] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3231 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, glutamine,
alanine, serine, C24:1 fatty acid (2-Hydroxy-tetracosenoic acid
(2-OH--C24:1)), starch, cellulose, glyceric acid, and/or pyruvate,
or mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11703] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3256 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of valine, phenylalanine,
palmitic acid (C16:0), linoleic acid (C18:cis[9,12]2), stearic acid
(C18:0) and/or campesterol, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11704] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3260 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of fructose and/or conferring a
decrease of a fine chemical, whereby the fine chemical is at least
one compound selected from the group consisting of proline, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11705] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3430 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of hexadecatrienoic acid
[11706] (C16:3) and/or myo-inositol, or mixtures thereof containing
at least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11707] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3457 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of palmitic acid (C16:0),
linoleic acid (C18:cis[9,12]2), linolenic acid (C18:cis[9,12,15]3),
stearic acid (C18:0) and/or glycerol (lipid fraction), or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11708] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3462 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, phenylalanine,
serine and/or linolenic acid (C18:cis[9,12,15]3), or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11709] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3578 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of palmitic acid (C16:0),
fructose, glucose and/or raffinose, or mixtures thereof containing
at least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11710] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3619 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamate, fructose and/or
glucose, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11711] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3644 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of proline, palmitic acid
(C16:0), linoleic acid (C18:cis[9,12]2), linolenic acid
(C18:cis[9,12,15]3), glycerol (lipid fraction) and/or coenzyme Q9
and/or conferring a decrease of a fine chemical, whereby the fine
chemical is at least one compound selected from the group
consisting of cerotic acid (C26:0), lignoceric acid (C24:0) and/or
fumarate, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11712] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3684 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of cryptoxanthin and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
malate, or mixtures thereof containing at least two, three, four or
five compounds selected from the aforementioned groups, preferably
6, 7, 8 or 9 compounds selected from the aforementioned groups,
more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11713] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3767 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of alanine and/or homoserine, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11714] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3791 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, glutamate and/or
glutamine, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11715] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3919 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of proline, phenylalanine,
starch and/or cellulose, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11716] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3926 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of citrulline and/or
cryptoxanthin and/or conferring a decrease of a fine chemical,
whereby the fine chemical is at least one compound selected from
the group consisting of glycerol-3-phosphate, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11717] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3936 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of arginine and/or conferring a
decrease of a fine chemical, whereby the fine chemical is at least
one compound selected from the group consisting of raffinose, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11718] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3938 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of valine, phenylalanine,
lignoceric acid (C24:0), beta-tocopherol, gamma-tocopherol and/or
glyceric acid, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11719] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3966 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, isoleucine and/or
leucine, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11720] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b3983 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of tryptophane, valine,
phenylalanine and/or tyrosine, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11721] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4004 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine and/or glutamine
and/or conferring a decrease of a fine chemical, whereby the fine
chemical is at least one compound selected from the group
consisting of coenzyme Q10, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11722] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4054 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of leucine, beta-tocopherol
and/or gamma-tocopherol and/or conferring a decrease of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of tyrosine, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11723] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4063 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of alpha-tocopherol, fumarate
and/or malate, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11724] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4074 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamine and/or
myo-inositol, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11725] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4122 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of fructose and/or fumarate, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11726] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4129 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of aspartic acid, palmitic acid
(C16:0), linoleic acid (C18:cis[9,12]2), beta-sitosterol,
myo-inositol, isopentenyl pyrophosphate and/or succinate and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
glycerol (polar fraction) or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11727] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4139 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of fumarate and/or conferring a
decrease of a fine chemical, whereby the fine chemical is at least
one compound selected from the group consisting of glyceric acid
and/or malate, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11728] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4232 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of methionine, starch and/or
cellulose and/or conferring a decrease of a fine chemical, whereby
the fine chemical is at least one compound selected from the group
consisting of lignoceric acid (C24:0) or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11729] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4239 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of fructose, glucose and/or
sucrose and/or conferring a decrease of a fine chemical, whereby
the fine chemical is at least one compound selected from the group
consisting of homoserine and/or succinate, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11730] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4327 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of isoleucine and/or fructose
and/or conferring a decrease of a fine chemical, whereby the fine
chemical is at least one compound selected from the group
consisting of glycerol (polar fraction), or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11731] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4346 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of glutamate and/or aspartic
acid and/or conferring a decrease of a fine chemical, whereby the
fine chemical is at least one compound selected from the group
consisting of 2,3-dimethyl-5-phytylquinol, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11732] In one embodiment in the process of the invention the
activity of the Escherichia coli K12 protein b4401 or its homologs,
preferably as indicated in the respective aforementioned paragraphs
[23.1.m.n] (where m and n can be one or more numbers between 0 to
25), e.g. the activity as defined in the respective aforementioned
paragraphs [22.0.m.n] (where m and n can be one or more numbers
between 0 to 25), is increased, conferring an increase of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of cryptoxanthin and/or lutein,
or mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11733] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YAL049C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of phenylalanine and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
threonine or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11734] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YBL015W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of methionine, alanine,
fumarate and/or malate, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11735] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YBR084W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of glycerol (polar
fraction) and/or malate, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11736] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YBR089C-A or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of palmitic acid
(C16:0), linoleic acid (C18:cis[9,12]2), stearic acid (C18:0)
and/or beta-carotene and/or conferring a decrease of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of methionine and/or
gamma-aminobutyric acid (GABA), or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11737] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YBR184W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of raffinose, ferulic
acid, coenzyme Q10 and/or malate, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11738] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YBR204C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of glutamate and/or
myo-inositol, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11739] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YCL038C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of malate and/or
succinate, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11740] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YCR012W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of fumarate and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
glycerol (lipid fraction) or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11741] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YCR059C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of fumarate and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
fructose and/or succinate, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11742] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YDL127W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of isoleucine and/or
5-oxoproline, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11743] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YDR271C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of isoleucine and/or
proline, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11744] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YDR316W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of arginine and/or
beta-carotene and/or conferring a decrease of a fine chemical,
whereby the fine chemical is at least one compound selected from
the group consisting of glycine or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11745] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YDR447C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of palmitic acid
(C16:0) and/or linoleic acid (C18:cis[9,12]2), or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11746] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YDR513W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of palmitic acid
(C16:0), stearic acid (C18:0), C20:1 fatty acid (Gadoleic acid),
heptadecanoic acid (C17:0), melissic acid (C30:0), glycerol (lipid
fraction), glycerol (polar fraction), sinapic acid, coenzyme Q9,
and/or beta-carotene and/or conferring a decrease of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of putrescine,
glycerol-3-phosphate, UDPglucose and/or succinate, or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11747] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YEL045C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of proline and/or
serine, or mixtures thereof containing at least two, three, four or
five compounds selected from the aforementioned groups, preferably
6, 7, 8 or 9 compounds selected from the aforementioned groups,
more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11748] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YEL046C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of methionine and/or
homoserine and/or conferring a decrease of a fine chemical, whereby
the fine chemical is at least one compound selected from the group
consisting of threonine, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11749] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YER152C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of serine and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
shikimic acid, C24:1 fatty acid (2-Hydroxy-tetracosenoic acid
(2-OH--C24:1)), cerotic acid (C26:0) and/or myo-inositol, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11750] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YER156C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of gamma-aminobutyric
acid (GABA), heptadecanoic acid (C17:0), melissic acid (C30:0),
beta-sitosterol, campesterol and/or coenzyme Q9, or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11751] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YER173W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of methionine,
tryptophane, leucine, valine, glutamine, proline, alanine, aspartic
acid, phenylalanine, linolenic acid (C18:cis[9,12,15]3),
2-hydroxy-palmitic acid, campesterol and/or
methylgalactopyranoside, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11752] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YER174C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of starch, cellulose,
and/or coenzyme Q9 and/or conferring a decrease of a fine chemical,
whereby the fine chemical is at least one compound selected from
the group consisting of phosphate (inorganic and from organic
phosphates) or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11753] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YFL019C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of valine, glutamate
and/or 2,3-dimethyl-5-phytylquinol, or mixtures thereof containing
at least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11754] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YFL050C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of threonine, proline,
alanine, glycine and/or tyrosine, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11755] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YFL053W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of beta-tocopherol
and/or gamma-tocopherol and/or conferring a decrease of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of shikimic acid, or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11756] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YFR007W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of lutein and/or
fumarate, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11757] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YFR042W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of leucine, glutamine,
gamma-aminobutyric acid (GABA), stearic acid (C18:0) and/or C24:1
fatty acid (2-Hydroxy-tetracosenoic acid (2-OH--C24:1)) and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
putrescine and/or iso-maltose, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11758] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YGL205W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of linolenic acid
(C18:cis[9,12,15]3), hexadecatrienoic acid (C16:3) and/or behenic
acid (C22:0), or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11759] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YGL237C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of glycerol (lipid
fraction) and/or conferring a decrease of a fine chemical, whereby
the fine chemical is at least one compound selected from the group
consisting of putrescine, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11760] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YGR101W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of valine and/or
phenylalanine, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11761] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YGR104C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of tryptophane,
isoleucine, glutamate and/or aspartic acid, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11762] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YGR261C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of myo-inositol and/or
raffinose, or mixtures thereof containing at least two, three, four
or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11763] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YHR072W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of glycerol (lipid
fraction) and/or methylgalactopyranoside and/or conferring a
decrease of a fine chemical, whereby the fine chemical is at least
one compound selected from the group consisting of campesterol, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11764] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YHR072W-A or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of myo-inositol and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
lignoceric acid (C24:0), or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11765] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YHR130C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of arginine,
phenylalanine and/or tyrosine, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11766] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YHR189W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of tryptophane and/or
conferring a decrease of a fine chemical, whereby the fine chemical
is at least one compound selected from the group consisting of
threonine, arginine, glutamine, citrulline, tyrosine, shikimic
acid, C24:1 fatty acid (2-Hydroxy-tetracosenoic acid
(2-OH--C24:1)), UDPglucose and/or isopentenyl pyrophosphate, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11767] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YHR201C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of cerotic acid (C26:0)
and/or lignoceric acid (C24:0) and/or conferring a decrease of a
fine chemical, whereby the fine chemical is at least one compound
selected from the group consisting of threonine, or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11768] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YIL150C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of threonine,
isoleucine, valine, proline, alanine, aspartic acid, serine,
tyrosine, linolenic acid (C18:cis[9,12,15]3), stearic acid (C18:0),
hexadecatrienoic acid (C16:3), glycerol (lipid fraction) and/or
myo-inositol and/or conferring a decrease of a fine chemical,
whereby the fine chemical is at least one compound selected from
the group consisting of zeaxanthin, lutein, coenzyme Q10,
beta-carotene, fumarate and/or malate, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11769] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YJL055W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of methionine,
threonine and/or fumarate, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11770] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YJL072C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of isoleucine,
phenylalanine, glucose and/or malate, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11771] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YJL099W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of myo-inositol,
fumarate and/or malate, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11772] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YKL132C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of hexadecatrienoic
acid (C16:3) and/or malate, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11773] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YKR057W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of threonine, valine,
arginine, glutamine, serine, beta-sitosterol and/or campesterol, or
mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11774] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YLL013C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of citrulline and/or
beta-carotene and/or conferring a decrease of a fine chemical,
whereby the fine chemical is at least one compound selected from
the group consisting of glycerol-3-phosphate, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11775] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YLR082C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of serine and/or
glycerol (polar fraction), or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11776] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YLR089C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of salicylic acid
and/or conferring a decrease of a fine chemical, whereby the fine
chemical is at least one compound selected from the group
consisting of myo-inositol and/or coenzyme Q10, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11777] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YLR224W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of glycerol (lipid
fraction) and/or methylgalactopyranoside, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11778] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YLR255C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of heptadecanoic acid
(C17:0), melissic acid (C30:0), glycerol (polar fraction) and/or
methylgalactopyranoside and/or conferring a decrease of a fine
chemical, whereby the fine chemical is at least one compound
selected from the group consisting of tryptophane and/or proline,
or mixtures thereof containing at least two, three, four or five
compounds selected from the aforementioned groups, preferably 6, 7,
8 or 9 compounds selected from the aforementioned groups, more
preferably 10, 11, 12, 13, 14, 15, 16, 17 or more compounds
selected from the aforementioned groups, most preferably conferring
a metabolic profile as indicated in Table X.
[11779] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YLR375W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of methionine and/or
shikimic acid and/or conferring a decrease of a fine chemical,
whereby the fine chemical is at least one compound selected from
the group consisting of threonine, or mixtures thereof containing
at least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11780] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YOR024W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of linoleic acid
(C18:cis[9,12]2) and/or stearic acid (C18:0), or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11781] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YOR044W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of campesterol,
myo-inositol and/or succinate, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11782] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YOR084W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of methionine,
gamma-aminobutyric acid (GABA) and/or beta-sitosterol, or mixtures
thereof containing at least two, three, four or five compounds
selected from the aforementioned groups, preferably 6, 7, 8 or 9
compounds selected from the aforementioned groups, more preferably
10, 11, 12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11783] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YOR245C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of threonine and/or
citrulline and/or conferring a decrease of a fine chemical, whereby
the fine chemical is at least one compound selected from the group
consisting of raffinose, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11784] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YOR317W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of hexadecadienoic acid
(C16:2) and/or conferring a decrease of a fine chemical, whereby
the fine chemical is at least one compound selected from the group
consisting of tryptophane, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11785] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YOR344C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of hexadecatrienoic
acid (C16:3) and/or glycerol (lipid fraction), or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11786] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YOR350C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of isoleucine, leucine,
tyrosine and/or myo-inositol, or mixtures thereof containing at
least two, three, four or five compounds selected from the
aforementioned groups, preferably 6, 7, 8 or 9 compounds selected
from the aforementioned groups, more preferably 10, 11, 12, 13, 14,
15, 16, 17 or more compounds selected from the aforementioned
groups, most preferably conferring a metabolic profile as indicated
in Table X.
[11787] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YPL099C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of palmitic acid
(C16:0) and/or glycerol (polar fraction), or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11788] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YPL268W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of glycerol (polar
fraction) and/or 2,3-dimethyl-5-phytylquinol, or mixtures thereof
containing at least two, three, four or five compounds selected
from the aforementioned groups, preferably 6, 7, 8 or 9 compounds
selected from the aforementioned groups, more preferably 10, 11,
12, 13, 14, 15, 16, 17 or more compounds selected from the
aforementioned groups, most preferably conferring a metabolic
profile as indicated in Table X.
[11789] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YPRO24W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of glutamate and/or
fumarate and/or conferring a decrease of a fine chemical, whereby
the fine chemical is at least one compound selected from the group
consisting of threonine, or mixtures thereof containing at least
two, three, four or five compounds selected from the aforementioned
groups, preferably 6, 7, 8 or 9 compounds selected from the
aforementioned groups, more preferably 10, 11, 12, 13, 14, 15, 16,
17 or more compounds selected from the aforementioned groups, most
preferably conferring a metabolic profile as indicated in Table
X.
[11790] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YPR138C or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of proline,
phenylalanine, lignoceric acid (C24:0), coenzyme Q10, fumarate
and/or malate, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11791] In one embodiment in the process of the invention the
activity of the Saccharomyces cerevisiae protein YPR172W or its
homologs, preferably as indicated in the respective aforementioned
paragraphs [23.1.m.n] (where m and n can be one or more numbers
between 0 to 25), e.g. the activity as defined in the respective
aforementioned paragraphs [22.0.m.n] (where m and n can be one or
more numbers between 0 to 25), is increased, conferring an increase
of a fine chemical, whereby the fine chemical is at least one
compound selected from the group consisting of campesterol and/or
coenzyme Q9, or mixtures thereof containing at least two, three,
four or five compounds selected from the aforementioned groups,
preferably 6, 7, 8 or 9 compounds selected from the aforementioned
groups, more preferably 10, 11, 12, 13, 14, 15, 16, 17 or more
compounds selected from the aforementioned groups, most preferably
conferring a metabolic profile as indicated in Table X.
[11792] [0040.0.0.26] In one embodiment, the process of the present
invention comprises one or more of the following steps:
a) stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptid of the invention or the nucleic acid molecule or the
polypeptide used in the method of the invention, e.g. of a
polypeptide having an activity of a protein as indicated in table X
and/or the same protein as indicated in Table II, column 3, or its
homologs activity, e.g. as indicated in Table II, columns 5 or 7,
having herein-mentioned the fine chemical-increasing and/or the
fine chemical-decreasing activity; b) stabilizing a mRNA conferring
the increased expression of a protein encoded by the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention, e.g. of a polypeptide having an activity
of a protein as indicated in table X and/or the same protein as
indicated in Table II, column 3, or its homologs activity, e.g. as
indicated in Table II, columns 5 or 7, or of a mRNA encoding the
polypeptide of the present invention having herein-mentioned fine
chemical-increasing and/or the fine chemical-decreasing activity;
c) increasing the specific activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention or of the polypeptide of the present
invention or the nucleic acid molecule or polypeptide used in the
method of the invention, having herein-mentioned fine
chemical-increasing and/or the fine chemical-decreasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in table X and/or the same protein as indicated in Table II, column
3, or its homologs activity, e.g. as indicated in Table II, columns
5 or 7, or decreasing the inhibitory regulation of the polypeptide
of the invention or the polypeptide used in the method of the
invention; d) generating or increasing the expression of an
endogenous or artificial transcription factor mediating the
expression of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention or of
the polypeptide of the invention or the polypeptide used in the
method of the invention having herein-mentioned fine
chemical-increasing and/or the fine chemical-decreasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in table X and/or the same protein as indicated in Table II, column
3, or its homologs activity, e.g. as indicated in Table II, columns
5 or 7; e) stimulating activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention having herein-mentioned fine chemical-increasing and/or
the fine chemical-decreasing activity, e.g. of a polypeptide having
an activity of a protein as indicated in table X and/or the same
protein as indicated in Table II, column 3, or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, by adding
one or more exogenous inducing factors to the organism or parts
thereof; f) expressing a transgenic gene encoding a protein
conferring the increased expression of a polypeptide encoded by the
nucleic acid molecule of the present invention or a polypeptide of
the present invention, having herein-mentioned fine
chemical-increasing and/or the fine chemical-decreasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in table X and/or the same protein as indicated in Table II, column
3, or its homologs activity, e.g. as indicated in Table II, columns
5 or 7; g) increasing the copy number of a gene conferring the
increased expression of a nucleic acid molecule encoding a
polypeptide encoded by the nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention or
the polypeptide of the invention or the polypeptide used in the
method of the invention having herein-mentioned fine
chemical-increasing and/or the fine chemical-decreasing activity,
e.g. of a polypeptide having an activity of a protein as indicated
in table X and/or the same protein as indicated in Table II, column
3, or its homologs activity, e.g. as indicated in Table II, columns
5 or 7; h) Increasing the expression of the endogenous gene
encoding the polypeptide of the invention or the polypeptide used
in the method of the invention, e.g. a polypeptide having an
activity of a protein as indicated in table X and/or the same
protein as 15 l indicated in Table II, column 3 or its homologs
activity, e.g. as indicated in Table II, columns 5 or 7, by adding
positive expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements.
[11793] Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced;
i) Modulating growth conditions of an organism in such a manner,
that the expression or activity of the gene encoding the protein of
the invention or the protein itself is enhanced for example
microorganisms or plants can be grown under a higher temperature
regime leading to an enhanced expression of heat shock proteins,
e.g. the heat shock protein of the invention, which can lead an
enhanced the fine chemical production; and/or j) selecting of
organisms with especially high activity of the proteins of the
invention from natural or from mutagenized resources and breeding
them into the target organisms, e.g. the elite crops.
[11794] [0041.0.0.26] Accordingly, in one embodiment, the process
according to the invention relates to a process which
comprises:
a) providing a non-human organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant; b) increasing an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
a homolog thereof, e.g. as indicated in table X and/or the same
protein as indicated in Table II, columns 5 or 7, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, i.e. conferring an increase
or decrease of the respective fine chemical according to table X in
the organism, preferably in a microorganism, a non-human animal, a
plant or animal cell, a plant or animal tissue or a plant, c)
growing the organism, preferably a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a
plant, under conditions which permit the production of the
respective fine chemical or of a defined metabolic profile in the
organism, preferably the microorganism, the plant cell, the plant
tissue or the plant; and d) if desired, recovering, optionally
isolating, one or more free and/or bound fine chemicals synthesized
by the organism, the microorganism, the non-human animal, the plant
or animal cell, the plant or animal tissue or the plant.
[11795] [0042.0.0.26] In a preferred embodiment, the present
invention relates to a process for the for the control of the
production of fine chemicals comprising or generating in an
organism or a part thereof the expression of at least one nucleic
acid molecule comprising a nucleic acid molecule selected from the
group consisting of:
a) nucleic acid molecule encoding, preferably at least the mature
form, of a polypeptide having a sequence as indicated in Table II,
columns 5 or 7, and selected from the group consisting of b0019,
b0050, b0057, b0112, b0124, b0138, b0149, b0161, b0175, b0196,
b0251, b0252, b0255, b0376, b0462, b0464, b0486, b0577, b0651,
b0695, b0730, b0828, b0847, b0849, b0880, b0970, b0986, b1097,
b1284, b1318, b1343, b1360, b1463, b1693, b1708, b1736, b1738,
b1829, b1886, b1896, b1926, b1961, b2023, b2078, b2095, b2211,
b2239, b2307, b2414, b2426, b2478, b2489, b2491, b2507, b2553,
b2576, b2597, b2599, b2664, b2699, b2703, b2710, b2753, b2796,
b2822, b3064, b3074, b3116, b3129, b3160, b3166, b3169, b3172,
b3231, b3256, b3260, b3430, b3457, b3462, b3578, b3619, b3644,
b3684, b3767, b3791, b3919, b3926, b3936, b3938, b3966, b3983,
b4004, b4054, b4063, b4074, b4122, b4129, b4139, b4232, b4239,
b4327, b4346, b4401, YAL049C, YBL015W, YBR084W, YBR089C-A, YBR184W,
YBR204C, YCL038C, YCR012W, YCR059C, YDL127W, YDR271C, YDR316W,
YDR447C, YDR513W, YEL045C, YEL046C, YER152C, YER156C, YER173W,
YER174C, YFL019C, YFL050C, YFL053W, YFR007W, YFR042W, YGL205W,
YGL237C, YGR101W, YGR104C, YGR261C, YHR072W, YHR072W-A, YHR130C,
YHR189W, YHR201C, YIL150C, YJL055W, YJL072C, YJL099W, YKL132C,
YKR057W, YLL013C, YLR082C, YLR089C, YLR224W, YLR255C, YLR375W,
YOR024W, YOR044W, YOR084W, YOR245C, YOR317W, YOR344C, YOR350C,
YPL099C, YPL268W, YPR024W, YPR138C and YPR172W, or a fragment
thereof, which confers an increase or a decrease in the amount of
the respective fine chemical as shown in table X in an organism or
a part thereof; b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table I, columns 5 or 7, and corresponding to the
polypeptide as defined in a) and named in table X; c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase or a
decrease in the amount of the respective fine chemical as shown in
table X in an organism or a part thereof; d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase or decrease in
the amount of the respective fine chemical as shown in table X in
an organism or a part thereof; e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase or a
decrease in the amount of the respective fine chemical as shown in
table X in an organism or a part thereof; f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c) and conferring an
increase or decrease in the amount of the respective fine chemical
as shown in table X in an organism or a part thereof; g) nucleic
acid molecule encoding a fragment or an epitope of a polypeptide
which is encoded by one of the nucleic acid molecules of (a) to
(e), preferably to (a) to (c) and conferring an increase or
decrease in the amount of the respective fine chemical as shown in
table X in an organism or a part thereof; h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers pairs having a sequence as indicated in Table
III, columns 7, and corresponding to a polypeptide as defined in a)
and named in table X, and conferring an increase or decrease in the
amount of the respective fine chemical as shown in table X in an
organism or a part thereof; i) nucleic acid molecule encoding a
polypeptide which is isolated, e.g. from an expression library,
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (h), preferably to
(a) to (c), and conferring an increase or decrease in the amount of
the respective fine chemical as shown in table X in an organism or
a part thereof; j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence having a sequences as
indicated in Table IV, columns 7, and corresponding to a
polypeptide as defined in a) and named in table X, and conferring
an increase or decrease in the amount of the respective fine
chemical as shown in table X in an organism or a part thereof; k)
nucleic acid molecule comprising one or more of the nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domain of a polypeptide indicated in Table II, columns 5 or 7,
and as defined in a) and named in table X, and conferring an
increase or decrease in the amount of the respective fine chemical
as shown in table X in an organism or a part thereof; and l)
nucleic acid molecule which is obtainable by screening a suitable
library under stringent conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase or decrease in the amount of
the respective fine chemical as shown in table X in an organism or
a part thereof; or which comprises a sequence which is
complementary thereto.
[11796] [0043.0.0.26] Accordingly, in one embodiment, the process
according to the invention comprises the following steps:
(a) introducing of a nucleic acid construct comprising the nucleic
acid molecule of the invention or used in the process of the
invention or encoding the polypeptide of the present invention or
used in the process of the invention; or (b) introducing of a
nucleic acid molecule, including regulatory sequences or factors,
which expression increases the expression of the nucleic acid
molecule of the invention or used in the process of the invention
or encoding the polypeptide of the present invention or used in the
process of the invention; in a cell, or an organism or a part
thereof, preferably in a plant, plant cell or a microorganism, and
(c) expressing of the gene product encoded by the nucleic acid
construct or the nucleic acid molecule mentioned under (a) or (b)
in the cell or the organism.
[11797] [0043.1.0.26] In a further embodiment the present invention
relates to a method for
[11798] the generation of a host or host cell, e.g. transgenic,
showing a metabolic profile as depicted in any one of the columns
of table X.
[11799] In a further embodiment the present invention relates to a
method for the generation of a transgenic host or host cell showing
a metabolic profile as depicted in any one of the columns of table
X and expressing an nucleic acid or a polypeptide of the invention
or use in the method of the invention.
[11800] In a further embodiment the present invention relates to a
method for the generation of a transgenic host or host cell showing
a metabolic profile 30% similar to a profile as depicted in any one
of the columns of table X and expressing the protein displayed in
the respective column of table X or a homolog thereof.
[11801] In a further embodiment the present invention relates to a
method for the generation of a transgenic host or host cell showing
a metabolic profile 50%, more preferred, 60%, even more preferred
70%, even more preferred 80% or even more preferred 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% similar to a profile as depicted
in any one of the columns of table X and expressing the protein
displayed in the respective column of table X or a homolog thereof.
% similarity is the above described context of metabolic profiles
is to be understood, that this percentage of metabolic changes
occur also in the transgenic host or host cell in the same
direction. For example a 80% similar metabolic profile displays 8
of 10 metabolic changes in the same direction as depicted in any
column of table X.
[11802] [0043.2.0.26] In a further embodiment the present invention
relates to the modification of the metabolic profile in plant seed,
resulting in grains with increased digestibility/nutrient
availability, with improved amino acid, fatty acids, glycerides,
lipids, vitamins, carotenoids, phytosterols, organic compounds,
preferably organic acids as disclosed in [0002.0.16.16],
[0002.0.17.17], [0002.0.19.19], [0002.0.20.20] and/or
[0002.0.23.23], glycerol and derivates, coenzymes, galactolipids
and/or carbohydrates, saccharides and/or sugars composition and
increased nutrient value, increased response to feed processing,
improved silage quality, increased efficiency of wet or dry
milling, and decreased allergenicity and/or toxicity.
[11803] [0044.0.0.26] Further embodiments of the present invention
are disclosed in the paragraphs [0014.l.m.n] to [0553.l.m.n],
whereby l, m and n is one or more numbers between zero to
twenty-five, as disclosed afore.
[11804] [0045.0.0.26] The process of the present invention may be
used for improving the quality of foodstuffs and animal feeds,
which is an important task of the food-and-feed industry.
Especially advantageous for the quality of foodstuffs and animal
feeds is as balanced as possible an metabolic profile since a great
excess of one fine chemical above a specific concentration in the
food has no further positive effect on the utilization of the food
since another fine chemical suddenly become limiting.
[11805] The manufacturing of high quality foodstuffs and animal
feeds is possible by the process of the present invention, which
controls the production of each fine chemical as depicted in table
X.
[11806] [0046.0.0.26] The process of the present invention stand
for an inexpensive process for the synthesis of a combination of
fine chemicals such as amino acids, fatty acids, carbohydrates,
vitamins, organic acids, carotenoids, preferably as mentioned in
table X, at the same time in an sufficient amount to provide
optimal growth and health benefit to animals or humans.
[11807] [0047.0.0.26] The products manufactered by the process of
the present invention can be used or can be incorporated without
great and expensive efforts as high quality food, nutraceuticals,
feed or additives there for. The fine chemical or the combination
of fine chemicals obtained in the process is suitable as starting
material for the synthesis of further products of value. For
example, they can be used in combination with other ingredients or
alone for the production of nutraceuticals, pharmaceuticals,
foodstuffs, animal feeds or cosmetics.
[11808] Accordingly, the present invention relates a method for the
production of a nutraceutical, pharmaceuticals, food stuff, animal
feeds, nutrients or cosmetics comprising the steps of the process
according to the invention, including the isolation of the fine
chemical or fine chemical composition produced and if desired
formulating the product with a pharmaceutical acceptable carrier or
formulating the product in a form acceptable for an application in
agriculture. A further embodiment according to the invention is the
use of the fine chemical produced in the process or of the
transgenic organisms in animal feeds, foodstuffs, medicines, food
supplements, cosmetics or pharmaceuticals.
[11809] [0048.0.0.26] In one embodiment the process of the
invention is used for the production of feed or functional feed by
overexpressing in a host cell the nucleic acid sequence coding for
the b0124, b0161, b0464, b0486, b1343, b1708, b1829, b2414, b2664,
b3074, b3966, b3983, YER173W, YGR104C, YIL150C and/or YKR057W
(and/or YPR172W-protein), or homologues thereof and isolating the
fine chemical or the combination of fine chemicals, preferably the
combination of amino acid or using the host cell as feed without
isolation of certain fine chemicals.
[11810] In a further embodiment the process of the invention is
used for the production of feed or functional feed by
overexpressing in a host cell the nucleic acid sequence coding for
the b0124, b0161, b0464, b0486, b1343, b1708, b1829, b2414, b2664,
b3074, b3966, b3983, YER173W, YGR104C, YIL150C and/or YKR057W or
homolgous thereof and using the host cell as feed containing an
enhanced amino acid profile. Enhanced amino acid profile means that
one or more amino acid, which in plants are limited with regard to
the supply of mammals, are increased in there amount. More
preferably, the nucleic acid sequence coding for the b0161 and/or
YER173W-protein, or homologues thereof are used, hence they confer
an increase in the important amino acids and in fatty acids,
organic acids, vitamins, phytosterols and saccharides.
[11811] [0049.0.0.26] In one embodiment the process of the
invention is used for the production of feed or functional feed by
overexpressing in a host cell the nucleic acid sequence coding for
the b0050, b0251, b0577, b0849, b2699, b2822, b3256, b3457, b3644,
b4129, YBR089C-A, YDR447C, YDR513W, YIL150C and/or YOR024W protein,
or homologues thereof and isolating the fine chemical or the
combination of fine chemicals, preferably the combination of
essential fatty acids or using the host cell as feed without
isolation of certain fine chemicals.
[11812] In a further embodiment the process of the invention is
used for the production of increased amounts of oil or and fat by
overexpressing in a host cell the nucleic acid sequence coding for
the b0050, b0251, b0577, b0849, b2699, b2822, b3256, b3457, b3644,
b4129, YBR089C-A, YDR447C, YDR513W, YIL150C and/or YOR024W protein,
or homologues thereof and optionally isolating the produced oil or
fat.
[11813] In a further embodiment the process of the invention is
used for the production of specific fatty acid compositions in oil
or fat by overexpressing in a host cell the nucleic acid sequence
coding for the b0050, b0251, b0577, b0849, b2699, b2822, b3256,
b3457, b3644, b4129, YBR089C-A, YDR447C, YDR513W, YIL150C and/or
YOR024W protein, or homologues thereof and optionally isolating the
produced oil or fat.
[11814] In a further embodiment the process of the invention is
used for the production of increased amounts LC-PUFAs (e.g.
arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid) by
overexpressing in a host cell the nucleic acid sequence coding for
the b0050, b0251, b0577, b0849, b2699, b2822, b3256, b3457, b3644,
b4129, YBR089C-A, YDR447C, YDR513W, YIL150C and/or YOR024W protein,
or homologues thereof and additionally genes for LC-PUFA
biosynthesis als for example disclosed in WO 2005/083093 or WO
2005/08305 and optionally isolating the produced LC-PUFAs.
[11815] More preferably, the nucleic acid sequence coding for the
b0050, YDR513W and/or b2699-protein, or homologues thereof are
used, hence they confer an increase in the important amino acids
and in fatty acids, organic acids, vitamins, phytosterols and
saccharides.
[11816] [0050.0.0.26] In one embodiment the process of the
invention is used for the production of preparations comprising
phospholipids and/or polar lipids enriched with fatty acids,
preferably PUFAs, which may be bounded to the lipid backbone. These
preparations are particularly useful as nutraceuticals, food
additives and/or pharmaceutical agents for the treatment of various
conditions, in particular related to cognitive functions.
[11817] Preferably, the nucleic acid sequence coding for the b3457,
b6344, YDR513W and/or YIL150C-protein, or homologues thereof are
used, hence they confer an increase in fatty acids and glycerol, as
backbone of lipids.
[11818] [0051.0.0.26] In one embodiment the process of the
invention is used for the production of preparations comprising a
decreased level of raffinose and increased level of other,
beneficial fine chemicals. Raffinose saccharides are an obstacle to
the efficient utilization of some economically important crop
species. Raffinose saccharides are not digested directly by
animals, primarily because alpha-galactosidase is not present in
the intestinal mucosa.
[11819] Preferably, the nucleic acid sequence coding for the b0880,
b3936 and/or YOR245C-protein, or homologues thereof are used, hence
they confer an decrease in raffinose and an increase in fatty acids
and amino acids respectively.
[11820] [0052.0.0.26] In one embodiment the process of the
invention is used for the production of preparations comprising a
increased level of anti-oxidants, like 2,3-Dimethyl-5-phytylquinol,
Ferulic acid, and/or Coenzyme Q10 in combination with increased
level of fatty acids and preferably other, beneficial fine
chemicals. Its stability of the oils containing the fatty acids is
increased due to resistance to oxidation, which helps to prevent
rancidity of fats.
[11821] Preferably, the nucleic acid sequence coding for the b1829,
b0730, b1829, b2699 and/or YPR138C-protein, or homologues thereof
are used, hence they confer an increase in anti-oxidants and an
increase in fatty acids and amino acids respectively.
[11822] [0053.0.0.26] In one embodiment the process of the
invention is used for the production of preparations comprising an
decreased level of vitamin A or precursors there of, like
2,3-Dimethyl-5-phytylquinol and/or alpha, beta and/or gamma
Tocopherol and in combination with increased level of fatty acids
and preferably other, beneficial fine chemicals.
[11823] Fortification of foods with relatively high levels of a
small number of fine chemicals may cause an imbalance in dietary
intakes and the addition of one nutrient may lead to disturbances
in the utilisation of others.
[11824] For example, high levels of vitamin A during pregnancy may
increase the risk of birth defects. Excessive intakes of vitamin A
from overuse of supplements are toxic and can cause damage to the
liver and cell membranes.
[11825] Preferably, the nucleic acid sequence coding for
YIL150C-protein, or homologues thereof are used, hence they confer
a decrease in vitamin A or precursors and an increase in fatty
acids and amino acids respectively.
[11826] [0054.0.0.26] Generally, the preparations produced
according to the present invention can contribute as additives or
compounds in the treatment of health disorders like high blood
cholesterol levels, high triglycerides levels, high blood
fibrinogen levels, HDL/LDL ratio, diabetes, metabolic syndrome,
menopausal or post-menopausal conditions, hormone related
disorders, vision disorders, inflammatory disorders, immune
disorders, liver diseases, chronic hepatitis, steatosis,
phospholipid deficiency, lipid peroxidation, dysrhythmia of cell
regeneration, destabilization of cell membranes, coronary artery
disease, high blood pressure, cancer, hypertension, aging, kidney
disease, skin diseases, edema, gastrointestinal diseases,
peripheral vascular system diseases, allergies, neurodegenerative
and psychiatric diseases.
[11827] [0055.0.0.26] The nutraceuticals can be formulated for
administration by inhalation or insufflation (either through the
mouth or the nose) or oral, buccal, parenteral or rectal
administration. For oral administration, the nutraceuticals can be
added directly to foods so that the nutraceuticals are ingested
during normal meals. Any methods known to those skilled in the art
may be used to add or incorporate nutraceuticals to natural or
processed foods. Generally, the preparations produced according to
the present invention may be introduced in dairy products like
biscuits, soy products, bakery, pastry and bread, sauces, soups,
prepared foods, frozen foods, condiments, confectionary, oils and
fats, margarines, spreads, fillings, salad dressings, cereals,
instant products, teas, drinks and shakes, infant formulas, infant
foods (biscuits, mashed vegetables and fruits, cereals), bars,
extruded and/or puffed snack foods, candies, ice-creams, chocolate
products, and/or products containing corn sweeteners, cereals,
chips, puddings, candies, and breads.
[11828] [0056.0.0.26] Generally, the preparations produced
according to the present invention may be introduced in
cosmeticals.
[11829] The process of the present invention is further used for
the production of cosmeceuticals, a term which refers to personal
care products that contain substances that exert beneficial effects
such as anti-wrinkle, antioxidant, skin conditioning, analgesia,
sun protection, stimulation of hair growth. Furthermore, they may
also impart a desirable physiological effect such as stimulation of
microcirculation. Anti-ageing is a key target area, this includes
antioxidants and sun protection, which also help to prevent
diseases such as skin cancer.
[11830] [0057.0.0.26] According to the present invention, the term
"metabolic profile" encompasses and implies also a decrease of one
or more fine chemicals as disclosed in each line of table IX.
[11831] [0058.0.0.26]
[11832] The sequence of b0021 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as an insertion element IS1
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Escherichia coli insB protein superfamily, preferably a protein
with an insertion element IS1 protein activity or its homolog, e.g.
as shown herein, from Escherichia coli K12 or its homolog, e.g. as
shown herein, for a decrease of the fine chemical, meaning of
leucine and/or citramalate in a range as indicated in Table IX, in
free or bound form in an organism or a part thereof, as
mentioned.
[11833] The sequence of b0043 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a probable quinone reductase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Escherichia coli fixC protein superfamily, preferably a protein
with a probable quinone reductase activity or its homolog, e.g. as
shown herein, from Escherichia coli K12 or its homolog, e.g. as
shown herein, for a decrease of the fine chemical, meaning of
tryptophane and/or raffinose in a range as indicated in Table IX,
in free or bound form in an organism or a part thereof, as
mentioned.
[11834] The sequence of b0134 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a 3-methyl-2-oxobutanoate
hydroxymethyltransferase. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of the Escherichia coli
3-methyl-2-oxobutanoate hydroxymethyltransferase superfamily,
preferably a protein with a 3-methyl-2-oxobutanoate
hydroxymethyltransferase activity or its homolog, e.g. as shown
herein, from Escherichia coli K12 or its homolog, e.g. as shown
herein, for a decrease of the fine chemical, meaning of sinapic
acid in a range as indicated in Table IX, in free or bound form in
an organism or a part thereof, as mentioned.
[11835] The sequence of b0186 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a lysine decarboxylase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Escherichia coli probable ornithine/lysine/arginine
decarboxylase superfamily, preferably a protein with a lysine
decarboxylase activity or its homolog, e.g. as shown herein, from
Escherichia coli K12 or its homolog, e.g. as shown herein, for a
decrease of the fine chemical, meaning of tryptophane, fumarate
and/or glyceric acid in a range as indicated in Table IX, in free
or bound form in an organism or a part thereof, as mentioned.
[11836] The sequence of b0328 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative cytochrome subunit
of dehydrogenase; putative transport protein. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the Escherichia coli
hypothetical protein b1798 superfamily, preferably a protein with a
putative cytochrome subunit of dehydrogenase; putative transport
protein activity or its homolog, e.g. as shown herein, from
Escherichia coli K12 or its homolog, e.g. as shown herein, for a
decrease of the fine chemical, meaning of
2,3-dimethyl-5-phytylquinol, beta-tocopherol, gamma-tocopherol,
glucose and/or coenzyme Q10 in a range as indicated in Table IX, in
free or bound form in an organism or a part thereof, as
mentioned.
[11837] The sequence of b0677 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a N-acetylgl
ucosamine-6-phosphate deacetylase. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of a N-acetylglucosamine-6-phosphate
deacetylase from E. coli K12 or its homolog, e.g. as shown herein,
for a decrease of the fine chemical, meaning of citrulline and/or
glycine in a range as indicated in Table IX, in free or bound form
in an organism or a part thereof, as mentioned.
[11838] The sequence of b0734 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a cytochrome D terminal
oxidase, polypeptide subunit II. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of the Escherichia coli cytochrome d
ubiquinol oxidase superfamily, preferably a protein with a
cytochrome D terminal oxidase, polypeptide subunit II activity or
its homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for a decrease of the fine chemical,
meaning of .beta.-carotene in a range as indicated in Table IX, in
free or bound form in an organism or a part thereof, as
mentioned.
[11839] The sequence of b0763 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a molybdate transport protein
(ABC superfamily, peri_bind). Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of the Escherichia coli molybdate-binding
periplasmic protein superfamily, preferably a protein with a
molybdate transport protein (ABC superfamily, peri_bind) activity
or its homolog, e.g. as shown herein, from Escherichia coli K12 or
its homolog, e.g. as shown herein, for a decrease of the fine
chemical, meaning of zeaxanthin in a range as indicated in Table
IX, in free or bound form in an organism or a part thereof, as
mentioned.
[11840] The sequence of b0895 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as an anaerobic dimethyl
sulfoxide (dMS0) reductase, subunit B. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the Escherichia coli nrfC
protein superfamily, preferably a protein with an anaerobic
dimethyl sulfoxide (dMS0) reductase, subunit B activity or its
homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for a decrease of the fine chemical,
meaning of shikimic acid and/or threonic acid
(2,3,4-trihydroxybutyric acid) in a range as indicated in Table IX,
in free or bound form in an organism or a part thereof, as
mentioned.
[11841] The sequence of b1054 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a lauroyl transferase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
lauroyl transferase from E. coli K12 or its homolog, e.g. as shown
herein, for a decrease of the fine chemical, meaning of threonic
acid (2,3,4-trihydroxybutyric acid) in a range as indicated in
Table IX, in free or bound form in an organism or a part thereof,
as mentioned.
[11842] The sequence of b1183 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a mutagenesis and repair
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Escherichia coli lexA repressor superfamily, preferably a
protein with a mutagenesis and repair protein activity or its
homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for a decrease of the fine chemical,
meaning of glucose in a range as indicated in Table IX, in free or
bound form in an organism or a part thereof, as mentioned.
[11843] The sequence of b1217 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a cation transport regulator.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
cation transport regulator from E. coli K12 or its homolog, e.g. as
shown herein, for a decrease of the fine chemical, meaning of
hexadecatrienoic acid (C16:cis[7,10,13]3) in a range as indicated
in Table IX, in free or bound form in an organism or a part
thereof, as mentioned.
[11844] The sequence of b1249 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a cardiolipin synthase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Escherichia coli probable cardiolipin synthetase superfamily,
preferably a protein with a cardiolipin synthase activity or its
homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for a decrease of the fine chemical,
meaning of isopentenyl pyrophosphate in a range as indicated in
Table IX, in free or bound form in an organism or a part thereof,
as mentioned.
[11845] The sequence of b1292 from Escherichia coli K12 has been
published in Blattner et al.,
[11846] Science 277(5331), 1453-1474, 1997, and its activity is
being defined as a peptide transport system permease protein sapC.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Escherichia coli oligopeptide permease protein oppB
superfamily, preferably a protein with a peptide transport system
permease protein sapC activity or its homolog, e.g. as shown
herein, from Escherichia coli K12 or its homolog, e.g. as shown
herein, for a decrease of the fine chemical, meaning of
2,3-dimethyl-5-phytylquinol, beta-tocopherol and/or
gamma-tocopherol in a range as indicated in Table IX, in free or
bound form in an organism or a part thereof, as mentioned.
[11847] The sequence of b1874 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a copper homeostasis protein.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the copper homeostasis protein from E. coli K12 or its homolog,
e.g. as shown herein, for a decrease of the fine chemical, meaning
of threonine, shikimic acid, palmitic acid (C16:0), linoleic acid
(C18:cis[9,12]2), myo-inositol, sinapic acid, fumarate and/or
succinate in a range as indicated in Table IX, in free or bound
form in an organism or a part thereof, as mentioned.
[11848] The sequence of b2110 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative periplasmic pilin
chaperone similar to Papd. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of the Escherichia coli chaperone protein
papD superfamily, preferably a protein with a putative periplasmic
pilin chaperone similar to Papd activity or its homolog, e.g. as
shown herein, from Escherichia coli K12 or its homolog, e.g. as
shown herein, for a decrease of the fine chemical, meaning of
shikimic acid, raffinose, sucrose, isopentenyl pyrophosphate,
fumarate and/or glyceric acid in a range as indicated in Table IX,
in free or bound form in an organism or a part thereof, as
mentioned.
[11849] The sequence of b2696 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a carbon storage regulator,
post-translational activator of flhdC expression, regulates biofilm
formation, RNA-binding. Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of the Escherichia coli glycogen biosynthesis inhibitor
superfamily, preferably a protein with a carbon storage regulator,
post-translational activator of flhdC expression, regulates biofilm
formation, RNA-binding activity or its homolog, e.g. as shown
herein, from Escherichia coli K12 or its homolog, e.g. as shown
herein, for a decrease of the fine chemical, meaning of sucrose in
a range as indicated in Table IX, in free or bound form in an
organism or a part thereof, as mentioned.
[11850] The sequence of b2901 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a 6-phospho-beta-glucosidase
A. Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Escherichia coli beta-glucosidase superfamily, preferably a
protein with a 6-phospho-beta-glucosidase
[11851] A activity or its homolog, e.g. as shown herein, from
Escherichia coli K12 or its homolog, e.g. as shown herein, for a
decrease of the fine chemical, meaning of proline in a range as
indicated in Table IX, in free or bound form in an organism or a
part thereof, as mentioned.
[11852] The sequence of b3025 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a response regulator in
two-component regulatory system with QseC, regulates flagella and
motility by quorum sensing (OmpR family). Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the Escherichia coli ompR
protein superfamily, preferably a protein with a response regulator
in two-component regulatory system with QseC, regulates flagella
and motility by quorum sensing (OmpR family) activity or its
homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for a decrease of the fine chemical,
meaning of proline in a range as indicated in Table IX, in free or
bound form in an organism or a part thereof, as mentioned.
[11853] The sequence of b3091 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a altronate hydrolase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Escherichia coli Escherichia altronate dehydratase superfamily,
preferably a protein with a altronate hydrolase activity or its
homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for a decrease of the fine chemical,
meaning of tryptophane, glycerol-3-phosphate (polar fraction)
and/or succinate in a range as indicated in Table IX, in free or
bound form in an organism or a part thereof, as mentioned.
[11854] The sequence of b3335 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a bifunctional prepilin
peptidase: leader peptidase; methyl transferase (General Secretory
Pathway). Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the Escherichia coli type IV prepilin peptidase
superfamily, preferably a protein with a bifunctional prepilin
peptidase: leader peptidase; methyl transferase (General Secretory
Pathway) activity or its homolog, e.g. as shown herein, from
Escherichia coli K12 or its homolog, e.g. as shown herein, for a
decrease of the fine chemical, meaning of hexadecatrienoic acid
(C16:cis[7,10,13]3) putative and/or methylgalactopyranosid in a
range as indicated in Table IX, in free or bound form in an
organism or a part thereof, as mentioned.
[11855] The sequence of b3709 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a low-affinity tryptophan
permease (HAAAP family). Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of the Escherichia coli tyrosine-specific
transport protein superfamily, preferably a protein with a
low-affinity tryptophan permease (HAAAP family) activity or its
homolog, e.g. as shown herein, from Escherichia coli K12 or its
homolog, e.g. as shown herein, for a decrease of the fine chemical,
meaning of tryptophane, tyrosine, citramalate and/or threonic acid
(2,3,4-trihydroxybutyric acid) in a range as indicated in Table IX,
in free or bound form in an organism or a part thereof, as
mentioned.
[11856] The sequence of b3825 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a lysophospholipase L(2).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
lysophospholipase L(2) from E. coli K12 or its homolog, e.g. as
shown herein, for a decrease of the fine chemical, meaning of
threonic acid (2,3,4-trihydroxybutyric acid) in a range as
indicated in Table IX, in free or bound form in an organism or a
part thereof, as mentioned.
[11857] The sequence of b3924 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a ferredoxin-NADP reductase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
ferredoxin-NADP reductasefrom E. coli K12 or its homolog, e.g. as
shown herein, for a decrease of the fine chemical, meaning of
alpha-tocopherol in a range as indicated in Table IX, in free or
bound form in an organism or a part thereof, as mentioned.
[11858] The sequence of b4101 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a putative C--P
(carbon-phosphorous) lyase component. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of the Escherichia coli phnG
protein superfamily, preferably a protein with a putative C--P
(carbon-phosphorous) lyase component activity or its homolog, e.g.
as shown herein, from Escherichia coli K12 or its homolog, e.g. as
shown herein, for a decrease of the fine chemical, meaning of
tyrosine and/or malate in a range as indicated in Table IX, in free
or bound form in an organism or a part thereof, as mentioned.
[11859] The sequence of b4113 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a response regulator in
two-component regulatory system with BasS (OmpR family).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the Escherichia coli ompR protein superfamily, preferably a protein
with a response regulator in two-component regulatory system with
BasS (OmpR family) activity or its homolog, e.g. as shown herein,
from Escherichia coli K12 or its homolog, e.g. as shown herein, for
a decrease of the fine chemical, meaning of hexadecatrienoic acid
(C16:cis[7,10,13]3) putative and/or methylgalactopyranosid in a
range as indicated in Table IX, in free or bound form in an
organism or a part thereof, as mentioned.
[11860] The sequence of b4242 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a P-type ATPase, Mg2+ATPase
transport protein. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of the Escherichia coli Na+/K+-transporting ATPase alpha
chain, ATPase nucleotide-binding domain homology superfamily,
preferably a protein with a P-type ATPase, Mg2+ATPase transport
protein activity or its homolog, e.g. as shown herein, from
Escherichia coli K12 or its homolog, e.g. as shown herein, for a
decrease of the fine chemical, meaning of threonine, shikimic acid,
palmitic acid (C16:0), linoleic acid (C18:cis[9,12]2), linolenic
acid (C18:cis[9,12,15]3), hexadecadienoic acid (C16:cis[7,10]2),
hexadecatrienoic acid (C16:cis[7,10,13]3), glycerol (polar
fraction), raffinose and/or methylgalactopyranosid in a range as
indicated in Table IX, in free or bound form in an organism or a
part thereof, as mentioned.
[11861] The sequence of b4359 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a phosphoglycerol transferase
I. Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
phosphoglycerol transferase I from E. coli K12 or its homolog, e.g.
as shown herein, for a decrease of the fine chemical, meaning of
threonic acid (2,3,4-trihydroxybutyric acid) in a range as
indicated in Table IX, in free or bound form in an organism or a
part thereof, as mentioned.
[11862] The sequence of YGL005C from Saccharomyces cerevisiae has
been published in Goffeau, Science 274 (5287), 546-547, 1996, and
its activity has been defined as a Component of the conserved
oligomeric Golgi complex. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with an activity of a Component of the conserved oligomeric
Golgi complex from Saccaromyces cerevisiae or its homolog, e.g. as
shown herein, for a decrease of the fine chemical, meaning of
zeaxanthin in a range as indicated in Table IX, in free or bound
form in an organism or a part thereof, as mentioned.
[11863] The sequence of YML005W from Saccharomyces cerevisiae has
been published in Goffeau, Science 274 (5287), 546-547, 1996, and
its activity has been defined as a putative
S-adenosylmethionine-dependent methyltransferase of the seven
beta-strand family. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of a putative S-adenosylmethionine-dependent
methyltransferase of the seven beta-strand family from Saccaromyces
cerevisiae or its homolog, e.g. as shown herein, for a decrease of
the fine chemical, meaning of 2,3-dimethyl-5-phytylquinol,
beta-tocopherol and/or gamma-tocopherol in a range as indicated in
Table IX, in free or bound form in an organism or a part thereof,
as mentioned. [0059.0.0.26]
[11864] In case the activity of the Escherichia coli K12 protein
b0021 or its homologs, as indicated in Table V, columns 5 or 7,
lines 1 to 2, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of leucine and/or
citramalate is conferred.
[11865] In case the activity of the Escherichia coli K12 protein
b0043 or its homologs, as indicated in Table V, columns 5 or 7,
lines 3 to 4, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of tryptophane and/or
raffinose is conferred.
[11866] In case the activity of the Escherichia coli K12 protein
b0134 or its homologs, as indicated in Table V, columns 5 or 7,
line 5, e.g. protein with an activity as defined in [0058.0.0.26],
is increased, preferably, in one embodiment the decrease of the
fine chemical, preferably of sinapic acid is conferred.
[11867] In case the activity of the Escherichia coli K12 protein
b0186 or its homologs, as indicated in Table V, columns 5 or 7,
lines 6 to 8, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of tryptophane, fumarate
and/or glyceric acid is conferred.
[11868] In case the activity of the Escherichia coli K12 protein
b0328 or its homologs, as indicated in Table V, columns 5 or 7,
lines 9 to 13, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of
2,3-dimethyl-5-phytylquinol, beta-tocopherol, gamma-tocopherol,
glucose and/or coenzyme Q10 is conferred.
[11869] In case the activity of the Escherichia coli K12 protein
b0677 or its homologs, as indicated in Table V, columns 5 or 7,
lines 14 to 15, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of citrulline and/or
glycine is conferred.
[11870] In case the activity of the Escherichia coli K12 protein
b0734 or its homologs, as indicated in Table V, columns 5 or 7,
line 16, e.g. protein with an activity as defined in [0058.0.0.26],
is increased, preferably, in one embodiment the decrease of the
fine chemical, preferably of .beta.-carotene is conferred.
[11871] In case the activity of the Escherichia coli K12 protein
b0763 or its homologs, as indicated in Table V, columns 5 or 7,
line 17, e.g. protein with an activity as defined in [0058.0.0.26],
is increased, preferably, in one embodiment the decrease of the
fine chemical, preferably of zeaxanthin is conferred.
[11872] In case the activity of the Escherichia coli K12 protein
b0895 or its homologs, as indicated in Table V, columns 5 or 7,
lines 18 to 19, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of shikimic acid and/or
threonic acid (2,3,4-trihydroxybutyric acid) is conferred.
[11873] In case the activity of the Escherichia coli K12 protein
b1054 or its homologs, as indicated in Table V, columns 5 or 7,
line 20, e.g. protein with an activity as defined in [0058.0.0.26],
is increased, preferably, in one embodiment the decrease of the
fine chemical, preferably of threonic acid (2,3,4-trihydroxybutyric
acid) is conferred.
[11874] In case the activity of the Escherichia coli K12 protein
b1183 or its homologs, as indicated in Table V, columns 5 or 7,
line 21, e.g. protein with an activity as defined in [0058.0.0.26],
is increased, preferably, in one embodiment the decrease of the
fine chemical, preferably of glucose is conferred.
[11875] In case the activity of the Escherichia coli K12 protein
1217 or its homologs, as indicated in Table V, columns 5 or 7, line
22, e.g. protein with an activity as defined in [0058.0.0.26], is
increased, preferably, in one embodiment the decrease of the fine
chemical, preferably of hexadecatrienoic acid (C16:cis[7,10,13]3)
putative is conferred.
[11876] In case the activity of the Escherichia coli K12 protein
b1249 or its homologs, as indicated in Table V, columns 5 or 7,
line 23, e.g. protein with an activity as defined in [0058.0.0.26],
is increased, preferably, in one embodiment the decrease of the
fine chemical, preferably of isopentenyl pyrophosphate is
conferred.
[11877] In case the activity of the Escherichia coli K12 protein
b1292 or its homologs, as indicated in Table V, columns 5 or 7,
lines 24 to 26, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of
2,3-dimethyl-5-phytylquinol, beta-tocopherol and/or
gamma-tocopherol is conferred.
[11878] In case the activity of the Escherichia coli K12 protein
b1874 or its homologs, as indicated in Table V, columns 5 or 7,
lines 27 to 34, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of threonine, shikimic
acid, palmitic acid (C16:0), linoleic acid (C18:cis[9,12]2),
myo-inositol, sinapic acid, fumarate and/or succinate is
conferred.
[11879] In case the activity of the Escherichia coli K12 protein
b2110 or its homologs, as indicated in Table V, columns 5 or 7,
lines 35 to 40, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of shikimic acid,
raffinose, sucrose, isopentenyl pyrophosphate, fumarate and/or
glyceric acid is conferred.
[11880] In case the activity of the Escherichia coli K12 protein
b2696 or its homologs, as indicated in Table V, columns 5 or 7,
line 41, e.g. protein with an activity as defined in [0058.0.0.26],
is increased, preferably, in one embodiment the decrease of the
fine chemical, preferably of sucrose is conferred.
[11881] In case the activity of the Escherichia coli K12 protein
b2901 or its homologs, as indicated in Table V, columns 5 or 7,
line 42, e.g. protein with an activity as defined in [0058.0.0.26],
is increased, preferably, in one embodiment the decrease of the
fine chemical, preferably of proline is conferred.
[11882] In case the activity of the Escherichia coli K12 protein
b3025 or its homologs, as indicated in Table V, columns 5 or 7,
line 43, e.g. protein with an activity as defined in [0058.0.0.26],
is increased, preferably, in one embodiment the decrease of the
fine chemical, preferably of proline is conferred.
[11883] In case the activity of the Escherichia coli K12 protein
b3091 or its homologs, as indicated in Table V, columns 5 or 7,
lines 44 to 46, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of tryptophane,
glycerol-3-phosphate (polar fraction) and/or succinate is
conferred.
[11884] In case the activity of the Escherichia coli K12 protein
b3335 or its homologs, as indicated in Table V, columns 5 or 7,
lines 47 to 48, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of hexadecatrienoic acid
(C16:cis[7,10,13]3) and/or methylgalactopyranosid is conferred.
[11885] In case the activity of the Escherichia coli K12 protein
b3709 or its homologs, as indicated in Table V, columns 5 or 7,
lines 49 to 52, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of tryptophane, tyrosine,
citramalate and/or threonic acid (2,3,4-trihydroxybutyric acid) is
conferred.
[11886] In case the activity of the Escherichia coli K12 protein
3825 or its homologs, as indicated in Table V, columns 5 or 7, line
53, e.g. protein with an activity as defined in [0058.0.0.26], is
increased, preferably, in one embodiment the decrease of the fine
chemical, preferably of threonic acid (2,3,4-trihydroxybutyric
acid) is conferred.
[11887] In case the activity of the Escherichia coli K12 protein
b3924 or its homologs, as indicated in Table V, columns 5 or 7,
line 54, e.g. protein with an activity as defined in [0058.0.0.26],
is increased, preferably, in one embodiment the decrease of the
fine chemical, preferably of alpha-tocopherol is conferred.
[11888] In case the activity of the Escherichia coli K12 protein
b4101 or its homologs, as indicated in Table V, columns 5 or 7,
lines 55 to 56, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of tyrosine and/or malate
is conferred.
[11889] In case the activity of the Escherichia coli K12 protein
b4113 or its homologs, as indicated in Table V, columns 5 or 7,
lines 57 to 58, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of hexadecatrienoic acid
(C16:cis[7,10,13]3) and/or methylgalactopyranosid is conferred.
[11890] In case the activity of the Escherichia coli K12 protein
b4242 or its homologs, as indicated in Table V, columns 5 or 7,
lines 59 to 68, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of threonine, shikimic
acid, palmitic acid (C16:0), linoleic acid (C18:cis[9,12]2),
linolenic acid (C18:cis[9,12,15]3), hexadecadienoic acid
(C16:cis[7,10]2) putative, hexadecatrienoic acid
(C16:cis[7,10,13]3, glycerol (polar fraction), raffinose and/or
methylgalactopyranosid is conferred.
[11891] In case the activity of the Escherichia coli K12 protein
b4359 or its homologs, as indicated in Table V, columns 5 or 7,
line 69, e.g. protein with an activity as defined in [0058.0.0.26],
is increased, preferably, in one embodiment the decrease of the
fine chemical, preferably of threonic acid (2,3,4-trihydroxybutyric
acid) is conferred.
[11892] In case the activity of the Saccharomyces cerevisiae
protein YGL005C or its homologs, as indicated in Table V, columns 5
or 7, line 70, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of zeaxanthin is
conferred.
[11893] In case the activity of the Saccharomyces cerevisiae
protein YML005W or its homologs, as indicated in Table V, columns 5
or 7, lines 71 to 73, e.g. protein with an activity as defined in
[0058.0.0.26], is increased, preferably, in one embodiment the
decrease of the fine chemical, preferably of
2,3-dimethyl-5-phytylquinol, beta-tocopherol and/or
gamma-tocopherol is conferred.
[11894] [0060.0.0.26] In one embodiment the process of the
invention is used for the production of stable oil, for example for
deep fat frying, with a decreased content of linoleic acid and
alpha-linolic acid, by overexpressing in a host cell the nucleic
acid sequence coding for the b0424 protein, or homologues
thereof.
[11895] [0061.0.0.26] In one embodiment the present invention is
directed to a method of diagnosis of the level of expression of a
nucleic acid molecule of claim 3 a) or claim 6 in a by measuring in
a first step the metabolic content, preferably the concentration of
one or more fine chemicals as depicted in table IX and/or X, in a
microorganism, a plant cell, a plant, a plant tissue or in one or
more parts thereof by known methods, preferably by GC/MS, more
preferably as disclosed in paragraph [0497.0.m.n] and/or
[11896] [0530.3.m.n].
[11897] In a second step these results are compared with the
metabolic content with the relative metabolic profile as depicted
in table X and/or IX.
[11898] If the measured relative metabolic profile is identical
with the relative metabolic profile as depicted in table X and/or
IX in at least two, preferably 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more fine
chemicals, the expression level of the nucleic acid molecule or its
homologues coding for a protein as depicted in table X and/or IX is
alterd, preferably the expression level of said nucleic acid
molecul is increased.
[11899] [0062.0.0.26] This method allows a direct conclusion of the
expression level of genes from analysis of the metabolic profile.
The diagnosis of the expression level is important for example for
breeders. With regard to the aforementioned side effect induced by
genetical manipulation in the genom of cells, it is important to
assign each alteration in the content of a metabolite to the
expression of a certain gene, in ordeer to avoid the expression of
genes which are coupled with production of undesired compound, e.g
fine chemicals with toxic properties.
[11900] The diagnosis of the expression level of genes allows the
selection of plant with desired features in order to hybidize them
with others.
[11901] [0063.0.0.26] By increasing or generating the activity of
one or more of the proteins of the invention as named in table I, V
and/or X, any possible combination of metabolic profiles,
preferably any combination of the relative metabolic profiles as
shown in table X and/or IX can be achieved.
[11902] An ther way to alter the metabolic profile in a defined
manner is as described above the hybridisation of plants with
defined expression levels of genes diagnosted by the method of the
present invention.
[11903] [0064.0.0.26] claims [11904] 1. Process for the control of
the production of fine chemicals comprising [11905] (a) increasing
or generating the activity of one or more [11906] b0019, b0050,
b0057, b0112, b0124, b0138, b0149, b0161, b0175, b0196, b0251,
b0252, b0255, b0376, b0462, b0464, b0486, b0577, b0651, b0695,
b0730, b0828, b0847, b0849, b0880, b0970, b0986, b1097, b1284,
b1318, b1343, b1360, b1463, b1693, b1708, b1736, b1738, b1829,
b1886, b1896, b1926, b1961, b2023, b2078, b2095, b2211, b2239,
b2307, b2414, b2426, b2478, b2489, b2491, b2507, b2553, b2576,
b2597, b2599, b2664, b2699, b2703, b2710, b2753, b2796, b2822,
b3064, b3074, b3116, b3129, b3160, b3166, b3169, b3172, b3231,
b3256, b3260, b3430, b3457, b3462, b3578, b3619, b3644, b3684,
b3767, b3791, b3919, b3926, b3936, b3938, b3966, b3983, b4004,
b4054, b4063, b4074, b4122, b4129, b4139, b4232, b4239, b4327,
b4346, b4401, YAL049C, YBL015W, YBR084W, YBR089C-A,
[11907] YBR184W, YBR204C, YCL038C, YCR012W, YCR059C, YDL127W,
YDR271C, YDR316W, YDR447C, YDR513W, YEL045C, YEL046C, YER152C,
YER156C, YER173W, YER174C, YFL019C, YFL050C, YFL053W, YFR007W,
YFR042W, YGL205W, YGL237C, YGR101W, YGR104C, YGR261C, YHR072W,
YHR072W-A, YHR130C, YHR189W, YHR201C, YIL150C, YJL055W, YJL072C,
YJL099W, YKL132C, YKR057W, YLL013C, YLR082C, YLR089C, YLR224W,
YLR255C, YLR375W, YOR024W, YOR044W, YOR084W, YOR245C, YOR317W,
YOR344C, YOR350C, YPL099C, YPL268W, YPRO24W, YPR138C and/or YPR172W
and/or b0021, b0043, b0134, b0186, b0186, b0328, b0677, b0734,
b0763, b0895, b0895, b1054, b1183, b1217, b1249, b1292, b1874,
b2110, b2696, b2901, b3025, b3091, b3335, b3709, b3825, b3924,
b4101, b4113, b4242, b4359, YGL005C and/or YML005W protein(s) or
homologes thereof, preferably having the sequence of a polypeptide
encoded by a corresponding nucleic acid molecule indicated in Table
I, columns 5 or 7 or indicated in table V columns 5 or 7, in a
non-human organism or in one or more parts thereof and [11908] (b)
growing the organism under conditions which permit the production
of the fine chemical in said organism in a metabolic profile as
indicated in Table X and/or IX, wherein a numerical value greater
than "1" stands for an increase of a metabolite content, a
numerical value less than "1" stands for a decrease of a metabolite
content, compared to the wild type cell, microorganism, plant cell,
plant, plant tissue or one or more parts thereof and no number in
table X means a numerical value of "1" concerning the metabolite
profile, which is esentially identical to the metabolite profile of
the wild type. [11909] 2. Process for the control of the production
of fine chemicals comprising [11910] (a) increasing or generating
the activity of one or more [11911] b0019, b0050, b0057, b0112,
b0124, b0138, b0149, b0161, b0175, b0196, b0251, b0252, b0255,
b0376, b0462, b0464, b0486, b0577, b0651, b0695, b0730, b0828,
b0847, b0849, b0880, b0970, b0986, b1097, b1284, b1318, b1343,
b1360, b1463, b1693, b1708, b1736, b1738, b1829, b1886, b1896,
b1926, b1961, b2023, b2078, b2095, b2211, b2239, b2307, b2414,
b2426, b2478, b2489, b2491, b2507, b2553, b2576, b2597, b2599,
b2664, b2699, b2703, b2710, b2753, b2796, b2822, b3064, b3074,
b3116, b3129, b3160, b3166, b3169, b3172, b3231, b3256, b3260,
b3430, b3457, b3462, b3578, b3619, b3644, b3684, b3767, b3791,
b3919, b3926, b3936, b3938, b3966, b3983, b4004, b4054, b4063,
b4074, b4122, b4129, b4139, b4232, b4239, b4327, b4346, b4401,
YAL049C, YBL015W, YBR084W, YBR089C-A, YBR184W, YBR204C, YCL038C,
YCR012W, YCR059C, YDL127W, YDR271C, YDR316W, YDR447C, YDR513W,
YEL045C, YEL046C, YER152C, YER156C, YER173W, YER174C, YFL019C,
YFL050C, YFL053W, YFR007W, YFR042W, YGL205W, YGL237C, YGR101W,
YGR104C, YGR261C, YHR072W, YHR072W-A, YHR130C, YHR189W, YHR201C,
YIL150C, YJL055W, YJL072C, YJL099W, YKL132C, YKR057W, YLL013C,
YLR082C, YLR089C, YLR224W, YLR255C, YLR375W, YOR024W, YOR044W,
YOR084W, YOR245C, YOR317W, YOR344C, YOR350C, YPL099C, YPL268W,
YPRO24W, YPR138C and/or YPR172W and/or b0021, b0043, b0134, b0186,
b0186, b0328, b0677, b0734, b0763, b0895, b0895, b1054, b1183,
b1217, b1249, b1292, b1874, b2110, b2696, b2901, b3025, b3091,
b3335, b3709, b3825, b3924, b4101, b4113, b4242, b4359, YGL005C
and/or YML005W protein(s) or homologes thereof, preferably having
the sequence of a polypeptide encoded by a corresponding nucleic
acid molecule indicated in Table I, columns 5 or 7, or indicated in
table V columns 5 or 7, in a non-human organism or in one or more
parts thereof and [11912] (b) growing the organism under conditions
which permit the production fine chemicals in defined ratios in
said organism resulting in a defined metabolic profile. [11913] 3.
Process for the control of the production of fine chemicals
comprising or generating in an organism or a part thereof the
expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[11914] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide having a sequence as indicated in
Table II, columns 5 or 7, or indicated in table VI, or selected
from the group consisting of b0019, b0050, b0057, b0112, b0124,
b0138, b0149, b0161, b0175, b0196, b0251, b0252, b0255, b0376,
b0462, b0464, b0486, b0577, b0651, b0695, b0730, b0828, b0847,
b0849, b0880, b0970, b0986, b1097, b1284, b1318, b1343, b1360,
b1463, b1693, b1708, b1736, b1738, b1829, b1886, b1896, b1926,
b1961, b2023, b2078, b2095, b2211, b2239, b2307, b2414, b2426,
b2478, b2489, b2491, b2507, b2553, b2576, b2597, b2599, b2664,
b2699, b2703, b2710, b2753, b2796, b2822, b3064, b3074, b3116,
b3129, b3160, b3166, b3169, b3172, b3231, b3256, b3260, b3430,
b3457, b3462, b3578, b3619, b3644, b3684, b3767, b3791, b3919,
b3926, b3936, b3938, b3966, b3983, b4004, b4054, b4063, b4074,
b4122, b4129, b4139, b4232, b4239, b4327, b4346, b4401, YAL049C,
YBL015W, YBR084W, YBR089C-A, YBR184W, YBR204C, YCL038C, YCR012W,
YCR059C, YDL127W, YDR271C, YDR316W, YDR447C, YDR513W, YEL045C,
YEL046C, YER152C, YER156C, YER173W, YER174C, YFL019C, YFL050C,
YFL053W, YFR007W, YFR042W, YGL205W, YGL237C, YGR101W, YGR104C,
YGR261C, YHR072W, YHR072W-A, YHR130C, YHR189W, YHR201C, YIL150C,
YJL055W, YJL072C, YJL099W, YKL132C, YKR057W, YLL013C, YLR082C,
YLR089C, YLR224W, YLR255C, YLR375W, YOR024W, YOR044W, YOR084W,
YOR245C, YOR317W, YOR344C, YOR350C, YPL099C, YPL268W, YPRO24W,
YPR138C and YPR172W and/or b0021, b0043, b0134, b0186, b0186,
b0328, b0677, b0734, b0763, b0895, b0895, b1054, b1183, b1217,
b1249, b1292, b1874, b2110, b2696, b2901, b3025, b3091, b3335,
b3709, b3825, b3924, b4101, b4113, b4242, b4359, YGL005C and/or
YML005W, or a fragment thereof, which confers an increase or a
decrease in the amount of the respective fine chemical as shown in
table X and/or indicated in table IX in an organism or a part
thereof; [11915] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table I, columns 5 or 7, and encoding a polypeptide
as defined in a) and named in table Xao IX and/or a sequence as
indicated in table V columns 5 or 7, [11916] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [11917] d) nucleic acid molecule encoding a polypeptide
which has at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase or decrease in the amount of the respective
fine chemical in an organism or a part thereof; [11918] e) nucleic
acid molecule which hybridizes with a nucleic acid molecule of (a)
to (c) under stringent hybridisation conditions and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [11919] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c) and conferring an
increase or decrease in the amount of the respective fine chemical
in an organism or a part thereof; [11920] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase or decrease in the amount of
the respective fine chemical in an organism or a part thereof;
[11921] h) nucleic acid molecule comprising a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers pairs having a
sequence as indicated in Table III, columns 7, and corresponding to
a polypeptide as defined in a) and named in table X and/or IX,
and/or primers pairs as indicated in table VII, and conferring an
increase or decrease in the amount of the respective fine chemical
in an organism or a part thereof; [11922] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from an expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and conferring an increase or
decrease in the amount of the respective fine chemical in an
organism or a part thereof; [11923] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence having a
sequences as indicated in Table IV, columns 7, and corresponding to
a polypeptide as defined in a) and named in table X and/or IX,
and/or the consensus sequence having a sequences as indicated in
Table VIII, columns 7 and conferring an increase or decrease in the
amount of the respective fine chemical in an organism or a part
thereof; [11924] k) nucleic acid molecule comprising one or more of
the nucleic acid molecules encoding the amino acid sequence of a
polypeptide comprising a domain of a polypeptide indicated in Table
II, columns 5 or 7, and as defined in a) and named in table X,
and/o as indicated in Table VI columns 5 or 7, and conferring an
increase or decrease in the amount of the respective fine chemical
in an organism or a part thereof; and [11925] l) nucleic acid
molecule which is obtainable by screening a suitable library under
stringent conditions with a probe comprising one of the sequences
of the nucleic acid molecule of (a) to (k), preferably to (a) to
(c), or with a fragment of at least 15 nt, preferably 20 nt, 30 nt,
50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k), preferably to (a) to (c), and
conferring an increase or decrease in the amount of the respective
fine chemical in an organism or a part thereof; [11926] or which
comprises a sequence which is complementary thereto. [11927] 4. The
process of any one of claims 1 to 3, wherein one or more fine
chemicals are isolated. [11928] 5. The process of any one of claims
1 to 4, wherein the activity of said protein or the expression of
said nucleic acid molecule is increased or generated transiently or
stably. [11929] 6. An isolated nucleic acid molecule coding for a
polypeptide conferring a metabolic profile as shown in table X
and/or table IX and comprising a nucleic acid molecule selected
from the group consisting of: [11930] a) nucleic acid molecule
encoding, preferably at least the mature form, of a polypeptide
having a sequence as indicated in Table II, columns 5 or 7, and
selected from the group consisting of b0019, b0050, b0057, b0112,
b0124, b0138, b0149, b0161, b0175, b0196, b0251, b0252, b0255,
b0376, b0462, b0464, b0486, b0577, b0651, b0695, b0730, b0828,
b0847, b0849, b0880, b0970, b0986, b1097, b1284, b1318, b1343,
b1360, b1463, b1693, b1708, b1736, b1738, b1829, b1886, b1896,
b1926, b1961, b2023, b2078, b2095, b2211, b2239, b2307, b2414,
b2426, b2478, b2489, b2491, b2507, b2553, b2576, b2597, b2599,
b2664, b2699, b2703, b2710, b2753, b2796, b2822, b3064, b3074,
b3116, b3129, b3160, b3166, b3169, b3172, b3231, b3256, b3260,
b3430, b3457, b3462, b3578, b3619, b3644, b3684, b3767, b3791,
b3919, b3926, b3936, b3938, b3966, b3983, b4004, b4054, b4063,
b4074, b4122, b4129, b4139, b4232, b4239, b4327, b4346, b4401,
YAL049C, YBL015W, YBR084W, YBR089C-A, YBR184W, YBR204C, YCL038C,
YCR012W, YCR059C, YDL127W, YDR271C, YDR316W, YDR447C, YDR513W,
YEL045C, YEL046C, YER152C, YER156C, YER173W, YER174C, YFL019C,
YFL050C, YFL053W, YFR007W, YFR042W, YGL205W, YGL237C, YGR101W,
YGR104C, YGR261C, YHR072W, YHR072W-A, YHR130C, YHR189W, YHR201C,
YIL150C, YJL055W, YJL072C, YJL099W, YKL132C, YKR057W, YLL013C,
YLR082C, YLR089C, YLR224W, YLR255C, YLR375W, YOR024W, YOR044W,
YOR084W, YOR245C, YOR317W, YOR344C, YOR350C, YPL099C, YPL268W,
YPRO24W, YPR138C and YPR172W and/or b0021, b0043, b0134, b0186,
b0186, b0328, b0677, b0734, b0763, b0895, b0895, b1054, b1183,
b1217, b1249, b1292, b1874, b2110, b2696, b2901, b3025, b3091,
b3335, b3709, b3825, b3924, b4101, b4113, b4242, b4359, YGL005C
and/or YML005W, or as indicated in Table VI columns 5 or 7, or a
fragment thereof, which confers an increase or a decrease in the
amount of the respective fine chemical as shown in table X and/or
or table IX respectively in an organism or a part thereof; [11931]
b) nucleic acid molecule comprising, preferably at least the mature
form, of a nucleic acid molecule having a sequence as indicated in
Table I, columns 5 or 7, and corresponding to the polypeptide as
defined in a) and named in table X and/or a nucleic acid molecule
having a sequence as indicated in Table V columns 5 or 7; [11932]
c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [11933] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase or decrease in
the amount of the respective fine chemical in an organism or a part
thereof; [11934] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [11935]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase or decrease in the amount of the
respective fine chemical in an organism or a part thereof; [11936]
g) nucleic acid molecule encoding a fragment or an epitope of a
polypeptide which is encoded by one of the nucleic acid molecules
of (a) to (e), preferably to (a) to (c) and conferring an increase
or decrease in the amount of the respective fine chemical in an
organism or a part thereof; [11937] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers pairs having a sequence as indicated in Table
III, columns 7, and corresponding to a polypeptide as defined in a)
and named in table X, and/or primers pairs having a sequence as
indicated in Table VII, column 7 and conferring an increase or
decrease in the amount of the respective fine chemical in an
organism or a part thereof; [11938] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from an expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and conferring an increase or
decrease in the amount of the respective fine chemical in an
organism or a part thereof;
[11939] j) nucleic acid molecule which encodes a polypeptide
comprising the consensus sequence having a sequences as indicated
in Table IV, column 7, and corresponding to a polypeptide as
defined in a) and named in table X, and/or the consensus sequence
having a sequences as indicated in Table VIII, column 7 and
conferring an increase or decrease in the amount of the respective
fine chemical in an organism or a part thereof; [11940] k) nucleic
acid molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide comprising a
domain of a polypeptide indicated in Table II, columns 5 or 7, and
as defined in a) and named in table X, and/or encoding a domain of
a polypeptide indicated in Table VI, columns 5 or 7, and conferring
an increase or decrease in the amount of the respective fine
chemical in an organism or a part thereof; and [11941] l) nucleic
acid molecule which is obtainable by screening a suitable library
under stringent conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k), preferably to
(a) to (c), or with a fragment of at least 15 nt, preferably 20 nt,
30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k), preferably to (a) to (c), and
conferring an increase or decrease in the amount of the respective
fine chemical in an organism or a part thereof; [11942] whereby the
nucleic acid molecule distinguishes over the sequence as indicated
in Table I, columns 5 or 7, or over the sequence as indicated in
Table V, columns 5 or 7 and defined in claim 6 a)., by one or more
nucleotides. [11943] 7. A nucleic acid construct which confers the
expression of the nucleic acid molecule of claim 6, comprising one
or more regulatory elements. [11944] 8. A vector comprising the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7. [11945] 9. The vector as claimed in claim 8,
wherein the nucleic acid molecule is in operable linkage with
regulatory sequences for the expression in a prokaryotic or
eukaryotic, host. [11946] 10. A host cell which exhibits a
metabolic profile according to any of the column as depicted in
table X and/or to any line as depicted in table IX. [11947] 11. A
host cell, which has been transformed stably or transiently with
the vector as claimed in claim 8 or 9 or the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 1 to 5. [11948]
12. The host cell of claim 10 or 11, which is a transgenic host
cell. [11949] 13 The host cell of in any one of claims 10 to 12,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [11950] 14. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 13. [11951] 15. A plant tissue, propagation
material, harvested material or a plant comprising the host cell as
claimed in claim 10 or 11 which is plant cell or an Agrobacterium.
[11952] 16. A process for the identification of a compound
conferring a metabolic profile as shown in table X or in table IX
in a cell, plant or microorganism, comprising the steps: [11953]
(a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 3 a) or claim 6 conferring an
increase or decrease in the amount of the respective fine chemicals
in an organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 3 a)
conferring an increase or decrease in the amount of the respective
fine chemicals in an organism or a part thereof; [11954] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [11955] 17. A method for the identification of a
gene product conferring an increase or decrease in fine chemical
production in a cell according to the metabolic profile disclosed
in table X and/or in table IX, comprising the following steps:
[11956] (a) contacting the nucleic acid molecules of a sample,
which can contain a candidate gene encoding a gene product
conferring an increase or decrease in one or more fine chemicals
according to table X and/or table IX after expression with the
nucleic acid molecule of claim 3 a) or claim 6; [11957] (b)
identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 3 a) or claim 6; [11958] (c) introducing the candidate
nucleic acid molecules in host cells appropriate for producing one
or more fine chemicals according to table X; [11959] (d) expressing
the identified nucleic acid molecules in the host cells; [11960]
(e) assaying the level of one or more fine chemicals according to
table X and/or table IX in the host cells; and [11961] (f)
identifying nucleic acid molecule and its gene product which
expression confers an increase or decrease in the level of one or
more fine chemicals according to table X and/or table IX in the
host cell in the host cell after expression compared to the wild
type. [11962] 18. A method for the identification of a gene product
conferring an increase or decrease in fine chemical production in a
cell according to the metabolic profile disclosed in table X and/or
table IX in a cell, comprising the following steps: [11963] (a)
identifying in a data bank nucleic acid molecules of an organism;
which can contain a candidate gene encoding a gene product
conferring an increase or decrease in the level of one or more fine
chemicals according to table X and/or table IX in an organism or a
part thereof after expression, and which are at least 20%
homologous to the nucleic acid molecule of claim 3 a) or claim 6;
[11964] (b) introducing the candidate nucleic acid molecules in
host cells appropriate for producing a metabolic profile according
to table X and/or table IX; [11965] (c) expressing the identified
nucleic acid molecules in the host cells; [11966] (d) assaying the
metabolic profil in the host cells; and [11967] (e) identifying
nucleic acid molecule and its gene product which expression confers
the desired metabolic profile according to table X and/or table IX
in the host cell after expression compared to the wild type.
[11968] 19. Process for the production of a composition of fine
chemicals comprising [11969] (a) increasing or generating the
activity of one or more b0019, b0050, b0057, b0112, b0124, b0138,
b0149, b0161, b0175, b0196, b0251, b0252, b0255, b0376, b0462,
b0464, b0486, b0577, b0651, b0695, b0730, b0828, b0847, b0849,
b0880, b0970, b0986, b1097, b1284, b1318, b1343, b1360, b1463,
b1693, b1708, b1736, b1738, b1829, b1886, b1896, b1926, b1961,
b2023, b2078, b2095, b2211, b2239, b2307, b2414, b2426, b2478,
b2489, b2491, b2507, b2553, b2576, b2597, b2599, b2664, b2699,
b2703, b2710, b2753, b2796, b2822, b3064, b3074, b3116, b3129,
b3160, b3166, b3169, b3172, b3231, b3256, b3260, b3430, b3457,
b3462, b3578, b3619, b3644, b3684, b3767, b3791, b3919, b3926,
b3936, b3938, b3966, b3983, b4004, b4054, b4063, b4074, b4122,
b4129, b4139, b4232, b4239, b4327, b4346, b4401, YAL049C, YBL015W,
YBR084W, YBR089C-A, YBR184W, YBR204C, YCL038C, YCR012W, YCR059C,
YDL127W, YDR271C, YDR316W, YDR447C, YDR513W, YEL045C, YEL046C,
YER152C, YER156C, YER173W, YER174C, YFL019C, YFL050C, YFL053W,
YFR007W, YFR042W, YGL205W, YGL237C, YGR101W, YGR104C, YGR261C,
YHR072W, YHR072W-A, YHR130C, YHR189W, YHR201C, YIL150C, YJL055W,
YJL072C, YJL099W, YKL132C, YKR057W, YLL013C, YLR082C, YLR089C,
YLR224W, YLR255C, YLR375W, YOR024W, YOR044W, YOR084W, YOR245C,
YOR317W, YOR344C, YOR350C, YPL099C, YPL268W, YPRO24W, YPR138C
and/or YPR172W and/or b0021, b0043, b0134, b0186, b0186, b0328,
b0677, b0734, b0763, b0895, b0895, b1054, b1183, b1217, b1249,
b1292, b1874, b2110, b2696, b2901, b3025, b3091, b3335, b3709,
b3825, b3924, b4101, b4113, b4242, b4359, YGL005C and/or YML005W
protein(s) or homologes thereof, preferably having the sequence of
a polypeptide encoded by a corresponding nucleic acid molecule
indicated in Table I, columns 5 or 7 or indicated in table V
columns 5 or 7, in a non-human organism or in one or more parts
thereof and [11970] (b) growing the organism under conditions which
permit the production of the fine chemical in said organism in a
relative ratio as indicated in Table X and/or IX, wherein a
numerical value greater than "1" stands for an increase of a
metabolite content, a numerical value less than "1" stands for a
decrease of a metabolite content, compared to the wild type cell,
microorganism, plant cell, plant, plant tissue or one or more parts
thereof and no number in table X means a numerical value of "1"
concerning the metabolite profile, which is esentially identical to
the metabolite profile of the wild type and. [11971] (c) said
composition is a biological composition. [11972] 20. Biological
composition of fine chemicals in a defined ratio, preferably in a
relative ratio as indicated in table X and/or IX, produced by the
process of claim 19. [11973] 21. Method of diagnosis of the level
of expression of a nucleic acid molecule of claim 3 a) or claim 6
in a by [11974] (a) measuring the metabolic content, preferably the
concentration of one or more fine chemicals as depicted in table IX
and/or X, in a microorganism, a plant cell, a plant, a plant tissue
or in one or more parts thereof and [11975] (b) comparing the
metabolic content with the relative metabolic profile as depicted
in table X and/or IX.
NEW GENES RELATED TO A PROCESS FOR THE PRODUCTION OF FINE
CHEMICALS
[11976] [0001.0.0.27] The present invention relates to a process
for the production of a fine chemical in a microorgansm, a plant
cell, a plant, a plant tissue or in one or more parts thereof. The
in vention furthermore relates to nucleic acid molecules,
polypeptides, nucleic acid constructs, vectors, antisense
molecules, antibodies, host cells, plant tissue, propagtion
material, harvested material, plants, microorganisms as well as
agricultural compositions and to their use.
[11977] [0002.0.0.27] Amino acids are used in many branches of
industry, including the food, animal feed, cosmetics,
pharmaceutical and chemical industries. Amino acids such as
D,L-methionine, L-lysine or L-threonine are used in the animal feed
industry. The essential amino acids valine, leucine, isoleucine,
lysine, threonine, methionine, tyrosine, phenylalanine and
tryptophan are particularly important for the nutrition of humans
and a number of livestock species. Glycine, L-methionine and
tryptophan are all used in the pharmaceutical industry. Glutamine,
valine, leucine, isoleucine, histidine, arginine, proline, serine
and alanine are used in the pharmaceutical and cosmetics
industries. Threonine, tryptophan and D,L-methionine are widely
used feed additives (Leuchtenberger, W. (1996) Amino
acids--technical production and use, pp. 466-502 in Rehm et al.,
(Ed.) Biotechnology vol. 6, chapter 14a, VCH Weinheim). Moreover,
amino acids are suitable for the chemical industry as precursors
for the synthesis of synthetic amino acids and proteins, such as
N-acetylcysteine, S-carboxymethyl-L-cysteine,
(S)-5-hydroxytryptophan and other substances described in Ullmann's
Encyclopedia of Industrial Chemistry, vol. A2, pp. 57-97, VCH
Weinheim, 1985.
[11978] [0003.0.0.27] Over one million tons of amino acids are
currently produced annually; their market value amounts to over 2.5
billion US dollars. They are currently produced by four competing
processes: Extraction from protein hydrolysates, for example
L-cystine, L-leucine or L-tyrosine, chemical synthesis, for example
of D-, L-methionine, conversion of chemical precursors in an enzyme
or cell reactor, for example L-phenylalanine, and fermentative
production by growing, on an industrial scale, bacteria which have
been developed to produce and secrete large amounts of the desired
molecule in question. An organism, which is particularly suitable
for this purpose is Corynebacterium glutamicum, which is used for
example for the production of L-lysine or L-glutamic acid. Other
amino acids which are produced by fermentation are, for example,
L-threonine, L-tryptophan, L-aspartic acid and L-phenylalanine.
[11979] [0004.0.0.27] The biosynthesis of the natural amino acids
in organisms capable of producing them, for example bacteria, has
been characterized thoroughly; for a review of the bacterial amino
acid biosynthesis and its regulation, see Umbarger, H. E. (1978)
Ann. Rev. Biochem. 47: 533-606.
[11980] [0005.0.0.27] It is known that amino acids are produced by
fermentation of strains of coryneform bacteria, in particular
Corynebacterium glutamicum. Due to their great importance, the
production processes are constantly being improved. Process
improvements can relate to measures regarding technical aspects of
the fermentation, such as, for example, stirring and oxygen supply,
or the nutrient media composition, such as, for example, the sugar
concentration during fermentation, or to the work-up to give the
product, for example by ion exchange chromatography, or to the
intrinsic performance properties of the microorganism itself.
Bacteria from other genera such as Escherichia or Bacillus are also
used for the production of amino acids. A number of mutant strains,
which produce an assortment of desirable compounds from the group
of the sulfur-containing fine chemicals, have been developed via
strain selection. The performance properties of said microorganisms
are improved with respect to the production of a particular
molecule by applying methods of mutagenesis, selection and mutant
selection. Methods for the production of methionine have also been
developed. In this manner, strains are obtained which are, for
example, resistant to antimetabolites, such as, for example, the
methionine analogues--methylmethionine, ethionine, norleucine,
N-acetylnorleucine, S-trifluoromethylhomocysteine,
2-amino-5-heprenoitic acid, selenomethionine, methionine
sulfoximine, methoxine, 1-aminocyclopentanecarboxylic acid or which
are auxotrophic for metabolites with regulatory importance and
which produce sulfur-containing fine chemicals such as, for
example, L-methionine. However, such processes developed for the
production of methionine have the disadvantage that their yields
are too low for being economically exploitable and that they are
therefore not yet competitive with regard to chemical
synthesis.
[11981] [0006.0.0.27] Zeh (Plant Physiol., Vol. 127, 2001: 792-802)
describes increasing the methionine content in potato plants by
inhibiting threonine synthase by what is known as antisense
technology. This leads to a reduced threonine synthase activity
without the threonine content in the plant being reduced. This
technology is highly complex; the enzymatic activity must be
inhibited in a very differentiated manner since otherwise
auxotrophism for the amino acid occurs and the plant will no longer
grow.
[11982] [0007.0.0.27] U.S. Pat. No. 5,589,616 teaches the
production of higher amounts of amino acids in plants by
overexpressing a monocot storage protein in dicots. WO 96/38574, WO
97/07665, WO 97/28247, U.S. Pat. No. 4,886,878, U.S. Pat. No.
5,082,993 and U.S. Pat. No. 5,670,635 are following this approach.
That means in all the aforementioned intellectual property rights
different proteins or polypeptides are expressed in plants. Said
proteins or polypeptides should function as amino acid sinks. Other
methods for increasing amino acids such as lysine are disclosed in
WO 95/15392, WO 96/38574, WO 89/11789 or WO 93/19190. In this cases
special enzymes in the amino acid biosynthetic pathway such as the
diphydrodipicolinic acid synthase are deregulated. This leads to an
increase in the production of lysine in the different plants.
Another approach to increase the level of amino acids in plants is
disclosed in EP-A-0 271 408. EP-A-0 271 408 teaches the mutagenesis
of plant and selection afterwards with inhibitors of certain
enzymes of amino acid biosynthetic pathway.
[11983] [0008.0.0.27] Methods of recombinant DNA technology have
also been used for some years to improve Corynebacterium strains
producing L-amino acids by amplifying individual amino acid
biosynthesis genes and investigating the effect on amino acid
production.
[11984] [0009.0.0.27] As described above, the essential amino acids
are necessary for humans and many mammals, for example for
livestock. L-methionine is important as methyl group donor for the
biosynthesis of, for example, choline, creatine, adrenaline, bases
and RNA and DNA, histidine, and for the transmethylation following
the formation of S-adenosylmethionine or as a sulfhydryl group
donor for the formation of cysteine. Moreover, L-methionine appears
to have a positive effect in depression.
[11985] [0010.0.0.27] Improving the quality of foodstuffs and
animal feeds is an important task of the food-and-feed industry.
This is necessary since, for example, certain amino acids, which
occur in plants are limited with regard to the supply of mammals.
Especially advantageous for the quality of foodstuffs and animal
feeds is as balanced as possible an amino acid profile since a
great excess of an amino acid above a specific concentration in the
food has no further positive effect on the utilization of the food
since other amino acids suddenly become limiting. A further
increase in quality is only possible via addition of further amino
acids, which are limiting under these conditions. The targeted
addition of the limiting amino acid in the form of synthetic
products must be carried out with extreme caution in order to avoid
amino acid imbalance. For example, the addition of an essential
amino acid stimulates protein digestion, which may cause deficiency
situations for the second or third limiting amino acid, in
particular. In feeding experiments, for example casein feeding
experiments, the additional provision of methionine, which is
limiting in casein, has revealed the fatty degeneration of liver,
which could only be alleviated after the additional provision of
tryptophan.
[11986] [0011.0.0.27] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add a plurality of amino acids
in a balanced manner to suit the organism.
[11987] [0012.0.0.27] It is an object of the present invention to
develop an inexpensive process for the synthesis of L-methionine.
L-methionine is with lysine or threonine (depending on the
organism) one of the two amino acids which are most frequently
limiting
[11988] [0013.0.0.27] It was now found that this object is achieved
by providing the process according to the invention described
herein and the embodiments characterized in the claims.
[11989] [0014.0.0.27] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is methionine Accordingly, in
the present invention, the term "the fine chemical" as used herein
relates to "methadone". Further, in another embodiment the term
"the fine chemicals" as used herein also relates to compositions of
fine chemicals comprising methionine.
[11990] [0015.0.0.27] In one embodiment, the term "the fine
chemical" or "the respective fine chemical" means L-methionine.
Throughout the specification the term "the fine chemical" or "the
respective fine chemical" means methionine, preferably
L-methionine, its salts, ester or amids in free form or bound to
proteins. In a preferred embodiment, the term "the fine chemical"
means L-methionine in free form or its salts or bound to proteins.
In one embodiment, the term "the fine chemical" and the term "the
respective fine chemical" mean at least one chemical compound with
an activity of the above mentioned fine chemical.
[11991] [0016.0.0.27] Accordingly, the present invention relates to
a process for the production of the respective fine chemical
comprising [11992] (a) increasing or generating the activity of one
or more [11993] of a protein as shown in table XII, application no.
27, column 3 encoded by the nucleic acid sequences as shown in
table XI, application no. 27, column 5, in a non-human organism or
in one or more parts thereof or [11994] (b) growing the organism
under conditions which permit the production of the fine chemical,
thus amino acid of the invention or fine chemicals comprising amino
acids of the invention, in said organism or in the culture medium
surrounding the organism.
[11995] [0017.0.0.27] Comprises/comprising and grammatical
variations thereof when used in this specification are to be taken
to specify the presence of stated features, integers, steps or
components or groups thereof, but not to preclude the presence or
addition of one or more other features, integers, steps, components
or groups thereof. The term "Table Xl" used in this specification
is to be taken to specify the content of Table XI A and Table XI B.
The term "Table XII" used in this specification is to be taken to
specify the content of Table XII A and Table XII B. The term "Table
XI A" used in this specification is to be taken to specify the
content of Table XI A. The term "Table XI B" used in this
specification is to be taken to specify the content of Table XI B.
The term "Table XII A" used in this specification is to be taken to
specify the content of Table XII A. The term "Table XII B" used in
this specification is to be taken to specify the content of Table
XII B. In one preferred embodiment, the term "Table Xl" means Table
XI B. In one preferred embodiment, the term "Table XII" means Table
XII B.
[11996] [0018.0.0.27] Preferably, this process further comprises
the step of recovering the fine chemical, which is synthesized by
the organism from the organism and/or from the culture medium used
for the growth or maintenance of the organism. The term
"recovering" means the isolation of the fine chemical in different
purities, that means on the one hand harvesting of the biological
material, which contains the fine chemical without further
purification and on the other hand purities of the fine chemical
between 5% and 100% purity, preferred purities are in the range of
10% and 99%. In one embodiment, the purities are 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95% or 99%.
[11997] [0019.0.0.27] Advantageously the process for the production
of the respective fine chemical leads to an enhanced production of
the fine respective chemical. The terms "enhanced" or "increase"
mean at least a 10%, 20%, 30%, 40% or 50%, preferably at least 60%,
70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%, 400% or
500% or more percent higher production of the respective fine
chemical in comparison to the reference as defined below, e.g. that
means in comparison to an organism without the aforementioned
modification of the activity of a protein having the activity of a
protein indicated in Table XII, column 3, or encoded by nucleic
acid molecule indicated in Table XI, columns 5 or 7.
[11998] [0020.0.0.27] Surprisingly it was found, that the
transgenic expression of the Linum usitatissimum protein as
indicated in Table XII, application no. 27, column 5, line 1 in a
plant conferred an increase in methionine content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of Methionine.
[11999] Surprisingly it was found, that the transgenic expression
of the Glycine max protein as indicated in Table XII, application
no. 27, column 5, line 2 in a plant conferred an increase in
methionine content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of methionine.
[12000] [0021.0.0.27] In accordance with the invention, the term
"organism" as understood herein relates always to a non-human
organism, in particular to an animal or plant organism or to a
microorganism. Further, the term "animal" as understood herein
relates always to a non-human animal.
[12001] In accordance with the invention it is known to the skilled
that anionic compounds such as acids are present in aqueous
solutions in an equilibrium between the acid and its salts
according to the pH present in the respective compartment of the
cell or organism and the pK of the acid. Depending on the strength
of the acid (pK) and the pH the salt or the free acid are
predominant. Thus, the term "the fine chemical", the term "the
respective fine chemical", or the term "acid" or the use of a
denomination referring to a neutralized anionic compound relates to
the anionic form as well as the neutralised status of that compound
according to the milieu of the aqueous solution in which they are
present.
[12002] [0022.0.0.27] The sequence of b1343 from Escherichia coli
K12 has been published in Blattner, Science 277(5331), 1453-1474,
1997, and its activity is being defined as an ATP-dependent RNA
helicase, stimulated by 23S rRNA. Accordingly, in one embodiment,
the process of the present invention comprises the use of an
"ATP-dependent RNA helicase, stimulated by 23S rRNA" from E. coli
or its homolog, e.g. as shown herein, for the production of the
fine chemical, meaning of methionine, in particular for increasing
the amount of methionine in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a protein having an activity
in rRNA processing or translation is increased or generated, e.g.
from E. coli or a plant or a homolog thereof. Accordingly, in one
embodiment, in the process of the present invention the activity of
a ATP-dependent RNA helicase, stimulated by 23S rRNA or its homolog
is increased for the production of the fine chemical, meaning of
methionine, in particular for increasing the amount of methionine
in free or bound form in an organism or a part thereof, as
mentioned.
[12003] The sequence of b4232 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a fructose-1,6-bisphosphatase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a "fructose-1,6-bisphosphatase" from
E. coli or a plant or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of methionine, in
particular for increasing the amount of methionine in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a protein of the superfamily "fructose-bisphosphatase", preferably
having an activity in C-compound and carbohydrate metabolism,
C-compound and carbohydrate utilization, energy, glycolysis and
gluconeogenesis, plastid, photosynthesis, more preferred having an
"fructose-1,6-bisphosphatase"-activity, is increased or generated,
e.g. from E. coli or a homolog thereof. Accordingly, in one
embodiment, in the process of the present invention the activity of
a "fructose-1,6-bisphosphatase" or its homolog is increased for the
production of the fine chemical, meaning of methionine, in
particular for increasing the amount of methionine in free or bound
form in an organism or a part thereof, as mentioned.
[12004] [0022.1.0.27] In one embodiment of the invention the
polypeptides as shown in table XII, column 5 and 7, or encoded by a
nucleic acid molecule as shown in table XI, column 5 and 7, and
conferring an increase in the content of the fine chemical as shown
in table XI to XIV, column 6 respectively, has preferably the
activity of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[12005] [0023.0.0.27] Homologues (=homologs) of the present gene
products can be derived from any organisms as long as the homologue
confers the herein mentioned activity, in particular, confers an
increase in the respective fine chemical amount or content.
[12006] In one embodiment, the homolog of the any one of the
polypeptides indicated in Table XII, application no. 27, column 3,
is a homolog having the same or a similar activity. In particular
an increase of activity confers an increase in the content of the
respective fine chemical in the organisms. In one embodiment, the
homolog is a homolog with a sequence as indicated in Table XI or
XII, application no. 27, column 7. In one embodiment, the homolog
of one of the polypeptides indicated in Table XII, application no.
27, column 3, is derived from an eukaryotic. In one embodiment, the
homolog is derived from plants. In one embodiment, the homolog of a
polypeptide indicated in Table XII, application no. 27, column 3,
is derived from a monocotyledonous plant. In one embodiment, the
homolog of a polypeptide indicated in Table XII, application no.
27, column 3, is derived from a dicotyledonous plant.
[12007] [0023.1.0.27] Homologs of the polypeptide disclosed in
table XII, application no. 27, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 27, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 27, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 27,
column 7, resp.
[12008] [0024.0.0.27] Further homologs of are described herein
below.
[12009] [0025.0.0.27] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 27, column 3 "if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 27, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[12010] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 27,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 27,
column 3 of a a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[12011] [0025.1.0.27] In one embodiment, the polypeptide of the
invention or the polypeptide used in the method of the invention
confers said activity, e.g. the increase of the fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary distant to the organism in which it is
expressed. For example origin and expressing organism are derived
from different families, orders, classes or phylums.
[12012] [0025.2.0.27] In one embodiment, the polypeptide of the
invention or the polypeptide used in the method of the invention
confers said activity, e.g. the increase of the fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table XI,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table XI,
column 4 are derived from the same families, orders, classes or
phylums.
[12013] [0026.0.0.27] The terms "increased", "rose", "extended",
"enhanced", "improved" or "amplified" relate to a corresponding
change of a property in an organism, a part of an organism such as
a tissue, seed, root, leave, flower etc. or in a cell and are
interchangeable. Preferably, the overall activity in the volume is
increased or enhanced in cases if the increase or enhancement is
related to the increase or enhancement of an activity of a gene
product, independent whether the amount of gene product or the
specific activity of the gene product or both is increased or
enhanced or whether the amount, stability or translation efficacy
of the nucleic acid sequence or gene encoding for the gene product
is increased or enhanced. The terms "reduction", "decrease" or
"deletion" relate to a corresponding change of a property in an
organism, a part of an organism such as a tissue, seed, root,
leave, flower etc. or in a cell. Preferably, the overall activity
in the volume is reduced, decreased or deleted in cases if the
reduction, decrease or deletion is related to the reduction,
decrease or deletion of an activity of a gene product, independent
whether the amount of gene product or the specific activity of the
gene product or both is reduced, decreased or deleted or whether
the amount, stability or translation efficacy of the nucleic acid
sequence or gene encoding for the gene product is reduced,
decreased or deleted.
[12014] [0027.0.0.27] The terms "increase" or "decrease" relate to
a corresponding change of a property an organism or in a part of an
organism, such as a tissue, seed, root, leave, flower etc. or in a
cell. Preferably, the overall activity in the volume is increased
in cases the increase relates to the increase of an activity of a
gene product, independent whether the amount of gene product or the
specific activity of the gene product or both is increased or
generated or whether the amount, stability or translation efficacy
of the nucleic acid sequence or gene encoding for the gene product
is increased.
[12015] [0028.0.0.27] Under "change of a property" it is understood
that the activity, expression level or amount of a gene product or
the metabolite content is changed in a specific volume relative to
a corresponding volume of a control, reference or wild type,
including the de novo creation of the activity or expression.
[12016] [0029.0.0.27] The terms "increase" or "decrease" include
the change or the modulation of said property in only parts of the
subject of the present invention, for example, the modification can
be found in compartment of a cell, like a organelle, or in a part
of a plant, like tissue, seed, root, leave, flower etc. but is not
detectable Xlf the overall subject, i.e. complete cell or plant, is
tested. Preferably, the increase or decrease is found cellular,
thus the term "increase of an activity" or "increase of a
metabolite content" relates to the cellular increase compared to
the wild type cell. However, the terms increase or decrease as used
herein also include the change or modulation of a property in the
whole organism as mentioned.
[12017] [0030.0.0.27] Accordingly, the term "increase" or
"decrease" means that the specific activity of an enzyme,
preferably the amount of a compound or metabolite, e.g. of a
polypeptide, a nucleic acid molecule or of the respective fine
chemical of the invention or an encoding mRNA or DNA, can be
increased or decreased in a volume.
[12018] [0031.0.0.27] The terms "wild type", "control" or
"reference" are exchangeable and can be a cell or a part of
organisms such as an organelle or a tissue, or an organism, in
particular a microorganism or a plant, which was not modified or
treated according to the herein described process according to the
invention. Accordingly, the cell or a part of organisms such as an
organelle or a tissue, or an organism, in particular a
microorganism or a plant used as wild type, control or reference
corresponds to the cell, organism or part thereof as much as
possible and is in any other property but in the result of the
process of the invention as identical to the subject matter of the
invention as possible. Thus, the wild type, control, or reference
is treated identically or as identical as possible, saying that
only conditions or properties might be different which do not
influence the quality of the tested property.
[12019] [0032.0.0.27] Preferably, any comparison is carried out
under analogous conditions. The term "analogous conditions" means
that all conditions such as, for example, culture or growing
conditions, assay conditions (such as buffer composition,
temperature, substrates, pathogen strain, concentrations and the
like) are kept identical between the experiments to be
compared.
[12020] [0033.0.0.27] The "reference", "control", or "wild type" is
preferably a subject, e.g. an organelle, a cell, a tissue, an
organism, in particular a plant or a microorganism, which was not
modified or treated according to the herein described process of
the invention and is in any other property as similar to the
subject matter of the invention as possible. The reference,
control, or wild type is in its genome, transcriptome, proteome or
meta-bolome as similar as possible to the subject of the present
invention. Preferably, the term "reference-" "control-" or "wild
type-"-organelle, -cell, -tissue or -organism, in particular plant
or microorganism, relates to an organelle, cell, tissue or
organism, in particular plant or microorganism, which is nearly
genetically identical to the organelle, cell, tissue or organism,
in particular microorganism or plant, of the present invention or a
part thereof preferably 95%, more preferred are 98%, even more
preferred are 99.00%, in particular 99.10%, 99.30%, 99.50%, 99.70%,
99.90%, 99.99%, 99, 999% or more. Most preferable the "reference",
"control", or "wild type" is a subject, e.g. an organelle, a cell,
a tissue, an organism, which is genetically identical to the
organism, cell or organelle used according to the process of the
invention except that the responsible or activity conferring
nucleic acid molecules or the gene product encoded by them are
amended, manipulated, exchanged or introduced according to the
inventive process.
[12021] [0034.0.0.27] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 27, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[12022] [0035.0.0.27] In case, a control, reference or wild type
differing from the subject of the present invention only by not
being subject of the process of the invention can not be provided,
a control, reference or wild type can be an organism in which the
cause for the modulation of an activity conferring the increase of
the fine chemical or expression of the nucleic acid molecule as
described herein has been switched back or off, e.g. by knocking
out the expression of responsible gene product, e.g. by antisense
inhibition, by inactivation of an activator or agonist, by
activation of an inhibitor or antagonist, by inhibition through
adding inhibitory antibodies, by adding active compounds as e.g.
hormones, by introducing negative dominant mutants, etc. A gene
production can for example be knocked out by introducing
inactivating point mutations, which lead to an enzymatic activity
inhibition or a destabilization or an inhibition of the ability to
bind to cofactors etc.
[12023] [0036.0.0.27] Accordingly, preferred reference subject is
the starting subject of the present process of the invention.
Preferably, the reference and the subject matter of the invention
are compared after standardization and normalization, e.g. to the
amount of total RNA, DNA, or Protein or activity or expression of
reference genes, like housekeeping genes, such as ubiquitin, actin
or ribosomal proteins.
[12024] [0037.0.0.27] A series of mechanisms exists via which a
modification of a protein, e.g. the polypeptide of the invention or
the polypeptide used in the method of the invention can directly or
indirectly affect the yield, production and/or production
efficiency of the fine chemical.
[12025] [0038.0.0.27] For example, the molecule number or the
specific activity of the polypeptide or the nucleic acid molecule
may be increased. Larger amounts of the fine chemical can be
produced if the polypeptide or the nucleic acid of the invention is
expressed de novo in an organism lacking the activity of said
protein. However, it is also possible to increase the expression of
the gene which is naturally present in the organisms, for example
by amplifying the number of gene(s), by modifying the regulation of
the gene, or by increasing the stability of the corresponding mRNA
or of the corresponding gene product encoded by the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention, or by introducing homologous genes from
other organisms which are differently regulated, e.g. not feedback
sensitive.
[12026] [0039.0.0.27] This also applies analogously to the combined
increased expression of the nucleic acid molecule of the present
invention or its gene product with that of further enzymes or
regulators of the biosynthesis pathways of the respective fine
chemical, e.g. which are useful for the synthesis of the respective
fine chemicals.
[12027] [0040.0.0.27] The increase, decrease or modulation
according to this invention can be constitutive, e.g. due to a
stable permanent transgenic expression or to a stable mutation in
the corresponding endogenous gene encoding the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention or to a modulation of the expression or of
the behaviour of a gene conferring the expression of the
polypeptide of the invention or the polypeptide used in the method
of the invention, or transient, e.g. due to an transient
transformation or temporary addition of a modulator such as a
agonist or antagonist or inducible, e.g. after transformation with
a inducible construct carrying the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention under control of a inducible promoter and adding the
inducer, e.g. tetracycline or as described herein below.
[12028] [0041.0.0.27] The increase in activity of the polypeptide
amounts in a cell, a tissue, a organelle, an organ or an organism
or a part thereof preferably to at least 5%, preferably to at least
20% or at to least 50%, especially preferably to at least 70%, 80%,
90% or more, very especially preferably are to at least 200%, most
preferably are to at least 500% or more in comparison to the
control, reference or wild type.
[12029] [0042.0.0.27] The specific activity of a polypeptide
encoded by a nucleic acid molecule of the present invention or of
the polypeptide of the present invention can be tested as described
in the examples. In particular, the expression of a protein in
question in a cell, e.g. a plant cell or a microorganism and the
detection of an increase the respective fine chemical level in
comparison to a control is an easy test and can be performed as
described in the state of the art.
[12030] [0043.0.0.27] The term "increase" includes, that a compound
or an activity is introduced into a cell de novo or that the
compound or the activity has not been detectable before, in other
words it is "generated".
[12031] [0044.0.0.27] Accordingly, in the following, the term
"increasing" also comprises the term "generating" or "stimulating".
The increased activity manifests itself in an increase of the fine
chemical.
[12032] [0045.0.0.27] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
27, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 27, column 6 of
the respective line, between 10% and 50%, 10% and 100%, 10% and
200%, 10% and 300%, 10% and 400%, 10% and 500%, 20% and 50%, 20%
and 250%, 20% and 500%, 50% and 100%, 50% and 250%, 50% and 500%,
500% and 1000% or more
[12033] [0046.0.0.27] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
27, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 27, column 6 of
the respective line confers an increase of the respective fine
chemical and of further amino acid or their precursors.
[12034] [0047.0.0.27] In this context, the respective fine chemical
amount in a cell, preferably in a tissue, more preferred in a
organism as a plant or a microorganism or part thereof, is
increased by 3% or more, especially preferably are 10% or more,
very especially preferably are more than 30% and most preferably
are 70% or more, such as 100%, 300% or 500%.
[12035] [0048.0.0.27] The respective fine chemical can be contained
in the organism either in its free form and/or bound to proteins or
polypeptides or mixtures thereof. Accordingly, in one embodiment,
the amount of the free form in a cell, preferably in a tissue, more
preferred in a organism as a plant or a microorganism or part
thereof, is increased by 3% or more, especially preferably are 10%
or more, very especially preferably are more than 30% and most
preferably are 70% or more, such as 100%, 300% or 500%.
Accordingly, in an other embodiment, the amount of the bound the
respective fine chemical in a cell, preferably in a tissue, more
preferred in a organism as a plant or a microorganism or part
thereof, is increased by 3% or more, especially preferably are 10%
or more, very especially preferably are more than 30% and most
preferably are 70% or more, such as 100%, 300% or 500%.
[12036] [0049.0.0.27] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 27, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 27, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 27, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[12037] [0050.0.0.27] For the purposes of the present invention,
the terms "L-methionine", "methionine", "homocysteine",
"S-adenosylmethionine" and "threonine" also encompass the
corresponding salts, such as, for example, methionine hydrochloride
or methionine sulfate. Preferably the terms methionine or threonine
are intended to encompass the terms L-methionine or
L-threonine.
[12038] [0051.0.0.27] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g. fine chemical compositions. Depending
on the choice of the organism used for the process according to the
present invention, for example a microorganism or a plant,
compositions or mixtures of various fine chemicals, e.g. comprising
further distinct amino acids, fatty acids, vitamins, hormones,
sugars, lipids, etc. can be produced.
[12039] [0052.0.0.27] The term "expression" refers to the
transcription and/or translation of a codogenic gene segment or
gene. As a rule, the resulting product is an mRNA or a protein.
However, expression products can also include functional RNAs such
as, for example, antisense, nucleic acids, tRNAs, snRNAs, rRNAs,
RNAi, siRNA, ribozymes etc. Expression may be systemic, local or
temporal, for example limited to certain cell types, tissues organs
or time periods.
[12040] [0053.0.0.27] In one embodiment, the process of the present
invention comprises one or more of the following steps: [12041] a)
stabilizing a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the invention, e.g. of a polypeptide having the
activity of a protein as indicated in table XII, application no.
27, columns 5 and 7 or its homologs activity having
herein-mentioned amino acid of the invention increasing activity;
and/or [12042] b) stabilizing a mRNA conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention, as shown in table XI, application no. 27, columns 5 and
7, e.g. a nucleic acid sequence encoding a polypeptide having the
activity of a protein as indicated in table XII, application no.
27, columns 5 and 7 or its homologs activity or of a mRNA encoding
the polypeptide of the present invention having herein-mentioned
amino acid of the invention increasing activity; and/or [12043] c)
increasing the specific activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention or of the polypeptide of the present
invention having herein-mentioned amino acid increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 27, columns 5 and 7 or its homologs
activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [12044] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned amino acid of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 27, columns 5
and 7 or its homologs activity; and/or [12045] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned amino acid of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 27, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [12046] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned amino acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 27, columns 5 and 7 or its
homologs activity, and/or [12047] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned amino acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 27, columns 5 and 7 or its
homologs activity; and/or [12048] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 27, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[12049] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [12050] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[12051] [0054.0.0.27] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 27 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 27, column 3 or its homologs. In general, the
amount of mRNA or polypeptide in a cell or a compartment of a
organism correlates with the amount of encoded protein and thus
with the overall activity of the encoded protein in said volume.
Said correlation is not always linear, the activity in the volume
is dependent on the stability of the molecules or the presence of
activating or inhibiting co-factors. Further, product and educt
inhibitions of enzymes are well known and described in Textbooks,
e.g. Stryer, Biochemistry.
[12052] [0055.0.0.27] In general, the amount of mRNA or polypeptide
in a cell or a compartment of a organism correlates with the amount
of encoded protein and thus with the overall activity of the
encoded protein in said volume. Said correlation is not always
linear, the activity in the volume is dependent on the stability of
the molecules or the presence of activating or inhibiting
co-factors. Further, product and educt inhibitions of enzymes are
well known and described in Textbooks, e.g. Stryer,
Biochemistry.
[12053] [0056.0.0.27] In general, the amount of mRNA,
polynucleotide or nucleic acid molecule in a cell or a compartment
of an organism correlates with the amount of encoded protein and
thus with the overall activity of the encoded protein in said
volume. Said correlation is not always linear, the activity in the
volume is dependent on the stability of the molecules, the
degradation of the molecules or the presence of activating or
inhibiting co-factors. Further, product and educt inhibitions of
enzymes are well known, e.g. Zinser et al.
"Enzyminhibitoren"/Enzyme inhibitors".
[12054] [0057.0.0.27] The activity of the abovementioned proteins
and/or polypeptide encoded by the nucleic acid molecule of the
present invention can be increased in various ways. For example,
the activity in an organism or in a part thereof, like a cell, is
increased via increasing the gene product number, e.g. by
increasing the expression rate, like introducing a stronger
promoter, or by increasing the stability of the mRNA expressed,
thus increasing the translation rate, and/or increasing the
stability of the gene product, thus reducing the proteins decayed.
Further, the activity or turnover of enzymes can be influenced in
such a way that a reduction or increase of the reaction rate or a
modification (reduction or increase) of the affinity to the
substrate results, is reached. A mutation in the catalytic centre
of an polypeptide of the invention or the polypeptide used in the
method of the invention, e.g. as enzyme, can modulate the turn over
rate of the enzyme, e.g. a knock out of an essential amino acid can
lead to a reduced or completely knock out activity of the enzyme,
or the deletion or mutation of regulator binding sites can reduce a
negative regulation like a feedback inhibition (or a substrate
inhibition, if the substrate level is also increased). The specific
activity of an enzyme of the present invention can be increased
such that the turn over rate is increased or the binding of a
co-factor is improved. Improving the stability of the encoding mRNA
or the protein can also increase the activity of a gene product.
The stimulation of the activity is also under the scope of the term
"increased activity".
[12055] [0058.0.0.27] Moreover, the regulation of the
abovementioned nucleic acid sequences may be modified so that gene
expression is increased. This can be achieved advantageously by
means of heterologous regulatory sequences or by modifying, for
example mutating, the natural regulatory sequences which are
present. The advantageous methods may also be combined with each
other.
[12056] [0059.0.0.27] In general, an activity of a gene product in
an organism or part thereof, in particular in a plant cell, a
plant, or a plant tissue or a part thereof or in a microorganism
can be increased by increasing the amount of the specific encoding
mRNA or the corresponding protein in said organism or part thereof.
"Amount of protein or mRNA" is understood as meaning the molecule
number of polypeptides or mRNA molecules in an organism, a tissue,
a cell, or a cell compartment. "Increase" in the amount of a
protein means the quantitative increase of the molecule number of
said protein in an organism, a tissue, a cell or a cell compartment
or part thereof--for example by one of the methods described herein
below--in comparison to a wild type, control or reference.
[12057] [0060.0.0.27] The increase in molecule number amounts
preferably to at least 1%, preferably to more than 10%, more
preferably to 30% or more, especially preferably to 50%, 70% or
more, very especially preferably to 100%, most preferably to 500%
or more. However, a de novo expression is also regarded as subject
of the present invention.
[12058] [0061.0.0.27] A modification, i.e. an increase or decrease,
can be caused by endogenous or exogenous factors. For example, an
increase in activity in an organism or a part thereof can be caused
by adding a gene product or a precursor or an activator or an
agonist to the media or nutrition or can be caused by introducing
said subjects into a organism, transient or stable.
[12059] [0062.0.0.27] In one embodiment the increase in the amount
of the fine chemical in the organism or a part thereof, e.g. in a
cell, a tissue, a organ, an organelle etc., is achieved by
increasing the endogenous level of the polypeptide of the invention
or the polypeptide used in the method of the invention.
Accordingly, in an embodiment of the present invention, the present
invention relates to a process wherein the gene copy number of a
gene encoding the polynucleotide or nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention as herein described is increased. Further, the endogenous
level of the polypeptide of the invention or the polypeptide used
in the method of the invention as described can for example be
increased by modifying the transcriptional or translational
regulation of the polypeptide.
[12060] [0063.0.0.27] In one embodiment the amount of the fine
chemical in the organism or part thereof can be increase by
targeted or random mutagenesis of the endogenous genes of the
invention. For example homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. In addition gene conversion like methods
described by Kochevenko and Willmitzer (Plant Physiol. 2003 May;
132(1): 174-84) and citations therein can be used to disrupt
repressor elements or to enhance to activity of positive regulatory
elements.
[12061] Furthermore positive elements can be randomly introduced in
(plant) genomes by T-DNA or transposon mutagenesis and lines can be
screened for, in which the positive elements has be integrated near
to a gene of the invention, the expression of which is thereby
enhanced. The activation of plant genes by random integrations of
enhancer elements has been described by Hayashi et al., 1992
(Science 258:1350-1353) or Weigel et al., 2000 (Plant Physiol. 122,
1003-1013) and others citied therein. Reverse genetic strategies to
identify insertions (which eventually carrying the activation
elements) near in genes of interest have been described for various
cases e.g. Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290);
Sessions et al., 2002 (Plant Cell 2002, 14, 2985-2994); Young et
al., 2001, (Plant Physiol. 2001, 125, 513-518); Koprek et al., 2000
(Plant J. 2000, 24, 253-263); Jeon et al., 2000 (Plant J. 2000, 22,
561-570); Tissier et al., 1999 (Plant Cell 1999, 11, 1841-1852);
Speulmann et al., 1999 (Plant Cell 1999,11, 1853-1866). Briefly
material from all plants of a large T-DNA or transposon mutagenized
plant population is harvested and genomic DNA prepared. Then the
genomic DNA is pooled following specific architectures as described
for example in Krysan et al., 1999 (Plant Cell 1999, 11,
2283-2290). Pools of genomics DNAs are then screened by specific
multiplex PCR reactions detecting the combination of the
insertional mutagen (e.g. T-DNA or Transposon) and the gene of
interest. Therefore PCR reactions are run on the DNA pools with
specific combinations of T-DNA or transposon border primers and
gene specific primers. General rules for primer design can again be
taken from Krysan et al., 1999 (Plant Cell 1999, 11, 2283-2290)
Rescreening of lower levels DNA pools lead to the identification of
individual plants in which the gene of interest is disrupted by the
insertional mutagen.
[12062] The enhancement of positive regulatory elements or the
disruption or weaking of negative regulatory elements can also be
achieved through common mutagenesis techniques: The production of
chemically or radiation mutated populations is a common technique
and known to the skilled worker. Methods for plants are described
by Koorneef et al. 1982 and the citations therein and by Lightner
and Caspar in "Methods in Molecular Biology" Vol 82. These
techniques usually induce pointmutations that can be identified in
any known gene using methods such as tilling (Colbert et al.
2001).
[12063] Accordingly, the expression level can be increased if the
endogenous genes encoding a polypeptide conferring an increased
expression of the polypeptide of the present invention, in
particular genes comprising the nucleic acid molecule of the
present invention, are modified via homologous recombination,
tilling approaches or gene conversion
[12064] [0064.0.0.27] Regulatory sequences can be operatively
linked to the coding region of an endogenous protein and control
its transcription and translation or the stability or decay of the
encoding mRNA or the expressed protein. In order to modify and
control the expression, promoter, UTRs, splicing sites, processing
signals, polyadenylation sites, terminators, enhancers, repressors,
post transcriptional or posttranslational modification sites can be
changed, added or amended for example, the activation of plant
genes by random integrations of enhancer elements has been
described by Hayashi et al., 1992 (Science 258:1350-1353) or Weigel
et al., 2000 (Plant Physiol. 122, 1003-1013) and others citied
therein. For example, the expression level of the endogenous
protein can be modulated by replacing the endogenous promoter with
a stronger transgenic promoter or by replacing the endogenous 3'UTR
with a 3'UTR, which provides more stability without amending the
coding region. Further, the transcriptional regulation can be
modulated by introduction of an artificial transcription factor as
described in the examples. Alternative promoters, terminators and
UTR are described below.
[12065] [0065.0.0.27] The activation of an endogenous polypeptide
having above-mentioned activity, of the polypeptide of the
invention or the polypeptide used in the method of the invention,
e.g. conferring the increase of the respective fine chemical after
increase of expression or activity can also be increased by
introducing a synthetic transcription factor, which binds close to
the coding region of an endogenous polypeptide of the invention or
the polypeptide used in the method of the invention- or used in the
process of the invention or its endogenous homolog-encoding gene
and the synthetic transcription factor activates its transcription.
A chimeric zinc finger protein can be construed, which comprises a
specific DNA-binding domain and an activation domain as e.g. the
VP16 domain of Herpes Simplex virus. The specific binding domain
can bind to the regulatory region of the endogenous protein coding
region. The expression of the chimeric transcription factor in a
organism, in particular in a plant, leads to a specific expression
of an endogenous polypeptid of the invention or used in the process
of the invention, in particular a plant homolog thereof, see e.g.
in WO01/52620, Oriz, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99,
13290 or Guan, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99,
13296.
[12066] [0066.0.0.27] In one further embodiment of the process
according to the invention, organisms are used in which one of the
abovementioned genes, or one of the abovementioned nucleic acids,
is mutated in a way that the activity of the encoded gene products
is less influenced by cellular factors, or not at all, in
comparison with the unmutated proteins. For example, well known
regulation mechanism of enzymic activity are substrate inhibition
or feed back regulation mechanisms. Ways and techniques for the
introduction of substitutions, deletions and additions of one or
more bases, nucleotides or amino acids of a corresponding sequence
are described herein below in the corresponding paragraphs and the
references listed there, e.g. in Sambrook et al., Molecular
Cloning, Cold Spring Habour, N.Y., 1989. The person skilled in the
art will be able to identify regulation domains and binding sites
of regulators by comparing the sequence of the nucleic acid
molecule of the present invention or the expression product thereof
with the state of the art by computer software means which comprise
algorithms for the identifying of binding sites and regulation
domains or by introducing into a nucleic acid molecule or in a
protein systematically mutations and assaying for those mutations
which will lead to an increased specific activity or an increased
activity per volume, in particular per cell.
[12067] [0067.0.0.27] It is therefore advantageously to express in
an organism a nucleic acid molecule of the invention or the nucleic
acid molecule used in the method of the invention or a polypeptide
of the invention or the polypeptide used in the method of the
invention derived from a evolutionary distantly related organism,
as e.g. using a prokaryotic gene in an eukaryotic host, as in these
cases the regulation mechanism of the host cell may not weaken the
activity (cellular or specific) of the gene or its expression
product
[12068] [0068.0.0.27] The mutation is introduced in such a way that
the production of the amino acids is not adversely affected.
[12069] [0069.0.0.27] Less influence on the regulation of a gene or
its gene product is understood as meaning a reduced regulation of
the enzymatic activity leading to an increased specific or cellular
activity of the gene or its product. An increase of the enzymatic
activity is understood as meaning an enzymatic activity, which is
increased by at least 10%, advantageously at least 20, 30 or 40%,
especially advantageously by at least 50, 60 or 70% in comparison
with the starting organism. This leads to an increased productivity
of the desired respective fine chemical(s).
[12070] [0070.0.0.27] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention or the polypeptide of the invention or the
polypeptide used in the method of the invention as described below,
for example the nucleic acid construct mentioned below, into an
organism alone or in combination with other genes, it is possible
not only to increase the biosynthetic flux towards the end product,
but also to increase, modify or create de novo an advantageous,
preferably novel metabolites composition in the organism, e.g. an
advantageous amino acid composition comprising a higher content of
(from a viewpoint of nutritional physiology limited) respective
fine chemicals, in particular amino acids, likewise the fine
chemical.
[12071] [0071.0.0.27] Preferably the composition further comprises
higher amounts of metabolites positively affecting or lower amounts
of metabolites negatively affecting the nutrition or health of
animals or humans provided with said compositions or organisms of
the invention or parts thereof. Likewise, the number or activity of
further genes which are required for the import or export of
nutrients or metabolites, including amino acids or its precursors,
required for the cell's biosynthesis of amino acids may be
increased so that the concentration of necessary or relevant
precursors, cofactors or intermediates within the cell(s) or within
the corresponding storage compartments is increased. Owing to the
increased or novel generated activity of the polypeptide of the
invention or the polypeptide used in the method of the invention or
owing to the increased number of nucleic acid sequences of the
invention and/or to the modulation of further genes which are
involved in the biosynthesis of the amino acids, e.g. by increasing
the activity of enzymes synthesizing precursors or by destroying
the activity of one or more genes which are involved in the
breakdown of the amino acids, it is possible to increase the yield,
production and/or production efficiency of amino acids in the host
organism, such as the plants or the microorganisms.
[12072] [0072.0.0.27] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous sulfur-containing compounds, which contain at
least one sulfur atom bound covalently. Examples of such compounds
are, in addition to methionine, homocysteine, S-adenosylmethionine,
cysteine, advantageously methionine and S-adenosylmethionine.
[12073] [0073.0.0.27] Accordingly, in one embodiment, the process
according to the invention relates to a process which comprises:
[12074] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [12075] b) increasing an activity of a
polypeptide of the invention or the polypeptide used in the method
of the invention or a homolog thereof, e.g. as indicated in Table
XII, application no. 27, columns 5 or 7, or of a polypeptide being
encoded by the nucleic acid molecule of the present invention and
described below, i.e. conferring an increase of the respective fine
chemical in the organism, preferably in a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant, [12076] c) growing the organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, under conditions which permit the
production of the respective fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and [12077] d) if desired, recovering, optionally
isolating, the free and/or bound the respective fine chemical and,
optionally further free and/or bound amino acids synthesized by the
organism, the microorganism, the non-human animal, the plant or
animal cell, the plant or animal tissue or the plant.
[12078] [0074.0.0.27] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the respective fine chemical or the free and
bound the fine chemical but as option it is also possible to
produce, recover and, if desired isolate, other free or/and bound
amino acids, in particular lysine. Galili et al., Transgenic Res.,
200, 9, 2, 137-144 describes that the heterologous expression of a
bacterial gene for the amino acid biosynthesis confers the increase
of free as well as of protein-bound amino acids.
[12079] [0075.0.0.27] After the above-described increasing (which
as defined above also encompasses the generating of an activity in
an organism, i.e. a de novo activity), for example after the
introduction and the expression of the nucleic acid molecules of
the invention or described in the methods or processes according to
the invention, the organism according to the invention,
advantageously, a microorganism, a non-human animal, a plant, plant
or animal tissue or plant or animal cell, is grown and subsequently
harvested.
[12080] [0076.0.0.27] Suitable organisms or host organisms
(transgenic organism) for the nucleic acid molecule used according
to the invention and for the inventive process, the nucleic acid
construct or the vector (both as described below) are, in
principle, all organisms which are capable of synthesizing the
respective fine chemical, and which are suitable for the
activation, introduction or stimulation genes. Examples which may
be mentioned are plants, microorganisms such as fungi, bacteria,
yeasts, alga or diatom, transgenic or obtained by site directed
mutagenesis or random mutagenesis combined with specific selection
procedures. Preferred organisms are those which are naturally
capable of synthesizing the respective fine chemical in substantial
amounts, like fungi, yeasts, bactria or plants. In principle,
transgenic animals, for example Caenorhabditis elegans, are also
suitable as host organisms.
[12081] [0077.0.0.27] In the event that the transgenic organism is
a microorganism, such as a eukaryotic organism, for example a
fungus, an alga, diatom or a yeast in particular a fungus, alga,
diatom or yeast selected from the families Chaetomiaceae,
Choanephoraceae, Cryptococcaceae, Gun ninghamellaceae, Demetiaceae,
Moniliaceae, Mortierellaceae, Mucoraceae, Pythiaceae,
Sacharomycetaceae, Saprolegniaceae, Schizosacharomycetaceae,
Sodariaceae, Sporobolomycetaceae Tuberculariaceae, Adelotheciaceae,
Dinophyceae, Ditrichaceae or Prasinophyceae, or a prokaryotic
organism, for example a bacterium or blue alga, in particular a
bacterium from the families Actinomycetaceae, Bacillaceae,
Brevibacteriaceae, Corynebacteriaceae, Enterobacteriacae,
Gordoniaceae, Nocardiaceae, Micrococcaceae, Mycobacteriaceae,
Pseudomonaceae, Rhizobiaceae or Streptomycetaceae, this
microorganism is grown on a solid or in a liquid medium which is
known to the skilled worker and suits the organism. After the
growing phase, the organisms can be harvested.
[12082] [0078.0.0.27] The microorganisms or the recovered, and if
desired isolated, respective fine chemical can then be processed
further directly into foodstuffs or animal feeds or for other
applications, for example according to the disclosures made in
EP-B-0 533 039 or EP-A-0 615 693, which are expressly incorporated
herein by reference. The fermentation broth or fermentation
products can be purified in the customary manner by extraction and
precipitation or via ion exchangers and other methods known to the
person skilled in the art and described herein below. Products of
these different work-up procedures are amino acids or amino acid
compositions which still comprise fermentation broth and cell
components in different amounts, advantageously in the range of
from 0 to 99% by weight, preferably below 80% by weight, especially
preferably between below 50% by weight.
[12083] [0079.0.0.27] Preferred microorganisms are selected from
the group consisting of Chaetomiaceae such as the genera Chaetomium
e.g. the species Chaetomidium fimeti; Choanephoraceae such as the
genera Blakeslea, Choanephora e.g. the species Blakeslea trispora,
Choanephora cucurbitarum or Choanephora infundibulifera var.
cucurbitarum; Cryptococcaceae such as the genera Candida,
Crytococcus, Rhodotorula, Torulopsis e.g. the species Candida
albicans, Candida albomarginata, Candida antarctica, Candida
bacarum, Candida bogoriensis, Candida boidinii, Candida bovina,
Candida brumptii, Candida cacaoi, Candida cariosifignicola, Candida
catenulata, Candida chalmersii, Candida ciferrii, Candida
cylindracea, Candida edax, Candida emobii, Candida famata, Candida
freyschussii, Candida friedrichii, Candida glabrata, Candida
guilfiermondii, Candida haemulonii, Candida humicola, Candida
inconspicua, Candida ingens, Candida intermedia, Candida kefyr,
Candida krusei, Candida lactiscondensi, Candida lambica, Candida
fipolyfica, Candida lusitaniae, Candida macedoniensis, Candida
magnoliae, Candida membranaefaciens, Candida mesenterica, Candida
multigemmis, Candida mycoderma, Candida nemodendra, Candida
nitratophila, Candida norvegensis, Candida norvegica, Candida
parapsilosis, Candida pelliculosa, Candida peltata, Candida pini,
Candida pseudotropicalis, Candida pulcherrima, Candida punicea,
Candida pustula, Candida ravautii, Candida reukaufii, Candida
rugosa, Candida sake, Candida silvicola, Candida solani, Candida
sp., Candida spandovensis, Candida succiphila, Candida tropicalis,
Candida utilis, Candida valida, Candida versatilis, Candida vini,
Candida zeylanoides, Cryptococcus albidus, Cryptococcus curvatus,
Cryptococcus flavus, Cryptococcus humicola, Cryptococcus
hungaricus, Cryptococcus kuetzingii, Cryptococcus laurentii,
Cryptococcus macerans, Cryptococcus neoformans, Cryptococcus
terreus, Cryptococcus uniguttulatus, Rhodotorula acheniorum,
Rhodotorula bacarum, Rhodotorula bogoriensis, Rhodotorula flava,
Rhodotorula glutinis, Rhodotorula macerans, Rhodotorula minuta,
Rhodotorula mucilaginosa, Rhodotorula pilimanae, Rhodotorula
pustula, Rhodotorula rubra, Rhodotorula tokyoensis, Torulopsis
colliculosa, Torulopsis dattila or Torulopsis neoformans;
Cunninghamellaceae such as the genera Cunninghamella e.g. the
species Cunninghamella blakesleeana, Cunninghamella echinulata,
Cunninghamella echinulata var. elegans, Cunninghamella elegans or
Cunninghamella homothalfica; Demetiaceae such as the genera
Alternaria, Bipolaris, Cercospora, Chalara, Cladosporium,
Curvularia, Exophilia, Helicosporium, Helminthosporium, Orbimyces,
Philalophora, Pithomyces, Spilocaea, Thielaviopsis, Wangiella e.g.
the species Curvularia affinis, Curvularia c/avata, Curvularia
fallax, Curvularia inaequalis, Curvularia indica, Curvularia
lunata, Curvularia pallescens, Curvularia verruculosa or
Helminothosporium sp.; Moniliaceae such as the genera Arthrobotrys,
Aspergillus, Epidermophyton, Geotrichum, Gliocladium, Histoplasma,
Microsporum, Monilia, Oedocephalum, Oidium, Penicillium,
Trichoderma, Trichophyton, Thrichoteclum, Verticillium e.g. the
species Aspergillus aculeatus, Aspergillus a/bus, Aspergillus
alliaceus, Aspergillus asperescens, Aspergillus awamori,
Aspergillus candidus, Aspergillus carbonarius, Aspergillus carneus,
Aspergillus chevalieri, Aspergillus chevalieri var. intermedius,
Aspergillus clavatus, Aspergillus ficuum, Aspergillus flavipes,
Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus,
Aspergillus giganteus, Aspergillus humicola, Aspergillus
intermedius, Aspergillus japonicus, Aspergillus nidulans,
Aspergillus niger, Aspergillus niveus, Aspergillus ochraceus,
Aspergillus oryzae, Aspergillus ostianus, Aspergillus parasiticus,
Aspergillus parasiticus var. globosus, Aspergillus penicillioides,
Aspergillus phoenicis, Aspergillus rugulosus, Aspergillus
sclerotiorum, Aspergillus sojae var. gymnosardae, Aspergillus
sydowii, Aspergillus tamarii, Aspergillus terreus, Aspergillus
terricola, Aspergillus toxicarius, Aspergillus unguis, Aspergillus
ustus, Aspergillus versicolor, Aspergillus vitricolae, Aspergillus
wentii, .cndot.Penicillium adametzi, .cndot.Penicillium albicans,
.cndot.Penicillium arabicum, .cndot.Penicillium arenicola,
.cndot.Penicillium argillaceum, .cndot.Penicillium arvense,
.cndot.Penicillium asperosporum, .cndot.Penicillium
aurantiogriseum, .cndot.Penicillium avellaneum, .cndot.Penicillium
baamense, .cndot.Penicillium bacillisporum, .cndot.Penicillium
brasilianum, .cndot.Penicillium brevicompactum, .cndot.Penicillium
camemberti, .cndot.Penicillium canadense, .cndot.Penicillium
canescens, .cndot.Penicillium caperatum, .cndot.Penicillium
capsulatum, .cndot.Penicillium caseicolum, .cndot.Penicillium
chrysogenum, .cndot.Penicillium citreonigrum, .cndot.Penicillium
citrinum, .cndot.Penicillium claviforme, .cndot.Penicillium
commune, .cndot.Penicillium corylophilum, .cndot.Penicillium
corymbiferum, .cndot.Penicillium crustosum, .cndot.Penicillium
cyclopium, .cndot.Penicillium daleae, .cndot.Penicillium decumbens,
.cndot.Penicillium dierckxii, .cndot.Penicillium digitatum,
.cndot.Penicillium digitatum var. latum, .cndot.Penicillium
divaricatum, .cndot.Penicillium diversum, .cndot.Penicillium
duclauxii, .cndot.Penicillium echinosporum, .cndot.Penicillium
expansum, .cndot.Penicillium fellutanum, .cndot.Penicillium
frequentans, .cndot.Penicillium funiculosum, .cndot.Penicillium
glabrum, .cndot.Penicillium gladioli, .cndot.Penicillium
griseofulvum, .cndot.Penicillium hirsutum, .cndot.Penicillium
hispanicum, .cndot.Penicillium islandicum, .cndot.Penicillium
italicum, .cndot.Penicillium italicum var. avellaneum,
.cndot.Penicillium janczewskii, .cndot.Penicillium janthinellum,
.cndot.Penicillium japonicum, .cndot.Penicillium lavendulum,
.cndot.Penicillium lilacinum, .cndot.Penicillium lividum,
.cndot.Penicillium martensii, .cndot.Penicillium megasporum,
.cndot.Penicillium miczynskii, .cndot.Penicillium nalgiovense,
.cndot.Penicillium nigricans, .cndot.Penicillium notatum,
.cndot.Penicillium ochrochloron, .cndot.Penicillium odoratum,
.cndot.Penicillium oxalicum, .cndot.Penicillium paraherquei,
.cndot.Penicillium patulum, .cndot.Penicillium pinophilum,
.cndot.Penicillium piscarium, .cndot.Penicillium pseudostromaticum,
.cndot.Penicillium puberulum, .cndot.Penicillium purpurogenum,
.cndot.Penicillium raciborskii, .cndot.Peniciffium roqueforti,
.cndot.Penicillium rotundum, .cndot.Penicillium rubrum,
.cndot.Penicillium sacculum, .cndot.Penicillium simplicissimum,
Penicillium sp., Penicillium spinulosum, Penicillium steckii,
Penicillium stoloniferum, Penicillium striatisporum, Penicillium
striatum, Penicillium tardum, Penicillium thomii, Penicillium
turbatum, Penicillium variabile, Penicillium vermiculatum,
Penicillium vermoesenii, Penicillium verrucosum, Penicillium
verrucosum var. corymbiferum, Penicillium verrucosum var.
cyclopium, Penicillium verruculosum, Penicillium vinaceum,
Penicillium violaceum, Penicillium viridicatum, Penicillium
vulpinum, Trichoderma hamatum, Trichoderma harzianum, Trichoderma
koningii, Trichoderma longibrachiatum, Trichoderma polysporum,
Trichoderma reesei, Trichoderma virens or Trichoderma viride;
Mortierellaceae such as the genera Mortierella e.g. the species
Mortierella isabellina, Mortierella polycephala, Mortierella
ramanniana, Mortierella vinacea or Mortierella zonata; Mucoraceae
such as the genera Actinomucor, Mucor, Phycomyces, Rhizopus,
Zygorhynchus e.g. the species Mucor amphibiorum, Mucor
circinelloides f. circinelloides, Mucor circinelloides var.
griseocyanus, Mucor flavus, Mucor fuscus, Mucor griseocyanus, Mucor
heterosporus, Mucor hiemalis, Mucor hiemalis f. hiemalis, Mucor
inaequisporus, Mucor indicus, Mucorjavanicus, Mucor mucedo, Mucor
mucilagineus, Mucor piriformis, Mucor plasmaticus, Mucor plumbeus,
Mucor racemosus, Mucor racemosus f. racemosus, Mucor racemosus f.
sphaerosporus, Mucor rouxianus, Mucor Mucor sinensis, Mucor sp.,
Mucor spinosus, Mucor tuberculisporus, Mucor variisporus, Mucor
variosporus, Mucor wosnessenskii, Phycomyces blakesleeanus,
Rhizopus achlamydosporus, Rhizopus arrhizus, Rhizopus chinensis,
Rhizopus delemar, Rhizopus formosaensis, Rhizopus japonicus,
Rhizopus javanicus, Rhizopus microsporus, Rhizopus microsporus var.
chinensis, Rhizopus microsporus var. oligosporus, Rhizopus
microsporus var. rhizopodiformis, Rhizopus nigricans, Rhizopus
niveus, Rhizopus oligosporus, Rhizopus oryzae, Rhizopus pygmaeus,
Rhizopus rhizopodiformis, Rhizopus semarangensis, Rhizopus sontii,
Rhizopus stolonifer, Rhizopus thermosus, Rhizopus tonkinensis,
Rhizopus tritici or Rhizopus usamii; Pythiaceae such as the genera
Phytium, Phytophthora e.g. the species Pythium debaryanum, Pythium
intermedium, Pythium irregulars, Pythium megalacanthum, Pythium
paroecandrum, Pythium sylvaticum, Pythium ultimum, Phytophthora
cactorum, Phytophthora cinnamomi, Phytophthora citricola,
Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora
drechsleri, Phytophthora erythroseptica, Phytophthora lateralis,
Phytophthora megasperma, Phytophthora nicotianae, Phytophthora
nicotianae var. parasitica, Phytophthora palmivora, Phytophthora
parasitica or Phytophthora syringae; Sacharomycetaceae such as the
genera Hansenula, Pichia, Saccharomyces, Saccharomycodes, Yarrowia
e.g. the species Hansenula anomala, Hansenula californica,
Hansenula canadensis, Hansenula capsulata, Hansenula ciferrii,
Hansenula glucozyma, Hansenula henricii, Hansenula holstii,
Hansenula minuta, Hansenula nonfermentans, Hansenula philodendri,
Hansenula polymorpha, Hansenula saturnus, Hansenula subpelliculosa,
Hansenula wickerhamii, Hansenula wingei, Pichia alcoholophila,
Pichia angusta, Pichia anomala, Pichia bispora, Pichia burtonii,
Pichia canadensis, Pichia capsulata, Pichia carsonii, Pichia
cellobiosa, Pichia ciferrii, Pichia farinosa, Pichia fermentans,
Pichia finlandica, Pichia glucozyma, Pichia guilfiermondii, Pichia
haplophila, Pichia henricii, Pichia holstfi, Pichia jadinii, Pichia
findnerii, Pichia membranaefaciens, Pichia methanolica, Pichia
minuta var. minuta, Pichia minuta var. nonfermentans, Pichia
norvegensis, Pichia ohmeri, Pichia pastoris, Pichia philodendri,
Pichia pini, Pichia polymorpha, Pichia quercuum, Pichia
rhodanensis, Pichia sargentensis, Pichia stipitis, Pichia
strasburgensis, Pichia subpelliculosa, Pichia toletana, Pichia
trehalophila, Pichia vini, Pichia xylosa, Saccharomyces aceti,
Saccharomyces bailiff, Saccharomyces bayanus, Saccharomyces
bisporus, Saccharomyces capensis, Saccharomyces carlsbergensis,
Saccharomyces cerevisiae, Saccharomyces cerevisiae var.
ellipsoideus, Saccharomyces chevalieri, Saccharomyces delbrueckii,
Saccharomyces diastaticus, Saccharomyces drosophilarum,
Saccharomyces elegans, Saccharomyces ellipsoideus, Saccharomyces
fermentati, Saccharomyces florentinus, Saccharomyces fragilis,
Saccharomyces heterogenicus, Saccharomyces hienipiensis,
Saccharomyces inusitatus, Saccharomyces italicus, Saccharomyces
kluyveri, Saccharomyces krusei, Saccharomyces lactis, Saccharomyces
marxianus, Saccharomyces microellipsoides, Saccharomyces montanus,
Saccharomyces norbensis, Saccharomyces oleaceus, Saccharomyces
paradoxus, Saccharomyces pastorianus, Saccharomyces pretoriensis,
Saccharomyces rosei, Saccharomyces rouxii, Saccharomyces uvarum,
Saccharomycodes ludwigii or Yarrowia lipolytica; Saprolegniaceae
such as the genera Saprolegnia e.g. the species Saprolegnia ferax;
Schizosacharomycetaceae such as the genera Schizosaccharomyces e.g.
the species Schizosaccharomyces japonicus var. japonicus,
Schizosaccharomyces japonicus var. versatilis, Schizosaccharomyces
malidevorans, Schizosaccharomyces octosporus, Schizosaccharomyces
pombe var. malidevorans or Schizosaccharomyces pombe var. pombe;
Sodariaceae such as the genera Neurospora, Sordaria e.g. the
species Neurospora africana, Neurospora crassa, Neurospora
intermedia, Neurospora sitophila, Neurospora tetrasperma, Sordaria
fimicola or Sordaria macrospora; Tuberculariaceae such as the
genera Epicoccum, Fusarium, Myrothecium, Sphacelia, Starkeyomyces,
Tubercularia e.g. the species Fusarium acuminatum, Fusarium
anthophilum, Fusarium aquaeductuum, Fusarium aquaeductuum var.
medium, Fusarium avenaceum, Fusarium buharicum, Fusarium
camptoceras, Fusarium cerealis, Fusarium chlamydosporum, Fusarium
ciliatum, Fusarium coccophilum, Fusarium coeruleum, Fusarium
concolor, Fusarium crookwellense, Fusarium culmorum, Fusarium
dimerum, Fusarium diversisporum, Fusarium equiseti, Fusarium
equiseti var. bullatum, Fusarium eumartii, Fusarium flocciferum,
Fusarium fujikuroi, Fusarium graminearum, Fusarium graminum,
Fusarium heterosporum, Fusarium incarnatum, Fusarium inflexum,
Fusarium javanicum, Fusarium lateritium, Fusarium lateritium var.
majus, Fusarium longipes, Fusarium melanochlorum, Fusarium
merismoides, Fusarium merismoides var. chlamydosporale, Fusarium
moniliforme, Fusarium moniliforme var. anthophilum, Fusarium
moniliforme var. subglutinans, Fusarium nivale, Fusarium nivale
var. majus, Fusarium oxysporum, Fusarium oxysporum f. sp. aechmeae,
Fusarium oxysporum f. sp. cepae, Fusarium oxysporum f. sp.
conglutinans, Fusarium oxysporum f. sp. cucumerinum, Fusarium
oxysporum f. sp. cyclaminis, Fusarium oxysporum f. sp. dianthi,
Fusarium oxysporum f. sp. lycopersici, Fusarium oxysporum f. sp.
melonis, Fusarium oxysporum f. sp. passiflorae, Fusarium oxysporum
f. sp. pisi, Fusarium oxysporum f. sp. tracheiphilum, Fusarium
oxysporum f. sp. tuberosi, Fusarium oxysporum f. sp. tulipae,
Fusarium oxysporum f. sp. vasinfectum, Fusarium pallidoroseum,
Fusarium poae, Fusarium proliferatum, Fusarium proliferatum var.
minus, Fusarium redolens, Fusarium redolens f. sp. dianthi,
Fusarium reticulatum, Fusarium roseum, Fusarium sacchari var.
elongatum, Fusarium sambucinum, Fusarium sambucinum var. coeruleum,
Fusarium semitectum, Fusarium semitectum var. majus, Fusarium
solani, Fusarium solani f. sp. pisi, Fusarium sporotrichioides,
Fusarium sporotrichioides var. minus, Fusarium sublunatum, Fusarium
succisae, Fusarium sulphureum, Fusarium tabacinum, Fusarium
tricinctum, Fusarium udum, Fusarium ventricosum, Fusarium
verticillioides, Fusarium xylarioides or Fusarium zonatum;
Sporobolomycetaceae such as the genera Bullera, Sporobolomyces,
Itersonilia e.g. the species Sporobolomyces holsaticus,
Sporobolomyces odorus, Sporobolomyces puniceus, Sporobolomyces
salmonicolor, Sporobolomyces singularis or Sporobolomyces tsugae;
Adelotheciaceae such as the genera e.g. the species Physcomitrella
patens; Dinophyceae such as the genera Crypthecodinium,
Phaeodactylum e.g. the species Crypthecodinium cohnii or
Phaeodactylum tricornutum; Ditrichaceae such as the genera
Ceratodon, Pleuridium, Astomiopsis, Ditrichum, Philibertiella,
Ceratodon, Distichium, Skottsbergia e.g. the species Ceratodon
antarcticus, Ceratodon purpureus, Ceratodon purpureus ssp.
convolutes or Ceratodon purpureus ssp. stenocarpus; Prasinophyceae
such as the genera
Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis, Mantoniella,
Ostreococcus e.g. the species Nephroselmis olivacea, Prasinococcus
capsulatus, Scherffelia dubia, Tetraselmis chui, Tetraselmis
suecica, Mantoniella squamata or Ostreococcus tauri;
Actinomycetaceae such as the genera Actinomyces, Actinobaculum,
Arcanobacterium, Mobiluncus e.g. the species Actinomyces bemardiae,
Actinomyces bovis, Actinomyces bowdenii, Actinomyces canis,
Actinomyces cardiffensis, Actinomyces catuli, Actinomyces
coleocanis, Actinomyces denticolens, Actinomyces europaeus,
Actinomyces funkei, Actinomyces georgiae, Actinomyces gerencseriae,
Actinomyces hordeovulneris, Actinomyces howellii, Actinomyces
humiferus, Actinomyces hyovaginalis, Actinomyces israelii,
Actinomyces marimammalium, Actinomyces meyeri, Actinomyces
naeslundii, Actinomyces nasicola, Actinomyces neuii subsp.
anitratus, Actinomyces neuii subsp. neuii, Actinomyces
odontolyticus, Actinomyces oricola, Actinomyces pyogenes,
Actinomyces radicidentis, Actinomyces radingae, Actinomyces
slackii, Actinomyces suimastitidis, Actinomyces suis, Actinomyces
turicensis, Actinomyces urogenitalis, Actinomyces vaccimaxillae,
Actinomyces viscosus, Actinobaculum schaalii, Actinobaculum suis,
Actinobaculum urinale, Arcanobacterium bemardiae, Arcanobacterium
haemolyticum, Arcanobacterium hippocoleae, Arcanobacterium phocae,
Arcanobacterium pluranimalium, Arcanobacterium pyogenes, Mobiluncus
curtisfi subsp. Mobiluncus curtisfi subsp. holmesfi or Mobiluncus
mulieris; Bacillaceae such as the genera Amphibacillus,
Anoxybacillus, Bacillus, Exiguobacterium, Gracilibacillus,
Holobacillus, Saccharococcus, Salibacillus, Virgibacillus e.g. the
species Amphibacillus fermentum, Amphibacillus tropicus,
Amphibacillus xylanus, Anoxybacillus flavithermus, Anoxybacillus
gonensis, Anoxybacillus pushchinoensis, Bacillus acidocaldarius,
Bacillus acidoterrestris, Bacillus aeolius, Bacillus agaradhaerens,
Bacillus agri, Bacillus alcalophilus, Bacillus alginolyticus,
Bacillus alvei, Bacillus amyloliquefaciens, Bacillus amylolyticus,
Bacillus aneurinilyticus, Bacillus aquimaris, Bacillus
arseniciselenatis, Bacillus atrophaeus, Bacillus azotofixans,
Bacillus azotoformans, Bacillus badius, Bacillus barbaricus,
Bacillus benzoevorans, Bacillus borstelensis, Bacillus brevis,
Bacillus carboniphilus, Bacillus centrosporus, Bacillus cereus,
Bacillus chitinolyticus, Bacillus chondroitinus, Bacillus
choshinensis, Bacillus circulans, Bacillus clarkii, Bacillus
clausii, Bacillus coagulans, Bacillus cohnii, Bacillus
curdlanolyticus, Bacillus cycloheptanicus, Bacillus decolorationis,
Bacillus dipsosauri, Bacillus edaphicus, Bacillus ehimensis,
Bacillus endophyticus, Bacillus fastidiosus, Bacillus firmus,
Bacillus flexus, Bacillus formosus, Bacillus fumarioli, Bacillus
funiculus, Bacillus fusiformis, Bacillus sphaericus subsp.
fusiformis, Bacillus galactophilus, Bacillus globisporus, Bacillus
globisporus subsp. marinus, Bacillus glucanolyticus, Bacillus
gordonae, Bacillus halmapalus, Bacillus haloalkaliphilus, Bacillus
halodenitrificans, Bacillus halodurans, Bacillus halophilus,
Bacillus horikoshii, Bacillus horti, Bacillus infernos, Bacillus
insolitus, Bacillus jeotgali, Bacillus kaustophilus, Bacillus
kobensis, Bacillus krulwichiae, Bacillus laevolacticus, Bacillus
larvae, Bacillus laterosporus, Bacillus lautus, Bacillus
lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus
luciferensis, Bacillus macerans, Bacillus macquariensis, Bacillus
marinus, Bacillus marisflavi, Bacillus marismortui, Bacillus
megaterium, Bacillus methanolicus, Bacillus migulanus, Bacillus
mojavensis, Bacillus mucilaginosus, Bacillus mycoides, Bacillus
naganoensis, Bacillus nealsonii, Bacillus neidei, Bacillus niacini,
Bacillus okuhidensis, Bacillus oleronius, Bacillus pabuli, Bacillus
pallidus, Bacillus pantothenticus, Bacillus parabrevis, Bacillus
pasteurii, Bacillus peoriae, Bacillus polymyxa, Bacillus popilliae,
Bacillus pseudalcaliphilus, Bacillus pseudofirmus, Bacillus
pseudomycoides, Bacillus psychrodurans, Bacillus psychrophilus,
Bacillus psychrosaccharolyticus, Bacillus psychrotolerans, Bacillus
pulvifaciens, Bacillus pumilus, Bacillus pycnus, Bacillus reuszeri,
Bacillus salexigens, Bacillus schlegelii, Bacillus
selenitireducens, Bacillus silvestris, Bacillus simplex, Bacillus
siralis, Bacillus smithii, Bacillus sonorensis, Bacillus
sphaericus, Bacillus sporothermodurans, Bacillus
stearothermophilus, Bacillus subterraneus, Bacillus subtilis subsp.
spizizenii, Bacillus subtilis subsp. subtilis, Bacillus
thermantarcticus, Bacillus thermoaerophilus, Bacillus
thermoamylovorans, Bacillus thermoantarcticus, Bacillus
thermocatenulatus, Bacillus thermocloacae, Bacillus
thermodenitrificans, Bacillus thermoglucosidasius, Bacillus
thermoleovorans, Bacillus thermoruber, Bacillus thermosphaericus,
Bacillus thiaminolyticus, Bacillus thuringiensis, Bacillus tusciae,
Bacillus validus, Bacillus vallismortis, Bacillus vedderi, Bacillus
vulcani, Bacillus weihenstephanensis, Exiguobacterium acetylicum,
Exiguobacterium antarcticum, Exiguobacterium aurantiacum,
Exiguobacterium undae, Gracilibacillus dipsosauri, Gracilibacillus
halotolerans, Halobacillus halophilus, Halobacillus karajensis,
Halobacillus litoralis, Halobacillus salinus, Halobacillus
trueperi, Saccharococcus caldoxylosilyticus, Saccharococcus
thermophilus, Salibacillus marismortui, Salibacillus salexigens,
Virgibacillus carmonensis, Virgibacillus marismortui, Virgibacillus
necropolis, Virgibacillus pantothenticus, Virgibacillus picturae,
Virgibacillus proomii or Virgibacillus salexigens,
Brevibacteriaceae such as the genera Brevibacterium e.g. the
species Brevibacterium acetylicum, Brevibacterium albidum,
Brevibacterium ammoniagenes, Brevibacterium avium, Brevibacterium
casei, Brevibacterium citreum, Brevibacterium divaricatum,
Brevibacterium epidermidis, Brevibacterium fermentans,
Brevibacterium frigoritolerans, Brevibacterium halotolerans,
Brevibacterium imperiale, Brevibacterium incertum, Brevibacterium
iodinum, Brevibacterium linens, Brevibacterium liquefaciens,
Brevibacterium lutescens, Brevibacterium luteum, Brevibacterium
lyticum, Brevibacterium mcbrellneri, Brevibacterium otitidis,
Brevibacterium oxydans, Brevibacterium paucivorans, Brevibacterium
protophormiae, Brevibacterium pusillum, Brevibacterium saperdae,
Brevibacterium stationis, Brevibacterium testaceum or
Brevibacterium vitaeruminis; Corynebacteriaceae such as the genera
Corynebacterium e.g. the species Corynebacterium accolens,
Corynebacterium afermentans subsp. afermentans, Corynebacterium
afermentans subsp. lipophilum, Corynebacterium ammoniagenes,
Corynebacterium amycolatum, Corynebacterium appendicis,
Corynebacterium aquilae, Corynebacterium argentoratense,
Corynebacterium atypicum, Corynebacterium aurimucosum,
Corynebacterium auris, Corynebacterium auriscanis, Corynebacterium
betae, Corynebacterium beticola, Corynebacterium bovis,
Corynebacterium callunae, Corynebacterium camporealensis,
Corynebacterium capitovis, Corynebacterium casei, Corynebacterium
confusum, Corynebacterium coyleae, Corynebacterium cystitidis,
Corynebacterium durum, Corynebacterium efficiens, Corynebacterium
equi, Corynebacterium falsenii, Corynebacterium fascians,
Corynebacterium felinum, Corynebacterium flaccumfaciens,
Corynebacterium flavescens, Corynebacterium freneyi,
Corynebacterium glaucum, Corynebacterium glucuronolyticum,
Corynebacterium glutamicum, Corynebacterium Corynebacterium ilicis,
Corynebacterium imitans, Corynebacterium insidiosum,
Corynebacterium iranicum, Corynebacterium jeikeium, Corynebacterium
kroppenstedtii, Corynebacterium kutscheri, Corynebacterium Ilium,
Corynebacterium lipophiloflavum, Corynebacterium macginleyi,
Corynebacterium mastitidis, Corynebacterium matruchotii,
Corynebacterium michiganense, Corynebacterium michiganense subsp.
tessellarius, Corynebacterium minutissimum, Corynebacterium
mooreparkense, Corynebacterium mucifaciens, Corynebacterium
mycetoides, Corynebacterium nebraskense, Corynebacterium oortii,
Corynebacterium paurometabolum, Corynebacterium phocae,
Corynebacterium pilosum, Corynebacterium poinsettiae,
Corynebacterium propinquum, Corynebacterium pseudodiphtheriticum,
Corynebacterium pseudotuberculosis, Corynebacterium pyogenes,
Corynebacterium rathayi, Corynebacterium renale, Corynebacterium
riegelii, Corynebacterium seminale, Corynebacterium sepedonicum,
Corynebacterium simulans, Corynebacterium singulare,
Corynebacterium sphenisci, Corynebacterium spheniscorum,
Corynebacterium striatum, Corynebacterium suicordis,
Corynebacterium sundsvallense, Corynebacterium terpenotabidum,
Corynebacterium testudinoris, Corynebacterium thomssenii,
Corynebacterium tritici, Corynebacterium ulcerans, Corynebacterium
urealyticum, Corynebacterium variable, Corynebacterium vitaeruminis
or Corynebacterium xerosis; Enterobacteriacae such as the genera
Alterococcus, Arsenophonus, Brenneria, Buchnera, Budvicia,
Buttiauxella, Calymmatobacterium, Cedecea, Citrobacter,
Edwardsiella, Enterobacter, Erwinia, Escherichia, Ewingella,
Hafnia, Klebsiella, Kluyvera, Leclercia, Leminorella, Moellerella,
Morganella, Obesumbacterium, Pantoea, Pectobacterium, Photorhabdus,
Plesiomonas, Pragia, Proteus, Providencia, Rahnella,
Saccharobacter, Salmonella, Shigella, Serratia, Sodalis, Tatumella,
Trabulsiella, Wigglesworthia, Xenorhabdus, Yersinia and Yokenella
e.g. the species Arsenophonus nasoniae, Brenneria alni, Brenneria
nigrifluens, Brenneria quercina, Brenneria rubrifaciens, Brenneria
salicis, Budvicia aquatica, Buttiauxella agrestis, Buttiauxella
brennerae, Buttiauxella ferragutiae, Buttiauxella gaviniae,
Buttiauxella izardii, Buttiauxella noackiae, Buttiauxella
warmboldiae, Cedecea davisae, Cedecea lapagei, Cedecea neteri,
Citrobacter amalonaticus, Citrobacter diversus, Citrobacter
freundii, Citrobacter genomospecies, Citrobacter gillenii,
Citrobacter intermedium, Citrobacter koseri, Citrobacter murliniae,
Citrobacter sp., Edwardsiella hoshinae, Edwardsiella ictaluri,
Edwardsiella tarda, Erwinia alni, Erwinia amylovora, Erwinia
ananatis, Erwinia aphidicola, Erwinia billingiae, Erwinia
cacticida, Erwinia cancerogena, Erwinia camegieana, Erwinia
carotovora subsp. atroseptica, Erwinia carotovora subsp.
betavasculorum, Erwinia carotovora subsp. odorifera, Erwinia
carotovora subsp. wasabiae, Erwinia chrysanthemi, Erwinia
cypripedii, Erwinia dissolvens, Erwinia herbicola, Erwinia
mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia
nimipressuralis, Erwinia persicina, Erwinia psidii, Erwinia
pyrifoliae, Erwinia quercina, Erwinia rhapontici, Erwinia
rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwinia
tracheiphila, Erwinia uredovora, Escherichia adecarboxylata,
Escherichia anindolica, Escherichia aurescens, Escherichia blattae,
Escherichia coli, Escherichia coli var. communior, Escherichia
coli-mutabile, Escherichia fergusonii, Escherichia hermannii,
Escherichia sp., Escherichia vulneris, Ewingella americana, Hafnia
alvei, Klebsiella aerogenes, Klebsiella edwardsii subsp. atlantae,
Klebsiella omithinolytica, Klebsiella oxytoca, Klebsiella
planticola, Klebsiella pneumoniae, Klebsiella pneumoniae subsp.
pneumoniae, Klebsiella sp., Klebsiella terrigena, Klebsiella
trevisanii, Kluyvera ascorbata, Kluyvera citrophila, Kluyvera
cochleae, Kluyvera cryocrescens, Kluyvera georgiana, Kluyvera
noncitrophila, Kluyvera sp., Leclercia adecarboxylata, Leminorella
grimontii, Leminorella richardii, Moellerella wisconsensis,
Morganella morganii, Morganella morganii subsp. morganii,
Morganella morganii subsp. Obesumbaterium proteus, Pantoea
agglomerans, Pantoea ananatis, Pantoea citrea, Pantoea dispersa,
Pantoea punctata, Pantoea stewartii subsp. stewartii, Pantoea
terrea, Pectobacterium atrosepticum, Pectobacterium carotovorum
subsp. atrosepticum, Pectobacterium carotovorum subsp. carotovorum,
Pectobacterium chrysanthemi, Pectobacterium cypripedii,
Photorhabdus asymbiotica, Photorhabdus luminescens, Photorhabdus
luminescens subsp. akhurstii, Photorhabdus luminescens subsp.
laumondii, Photorhabdus luminescens subsp. luminescens,
Photorhabdus sp., Photorhabdus temperata, Plesiomonas shigelloides,
Pragia fontium, Proteus hauseri, Proteus ichthyosmius, Proteus
inconstans, Proteus mirabilis, Proteus morganii, Proteus
myxofaciens, Proteus penneri, Proteus rettgeri, Proteus
shigelloides, Proteus vulgaris, Providencia alcalifaciens,
Providencia friedericiana, Providencia heimbachae, Providencia
rettgeri, Providencia rustigianii, Providencia stuartii, Rahnella
aquatilis, Salmonella abony, Salmonella arizonae, Salmonella
bongori, Salmonella choleraesuis subsp. arizonae, Salmonella
choleraesuis subsp. bongori, Salmonella choleraesuis subsp.
cholereasuis, Salmonella choleraesuis subsp. diarizonae, Salmonella
choleraesuis subsp. houtenae, Salmonella choleraesuis subsp.
indica, Salmonella choleraesuis subsp. salamae, Salmonella
daressalaam, Salmonella enterica subsp. houtenae, Salmonella
enterica subsp. salamae, Salmonella enteritidis, Salmonella
gallinarum, Salmonella heidelberg, Salmonella panama, Salmonella
senftenberg, Salmonella typhimurium, Serratia entomophila, Serratia
ficaria, Serratia fonticola, Serratia Serratia liquefaciens,
Serratia marcescens, Serratia marcescens subsp. marcescens,
Serratia marinorubra, Serratia odorifera, Serratia plymouthensis,
Serratia plymuthica, Serratia proteamaculans, Serratia
proteamaculans subsp. quinovora, Serratia quinivorans, Serratia
rubidaea, Shigella boydii, Shigella flexneri, Shigella
paradysenteriae, Shigella sonnei, Tatumella ptyseos, Xenorhabdus
beddingii, Xenorhabdus bovienii, Xenorhabdus luminescens,
Xenorhabdus nematophila, Xenorhabdus nematophila subsp. beddingii,
Xenorhabdus nematophila subsp. bovienii, Xenorhabdus nematophila
subsp. poinarii or Xenorhabdus poinarii; Gordoniaceae such as the
genera Gordonia, Skermania e.g. the species Gordonia aichiensis,
Gordonia alkanivorans, Gordonia amarae, Gordonia amicalis, Gordonia
bronchialis, Gordonia desulfuricans, Gordonia hirsuta, Gordonia
hydrophobica, Gordonia namibiensis, Gordonia nitida, Gordonia
paraffinivorans, Gordonia polyisoprenivorans, Gordonia rhizosphera,
Gordonia rubripertincta, Gordonia sihwensis, Gordonia sinesedis,
Gordonia sputi, Gordonia terrae or Gordonia westfalica;
Micrococcaceae such as the genera Micrococcus, Arthrobacter,
Kocuria, Nesterenkonia, Renibacterium, Rothia, Stomatococcus e.g.
the species Micrococcus agilis, Micrococcus antarcticus,
Micrococcus halobius, Micrococcus kristinae, Micrococcus luteus,
Micrococcus lylae, Micrococcus nishinomiyaensis, Micrococcus
roseus, Micrococcus sedentarius, Micrococcus varians, Arthrobacter
agilis, Arthrobacter albus, Arthrobacter atrocyaneus, Arthrobacter
aurescens, Arthrobacter chlorophenolicus, Arthrobacter citreus,
Arthrobacter creatinolyticus, Arthrobacter crystallopoietes,
Arthrobacter cumminsii, Arthrobacter duodecadis, Arthrobacter
flavescens, Arthrobacter flavus, Arthrobacter gandavensis,
Arthrobacter globiformis, Arthrobacter histidinolovorans,
Arthrobacter Arthrobacter koreensis, Arthrobacter luteolus,
Arthrobacter methylotrophus, Arthrobacter mysorens, Arthrobacter
nasiphocae, Arthrobacter nicotianae, Arthrobacter nicotinovorans,
Arthrobacter oxydans, Arthrobacter pascens, Arthrobacter
picolinophilus, Arthrobacter polychromogenes, Arthrobacter
protophormiae, Arthrobacter psychrolactophilus, Arthrobacter
radiotolerans, Arthrobacter ramosus, Arthrobacter rhombi,
Arthrobacter roseus, Arthrobacter siderocapsulatus, Arthrobacter
simplex, Arthrobacter sulfonivorans, Arthrobacter sulfureus,
Arthrobacter terregens, Arthrobacter tumescens, Arthrobacter
uratoxydans, Arthrobacter ureafaciens, Arthrobacter variabilis,
Arthrobacter viscosus, Arthrobacter woluwensis, Kocuria
erythromyxa, Kocuria kristinae, Kocuria palustris, Kocuria polaris,
Kocuria rhizophila, Kocuria rosea, Kocuria varians, Nesterenkonia
halobia, Nesterenkonia lacusekhoensis, Renibacterium salmoninarum,
Rothia amarae, Rothia dentocariosa, Rothia mucilaginosa, Rothia
nasimurium
or Stomatococcus mucilaginosus; Mycobacteriaceae such as the genera
Mycobacterium e.g. the species Mycobacterium africanum,
Mycobacterium agri, Mycobacterium aichiense, Mycobacterium alvei,
Mycobacterium asiaticum, Mycobacterium aurum, Mycobacterium
austroafricanum, Mycobacterium bohemicum, Mycobacterium botniense,
Mycobacterium brumae, Mycobacterium chelonae subsp. abscessus,
Mycobacterium chitae, Mycobacterium chlorophenolicum, Mycobacterium
chubuense, Mycobacterium confluentis, Mycobacterium cookii,
Mycobacterium diernhoferi, Mycobacterium doricum, Mycobacterium
duvalii, Mycobacterium fallax, Mycobacterium farcinogenes,
Mycobacterium flavescens, Mycobacterium frederiksbergense,
Mycobacterium gadium, Mycobacterium gilvum, Mycobacterium gordonae,
Mycobacterium hassiacum, Mycobacterium hiberniae, Mycobacterium
hodleri, Mycobacterium holsaticum, Mycobacterium komossense,
Mycobacterium lacus, Mycobacterium madagascariense, Mycobacterium
mageritense, Mycobacterium montefiorense, Mycobacterium
moriokaense, Mycobacterium murale, Mycobacterium neoaurum,
Mycobacterium nonchromogenicum, Mycobacterium obuense,
Mycobacterium palustre, Mycobacterium parafortuitum, Mycobacterium
peregrinum, Mycobacterium phlei, Mycobacterium pinnipedii,
Mycobacterium poriferae, Mycobacterium pulveris, Mycobacterium
rhodesiae, Mycobacterium shottsii, Mycobacterium sphagni,
Mycobacterium terrae, Mycobacterium the rmoresistibile,
Mycobacterium tokaiense, Mycobacterium triviale, Mycobacterium
tusciae or Mycobacterium vanbaalenii; Nocardiaceae such as the
genera Nocardia, Rhodococcus e.g. the species Nocardia abscessus,
Nocardia africana, Nocardia amarae, Nocardia asteroides, Nocardia
autotrophica, Nocardia beijingensis, Nocardia brasiliensis,
Nocardia brevicatena, Nocardia caishijiensis, Nocardia calcarea,
Nocardia carnea, Nocardia cellulans, Nocardia cerradoensis,
Nocardia coeliaca, Nocardia corynebacterioides, Nocardia
crassostreae, Nocardia cummidelens, Nocardia cyriacigeorgica,
Nocardia farcinica, Nocardia flavorosea, Nocardia fluminea,
Nocardia globerula, Nocardia hydrocarbonoxydans, Nocardia ignorata,
Nocardia mediterranei, Nocardia nova, Nocardia orientalis, Nocardia
otitidis-caviarum, Nocardia otitidiscaviarum, Nocardia paucivorans,
Nocardia petroleophila, Nocardia pinensis, Nocardia
pseudobrasiliensis, Nocardia pseudovaccinii, Nocardia purls,
Nocardia restricta, Nocardia rugosa, Nocardia salmonicida, Nocardia
saturnea, Nocardia serioiae, Nocardia soli, Nocardia sulphurea,
Nocardia transvaiensis, Nocardia uniformis, Nocardia vaccinii,
Nocardia veterana or Nocardia vinacea; Pseudomonaceae such as the
genera Azomonas, Azotobacter, Cellvibrio, Chryseomonas,
Flaviomonas, Lampropedia, Mesophilobacter, Morococcus, Oligella,
Pseudomonas, Rhizobacter, Rugamonas, Serpens, Thermoleophilum,
Xylophilus e.g. the species Azomonas agilis, Azomonas insignis,
Azomonas macro cytogenes, Azotobacter agilis, Azotobacter agilis
subsp. armeniae, Azotobacter armeniacus, Azotobacter beijerinckii,
Azotobacter chroococcum, Azotobacter indicum, Azotobacter
macrocytogenes, Azotobacter miscellum, Azotobacter nigricans subsp.
nigricans, Azotobacter paspali, Azotobacter salinestris,
Azotobacter sp., Azotobacter vineiandii, Fiavimonas oryzihabitans,
Mesophilobacter marinus, Oligella urethralis, Pseudomonas
acidovorans, Pseudomonas aeruginosa, Pseudomonas agarici,
Pseudomonas alcaligenes, Pseudomonas aminovorans, Pseudomonas
amygdali, Pseudomonas andropogonis, Pseudomonas anguilliseptica,
Pseudomonas antarctica, Pseudomonas antimicrobica, Pseudomonas
antimycetica, Pseudomonas aptata, Pseudomonas arvilla, Pseudomonas
asplenii, Pseudomonas atiantica, Pseudomonas atrofaciens,
Pseudomonas aureofaciens, Pseudomonas avellanae, Pseudomonas
azeiaica, Pseudomonas azotocolligans, Pseudomonas baiearica,
Pseudomonas barkeri, Pseudomonas bathycetes, Pseudomonas
beijerinckii, Pseudomonas brassicacearum, Pseudomonas brenneri,
Pseudomonas butanovora, Pseudomonas carboxydoflava, Pseudomonas
carboxydohydrogena, Pseudomonas carboxydovorans, Pseudomonas
carrageenovora, Pseudomonas caryophylli, Pseudomonas cepacia,
Pseudomonas chloritidismutans, Pseudomonas chlororaphis,
Pseudomonas cichorii, Pseudomonas citronellolis, Pseudomonas
cocovenenans, Pseudomonas compransoris, Pseudomonas congeians,
Pseudomonas coronafaciens, Pseudomonas corrugata, Pseudomonas
dacunhae, Pseudomonas deiafieidii, Pseudomonas delphinii,
Pseudomonas denitrificans, Pseudomonas desmolytica, Pseudomonas
diminuta, Pseudomonas doudoroffii, Pseudomonas echinoides,
Pseudomonas eiongata, Pseudomonas extorquens, Pseudomonas
extremorientalis, Pseudomonas facilis, Pseudomonas ficuserectae,
Pseudomonas flava, Pseudomonas flavescens, Pseudomonas fluorescens,
Pseudomonas fragi, Pseudomonas frederiksbergensis, Pseudomonas
fulgida, Pseudomonas fuscovaginae, Pseudomonas gazotropha,
Pseudomonas gladioli, Pseudomonas glathei, Pseudomonas glumae,
Pseudomonas graminis, Pseudomonas halophila, Pseudomonas helianthi,
Pseudomonas huttiensis, Pseudomonas hydrogenothermophila,
Pseudomonas hydrogenovora, Pseudomonas indica, Pseudomonas
indigofera, Pseudomonas iodinum, Pseudomonas kilonensis,
Pseudomonas lachrymans, Pseudomonas lapsa, Pseudomonas lemoignei,
Pseudomonas lemonnieri, Pseudomonas lundensis, Pseudomonas luteola,
Pseudomonas maltophilia, Pseudomonas marginalis, Pseudomonas
marginata, Pseudomonas marina, Pseudomonas meliae, Pseudomonas
mendocina, Pseudomonas mesophilica, Pseudomonas mixta, Pseudomonas
monteilii, Pseudomonas morsprunorum, Pseudomonas multivorans,
Pseudomonas natriegens, Pseudomonas nautica, Pseudomonas
nitroreducens, Pseudomonas oleovorans, Pseudomonas oryzihabitans,
Pseudomonas ovalis, Pseudomonas oxalaticus, Pseudomonas palleronii,
Pseudomonas paucimobilis, Pseudomonas phaseolicola, Pseudomonas
phenazinium, Pseudomonas pickettii, Pseudomonas pisi, Pseudomonas
plantarii, Pseudomonas plecoglossicida, Pseudomonas poae,
Pseudomonas primulae, Pseudomonas proteolytica, Pseudomonas
pseudoalcaligenes, Pseudomonas pseudoalcaligenes subsp. konjaci,
Pseudomonas pseudoalcaligenes subsp. pseudoalcaligenes, Pseudomonas
pseudoflava, Pseudomonas putida, Pseudomonas putida var. naraensis,
Pseudomonas putrefaciens, Pseudomonas pyrrocinia, Pseudomonas
radiora, Pseudomonas reptilivora, Pseudomonas rhodesiae,
Pseudomonas rhodos, Pseudomonas riboflavina, Pseudomonas rubescens,
Pseudomonas rubrisubalbicans, Pseudomonas ruhlandii, Pseudomonas
saccharophila, Pseudomonas savastanoi, Pseudomonas savastanoi pvar.
glycinea, Pseudomonas savastanoi pvar. phaseolicola, Pseudomonas
solanacearum, Pseudomonas sp., Pseudomonas spinosa, Pseudomonas
stanieri, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas
syringae pvar. aptata, Pseudomonas syringae pvar. atrofaciens,
Pseudomonas syringae pvar. coronafaciens, Pseudomonas syringae
pvar. delphinii, Pseudomonas syringae pvar. glycinea, Pseudomonas
syringae pvar. helianthi, Pseudomonas syringae pvar. lachrymans,
Pseudomonas syringae pvar. lapsa, Pseudomonas syringae pvar.
morsprunorum, Pseudomonas syringae pvar. phaseolicola, Pseudomonas
syringae pvar. primulae, Pseudomonas syringae pvar. syringae,
Pseudomonas syringae pvar. tabaci, Pseudomonas syringae pvar.
tomato, Pseudomonas syringae subsp. glycinea, Pseudomonas syringae
subsp. savastanoi, Pseudomonas syringae subsp. syringae,
Pseudomonas syzygii, Pseudomonas tabaci, Pseudomonas
taeniospiralis, Pseudomonas testosteroni, Pseudomonas
thermocarboxydovorans, Pseudomonas thermotolerans, Pseudomonas
thivervalensis, Pseudomonas tomato, Pseudomonas trivialis,
Pseudomonas veronii, Pseudomonas vesicularis, Pseudomonas
viridiflava, Pseudomonas viscogena, Pseudomonas woodsii,
Rhizobacter dauci, Rhizobacter daucus or Xylophilus ampelinus;
Rhizobiaceae such as the genera Agrobacterium, Carbophilus,
Chelatobacter, Ensifer, Rhizobium, Sinorhizobium e.g. the species
Agrobacterium atlanticum, Agrobacterium ferrugineum, Agrobacterium
gelatinovorum, Agrobacterium larrymoorei, Agrobacterium meteori,
Agrobacterium radiobacter, Agrobacterium rhizogenes, Agrobacterium
rubi, Agrobacterium stellulatum, Agrobacterium tumefaciens,
Agrobacterium vitis, Carbophilus carboxidus, Chelatobacter
heintzii, Ensifer adhaerens, Ensifer arboris, Ensifer fredii,
Ensifer kostiensis, Ensifer kummerowiae, Ensifer medicae, Ensifer
meliloti, Ensifer saheli, Ensifer terangae, Ensifer xinjiangensis,
Rhizobium ciceri Rhizobium etli, Rhizobium fredii, Rhizobium
galegae, Rhizobium gallicum, Rhizobium giardinii, Rhizobium
hainanense, Rhizobium huakuii, Rhizobium huautlense, Rhizobium
indigoferae, Rhizobium japonicum, Rhizobium leguminosarum,
Rhizobium loessense, Rhizobium loti, Rhizobium lupini, Rhizobium
mediterraneum, Rhizobium meliloti, Rhizobium mongolense, Rhizobium
phaseoli, Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium
rubi, Rhizobium sullae, Rhizobium tianshanense, Rhizobium trifolii,
Rhizobium tropici, Rhizobium undicola, Rhizobium vitis,
Sinorhizobium adhaerens, Sinorhizobium arboris, Sinorhizobium
fredii, Sinorhizobium kostiense, Sinorhizobium kummerowiae,
Sinorhizobium medicae, Sinorhizobium meliloti, Sinorhizobium
morelense, Sinorhizobium saheli or Sinorhizobium xinjiangense;
Streptomycetaceae such as the genera Kitasatosprora, Streptomyces,
Streptoverticillium e.g. the species Streptomyces abikoensis,
Streptomyces aburaviensis, Streptomyces achromogenes subsp.
achromogenes, Streptomyces achromogenes subsp. rubradiris,
Streptomyces acid iscabies, Streptomyces acrimycini, Streptomyces
aculeolatus, Streptomyces afghaniensis, Streptomyces alanosinicus,
Streptomyces albaduncus, Streptomyces albiaxialis, Streptomyces
albidochromogenes, Streptomyces albidoflavus, Streptomyces
albireticuli, Streptomyces albofaciens, Streptomyces alboflavus,
Streptomyces albogriseolus, Streptomyces albolongus, Streptomyces
alboniger, Streptomyces albospinus, Streptomyces albosporeus subsp.
albosporeus, Streptomyces albosporeus subsp. labilomyceticus,
Streptomyces alboverticillatus, Streptomyces albovinaceus,
Streptomyces alboviridis, Streptomyces albulus, Streptomyces albus
subsp. albus, Streptomyces albus subsp. pathocidicus, Streptomyces
almquistii, Streptomyces althioticus, Streptomyces amakusaensis,
Streptomyces ambofaciens, Streptomyces aminophilus, Streptomyces
anandii, Streptomyces anthocyanicus, Streptomyces antibioticus,
Streptomyces antimycoticus, Streptomyces anulatus, Streptomyces
arabicus, Streptomyces ardus, Streptomyces arenae, Streptomyces
argenteolus, Streptomyces armeniacus, Streptomyces asiaticus,
Streptomyces asterosporus, Streptomyces atratus, Streptomyces
atroaurantiacus, Streptomyces atroolivaceus, Streptomyces
atrovirens, Streptomyces aurantiacus, Streptomyces aurantiogriseus,
Streptomyces aureocirculatus, Streptomyces aureofaciens,
Streptomyces aureorectus, Streptomyces aureoversilis, Streptomyces
aureoverticillatus, Streptomyces aureus, Streptomyces avellaneus,
Streptomyces avermectinius, Streptomyces avermitilis, Streptomyces
avidinii, Streptomyces azaticus, Streptomyces azureus, Streptomyces
baarnensis, Streptomyces bacillaris, Streptomyces badius,
Streptomyces baldaccii, Streptomyces bambergiensis, Streptomyces
beijiangensis, Streptomyces bellus, Streptomyces bikiniensis,
Streptomyces biverticillatus, Streptomyces blastmyceticus,
Streptomyces bluensis, Streptomyces bobili, Streptomyces
bottropensis, Streptomyces brasiliensis, Streptomyces bungoensis,
Streptomyces cacaoi subsp. asoensis, Streptomyces cacaoi subsp.
cacaoi, Streptomyces caelestis, Streptomyces caeruleus,
Streptomyces califomicus, Streptomyces calvus, Streptomyces
canaries, Streptomyces candidus, Streptomyces canescens,
Streptomyces cangkringensis, Streptomyces caniferus, Streptomyces
canus, Streptomyces capillispiralis, Streptomyces capoamus,
Streptomyces carpaticus, Streptomyces carpinensis, Streptomyces
catenulae, Streptomyces caviscabies, Streptomyces cavourensis
subsp. cavourensis, Streptomyces cavourensis subsp.
washingtonensis, Streptomyces cellostaticus, Streptomyces
celluloflavus, Streptomyces cellulolyticus, Streptomyces
cellulosae, Streptomyces champavatii, Streptomyces chartreuses,
Streptomyces chattanoogensis, Streptomyces chibaensis, Streptomyces
chrestomyceticus, Streptomyces chromofuscus, Streptomyces chryseus,
Streptomyces chrysomallus subsp. chrysomallus, Streptomyces
chrysomallus subsp. fumigatus, Streptomyces cinereorectus,
Streptomyces cinereoruber subsp. cinereoruber, Streptomyces
cinereoruber subsp. fructofermentans, Streptomyces cinereospinus,
Streptomyces cinereus, Streptomyces cinerochromogenes, Streptomyces
cinnabarinus, Streptomyces cinnamonensis, Streptomyces cinnamoneus,
Streptomyces cinnamoneus subsp. albosporus, Streptomyces
cinnamoneus subsp. cinnamoneus, Streptomyces cinnamoneus subsp.
lanosus, Streptomyces cinnamoneus subsp. sparsus, Streptomyces
cirratus, Streptomyces ciscaucasicus, Streptomyces
citreofluorescens, Streptomyces clavifer, Streptomyces
clavuligerus, Streptomyces cochleatus, Streptomyces coelescens,
Streptomyces coelicoflavus, Streptomyces coelicolor, Streptomyces
coeruleoflavus, Streptomyces coeruleofuscus, Streptomyces
coeruleoprunus, Streptomyces coeruleorubidus, Streptomyces
coerulescens, Streptomyces collinus, Streptomyces colombiensis,
Streptomyces corchorusii, Streptomyces costaricanus, Streptomyces
cremeus, Streptomyces crystallinus, Streptomyces curacoi,
Streptomyces cuspidosporus, Streptomyces cyaneofuscatus,
Streptomyces cyaneus, Streptomyces cyanoalbus, Streptomyces
cystargineus, Streptomyces daghestanicus, Streptomyces diastaticus
subsp. ardesiacus, Streptomyces diastaticus subsp. diastaticus,
Streptomyces diastatochromogenes, Streptomyces distallicus,
Streptomyces djakartensis, Streptomyces durhamensis, Streptomyces
echinatus, Streptomyces echinoruber, Streptomyces ederensis,
Streptomyces ehimensis, Streptomyces endus, Streptomyces
enissocaesilis, Streptomyces erumpens, Streptomyces erythraeus,
Streptomyces erythrogriseus, Streptomyces eurocidicus, Streptomyces
europaeiscabiei, Streptomyces eurythermus, Streptomyces exfoliates,
Streptomyces felleus, Streptomyces fervens, Streptomyces fervens
subsp. fervens, Streptomyces fervens subsp. melrosporus,
Streptomyces filamentosus, Streptomyces filipinensis, Streptomyces
fimbriatus, Streptomyces fimicarius, Streptomyces finlayi,
Streptomyces flaveolus, Streptomyces flaveus, Streptomyces
flavidofuscus, Streptomyces flavidovirens, Streptomyces
flavisderoticus, Streptomyces flavofungini, Streptomyces
flavofuscus, Streptomyces flavogriseus, Streptomyces flavopersicus,
Streptomyces flavotricini, Streptomyces flavovariabilis,
Streptomyces flavovirens, Streptomyces flavoviridis, Streptomyces
flocculus, Streptomyces floridae, Streptomyces fluorescens,
Streptomyces fradiae, Streptomyces fragilis, Streptomyces
fulvissimus, Streptomyces fulvorobeus, Streptomyces fumanus,
Streptomyces fumigatiscleroticus, Streptomyces galbus, Streptomyces
galilaeus, Streptomyces gancidicus, Streptomyces gardneri,
Streptomyces gelaticus, Streptomyces geysiriensis, Streptomyces
ghanaensis, Streptomyces gibsonii, Streptomyces glaucescens,
Streptomyces glaucosporus, Streptomyces glaucus, Streptomyces
globisporus
subsp. caucasicus, Streptomyces globisporus subsp. flavofuscus,
Streptomyces globisporus subsp. globisporus, Streptomyces globosus,
Streptomyces glomeratus, Streptomyces glomeroaurantiacus,
Streptomyces gobitricini, Streptomyces goshikiensis, Streptomyces
gougerotii, Streptomyces graminearus, Streptomyces graminofaciens,
Streptomyces griseinus, Streptomyces griseoaurantiacus,
Streptomyces griseobrunneus, Streptomyces griseocarneus,
Streptomyces griseochromogenes, Streptomyces griseoflavus,
Streptomyces griseofuscus, Streptomyces griseoincarnatus,
Streptomyces griseoloalbus, Streptomyces griseolosporeus,
Streptomyces griseolus, Streptomyces griseoluteus, Streptomyces
griseomycini, Streptomyces griseoplanus, Streptomyces griseorubens,
Streptomyces griseoruber, Streptomyces griseorubiginosus,
Streptomyces griseosporeus, Streptomyces griseostramineus,
Streptomyces griseoverticillatus, Streptomyces griseoviridis,
Streptomyces griseus subsp. alpha, Streptomyces griseus subsp.
cretosus, Streptomyces griseus subsp. griseus, Streptomyces griseus
subsp. solvifaciens, Streptomyces hachijoensis, Streptomyces
halstedii, Streptomyces hawaiiensis, Streptomyces heliomycini,
Streptomyces helvaticus, Streptomyces herbaricolor, Streptomyces
hiroshimensis, Streptomyces hirsutus, Streptomyces humidus,
Streptomyces humiferus, Streptomyces hydrogenans, Streptomyces
hygroscopicus subsp. angustmyceticus, Streptomyces hygroscopicus
subsp. decoyicus, Streptomyces hygroscopicus subsp. glebosus,
Streptomyces hygroscopicus subsp. hygroscopicus, Streptomyces
hygroscopicus subsp. ossamyceticus, Streptomyces iakyrus,
Streptomyces indiaensis, Streptomyces indigoferus, Streptomyces
indonesiensis, Streptomyces intermedius, Streptomyces inusitatus,
Streptomyces ipomoeae, Streptomyces janthinus, Streptomyces
javensis, Streptomyces kanamyceticus, Streptomyces kashmirensis,
Streptomyces kasugaensis, Streptomyces katrae, Streptomyces
kentuckensis, Streptomyces kifunensis, Streptomyces kishiwadensis,
Streptomyces kunmingensis, Streptomyces kurssanovii, Streptomyces
labedae, Streptomyces laceyi, Streptomyces ladakanum, Streptomyces
lanatus, Streptomyces lateritius, Streptomyces laurentii,
Streptomyces lavendofoliae, Streptomyces lavendulae subsp.
grasserius, Streptomyces lavendulae subsp. lavendulae, Streptomyces
lavenduligriseus, Streptomyces lavendulocolor, Streptomyces levis,
Streptomyces libani subsp. libani, Streptomyces libani subsp.
rufus, Streptomyces lienomycini, Streptomyces lilacinus,
Streptomyces limosus, Streptomyces lincolnensis, Streptomyces
lipmanii, Streptomyces litmocidini, Streptomyces lomondensis,
Streptomyces longisporoflavus, Streptomyces longispororuber,
Streptomyces longisporus, Streptomyces longwoodensis, Streptomyces
lucensis, Streptomyces luridiscabiei, Streptomyces luridus,
Streptomyces lusitanus, Streptomyces luteireticuli, Streptomyces
luteogriseus, Streptomyces luteosporeus, Streptomyces
luteoverticillatus, Streptomyces lydicus, Streptomyces macrosporus,
Streptomyces malachitofuscus, Streptomyces malachitospinus,
Streptomyces malaysiensis, Streptomyces mashuensis, Streptomyces
massasporeus, Streptomyces matensis, Streptomyces mauvecolor,
Streptomyces mediocidicus, Streptomyces mediolani, Streptomyces
megasporus, Streptomyces melanogenes, Streptomyces
melanosporofaciens, Streptomyces mexicanus, Streptomyces
michiganensis, Streptomyces microflavus, Streptomyces
minutiscleroticus, Streptomyces mirabilis, Streptomyces
misakiensis, Streptomyces misionensis, Streptomyces mobaraensis,
Streptomyces monomycini, Streptomyces morookaensis, Streptomyces
murinus, Streptomyces mutabilis, Streptomyces mutomycini,
Streptomyces naganishii, Streptomyces narbonensis, Streptomyces
nashvillensis, Streptomyces netropsis, Streptomyces neyagawaensis,
Streptomyces niger, Streptomyces nigrescens, Streptomyces
nigrifaciens, Streptomyces nitrosporeus, Streptomyces
niveiciscabiei, Streptomyces niveoruber, Streptomyces niveus,
Streptomyces noboritoensis, Streptomyces nodosus, Streptomyces
nogalater, Streptomyces nojiriensis, Streptomyces noursei,
Streptomyces novaecaesareae, Streptomyces ochraceiscieroticus,
Streptomyces odorifer, Streptomyces olivaceiscieroticus,
Streptomyces olivaceoviridis, Streptomyces olivaceus, Streptomyces
olivochromogenes, Streptomyces olivomycini, Streptomyces
olivoreticuli, Streptomyces olivoreticuli subsp. cellulophilus,
Streptomyces olivoreticuli subsp. olivoreticuli, Streptomyces
olivoverticillatus, Streptomyces olivoviridis, Streptomyces
omiyaensis, Streptomyces orinoci, Streptomyces pactum, Streptomyces
paracochleatus, Streptomyces paradoxus, Streptomyces
parvisporogenes, Streptomyces parvulus, Streptomyces parvus,
Streptomyces peucetius, Streptomyces phaeochromogenes, Streptomyces
phaeofaciens, Streptomyces phaeopurpureus, Streptomyces
phaeoviridis, Streptomyces phosaiacineus, Streptomyces pilosus,
Streptomyces platensis, Streptomyces plicatus, Streptomyces
pluricolorescens, Streptomyces polychromogenes, Streptomyces
poonensis, Streptomyces praecox, Streptomyces prasinopilosus,
Streptomyces prasinosporus, Streptomyces prasinus, Streptomyces
prunicoior, Streptomyces psammoticus, Streptomyces
pseudoechinosporeus, Streptomyces pseudogriseolus, Streptomyces
pseudovenezuelae, Streptomyces pulveraceus, Streptomyces puniceus,
Streptomyces puniciscabiei, Streptomyces purpeofuscus, Streptomyces
purpurascens, Streptomyces purpureus, Streptomyces
purpurogeneiscieroticus, Streptomyces racemochromogenes,
Streptomyces rameus, Streptomyces ramulosus, Streptomyces
rangoonensis, Streptomyces recifensis, Streptomyces
rectiverticillatus, Streptomyces rectivioiaceus, Streptomyces
regensis, Streptomyces resistomycificus, Streptomyces
reticuliscabiei, Streptomyces rhizosphaericus, Streptomyces rimosus
subsp. paromomycinus, Streptomyces rimosus subsp. rimosus,
Streptomyces rishiriensis, Streptomyces rochei, Streptomyces
roseiscieroticus, Streptomyces roseodiastaticus, Streptomyces
roseoflavus, Streptomyces roseofulvus, Streptomyces roseolilacinus,
Streptomyces roseolus, Streptomyces roseosporus, Streptomyces
roseoverticillatus, Streptomyces roseovioiaceus, Streptomyces
roseoviridis, Streptomyces rubber, Streptomyces rubiginosohelvolus,
Streptomyces rubiginosus, Streptomyces rubrogriseus, Streptomyces
rutgersensis subsp. casteiarensis, Streptomyces rutgersensis subsp.
rutgersensis, Streptomyces salmonis, Streptomyces sampsonii,
Streptomyces sanglieri, Streptomyces sannanensis, Streptomyces
sapporonensis, Streptomyces scabiei, Streptomyces sclerotialus,
Streptomyces scopiformis, Streptomyces seoulensis, Streptomyces
septatus, Streptomyces setae, Streptomyces setonii, Streptomyces
showdoensis, Streptomyces sindenensis, Streptomyces sioyaensis,
Streptomyces somaliensis, Streptomyces sparsogenes, Streptomyces
spectabilis, Streptomyces speibonae, Streptomyces speleomycini,
Streptomyces spheroids, Streptomyces spinoverrucosus, Streptomyces
spiralis, Streptomyces spiroverticillatus, Streptomyces
spitsbergensis, Streptomyces sporocinereus, Streptomyces
sporoclivatus, Streptomyces spororaveus, Streptomyces
sporoverrucosus, Streptomyces stelliscabiei, Streptomyces
stramineus, Streptomyces subrutilus, Streptomyces sulfonofaciens,
Streptomyces sulphurous, Streptomyces syringium, Streptomyces
tanashiensis, Streptomyces tauricus, Streptomyces tendae,
Streptomyces termitum, Streptomyces the rmoalcalitolerans,
Streptomyces thermoautotrophicus, Streptomyces
thermocarboxydovorans, Streptomyces thermocarboxydus, Streptomyces
thermocoprophilus, Streptomyces the rmodiastaticus, Streptomyces
thermogriseus, Streptomyces thermolineatus, Streptomyces
thermonitrificans, Streptomyces thermospinosisporus, Streptomyces
thermoviolaceus subsp. apingens, Streptomyces thermoviolaceus
subsp. thermoviolaceus, Streptomyces thermovulgaris, Streptomyces
thioluteus, Streptomyces torulosus, Streptomyces toxytricini,
Streptomyces tricolor, Streptomyces tubercidicus, Streptomyces
tuirus, Streptomyces turgidiscabies, Streptomyces umbrinus,
Streptomyces variabilis, Streptomyces variegates, Streptomyces
varsoviensis, Streptomyces vastus, Streptomyces venezuelae,
Streptomyces vinaceus, Streptomyces vinaceusdrappus, Streptomyces
violaceochromogenes, Streptomyces violaceolatus, Streptomyces
violaceorectus, Streptomyces violaceoruber, Streptomyces
violaceorubidus, Streptomyces violaceus, Streptomyces
violaceusniger, Streptomyces violarus, Streptomyces violascens,
Streptomyces violatus, Streptomyces violens, Streptomyces virens,
Streptomyces virginiae, Streptomyces viridiflavus, Streptomyces
viridiviolaceus, Streptomyces viridobrunneus, Streptomyces
viridochromogenes, Streptomyces viridodiastaticus, Streptomyces
viridosporus, Streptomyces vitaminophileus, Streptomyces
vitaminophilus, Streptomyces wedmorensis, Streptomyces werraensis,
Streptomyces willmorei, Streptomyces xanthochromogenes,
Streptomyces xanthocidicus, Streptomyces xantholiticus,
Streptomyces xanthophaeus, Streptomyces yatensis, Streptomyces
yerevanensis, Streptomyces yogyakartensis, Streptomyces
yokosukanensis, Streptomyces yunnanensis, Streptomyces
zaomyceticus, Streptoverticillium abikoense, Streptoverticillium
albireticuli, Streptoverticillium alboverticillatum,
Streptoverticillium album, Streptoverticillium ardum,
Streptoverticillium aureoversale, Streptoverticillium aureoversile,
Streptoverticillium baldaccii, Streptoverticillium biverticillatum,
Streptoverticillium biastmyceticum, Streptoverticillium cinnamoneum
subsp. aibosporum, Streptomyces cinnamoneus subsp. aibosporus,
Streptoverticillium cinnamoneum subsp. cinnamoneum,
Streptoverticillium cinnamoneum subsp. lanosum, Streptoverticillium
cinnamoneum subsp. sparsum, Streptoverticillium distallicum,
Streptoverticillium ehimense, Streptoverticillium eurocidicum,
Streptoverticillium fervens subsp. fervens, Streptoverticillium
fervens subsp. melrosporus, Streptoverticillium flavopersicum,
Streptoverticillium griseocarneum, Streptoverticillium
griseoverticillatum, Streptoverticillium hachijoense,
Streptoverticillium hiroshimense, Streptoverticillium kashmirense,
Streptoverticillium kentuckense, Streptoverticillium kishiwadense,
Streptoverticillium ladakanum, Streptoverticillium
lavenduligriseum, Streptoverticillium lilacinum,
Streptoverticillium luteoverticillatum, Streptoverticillium
mashuense, Streptoverticillium mobaraense, Streptoverticillium
morookaense, Streptoverticillium netropsis, Streptoverticillium
olivomycini, Streptomyces olivomycini, Streptoverticillium
olivoreticuli subsp. cellulophilum, Streptoverticillium
olivoreticuli subsp. olivoreticuli, Streptoverticillium
olivoreticulum, Streptoverticillium olivoreticulum subsp.
cellulophilum, Streptoverticillium olivoverticillatum,
Streptoverticillium orinoci, Streptoverticillium parvisporogenes,
Streptoverticillium parvisporogenum, Streptoverticillium
rectiverticillatum, Streptoverticillium reticulum subsp.
protomycicum, Streptoverticillium roseoverticillatum,
Streptoverticillium salmonis, Streptoverticillium sapporonense,
Streptoverticillium septatum, Streptoverticillium syringium,
Streptoverticillium thioluteum, Streptoverticillium verticillium
subsp. quantum, Streptoverticillium verticillium subsp.
tsukushiense or Streptoverticillium viridoflavum.
[12084] [0080.0.0.27] Particular preferred strains are strains
selected from the group consisting of Bacillaceae,
Brevibacteriaceae, Corynebacteriaceae, Nocardiaceae,
Mycobacteriaceae, Streptomycetaceae, Enterobacteriaceae such as
Bacillus circulans, Bacillus subtilis, Bacillus sp., Brevibacterium
aibidum, Brevibacterium album, Brevibacterium cerinum,
Brevibacterium flavum, Brevibacterium glutamigenes, Brevibacterium
iodinum, Brevibacterium ketoglutamicum, Brevibacterium
lactofermentum, Brevibacterium linens, Brevibacterium roseum,
Brevibacterium saccharolyticum, Brevibacterium sp., Corynebacterium
acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium
ammoniagenes, Corynebacterium glutamicum (=Micrococcus glutamicum),
Corynebacterium meiassecoia, Corynebacterium sp., Nocardia
rhodochrous (Rhodococcus rhodochrous), Mycobacterium rhodochrous,
Streptomyces lividans and Escherichia coli especially Escherichia
coli K12.
[12085] [0081.0.0.27] In addition particular preferred strains are
strains selected from the group consisting of Cryptococcaceae,
Saccharomycetaceae, Schizosaccharo-mycetacease such as the genera
Candida, Hansenula, Pichia, Saccharomyces and Schizosaccharomyces
preferred are strains selected from the group consisting of the
species Rhodotorula rubra, Rhodotorula glutinis, Rhodotorula
graminis, Yarrowia lipolytica, Sporobolomyces salmonicolor,
Sporobolomyces shibatanus, Saccharomyces cerevisiae, Candida
boidinii, Candida bombicola, Candida cylindracea, Candida
parapsilosis, Candida rugosa, Candida tropicalis, Pichia
methanolica and Pichia pastoris.
[12086] [0082.0.0.27] Anacardiaceae such as the genera Pistacia,
Mangifera, Anacardium e.g. the species Pistacia vera [pistachios,
Pistazie], Mangifer indica [Mango] or Anacardium occidentale
[Cashew]; Asteraceae such as the genera Calendula, Carthamus,
Centaurea, Cichorium, Cynara, Helianthus, Lactuca, Locusta,
Tagetes, Valeriana e.g. the species Calendula officinalis
[Marigold], Carthamus tinctorius [safflower], Centaurea cyanus
[cornflower], Cichorium intybus [blue daisy], Cynara [Artichoke],
Helianthus annus [sunflower], Lactuca sativa, Lactuca crispa,
Lactuca esculenta, Lactuca scariola L. ssp. sativa, Lactuca
scariola L. var. integrata, Lactuca scariola L. var. integrifolia,
Lactuca sativa subsp. romana, Locusta communis, Valeriana locusta
[lettuce], Tagetes lucida, Tagetes erecta or Tagetes tenuifolia
[Marigold]; Apiaceae such as the genera Daucus e.g. the species
Daucus carota [carrot]; Betulaceae such as the genera Corylus e.g.
the species Corylus avellana or Corylus colurna [hazelnut];
Boraginaceae such as the genera Borago e.g. the species Borago
officinalis [borage]; Brassicaceae such as the genera Brassica,
Melanosinapis, Sinapis, Arabadopsis e.g. the species Brassica
napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape],
Sinapis arvensis Brassica juncea, Brassica juncea var. juncea,
Brassica juncea var. crispifolia, Brassica juncea var. foliosa,
Brassica nigra, Brassica sinapioides, Melanosinapis communis
[mustard], Brassica oleracea [fodder beet] or Arabidopsis thaliana;
Bromeliaceae such as the genera Anana, Bromelia e.g. the species
Anana comosus, Ananas ananas or Bromelia comosa [pineapple];
Caricaceae such as the genera Carica e.g. the species Carica papaya
[papaya]; Cannabaceae such as the genera Cannabis e.g. the species
Cannabis sative [hemp], Convolvulaceae such as the genera Ipomea,
Convolvulus e.g. the species Ipomoea batatus, Ipomoea pandurata,
Convolvulus batatas, Convolvulus tiliaceus, Ipomoea fastigiata,
Ipomoea tiliacea, Ipomoea triloba or Convolvulus panduratus [sweet
potato, Man of the Earth, wild potato], Chenopodiaceae such as the
genera Beta, i.e. the species Beta vulgaris, Beta vulgaris var.
altissima, Beta vulgaris var. Vulgaris, Beta maritima, Beta
vulgaris var. perennis, Beta vulgaris var. conditiva or Beta
vulgaris var. esculenta [sugar beet]; Cucurbitaceae such as the
genera Cucubita e.g. the species Cucurbita maxima, Cucurbita mixta,
Cucurbita pepo or Cucurbita moschata [pumpkin, squash];
Elaeagnaceae such as the genera Elaeagnus e.g. the species Olea
europaea [olive]; Ericaceae such as the genera Kalmia e.g. the
species Kalmia latifolia, Kalmia angustifolia, Kalmia microphylla,
Kalmia polifolia, Kalmia occidentalis, Cistus chamaerhodendros or
Kalmia lucida [American laurel, broad-leafed laurel, calico bush,
spoon wood, sheep laurel, alpine laurel, bog laurel, western
bog-laurel, swamp-laurel]; Euphorbiaceae such as the genera
Manihot, Janipha, Jatropha, Ricinus e.g. the species Manihot
utilissima, Janipha manihot, Jatropha manihot, Manihot aipil,
Manihot dulcis, Manihot manihot, Manihot melanobasis, Manihot
esculenta [manihot, arrowroot, tapioca, cassava] or Ricinus
communis [castor bean, Castor Oil Bush, Castor Oil Plant, Palma
Christi, Wonder Tree]; Fabaceae such as the genera Pisum, Albizia,
Cathormion, Feuillea, Inga, Pithecolobium, Acacia, Mimosa,
Medicajo, Glycine, Dolichos, Phaseolus, Soja e.g. the species Pisum
sativum, Pisum arvense, Pisum humile [pea], Albizia berteriana,
Albizia julibrissin, Albizia lebbeck, Acacia berteriana, Acacia
littoralis, Albizia berteriana, Albizzia berteriana, Cathormion
berteriana, Feuillea berteriana, Inga fragrans, Pithecellobium
berterianum, Pithecellobium fragrans, Pithecolobium berterianum,
Pseuda/bizzia berteriana, Acacia julibrissin, Acacia nemu, Albizia
nemu, Feuilleea julibrissin, Mimosa julibrissin, Mimosa speciosa,
Sericanrda julibrissin, Acacia lebbeck, Acacia macrophylla, Albizia
lebbek, Feuilleea lebbeck, Mimosa lebbeck, Mimosa speciosa [bastard
logwood, silk tree, East Indian Walnut], Medicago sativa, Medicago
falcata, Medicago varia [alfalfa] Glycine max Dolichos soja,
Glycine gracilis, Glycine hispida, Phaseolus max, Soja hispida or
Soja max [Soybean]; Geraniaceae such as the genera Pelargonium,
Cocos, Oleum e.g. the species Cocos nucifera, Pelargonium
grossularioides or Oleum cocois [coconut]; Gramineae such as the
genera Saccharum e.g. the species Saccharum officinarum;
Juglandaceae such as the genera Juglans, Wallia e.g. the species
Juglans regia, Juglans ailanthifolia, Juglans sieboldiana, Juglans
cinerea, Wallia cinerea, Juglans bixbyi, Juglans californica,
Juglans hindsii, Juglans intermedia, Juglans jamaicensis, Juglans
major, Juglans microcarpa, Juglans nigra or Wallia nigra [walnut,
black walnut, common walnut, persian walnut, white walnut,
butternut, black walnut]; Lauraceae such as the genera Persea,
Laurus e.g. the species laurel Laurus nobilis [bay, laurel, bay
laurel, sweet bay], Persea americana Persea americana, Persea
gratissima or Persea persea [avocado]; Leguminosae such as the
genera Arachis e.g. the species Arachis hypogaea [peanut]; Linaceae
such as the genera Linum, Adenolinum e.g. the species Linum
usitatissimum, Linum humile, Linum austriacum, Linum bienne, Linum
angustifolium, Linum catharticum, Linum flavum, Linum grandiflorum,
Adenolinum grandiflorum, Linum lewisii, Linum narbonense, Linum
perenne, Linum perenne var. lewisii, Linum pratense or Linum
trigynum [flax, linseed]; Lythrarieae such as the genera Punica
e.g. the species Punica granatum [pomegranate]; Malvaceae such as
the genera Gossypium e.g. the species Gossypium hirsutum, Gossypium
arboreum, Gossypium barbadense, Gossypium herbaceum or Gossypium
thurberi [cotton]; Musaceae such as the genera Musa e.g. the
species Musa nana, Musa acuminate, Musa paradisiaca, Musa spp.
[banana]; Onagraceae such as the genera Camissonia, Oenothera e.g.
the species Oenothera biennis or Camissonia brevipes [primrose,
evening primrose]; Palmae such as the genera Elacis e.g. the
species Elaeis guineensis [oil plam]; Papaveraceae such as the
genera Papaver e.g. the species Papaver orientale, Papaver rhoeas,
Papaver dubium [poppy, oriental poppy, corn poppy, field poppy,
shirley poppies, field poppy, long-headed poppy, long-pod poppy];
Pedaliaceae such as the genera Sesamum e.g. the species Sesamum
indicum [sesame]; Piperaceae such as the genera Piper, Artanthe,
Peperomia, Steffensia e.g. the species Piper aduncum, Piper
amalago, Piper angustifolium, Piper auritum, Piper betel, Piper
cubeba, Piper longum, Piper nigrum, Piper retrofractum, Artanthe
adunca, Artanthe elongate, Peperomia elongate, Piper elongatum,
Steffensia elongate. [Cayenne pepper, wild pepper]; Poaceae such as
the genera Hordeum, Secale, Avena, Sorghum, Andropogon, Holcus,
Panicum, Oryza, Zea, Triticum e.g. the species Hordeum vulgare,
Hordeum jubatum, Hordeum murinum, Hordeum secalinum, Hordeum
distichon Hordeum aegiceras, Hordeum hexastichon., Hordeum
hexastichum, Hordeum irregulare, Hordeum sativum, Hordeum secalinum
[barley, pearl barley, foxtail barley, wall barley, meadow barley],
Secale cereale [rye], Avena sativa, Avena fatua, Avena byzantine,
Avena fatua var. sativa, Avena hybrida [oat], Sorghum bicolor,
Sorghum halepense, Sorghum saccharatum, Sorghum vulgare, Andropogon
drummondii, Holcus bicolor, Holcus sorghum, Sorghum aethiopicum,
Sorghum arundinaceum, Sorghum caffrorum, Sorghum cemuum, Sorghum
dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense,
Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghum
subglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcus
halepensis, Sorghum miliaceum millet, Panicum militaceum [Sorghum,
Oryza sativa, Oryza latifolia [rice], Zea mays [corn, maize]
Triticum aestivum, Triticum durum, Triticum turgidum, Triticum
hybernum, Triticum macha, Triticum sativum or Triticum vulgare
[wheat, bread wheat, common wheat], Proteaceae such as the genera
Macadamia e.g. the species Macadamia intergrifolia [macadamia];
Rubiaceae such as the genera Coffea e.g. the species Cofea spp.,
Coffea arabica, Coffea canephora or Coffea liberica [coffee];
Scrophulariaceae such as the genera Verbascum e.g. the species
Verbascum bletteria, Verbascum Verbascum densiflorum, Verbascum
lagurus, Verbascum longifolium, Verbascum lychnitis, Verbascum
nigrum, Verbascum olympicum, Verbascum phlomoides, Verbascum
phoenicum, Verbascum pulverulentum or Verbascum thapsus [mullein,
white moth mullein, nettle-leaved mullein, dense-flowered mullein,
silver mullein, long-leaved mullein, white mullein, dark mullein,
greek mullein, orange mullein, purple mullein, hoary mullein, great
mullein]; Solanaceae such as the genera Capsicum, Nicotiana,
Solanum, Lycopersicon e.g. the species Capsicum annuum, Capsicum
annuum var. glabriusculum, Capsicum frutescens [pepper], Capsicum
annuum [paprika], Nicotiana tabacum, Nicotiana alata, Nicotiana
attenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotiana
obtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotiana
rustica, Nicotiana sylvestris [tobacco], Solanum tuberosum
[potato], Solanum melongena [egg-plant] (Lycopersicon esculentum,
Lycopersicon lycopersicum., Lycopersicon pyriforme, Solanum
integrifolium or Solanum lycopersicum [tomato]; Sterculiaceae such
as the genera Theobroma e.g. the species Theobroma cacao [cacao];
Theaceae such as the genera Camellia e.g. the species Camellia
sinensis) [tea].
[12087] All abovementioned organisms can in princible also function
as host organisms.
[12088] [0083.0.0.27] Particular preferred plants are plants
selected from the group consisting of Asteraceae such as the genera
Helianthus, Tagetes e.g. the species Helianthus annus [sunflower],
Tagetes lucida, Tagetes erecta or Tagetes tenuifolia [Marigold],
Brassicaceae such as the genera Brassica, Arabadopsis e.g. the
species Brassica napus, Brassica rapa ssp. [canola, oilseed rape,
turnip rape] or Arabidopsis thaliana. Fabaceae such as the genera
Glycine e.g. the species Glycine max, Soja hispida or Soja max
[soybean] (wobei ich nicht sicher bin, ob es Soja max uberhaupt
gibt, die heiBt eigentlich Glycine max). Linaceae such as the
genera Linum e.g. the species Linum usitatissimum, [flax, linseed];
Poaceae such as the genera Hordeum, Secale, Avena, Sorghum, Oryza,
Zea, Triticum e.g. the species Hordeum vulgare [barley]; Secale
cereale [rye], Avena sativa, Avena fatua, Avena byzantina, Avena
fatua var. sativa, Avena hybrida [oat], Sorghum bicolor [Sorghum,
millet], Oryza sativa, Oryza latifolia [rice], Zea mays [corn,
maize] Triticum aestivum, Triticum durum, Triticum turgidum,
Triticum hybernum, Triticum macha, Triticum sativum or Triticum
vulgare [wheat, bread wheat, common wheat]; Solanaceae such as the
genera Solanum, Lycopersicon e.g. the species Solanum tuberosum
[potato], Lycopersicon esculentum, Lycopersicon lycopersicum.,
Lycopersicon pyriforme, Solanum integrifolium or Solanum
lycopersicum [tomato].
[12089] [0084.0.0.27] All abovementioned organisms can in princible
also function as host organisms.
[12090] [0085.0.0.27] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [12091] a) a nucleic acid sequence as
indicated in Table XI, application no. 27, columns 5 or 7, or a
derivative thereof, or [12092] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 27, columns
5 or 7, or a derivative thereof, or [12093] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[12094] [0086.0.0.27] The use of the nucleic acid sequence
according to the invention or of the nucleic acid construct
according to the invention for the generation of transgenic plants
is therefore also subject matter of the invention.
[12095] [0087.0.0.27] The respective fine chemical, which is
synthesized in the organism, in particular the microorganism, the
cell, the tissue or the plant, of the invention can be isolated if
desired. Depending on the use of the respective fine chemical,
different purities resulting from the purification may be
advantageous as will be described herein below.
[12096] [0088.0.0.27] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose amino acid
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for monogastric animals is limited by a few essential amino acids
such as lysine, threonine or methionine.
[12097] [0088.1.0.27] In one embodiment, after an activity of a
polypeptide of the present invention or used in the process of the
present invention has been increased or generated, or after the
expression of a nucleic acid molecule or polypeptide according to
the invention has been generated or increased, the transgenic plant
generated can be grown on or in a nutrient medium or else in the
soil and subsequently harvested.
[12098] [0089.0.0.27] The plants or parts thereof, e.g. the leaves,
roots, flowers, and/or stems and/or other harvestable material as
described below, can then be used directly as foodstuffs or animal
feeds or else be further processed. Again, the amino acids can be
purified further in the customary manner via extraction and
precipitation or via ion exchangers and other methods known to the
person skilled in the art and described herein below. Products
which are suitable for various applications and which result from
these different processing procedures are amino acids or amino acid
compositions which can still comprise further plant components in
different amounts, advantageously in the range of from 0 to 99% by
weight, preferably from below 90% by weight, especially preferably
below 80% by weight. The plants can also advantageously be used
directly without further processing, e.g. as feed or for
extraction.
[12099] [0090.0.0.27] The chemically pure respective fine chemical
or chemically pure compositions comprising the respective fine
chemical may also be produced by the process described above. To
this end, the respective fine chemical or the compositions are
isolated in the known manner from an organism according to the
invention, such as the microorganisms, non-human animal or the
plants, and/or their culture medium in which or on which the
organisms had been grown. These chemically pure respective fine
chemical or said compositions are advantageous for applications in
the field of the food industry, the cosmetics industry or the
pharmaceutical industry.
[12100] [0091.0.0.27] Thus, the content of plant components and
preferably also further impurities is as low as possible, and the
abovementioned respective fine chemical is obtained in as pure form
as possible. In these applications, the content of plant components
advantageously amounts to less than 10%, preferably 1%, more
preferably 0.1%, very especially preferably 0.01% or less.
[12101] [0092.0.0.27] Accordingly, the respective fine chemical
produced by the present invention is at least 0.1% by weight pure,
preferably more than 1% by weight pure, more preferred 10% by
weight pure, even more preferred are more than 50, 60, 70 or 80% by
weight purity, even more preferred are more than 90 weight-%
purity, most preferred are 95% by weight, 99% by weight or
more.
[12102] [0093.0.0.27] In this context, the amount of the respective
fine chemical in a cell of the invention may be increased according
to the process of the invention by at least a factor of 1.1,
preferably at least a factor of 1.5; 2; or 5, especially preferably
by at least a factor of 10 or 30, very especially preferably by at
least a factor of 50, in comparison with the wild type, control or
reference. Preferably, said increase is found a tissue, more
preferred in an organism or in a harvestable part thereof.
[12103] [0094.0.0.27] In principle, the respective fine chemicals
produced can be increased in two ways by the process according to
the invention. The pool of free respective fine chemicals, in
particular of the free respective fine chemical, and/or the content
of protein-bound respective fine chemicals, in particular of the
protein-bound respective fine chemical may advantageously be
increased.
[12104] [0095.0.0.27] It may be advantageous to increase the pool
of free amino acids in the transgenic organisms by the process
according to the invention in order to isolate high amounts of the
pure respective fine chemical.
[12105] [0096.0.0.27] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid, which functions as a
sink for the desired amino acid for example methionine, lysine or
threonine in the organism is useful to increase the production of
the respective fine chemical (see U.S. Pat. No. 5,589,616, WO
96/38574, WO 97/07665, WO 97/28247, U.S. Pat. No. 4,886,878, U.S.
Pat. No. 5,082,993 and U.S. Pat. No. 5,670,635). Galili et al.,
Transgenic Res. 2000 showed, that enhancing the synthesis of
threonine by a feed back insensitive aspartate kinase did not lead
only to in increase in free threonine but also in protein bound
threonine.
[12106] [0097.0.0.27] In may also be advantageous to increase the
content of the protein-bound respective fine chemical.
[12107] [0098.0.0.27] In a preferred embodiment, the respective
fine chemical (methionine) and/or threonine are produced in
accordance with the invention and, if desired, are isolated. The
production of further amino acids such as lysine and of amino acid
mixtures by the process according to the invention is
advantageous.
[12108] [0099.0.0.27] In the case of the fermentation of
microorganisms, the abovementioned amino acids may accumulate in
the medium and/or the cells. If microorganisms are used in the
process according to the invention, the fermentation broth can be
processed after the cultivation. Depending on the requirement, all
or some of the biomass can be removed from the fermentation broth
by separation methods such as, for example, centrifugation,
filtration, decanting or a combination of these methods, or else
the biomass can be left in the fermentation broth. The fermentation
broth can subsequently be reduced, or concentrated, with the aid of
known methods such as, for example, rotary evaporator, thin-layer
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. This concentrated fermentation broth can
subsequently be processed by lyophilization, spray drying, spray
granulation or by other methods.
[12109] [0100.0.0.27] To purify an amino acid, a product-containing
fermentation broth from which the biomass has been separated may be
subjected to chromatography with a suitable resin such as ion
exchange resin for example anion or cation exchange resin,
hydrophobic resin or hydrophilic resin for example epoxy resin,
polyurethane resin or polyacrylamid resin, or resin for separation
according to the molecular weight of the compounds for example
polyvinyl chloride homopolymer resin or resins composed for example
of polymers of acrylic acid, crosslinked with polyalkenyl ethers or
divinyl glycol such as Carbopol.RTM., Pemulen.RTM. and Noveon.RTM..
If necessary these chromatography steps may be repeated using the
same or other chromatography resins. The skilled worker is familiar
with the choice of suitable chromatography resins and their most
effective use. The purified product may be concentrated by
filtration or ultrafiltration and stored at a temperature, which
ensures the maximum stability of the product.
[12110] [0101.0.0.27] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[12111] [0102.0.0.27] Amino acids can for example be detected
advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55). Amino acids can be extracted with hot water. After
filtration the extracts are diluted with water containing 20 mg/mL
sodium acide. The separation and detection of the amino acids is
performed using an anion exchange column and an electrochemical
detector. Technical details can be taken from Y. Ding et al., 2002,
Direct determination of free amino acids and sugars in green tea by
anion-exchange chromatography with integrated pulsed amperometric
detection, J Chromatogr A, (2002) 982; 237-244, or e.g. from Karchi
et al., 1993, Plant J. 3: 721-727; Matthews M J, 1997 (Lysine,
threonine and methionine biosynthesis. In BK Singh, ed, Plant Amino
Acids: Biochemistry and Biotechnology. Dekker, New York, pp
205-225; H Hesse and R Hoefgen. (2003) Molecular aspects of
methionine biosynthesis. TIPS 8(259-262.
[12112] [0103.0.0.27] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [12113] a) nucleic acid molecule encoding,
preferably at least the mature form, of a polypeptide having a
sequence as indicated in Table XII, application no. 27, columns 5
or 7, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [12114] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table XI, application no. 27, columns 5 or 7;
[12115] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [12116] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[12117] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [12118]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [12119] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[12120] h) nucleic acid molecule comprising a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers pairs having a
sequence as indicated in Table XIII, application no. 27, columns 7,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [12121] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from an
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [12122] j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence having a sequences as
indicated in Table XIV, application no. 27, columns 7, and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [12123] k) nucleic acid
molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of a polypeptide indicated in Table XII, application no. 27,
columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[12124] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[12125] [0103.1.0.27.] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table XI, application no. 27, columns 5 or 7,
by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence shown in indicated in Table XI, application no. 27,
columns 5 or 7. In one embodiment, the nucleic acid molecule used
in the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI,
application no. 27, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table XII, application no. 27, columns 5 or 7.
[12126] [0104.0.0.27] In one embodiment, the nucleic acid molecule
of the invention or used in the process of the invention
distinguishes over the sequence indicated in Table XI, application
no. 27, columns 5 or 7, by one or more nucleotides. In one
embodiment, the nucleic acid molecule of the invention or the
nucleic acid used in the process of the invention does not consist
of the sequence shown in indicated in Table XI, application no. 27,
columns 5 or 7: In one embodiment, the nucleic acid molecule of the
present invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table XI, application no. 27,
columns 5 or 7. In another embodiment, the nucleic acid molecule
does not encode a polypeptide of a sequence indicated in Table XII,
application no. 27, columns 5 or 7.
[12127] [0105.0.0.27] Unless otherwise specified, the terms
"polynucleotides", "nucleic acid" and "nucleic acid molecule" are
interchangeably in the present context. Unless otherwise specified,
the terms "peptide", "polypeptide" and "protein" are
interchangeably in the present context. The term "sequence" may
relate to polynucleotides, nucleic acids, nucleic acid molecules,
peptides, polypeptides and proteins, depending on the context in
which the term "sequence" is used. The terms "gene(s)",
"polynucleotide", "nucleic acid sequence", "nucleotide sequence",
or "nucleic acid molecule(s)" as used herein refers to a polymeric
form of nucleotides of any length, either ribonucleotides or
deoxyribonucleotides. The terms refer only to the primary structure
of the molecule.
[12128] [0106.0.0.27] Thus, the terms "gene(s)", "polynucleotide",
"nucleic acid sequence", "nucleotide sequence", or "nucleic acid
molecule(s)" as used herein include double- and single-stranded DNA
and RNA. They also include known types of modifications, for
example, methylation, "caps", substitutions of one or more of the
naturally occurring nucleotides with an analog. Preferably, the DNA
or RNA sequence of the invention comprises a coding sequence
encoding the herein defined polypeptide.
[12129] [0107.0.0.27] A "coding sequence" is a nucleotide sequence,
which is transcribed into mRNA and/or translated into a polypeptide
when placed under the control of appropriate regulatory sequences.
The boundaries of the coding sequence are determined by a
translation start codon at the 5'-terminus and a translation stop
codon at the 3'-terminus. A coding sequence can include, but is not
limited to mRNA, cDNA, recombinant nucleotide sequences or genomic
DNA, while introns may be present as well under certain
circumstances.
[12130] [0108.0.0.27] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 27, columns 5 or 7, nucleic
acid molecules which are derived from a amino acid sequences as
indicated in Table XII, application no. 27, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 27, columns 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of a polypeptide as indicated in Table XII, application
no. 27, column 3, 5 or 7, or e.g. conferring a increase of the
respective fine chemical after increasing its expression or
activity are advantageously increased in the process according to
the invention.
[12131] [0109.0.0.27] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemical produced in the process
according to the invention.
[12132] [0110.0.0.27] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
used in the process of the invention, e.g. of a protein as
indicated in Table XII, application no. 27, column 5, or being
encoded by a nucleic acid molecule indicated in
[12133] Table XI, application no. 27, column 5, or of its homologs,
e.g. as indicated in Table XII, application no. 27, column 7, can
be determined from generally accessible databases.
[12134] [0111.0.0.27] Those, which must be mentioned, in particular
in this context are general gene databases such as the EMBL
database (Stoesser G. et al., Nucleic Acids Res 2001, Vol. 29,
17-21), the GenBank database (Benson D. A. et al., Nucleic Acids
Res 2000, Vol. 28, 15-18), or the PIR database (Barker W. C. et
al., Nucleic Acids Res. 1999, Vol. 27, 39-43). It is furthermore
possible to use organism-specific gene databases for determining
advantageous sequences, in the case of yeast for example
advantageously the SGD database (Cherry J. M. et al., Nucleic Acids
Res. 1998, Vol. 26, 73-80) or the MIPS database (Mewes H. W. et
al., Nucleic Acids Res. 1999, Vol. 27, 44-48), in the case of E.
coli the GenProtEC database
(http://web.bham.ac.uk/bcm4ght6/res.html), and in the case of
Arabidopsis the TAIR-database (Huela, E. et al., Nucleic Acids Res.
2001 Vol. 29(1), 102-5) or the MIPS database.
[12135] [0112.0.0.27] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XI, application no. 27,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 27, columns 5 and 7, and conferring an
increase of the respective fine chemical.
[12136] [0113.0.0.27] The nucleic acid sequence(s) used in the
process for the production of the respective fine chemical in
transgenic organisms originate advantageously from an eukaryote but
may also originate from a prokaryote or an archebacterium, thus it
can derived from e.g. a microorganism, an animal or a plant.
[12137] [0114.0.0.27] For the purposes of the invention, as a rule
the plural is intended to encompass the singular and vice
versa.
[12138] [0115.0.0.27] In order to improve the introduction of the
nucleic acid sequences and the expression of the sequences in the
transgenic organisms, which are used in the process, the nucleic
acid sequences are incorporated into a nucleic acid construct
and/or a vector. In addition to the herein described sequences
which are used in the process according to the invention, further
nucleic acid sequences, advantageously of biosynthesis genes of the
respective fine chemical produced in the process according to the
invention, may additionally be present in the nucleic acid
construct or in the vector and may be introduced into the organism
together. However, these additional sequences may also be
introduced into the organisms via other, separate nucleic acid
constructs or vectors.
[12139] [0116.0.0.27] Using the herein mentioned cloning vectors
and transformation methods such as those which are published and
cited in: Plant Molecular Biology and Biotechnology (CRC Press,
Boca Raton, Fla.), chapter 6/7, pp. 71-119 (1993); F. F. White,
Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants,
vol. 1, Engineering and Utilization, Ed.: Kung and R. Wu, Academic
Press, 1993, 15-38; B. Jenes et al., Techniques for Gene Transfer,
in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.:
Kung and R. Wu, Academic Press (1993), 128-143; Potrykus, Annu.
Rev. Plant Physiol. Plant Molec. Biol. 42 (1991), 205-225)) and
further cited below, the nucleic acids may be used for the
recombinant modification of a wide range of organisms, in
particular prokaryotic or eukaryotic microorganisms or plants, so
that they become a better and more efficient producer of the
respective fine chemical produced in the process according to the
invention. This improved production, or production efficiency, of
the respective fine chemical or products derived there from, such
as modified proteins, can be brought about by a direct effect of
the manipulation or by an indirect effect of this manipulation.
[12140] [0117.0.0.27] In one embodiment, the nucleic acid molecule
according to the invention originates from a plant, such as a plant
selected from the families Aceraceae, Anacardiaceae, Apiaceae,
Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Euphorbiaceae,
Fabaceae, Malvaceae, Nymphaeaceae, Papaveraceae, Rosaceae,
Salicaceae, Solanaceae, Arecaceae, Bromeliaceae, Cyperaceae,
Iridaceae, Liliaceae, Orchidaceae, Gentianaceae, Labiaceae,
Magnoliaceae, Ranunculaceae, Carifolaceae, Rubiaceae,
Scrophulariaceae, Caryophyllaceae, Ericaceae, Polygonaceae,
Violaceae, Juncaceae or Poaceae and preferably from a plant
selected from the group of the families Apiaceae, Asteraceae,
Brassicaceae, Cucurbitaceae, Fabaceae, Papaveraceae, Rosaceae,
Solanaceae, Liliaceae or Poaceae. Preferred are crop plants and in
particular plants mentioned herein above as host plants such as the
families and genera mentioned above for example preferred the
species Anacardium occidentale, Calendula officinalis, Carthamus
tinctorius, Cichorium intybus, Cynara scolymus, Helianthus annus,
Tagetes lucida, Tagetes erecta, Tagetes tenuifolia; Daucus carota;
Corylus avellana, Corylus colurna, Borago officinalis; Brassica
napus, Brassica rapa ssp., Sinapis arvensis Brassica juncea,
Brassica juncea var. juncea, Brassica juncea var. crispifolia,
Brassica juncea var. foliosa, Brassica nigra, Brassica sinapioides,
Melanosinapis communis, Brassica oleracea, Arabidopsis thaliana,
Anana comosus, Ananas ananas, Bromelia comosa, Carica papaya,
Cannabis sative, Ipomoea batatus, Ipomoea pandurata, Convolvulus
batatas, Convolvulus tiliaceus, Ipomoea fastigiata, Ipomoea
tiliacea, Ipomoea triloba, Convolvulus panduratus, Beta vulgaris,
Beta vulgaris var. altissima, Beta vulgaris var. vulgaris, Beta
maritima, Beta vulgaris var. perennis, Beta vulgaris var.
conditiva, Beta vulgaris var. esculenta, Cucurbita maxima,
Cucurbita mixta, Cucurbita pepo, Cucurbita moschata, Olea europaea,
Manihot utilissima, Janipha manihotJatropha manihot., Manihot
aipil, Manihot dulcis, Manihot manihot, Manihot melanobasis,
Manihot esculenta, Ricinus communis, Pisum sativum, Pisum arvense,
Pisum humile, Medicago sativa, Medicago falcata, Medicago varia,
Glycine max Dolichos soja, Glycine gracilis, Glycine hispida,
Phaseolus max, Soja hispida, Soja max, Cocos nucifera, Pelargonium
grossularioides, Oleum cocoas, Laurus nobilis, Persea americana,
Arachis hypogaea, Linum usitatissimum, Linum humile, Linum
austriacum, Linum bienne, Linum angustifolium, Linum catharticum,
Linum flavum, Linum grandiflorum, Adenolinum grandiflorum, Linum
lewisii, Linum narbonense, Linum perenne, Linum perenne var.
lewisii, Linum pratense, Linum trigynum, Punica granatum, Gossypium
hirsutum, Gossypium arboreum, Gossypium barbadense, Gossypium
herbaceum, Gossypium thurberi, Musa nana, Musa acuminate, Musa
paradisiaca, Musa spp., Elaeis guineensis, Papaver orientale,
Papaver rhoeas, Papaver dubium, Sesamum indicum, Piper aduncum,
Piper amalago, Piper angustifolium, Piper auritum, Piper betel,
Piper cubeba, Piper longum, Piper nigrum, Piper retrofractum,
Artanthe adunca, Artanthe elongate, Peperomia elongate, Piper
elongatum, Steffensia elongata, Hordeum vulgare, Hordeum jubatum,
Hordeum murinum, Hordeum secalinum, Hordeum distichon Hordeum
aegiceras, Hordeum hexastichon., Hordeum hexastichum, Hordeum
irregulare, Hordeum sativum, Hordeum secalinum, Avena Avena fatua,
Avena byzantina, Avena fatua var. sativa, Avena hybrida, Sorghum
bicolor, Sorghum halepense, Sorghum saccharatum, Sorghum vulgare,
Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghum
aethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum
cemuum, Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum
guineense, Sorghum lanceolatum, Sorghum nervosum, Sorghum
saccharatum, Sorghum subglabrescens, Sorghum verticilliflorum,
Sorghum vulgare, Holcus halepensis, Sorghum miliaceum millet,
Panicum militaceum, Zea mays, Triticum aestivum, Triticum durum,
Triticum turgidum, Triticum hybemum, Triticum macha, Triticum
sativum or Triticum vulgare, Cofea spp., Coffea arabica, Coffea
canephora, Coffea liberica, Capsicum annuum, Capsicum annuum var.
glabriusculum, Capsicum frutescens, Capsicum annuum, Nicotiana
tabacum, Solanum tuberosum, Solanum melongena, Lycopersicon
esculentum, Lycopersicon lycopersicum., Lycopersicon pyriforme,
Solanum integrifolium, Solanum lycopersicum Theobroma cacao or
Camellia sinensis.
[12141] [0118.0.0.27] In one embodiment, the nucleic acid molecule
sequence originates advantageously from a microorganism as
mentioned above under host organism such as a fungus for example
the genera Aspergillus, Penicillium or Claviceps or from yeasts
such as the genera Pichia, Torulopsis, Hansenula,
Schizosaccharomyces, Candida, Rhodotorula or Saccharomyces, very
especially advantageously from the yeast of the family
Saccharomycetaceae, such as the advantageous genus Saccharomyces
and the very advantageous genus and species Saccharomyces
cerevisiae for the production of the respective fine chemical in
microorganims.
[12142] [0119.0.0.27] The skilled worker knows other suitable
sources for the production of respective fine chemicals, which
present also useful nucleic acid molecule sources. They include in
general all prokaryotic or eukaryotic cells, preferably unicellular
microorganisms, such as fungi like the genus Claviceps or
Aspergillus or gram-positive bacteria such as the genera Bacillus,
Corynebacterium, Micrococcus, Brevibacterium, Rhodococcus,
Nocardia, Caseobacter or Arthrobacter or gram-negative bacteria
such as the genera Escherichia, Flavobacterium or Salmonella, or
yeasts such as the genera Rhodotorula, Hansenula or Candida.
[12143] [0120.0.0.27] Production strains which are especially
advantageously selected in the process according to the invention
are microorganisms selected from the group of the families
Actinomycetaceae, Bacillaceae, Brevibacteriaceae,
Corynebacteriaceae, Enterobacteriacae, Gordoniaceae,
Micrococcaceae, Mycobacteriaceae, Nocardiaceae, Pseudomonaceae,
Rhizobiaceae, Streptomycetaceae, Chaetomiaceae, Choanephoraceae,
Cryptococcaceae, Cunninghamellaceae, Demetiaceae, Moniliaceae,
Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae,
Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae,
Sporobolomycetaceae, Tuberculariaceae, Adelotheciaceae,
Dinophyceae, Ditrichaceae and Prasinophyceaeor of the genera and
species consisting of Hansenula anomala, Candida utilis, Claviceps
purpurea, Bacillus circulans, Bacillus subtilis, Bacillus sp.,
Brevibacterium albidum, Brevibacterium album, Brevibacterium
cerinum, Brevibacterium flavum, Brevibacterium glutamigenes,
Brevibacterium iodinum, Brevibacterium ketoglutamicum,
Brevibacterium lactofermentum, Brevibacterium linens,
Brevibacterium roseum, Brevibacterium saccharolyticum,
Brevibacterium sp., Corynebacterium acetoacidophilum,
Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes,
Corynebacterium glutamicum (=Micrococcus glutamicum),
Corynebacterium melassecola, Corynebacterium sp. or Escherichia
coli, specifically Escherichia coli K12 and its described
strains.
[12144] [0121.0.0.27] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 27, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a increase of the respective fine
chemical after increasing its activity.
[12145] [0122.0.0.27] In the process according to the invention
nucleic acid sequences can be used, which, if appropriate, contain
synthetic, non-natural or modified nucleotide bases, which can be
incorporated into DNA or RNA. Said synthetic, non-natural or
modified bases can for example increase the stability of the
nucleic acid molecule outside or inside a cell. The nucleic acid
molecules of the invention can contain the same modifications as
aforementioned.
[12146] [0123.0.0.27] As used in the present context the term
"nucleic acid molecule" may also encompass the untranslated
sequence located at the 3' and at the 5' end of the coding gene
region, for example at least 500, preferably 200, especially
preferably 100, nucleotides of the sequence upstream of the 5' end
of the coding region and at least 100, preferably 50, especially
preferably 20, nucleotides of the sequence downstream of the 3' end
of the coding gene region. It is often advantageous only to choose
the coding region for cloning and expression purposes.
[12147] [0124.0.0.27] Preferably, the nucleic acid molecule used in
the process according to the invention or the nucleic acid molecule
of the invention is an isolated nucleic acid molecule.
[12148] [0125.0.0.27] An "isolated" polynucleotide or nucleic acid
molecule is separated from other polynucleotides or nucleic acid
molecules, which are present in the natural source of the nucleic
acid molecule. An isolated nucleic acid molecule may be a
chromosomal fragment of several kb, or preferably, a molecule only
comprising the coding region of the gene. Accordingly, an isolated
nucleic acid molecule of the invention may comprise chromosomal
regions, which are adjacent 5' and 3' or further adjacent
chromosomal regions, but preferably comprises no such sequences
which naturally flank the nucleic acid molecule sequence in the
genomic or chromosomal context in the organism from which the
nucleic acid molecule originates (for example sequences which are
adjacent to the regions encoding the 5'- and 3'-UTRs of the nucleic
acid molecule). In various embodiments, the isolated nucleic acid
molecule used in the process according to the invention may, for
example comprise less than approximately 5 kb, 4 kb, 3 kb, 2 kb, 1
kb, 0.5 kb or 0.1 kb nucleotide sequences which naturally flank the
nucleic acid molecule in the genomic DNA of the cell from which the
nucleic acid molecule originates.
[12149] [0126.0.0.27] The nucleic acid molecules used in the
process, for example the polynucleotides of the invention or of a
part thereof can be isolated using molecular-biological standard
techniques and the sequence information provided herein. Also, for
example a homologous sequence or homologous, conserved sequence
regions at the DNA or amino acid level can be identified with the
aid of comparison algorithms. The former can be used as
hybridization probes under standard hybridization techniques (for
example those described in Sambrook et al., Molecular Cloning: A
Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) for
isolating further nucleic acid sequences useful in this
process.
[12150] [0127.0.0.27] A nucleic acid molecule encompassing a
complete sequence of the nucleic acid molecules used in the
process, for example the polynucleotide of the invention, or a part
thereof may additionally be isolated by polymerase chain reaction,
oligonucleotide primers based on this sequence or on parts thereof
being used. For example, a nucleic acid molecule comprising the
complete sequence or part thereof can be isolated by polymerase
chain reaction using oligonucleotide primers which have been
generated on the basis of this sequence for example, mRNA can be
isolated from cells (for example by means of the guanidinium
thiocyanate extraction method of Chirgwin et al. (1979)
Biochemistry 18:5294-5299) and cDNA can be generated by means of
reverse transcriptase (for example Moloney MLV reverse
transcriptase, available from Gibco/BRL, Bethesda, Md., or AMV
reverse transcriptase, obtainable from Seikagaku America, Inc., St.
Petersburg, Fla.).
[12151] [0128.0.0.27] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 27, columns 7, by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table XI, application no.
27, columns 5 or 7, or the sequences derived from sequences as
indicated in Table XII, application no. 27, columns 5 or 7.
[12152] [0129.0.0.27] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention or the polypeptide used in the method of the invention,
from which conserved regions, and in turn, degenerate primers can
be derived. Conserved region for the polypeptide of the invention
or the polypeptide used in the method of the invention are
indicated in the alignments shown in the figures. Conserved regions
are those, which show a very little variation in the amino acid
sequence in one particular position of several homologs from
different origin. The consensus sequences indicated in Table XIV,
application no. 27, columns 7, are derived from said
alignments.
[12153] [0130.0.0.27] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of the
respective fine chemical after increasing its expression or
activity or further functional homologs of the polypeptide of the
invention or the polypeptide used in the method of the invention
from other organisms.
[12154] [0131.0.0.27] These fragments can then be utilized as
hybridization probe for isolating the complete gene sequence. As an
alternative, the missing 5' and 3' sequences can be isolated by
means of RACE-PCR (rapid amplification of cDNA ends). A nucleic
acid molecule according to the invention can be amplified using
cDNA or, as an alternative, genomic DNA as template and suitable
oligonucleotide primers, following standard PCR amplification
techniques. The nucleic acid molecule amplified thus can be cloned
into a suitable vector and characterized by means of DNA sequence
analysis. Oligonucleotides, which correspond to one of the nucleic
acid molecules used in the process, can be generated by standard
synthesis methods, for example using an automatic DNA
synthesizer.
[12155] [0132.0.0.27] Nucleic acid molecules which are
advantageously for the process according to the invention can be
isolated based on their homology to the nucleic acid molecules
disclosed herein using the sequences or part thereof as
hybridization probe and following standard hybridization techniques
under stringent hybridization conditions. In this context, it is
possible to use, for example, isolated nucleic acid molecules of at
least 15, 20, 25, 30, 35, 40, 50, 60 or more nucleotides,
preferably of at least 15, 20 or 25 nucleotides in length which
hybridize under stringent conditions with the above-described
nucleic acid molecules, in particular with those which encompass a
nucleotide sequence of the nucleic acid molecule used in the
process of the invention or encoding a protein used in the
invention or of the nucleic acid molecule of the invention. Nucleic
acid molecules with 30, 50, 100, 250 or more nucleotides may also
be used.
[12156] [0133.0.0.27] The term "homology" means that the respective
nucleic acid molecules or encoded proteins are functionally and/or
structurally equivalent. The nucleic acid molecules that are
homologous to the nucleic acid molecules described above and that
are derivatives of said nucleic acid molecules are, for example,
variations of said nucleic acid molecules which represent
modifications having the same biological function, in particular
encoding proteins with the same or substantially the same
biological function. They may be naturally occurring variations,
such as sequences from other plant varieties or species, or
mutations. These mutations may occur naturally or may be obtained
by mutagenesis techniques. The allelic variations may be naturally
occurring allelic variants as well as synthetically produced or
genetically engineered variants. Structurally equivalents can, for
example, be identified by testing the binding of said polypeptide
to antibodies or computer based predictions. Structurally
equivalent have the similar immunological characteristic, e.g.
comprise similar epitopes.
[12157] [0134.0.0.27] By "hybridizing" it is meant that such
nucleic acid molecules hybridize under conventional hybridization
conditions, preferably under stringent conditions such as described
by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N. Y. (1989)) or in Current Protocols in Molecular Biology, John
Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6.
[12158] [0135.0.0.27] According to the invention, DNA as well as
RNA molecules of the nucleic acid of the invention can be used as
probes. Further, as template for the identification of functional
homologues Northern blot assays as well as Southern blot assays can
be performed. The Northern blot assay advantageously provides
further information about the expressed gene product: e.g.
expression pattern, occurrence of processing steps, like splicing
and capping, etc. The Southern blot assay provides additional
information about the chromosomal localization and organization of
the gene encoding the nucleic acid molecule of the invention.
[12159] [0136.0.0.27] A preferred, nonlimiting example of stringent
hydridization conditions are hybridizations in 6.times. sodium
chloride/sodium citrate (=SSC) at approximately 45.degree. C.,
followed by one or more wash steps in 0.2.times.SSC, 0.1% SDS at 50
to 65.degree. C., for example at 50.degree. C., 55.degree. C. or
60.degree. C. The skilled worker knows that these hybridization
conditions differ as a function of the type of the nucleic acid
and, for example when organic solvents are present, with regard to
the temperature and concentration of the buffer. The temperature
under "standard hybridization conditions" differs for example as a
function of the type of the nucleic acid between 42.degree. C. and
58.degree. C., preferably between 45.degree. C. and 50.degree. C.
in an aqueous buffer with a concentration of 0.1.times.0.5.times.,
1.times., 2.times., 3.times., 4.times. or 5.times.SSC (pH 7.2). If
organic solvent(s) is/are present in the abovementioned buffer, for
example 50% formamide, the temperature under standard conditions is
approximately 40.degree. C., 42.degree. C. or 45.degree. C. The
hybridization conditions for DNA:DNA hybrids are preferably for
example 0.1.times.SSC and 20.degree. C., 25.degree. C., 30.degree.
C., 35.degree. C., 40.degree. C. or 45.degree. C., preferably
between 30.degree. C. and 45.degree. C. The hybridization
conditions for DNA:RNA hybrids are preferably for example
0.1.times.SSC and 30.degree. C., 35.degree. C., 40.degree. C.,
45.degree. C., 50.degree. C. or 55.degree. C., preferably between
45.degree. C. and 55.degree. C. The abovementioned hybridization
temperatures are determined for example for a nucleic acid
approximately 100 by (=base pairs) in length and a G+C content of
50% in the absence of formamide. The skilled worker knows to
determine the hybridization conditions required with the aid of
textbooks, for example the ones mentioned above, or from the
following textbooks: Sambrook et al., "Molecular Cloning", Cold
Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985,
"Nucleic Acids Hybridization: A Practical Approach", IRL Press at
Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential
Molecular Biology: A Practical Approach", IRL Press at Oxford
University Press, Oxford.
[12160] [0137.0.0.27] A further example of one such stringent
hybridization condition is hybridization at 4.times.SSC at
65.degree. C., followed by a washing in 0.1.times.SSC at 65.degree.
C. for one hour. Alternatively, an exemplary stringent
hybridization condition is in 50 formamide, 4.times.SSC at
42.degree. C. Further, the conditions during the wash step can be
selected from the range of conditions delimited by low-stringency
conditions (approximately 2.times.SSC at 50.degree. C.) and
high-stringency conditions (approximately 0.2.times.SSC at
50.degree. C., preferably at 65.degree. C.) (20.times.SSC: 0.3M
sodium citrate, 3M NaCl, pH 7.0). In addition, the temperature
during the wash step can be raised from low-stringency conditions
at room temperature, approximately 22.degree. C., to
higher-stringency conditions at approximately 65.degree. C. Both of
the parameters salt concentration and temperature can be varied
simultaneously, or else one of the two parameters can be kept
constant while only the other is varied. Denaturants, for example
formamide or SDS, may also be employed during the hybridization. In
the presence of 50% formamide, hybridization is preferably effected
at 42.degree. C. Relevant factors like i) length of treatment, ii)
salt conditions, iii) detergent conditions, iv) competitor DNAs, v)
temperature and vi) probe selection can be combined case by case so
that not all possibilities can be mentioned herein.
[12161] Thus, in a preferred embodiment, Northern blots are
prehybridized with Rothi-Hybri-Quick buffer (Roth, Karlsruhe) at
68.degree. C. for 2 h. Hybridization with radioactive labelled
probe is done overnight at 68.degree. C. Subsequent washing steps
are performed at 68.degree. C. with 1.times.SSC.
[12162] For Southern blot assays the membrane is prehybridized with
Rothi-Hybri-Quick buffer (Roth, Karlsruhe) at 68.degree. C. for 2
h. The hybridization with radioactive labelled probe is conducted
over night at 68.degree. C. Subsequently the hybridization buffer
is discarded and the filter shortly washed using 2.times.SSC; 0.1%
SDS. After discarding the washing buffer new 2.times.SSC; 0.1% SDS
buffer is added and incubated at 68.degree. C. for 15 minutes. This
washing step is performed twice followed by an additional washing
step using 1.times.SSC; 0.1% SDS at 68.degree. C. for 10 min.
[12163] [0138.0.0.27] Some further examples of conditions for DNA
hybridization (Southern blot assays) and wash step are shown herein
below: [12164] (1) Hybridization conditions can be selected, for
example, from the following conditions: [12165] a) 4.times.SSC at
65.degree. C., [12166] b) 6.times.SSC at 45.degree. C., [12167] c)
6.times.SSC, 100 mg/ml denatured fragmented fish sperm DNA at
68.degree. C., [12168] d) 6.times.SSC, 0.5% SDS, 100 mg/ml
denatured salmon sperm DNA at 68.degree. C., [12169] e)
6.times.SSC, 0.5% SDS, 100 mg/ml denatured fragmented salmon sperm
DNA, 50% formamide at 42.degree. C., [12170] f) 50% formamide,
4.times.SSC at 42.degree. C., [12171] g) 50% (vol/vol) formamide,
0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone,
50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 75 mM sodium
citrate at 42.degree. C., [12172] h) 2.times. or 4.times.SSC at
50.degree. C. (low-stringency condition), or [12173] i) 30 to 40%
formamide, 2.times. or 4.times.SSC at 42.degree. C. (low-stringency
condition). [12174] (2) Wash steps can be selected, for example,
from the following conditions: [12175] a) 0.015 M NaCl/0.0015 M
sodium citrate/0.1.degree. C. SDS at 50.degree. C. [12176] b)
0.1.times.SSC at 65.degree. C. [12177] c) 0.1.times.SSC, 0.5% SDS
at 68.degree. C. [12178] d) 0.1.times.SSC, 0.5% SDS, 50% formamide
at 42.degree. C. [12179] e) 0.2.times.SSC, 0.1% SDS at 42.degree.
C. [12180] f) 2.times.SSC at 65.degree. C. (low-stringency
condition).
[12181] [0139.0.0.27] Polypeptides having above-mentioned activity,
i.e. conferring the respective fine chemical increase, derived from
other organisms, can be encoded by other DNA sequences which
hybridize to a sequences indicated in Table XI, application no. 27,
columns 5 or 7, under relaxed hybridization conditions and which
code on expression for peptides having the methionine increasing
activity.
[12182] [0140.0.0.27] Further, some applications have to be
performed at low stringency hybridisation conditions, without any
consequences for the specificity of the hybridisation. For example,
a Southern blot analysis of total DNA could be probed with a
nucleic acid molecule of the present invention and washed at low
stringency (55.degree. C. in 2.times.SSPE0.1% SDS). The
hybridisation analysis could reveal a simple pattern of only genes
encoding polypeptides of the present invention or used in the
process of the invention, e.g. having herein-mentioned activity of
increasing the respective fine chemical. A further example of such
low-stringent hybridization conditions is 4.times.SSC at 50.degree.
C. or hybridization with 30 to 40% formamide at 42.degree. C. Such
molecules comprise those which are fragments, analogues or
derivatives of the polypeptide of the invention or used in the
process of the invention and differ, for example, by way of amino
acid and/or nucleotide deletion(s), insertion(s), substitution (s),
addition(s) and/or recombination (s) or any other modification(s)
known in the art either alone or in combination from the
above-described amino acid sequences or their underlying nucleotide
sequence(s). However, it is preferred to use high stringency
hybridisation conditions.
[12183] [0141.0.0.27] Hybridization should advantageously be
carried out with fragments of at least 5, 10, 15, 20, 25, 30, 35 or
40 bp, advantageously at least 50, 60, 70 or 80 bp, preferably at
least 90, 100 or 110 bp. Most preferably are fragments of at least
15, 20, 25 or 30 bp. Preferably are also hybridizations with at
least 100 by or 200, very especially preferably at least 400 by in
length. In an especially preferred embodiment, the hybridization
should be carried out with the entire nucleic acid sequence with
conditions described above.
[12184] [0142.0.0.27] The terms "fragment", "fragment of a
sequence" or "part of a sequence" mean a truncated sequence of the
original sequence referred to. The truncated sequence (nucleic acid
or protein sequence) can vary widely in length; the minimum size
being a sequence of sufficient size to provide a sequence with at
least a comparable function and/or activity of the original
sequence referred to or hybridising with the nucleic acid molecule
of the invention or the nucleic acid molecule used in the method of
the invention or used in the process of the invention under
stringent conditions, while the maximum size is not critical. In
some applications, the maximum size usually is not substantially
greater than that required to provide the desired activity and/or
function(s) of the original sequence.
[12185] [0143.0.0.27] Typically, the truncated amino acid sequence
will range from about 5 to about 310 amino acids in length. More
typically, however, the sequence will be a maximum of about 250
amino acids in length, preferably a maximum of about 200 or 100
amino acids. It is usually desirable to select sequences of at
least about 10, 12 or 15 amino acids, up to a maximum of about 20
or 25 amino acids.
[12186] [0144.0.0.27] The term "epitope" relates to specific
immunoreactive sites within an antigen, also known as antigenic
determinates. These epitopes can be a linear array of monomers in a
polymeric composition--such as amino acids in a protein--or consist
of or comprise a more complex secondary or tertiary structure.
Those of skill will recognize that immunogens (i.e., substances
capable of eliciting an immune response) are antigens; however,
some antigen, such as haptens, are not immunogens but may be made
immunogenic by coupling to a carrier molecule. The term "antigen"
includes references to a substance to which an antibody can be
generated and/or to which the antibody is specifically
immunoreactive.
[12187] [0145.0.0.27] In one embodiment the present invention
relates to a epitope of the polypeptide of the present invention or
used in the process of the present invention and conferring above
mentioned activity, preferably conferring an increase in the
respective fine chemical.
[12188] [0146.0.0.27] The term "one or several amino acids" relates
to at least one amino acid but not more than that number of amino
acids, which would result in a homology of below 50% identity.
Preferably, the identity is more than 70% or 80%, more preferred
are 85%, 90%, 91%, 92%, 93%, 94% or 95%, even more preferred are
96%, 97%, 98%, or 99% identity.
[12189] [0147.0.0.27] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 27, columns 5 or 7 is one which is sufficiently
complementary to one of said nucleotide sequences such that it can
hybridize to one of said nucleotide sequences thereby forming a
stable duplex. Preferably, the hybridisation is performed under
stringent hybridization conditions. However, a complement of one of
the herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[12190] [0148.0.0.27] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 27,
columns 5 or 7, or a functional portion thereof and preferably has
above mentioned activity, in particular has
the--fine-chemical-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homologs.
[12191] [0149.0.0.27] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridises, preferably
hybridises under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 27,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity and as indicated in indicated in Table
XII, application no. 27, columns 5 or 7, e.g. conferring an
increase of the respective fine chemical.
[12192] [0149.1.0.27] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table
XI, application no. 27, columns 5 or 7, has further one or more of
the activities annotated or known for the a protein as indicated in
Table XII, application no. 27, column 3.
[12193] [0150.0.0.27] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table XI, application no. 27, columns 5 or 7, for example a
fragment which can be used as a probe or primer or a fragment
encoding a biologically active portion of the polypeptide of the
present invention or of a polypeptide used in the process of the
present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of methionine if its activity is increased.
The nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences indicated in Table XI, application no. 27, columns
5 or 7, an anti-sense sequence of one of the sequences indicated in
Table XI, application no. 27, columns 5 or 7, or naturally
occurring mutants thereof. Primers based on a nucleotide sequence
of the invention can be used in PCR reactions to clone homologues
of the polypeptide of the invention or of the polypeptide used in
the process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primer pairs indicated in Table XIII, application no.
27, column 7, will result in a fragment of a polynucleotide
sequence as indicated in Table XI, application no. 27, columns 5 or
7.
[12194] [0151.0.0.27] Primer sets are interchangeable. The person
skilled in the art knows to combine said primers to result in the
desired product, e.g. in a full-length clone or a partial sequence.
Probes based on the sequences of the nucleic acid molecule of the
invention or used in the process of the present invention can be
used to detect transcripts or genomic sequences encoding the same
or homologous proteins. The probe can further comprise a label
group attached thereto, e.g. the label group can be a radioisotope,
a fluorescent compound, an enzyme, or an enzyme co-factor. Such
probes can be used as a part of a genomic marker test kit for
identifying cells which express an polypeptide of the invention or
used in the process of the present invention, such as by measuring
a level of an encoding nucleic acid molecule in a sample of cells,
e.g., detecting mRNA levels or determining, whether a genomic gene
comprising the sequence of the polynucleotide of the invention or
used in the processes of the present invention has been mutated or
deleted.
[12195] [0152.0.0.27] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 27, columns 5
or 7, such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular a methionine increasing activity as mentioned above
or as described in the examples in plants or microorganisms is
comprised.
[12196] [0153.0.0.27] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 27,
columns 5 or 7, such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 27, columns 5 or 7, has for
example an activity of a polypeptide indicated in Table XII,
application no. 27, column 3.
[12197] [0154.0.0.27] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 27, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[12198] [0155.0.0.27] Portions of proteins encoded by the nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention are preferably biologically active,
preferably having above-mentioned annotated activity, e.g.
conferring a increase the respective fine chemical after increase
of activity.
[12199] [0156.0.0.27] As mentioned herein, the term "biologically
active portion" is intended to include a portion, e.g., a
domain/motif, that confers increase of the respective fine chemical
or has an immunological activity such that it is binds to an
antibody binding specifically to the polypeptide of the present
invention or a polypeptide used in the process of the present
invention for producing the respective fine chemical;
[12200] [0157.0.0.27] The invention further relates to nucleic acid
molecules that differ from one of the nucleotide sequences
indicated in Table XI, application no. 27, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides comprising the consensus sequences as indicated
in Table XIV, application no. 27, columns 5 or 7, or of the
polypeptide as indicated in Table XII, application no. 27, columns
5 or 7, or their functional homologues. Advantageously, the nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention comprises, or in an other embodiment
has, a nucleotide sequence encoding a protein comprising, or in an
other embodiment having, a consensus sequences as indicated in
Table XIV, application no. 27, 7, or of the polypeptide as
indicated in Table XII, application no. 27, columns 5 or 7 or the
functional homologues. In a still further embodiment, the nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention encodes a full length protein which is
substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table XIV, application no. 27,
columns 7, or of a polypeptide as indicated in Table XII,
application no. 27, columns 5 or 7, or the functional homologues
thereof. However, in a one embodiment, the nucleic acid molecule of
the present invention does not consist of a sequence as indicated
in Table XI, application no. 27, columns 5 or 7,
[12201] [0158.0.0.27] In addition, it will be appreciated by those
skilled in the art that DNA sequence polymorphisms that lead to
changes in the amino acid sequences may exist within a population.
Such genetic polymorphism in the gene encoding the polypeptide of
the invention or the polypeptide used in the method of the
invention or comprising the nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
may exist among individuals within a population due to natural
variation.
[12202] [0159.0.0.27] As used herein, the terms "gene" and
"recombinant gene" refer to nucleic acid molecules comprising an
open reading frame encoding the polypeptide of the invention or the
polypeptide used in the method of the invention or comprising the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention or encoding the polypeptide
used in the process of the present invention, preferably from a
crop plant or from a microorganism useful for the production of
respective fine chemicals, in particular for the production of the
respective fine chemical. Such natural variations can typically
result in 1-5% variance in the nucleotide sequence of the gene. Any
and all such nucleotide variations and resulting amino acid
polymorphisms in genes encoding a polypeptide of the invention or
the polypeptide used in the method of the invention or comprising a
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention that are the result of
natural variation and that do not alter the functional activity as
described are intended to be within the scope of the invention.
[12203] [0160.0.0.27] Nucleic acid molecules corresponding to
natural variants homologues of a nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention, which can also be a cDNA, can be isolated based on their
homology to the nucleic acid molecules disclosed herein using the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, or a portion thereof, as a
hybridization probe according to standard hybridization techniques
under stringent hybridization conditions.
[12204] [0161.0.0.27] Accordingly, in another embodiment, a nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 27, columns 5 or
7. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[12205] [0162.0.0.27] The term "hybridizes under stringent
conditions" is defined above. In one embodiment, the term
"hybridizes under stringent conditions" is intended to describe
conditions for hybridization and washing under which nucleotide
sequences at least 30%, 40%, 50% or 65% identical to each other
typically remain hybridized to each other. Preferably, the
conditions are such that sequences at least about 70%, more
preferably at least about 75% or 80%, and even more preferably at
least about 85%, 90% or 95% or more identical to each other
typically remain hybridized to each other.
[12206] [0163.0.0.27] Preferably, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 27, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of an protein of the invention or used in
the process of the invention.
[12207] [0164.0.0.27] In addition to naturally-occurring variants
of the sequences of the polypeptide or nucleic acid molecule of the
invention as well as of the polypeptide or nucleic acid molecule
used in the process of the invention that may exist in the
population, the skilled artisan will further appreciate that
changes can be introduced by mutation into a nucleotide sequence of
the nucleic acid molecule encoding the polypeptide of the invention
or used in the process of the present invention, thereby leading to
changes in the amino acid sequence of the encoded said polypeptide,
without altering the functional ability of the polypeptide,
preferably not decreasing said activity.
[12208] [0165.0.0.27] For example, nucleotide substitutions leading
to amino acid substitutions at "non-essential" amino acid residues
can be made in a sequence of the nucleic acid molecule of the
invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 27, columns 5 or 7.
[12209] [0166.0.0.27] A "non-essential" amino acid residue is a
residue that can be altered from the wild-type sequence of one
without altering the activity of said polypeptide, whereas an
"essential" amino acid residue is required for an activity as
mentioned above, e.g. leading to an increase in the respective fine
chemical in an organism after an increase of activity of the
polypeptide. Other amino acid residues, however, (e.g., those that
are not conserved or only semi-conserved in the domain having said
activity) may not be essential for activity and thus are likely to
be amenable to alteration without altering said activity.
[12210] [0167.0.0.27] Further, a person skilled in the art knows
that the codon usage between organism can differ. Therefore, he may
adapt the codon usage in the nucleic acid molecule of the present
invention to the usage of the organism in which the polynucleotide
or polypeptide is expressed.
[12211] [0168.0.0.27] Accordingly, the invention relates to nucleic
acid molecules encoding a polypeptide having above-mentioned
activity, e.g. conferring an increase in the respective fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table XII, application no.
27, columns 5 or 7, yet retain said activity described herein. The
nucleic acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table XII, application no. 27, columns 5 or 7, and is
capable of participation in the increase of production of the
respective fine chemical after increasing its activity, e.g. its
expression. Preferably, the protein encoded by the nucleic acid
molecule is at least about 60% identical to a sequence as indicated
in Table XII, application no. 27, columns 5 or 7, more preferably
at least about 70% identical to one of the sequences as indicated
in Table XII, application no. 27, columns 5 or 7, even more
preferably at least about 80%, 90%, or 95% homologous to a sequence
as indicated in Table XII, application no. 27, columns 5 or 7, and
most preferably at least about 96%, 97%, 98%, or 99% identical to
the sequence as indicated in Table XII, application no. 27, columns
5 or 7.
[12212] [0169.0.0.27] To determine the percentage homology
(=identity) of two amino acid sequences or of two nucleic acid
molecules, the sequences are written one underneath the other for
an optimal comparison (for example gaps may be inserted into the
sequence of a protein or of a nucleic acid in order to generate an
optimal alignment with the other protein or the other nucleic
acid).
[12213] [0170.0.0.27] The amino acid residues or nucleic acid
molecules at the corresponding amino acid positions or nucleotide
positions are then compared. If a position in one sequence is
occupied by the same amino acid residue or the same nucleic acid
molecule as the corresponding position in the other sequence, the
molecules are homologous at this position (i.e. amino acid or
nucleic acid "homology" as used in the present context corresponds
to amino acid or nucleic acid "identity". The percentage homology
between the two sequences is a function of the number of identical
positions shared by the sequences (i.e. % homology=number of
identical positions/total number of positions.times.100). The terms
"homology" and "identity" are thus to be considered as
synonyms.
[12214] [0171.0.0.27] For the determination of the percentage
homology (=identity) of two or more amino acids or of two or more
nucleotide sequences several computer software programs have been
developed. The homology of two or more sequences can be calculated
with for example the software fasta, which presently has been used
in the version fasta 3 (W. R. Pearson and D. J. Lipman (1988),
Improved Tools for Biological Sequence Comparison. PNAS
85:2444-2448; W. R. Pearson (1990) Rapid and Sensitive Sequence
Comparison with FASTP and FASTA, Methods in Enzymology 183:63-98;
W. R. Pearson and D. J. Lipman (1988) Improved Tools for Biological
Sequence Comparison. PNAS 85:2444-2448; W. R. Pearson (1990); Rapid
and Sensitive Sequence Comparison with FASTP and FASTA Methods in
Enzymology 183:63-98). Another useful program for the calculation
of homologies of different sequences is the standard blast program,
which is included in the Biomax pedant software (Biomax, Munich,
Federal Republic of Germany). This leads unfortunately sometimes to
suboptimal results since blast does not always include complete
sequences of the subject and the query. Nevertheless as this
program is very efficient it can be used for the comparison of a
huge number of sequences. The following settings are typically used
for such a comparisons of sequences: -p Program Name [String]; -d
Database [String]; default=nr; -i Query File [File In];
default=stdin; -e Expectation value (E) [Real]; default=10.0; -m
alignment view options: 0=pairwise; 1=query-anchored showing
identities; 2=query-anchored no identities; 3=flat query-anchored,
show identities; 4=flat query-anchored, no identities;
5=query-anchored no identities and blunt ends; 6=flat
query-anchored, no identities and blunt ends; 7=XML Blast output;
8=tabular; 9 tabular with comment lines [Integer]; default=0; -o
BLAST report Output File [File Out] Optional; default=stdout; -F
Filter query sequence (DUST with blastn, SEG with others) [String];
default=T; -G Cost to open a gap (zero invokes default behavior)
[Integer]; default=0; -E Cost to extend a gap (zero invokes default
behavior) [Integer]; default=0; -X X dropoff value for gapped
alignment (in bits) (zero invokes default behavior); blastn 30,
megablast 20, tblastx 0, all others 15 [Integer]; default=0; -I
Show GI's in deflines [T/F]; default=F; -q Penalty for a nucleotide
mismatch (blastn only) [Integer]; default=-3; -r Reward for a
nucleotide match (blastn only) [Integer]; default=1; -v Number of
database sequences to show one-line descriptions for (V) [Integer];
default=500; -b Number of database sequence to show alignments for
(B) [Integer]; default=250; -f Threshold for extending hits,
default if zero; blastp 11, blastn 0, blastx 12, tblastn 13;
tblastx 13, megablast 0 [Integer]; default=0; -g Perfom gapped
alignment (not available with tblastx) [T/F]; default=T; -Q Query
Genetic code to use [Integer]; default=1; -D DB Genetic code (for
tblast[nx] only) [Integer]; default=1; -a Number of processors to
use [Integer]; default=1; -O SeqAlign file [File Out] Optional; -J
Believe the query defline [T/F]; default=F; -M Matrix [String];
default=BLOSUM62; -W Word size, default if zero (blastn 11,
megablast 28, all others 3) [Integer]; default=0; -z Effective
length of the database (use zero for the real size) [Real];
default=0; -K Number of best hits from a region to keep (off by
default, if used a value of 100 is recommended) [Integer];
default=0; -P 0 for multiple hit, 1 for single hit [Integer];
default=0; -Y Effective length of the search space (use zero for
the real size) [Real]; default=0; -S Query strands to search
against database (for blast[nx], and tblastx); 3 is both, 1 is top,
2 is bottom [Integer]; default=3; -T Produce HTML output [T/F];
default=F; -I Restrict search of database to list of GI's [String]
Optional; -U Use lower case filtering of FASTA sequence [T/F]
Optional; default=F; -y X dropoff value for ungapped extensions in
bits (0.0 invokes default behavior); blastn 20, megablast 10, all
others 7 [Real]; default=0.0; -Z X dropoff value for final gapped
alignment in bits (0.0 invokes default behavior); blastn/megablast
50, tblastx 0, all others 25 [Integer]; default=0; -R PSI-TBLASTN
checkpoint file [File In] Optional; -n MegaBlast search [T/F];
default=F; -L Location on query sequence [String] Optional; -A
Multiple Hits window size, default if zero (blastn/megablast 0, all
others 40 [Integer]; default=0; -w Frame shift penalty (OOF
algorithm for blastx) [Integer]; default=0; -t Length of the
largest intron allowed in tblastn for linking HSPs (0 disables
linking) [Integer]; default=0.
[12215] [0172.0.0.27] Results of high quality are reached by using
the algorithm of Needleman and Wunsch or Smith and Waterman.
Therefore programs based on said algorithms are preferred.
Advantageously the comparisons of sequences can be done with the
program PileUp (J. Mol. Evolution., 25, 351-360, 1987, Higgins et
al., CABIOS, 5 1989: 151-153) or preferably with the programs Gap
and BestFit, which are respectively based on the algorithms of
Needleman and Wunsch [J. Mol. Biol. 48; 443-453 (1970)] and Smith
and Waterman [Adv. Appl. Math. 2; 482-489 (1981)]. Both programs
are part of the GCG software-package [Genetics Computer Group, 575
Science Drive, Madison, Wis., USA 53711 (1991); Altschul et al.
(1997) Nucleic Acids Res. 25:3389 et seq.]. Therefore preferably
the calculations to determine the percentages of sequence homology
are done with the program Gap over the whole range of the
sequences. The following standard adjustments for the comparison of
nucleic acid sequences were used: gap weight: 50, length weight: 3,
average match: 10.000, average mismatch: 0.000.
[12216] [0173.0.0.27] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108199 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108199 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[12217] [0174.0.0.27] In the state of the art, homology between two
polypeptides is also understood as meaning the identity of the
amino acid sequence over in each case the entire sequence length
which is calculated by comparison with the aid of the program
algorithm GAP (Wisconsin Package Version 10.0, University of
Wisconsin, Genetics Computer Group (GCG), Madison, USA), setting
the following parameters:
TABLE-US-00146 Gap weight: 8 Length weight: 2 Average match: 2,912
Average mismatch: -2,003
[12218] [0175.0.0.27] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108200 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108200 by the above program algorithm with the
above parameter set, has a 80% homology.
[12219] [0176.0.0.27] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 27,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 27, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 27, columns
5 or 7.
[12220] [0177.0.0.27] Functional equivalents derived from a nucleic
acid sequence as indicated in Table XI, application no. 27, columns
5 or 7, according to the invention by substitution, insertion or
deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at
least 55%, 60%, 65% or 70% by preference at least 80%, especially
preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very
especially preferably at least 95%, 97%, 98% or 99% homology with
one of a polypeptide as indicated in Table XII, application no. 27,
columns 5 or 7, according to the invention and encode polypeptides
having essentially the same properties as a polypeptide as
indicated in Table XII, application no. 27, columns 5 or 7.
[12221] [0178.0.0.27] "Essentially the same properties" of a
functional equivalent is above all understood as meaning that the
functional equivalent has above mentioned activity, e.g. conferring
an increase in the respective fine chemical amount while increasing
the amount of protein, activity or function of said functional
equivalent in an organism, e.g. a microorganism, a plant or plant
or animal tissue, plant or animal cells or a part of the same.
[12222] [0179.0.0.27] A nucleic acid molecule encoding a homologous
to a protein sequence of as indicated in Table XII, application no.
27, columns 5 or 7, can be created by introducing one or more
nucleotide substitutions, additions or deletions into a nucleotide
sequence of the nucleic acid molecule of the present invention, in
particular as indicated in Table XI, application no. 27, columns 5
or 7, such that one or more amino acid substitutions, additions or
deletions are introduced into the encoded protein. Mutations can be
introduced into the encoding sequences for example into sequences
as indicated in Table XI, application no. 27, columns 5 or 7, by
standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[12223] [0180.0.0.27] Preferably, conservative amino acid
substitutions are made at one or more predicted non-essential amino
acid residues. A "conservative amino acid substitution" is one in
which the amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[12224] [0181.0.0.27] Thus, a predicted nonessential amino acid
residue in a polypeptide of the invention or a polypeptide used in
the process of the invention is preferably replaced with another
amino acid residue from the same family. Alternatively, in another
embodiment, mutations can be introduced randomly along all or part
of a coding sequence of a nucleic acid molecule of the invention or
used in the process of the invention, such as by saturation
mutagenesis, and the resultant mutants can be screened for activity
described herein to identify mutants that retain or even have
increased above mentioned activity, e.g. conferring an increase in
content of the respective fine chemical.
[12225] [0182.0.0.27] Following mutagenesis of one of the sequences
shown herein, the encoded protein can be expressed recombinantly
and the activity of the protein can be determined using, for
example, assays described herein (see Examples).
[12226] [0183.0.0.27] The highest homology of the nucleic acid
molecule used in the process according to the invention was found
for the following database entries by Gap search.
[12227] [0184.0.0.27] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 27,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 27, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, columns 5 or 7, lines 1
to 5 and/or lines 334 to 338, or from the derived nucleic acid
sequences, the intention being, however, that the enzyme activity
or the biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[12228] [0185.0.0.27] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 27, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotide sequences not shown in any one of
sequences as indicated in Table XI, application no. 27, columns 5
or 7. In one embodiment, the nucleic acid molecule comprises less
than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or 40 further
nucleotides. In a further embodiment, the nucleic acid molecule
comprises less than 30, 20 or 10 further nucleotides. In one
embodiment, a nucleic acid molecule used in the process of the
invention is identical to a sequences as indicated in Table XI,
application no. 27, columns 5 or 7.
[12229] [0186.0.0.27] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encode a
polypeptide comprising a sequence as indicated in Table XII,
application no. 27, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 27, columns 5 or 7.
[12230] [0187.0.0.27] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table XII, application no.
27, columns 5 or 7, comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence
encoding a sequences as indicated in Table XII, application no. 27,
columns 5 or 7.
[12231] [0188.0.0.27] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 27, columns 5 or 7, preferably compared to a
sequence as indicated in Table XII, application no. 27, column 5,
and expressed under identical conditions.
[12232] [0189.0.0.27] Homologues of a sequence as indicated in
Table XI, application no. 27, columns 5 or 7, or of a derived
sequence as indicated in Table XII, application no. 27, columns 5
or 7, also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[12233] [0190.0.0.27] In a further embodiment, the process
according to the present invention comprises the following steps:
[12234] (a) selecting an organism or a part thereof expressing the
polypeptide of this invention; [12235] (b) mutagenizing the
selected organism or the part thereof; [12236] (c) comparing the
activity or the expression level of said polypeptide in the
mutagenized organism or the part thereof with the activity or the
expression of said polypeptide in the selected organisms or the
part thereof; [12237] (d) selecting the mutagenized organisms or
parts thereof, which comprise an increased activity or expression
level of said polypeptide compared to the selected organism (a) or
the part thereof; [12238] (e) optionally, growing and cultivating
the organisms or the parts thereof; and [12239] (f) recovering, and
optionally isolating, the free or bound respective fine chemical
produced by the selected mutated organisms or parts thereof.
[12240] [0191.0.0.27] The organisms or part thereof produce
according to the herein mentioned process of the invention an
increased level of free and/or -bound respective fine chemical
compared to said control or selected organisms or parts
thereof.
[12241] [0191.1.0.27] In one embodiment, the organisms or part
thereof produce according to the herein mentioned process of the
invention an increased level of protein-bound respective fine
chemical compared to said control or selected organisms or parts
thereof.
[12242] [0192.0.0.27] Advantageously the selected organisms are
mutagenized according to the invention. According to the invention
mutagenesis is any change of the genetic information in the genome
of an organism, that means any structural or compositional change
in the nucleic acid preferably DNA of an organism that is not
caused by normal segregation or genetic recombination processes.
Such mutations may occur spontaneously, or may be induced by
mutagens as described below. Such change can be induced either
randomly or selectively. In both cases the genetic information of
the organism is modified. In general this lead to the situation
that the activity of the gene product of the relevant genes inside
the cells or inside the organism is increased.
[12243] [0193.0.0.27] In case of the specific or so called site
directed mutagenesis a distinct gene is mutated and thereby its
activity and/or the activity or the encoded gene product is
repressed, reduced or increased, preferably increased. In the event
of a random mutagenesis one or more genes are mutated by chance and
their activities and/or the activities of their gene products are
repressed, reduced or increased, preferably increased.
[12244] [0194.0.0.27] For the purpose of a mutagenesis of a huge
population of organisms, such population can be transformed with a
DNA construct, which is useful for the activation of as much as
possible genes of an organism, preferably all genes. For example
the construct can contain a strong promoter or one or more
enhancers, which are capable of transcriptionally activate genes in
the vicinity of their integration side. With this method it is
possible to statistically mutagenize, e.g. activate nearly all
genes of an organism by the random integration of an activation
construct. Afterwards the skilled worker can identify those
mutagenized lines in which a gene of the invention has been
activated, which in turns leads to the desired increase in the
respective fine chemical production.
[12245] [0195.0.0.27] The genes of the invention can also be
activated by mutagenesis, either of regulatory or coding regions.
In the event of a random mutagenesis a huge number of organisms are
treated with a mutagenic agent. The amount of said agent and the
intensity of the treatment will be chosen in such a manner that
statistically nearly every gene is mutated once. The process for
the random mutagenesis as well as the respective agens is well
known by the skilled person. Such methods are disclosed for example
by A. M. van Harten [(1998), "Mutation breeding: theory and
practical applications", Cambridge University Press, Cambridge,
UK], E Friedberg, G Walker, W Siede [(1995), "DNA Repair and
Mutagenesis", Blackwell Publishing], or K. Sankaranarayanan, J. M.
Gentile, L. R. Ferguson [(2000) "Protocols in Mutagenesis",
Elsevier Health Sciences]. As the skilled worker knows the
spontaneous mutation rate in the cells of an organism is very low
and that a large number of chemical, physical or biological agents
are available for the mutagenesis of organisms. These agents are
named as mutagens or mutagenic agents. As mentioned before three
different kinds of mutagens (chemical, physical or biological
agents) are available.
[12246] [0196.0.0.27] There are different classes of chemical
mutagens, which can be separated by their mode of action. For
example base analogues such as 5-bromouracil, 2-amino purin. Other
chemical mutagens are interacting with the DNA such as sulphuric
acid, nitrous acid, hydroxylamine; or other alkylating agents such
as monofunctional agents like ethyl methanesulfonate,
dimethylsulfate, methyl methanesulfonate), bifunctional like
dichloroethyl sulphide, Mitomycin,
Nitrosoguanidine-dialkylnitrosamine, N-Nitrosoguanidin derivatives,
N-alkyl-N-nitro-N-nitroso-guanidine-), intercalating dyes like
Acridine, ethidium bromide).
[12247] [0197.0.0.27] Physical mutagens are for example ionizing
irradiation (X ray), UV irradiation. Different forms of irradiation
are available and they are strong mutagens. Two main classes of
irradiation can be distinguished: a) non-ionizing irradiation such
as UV light or ionizing irradiation such as X ray. Biological
mutagens are for example transposable elements for example IS
elements such as IS100, transposons such as Tn5, Tn10, Tn916 or
Tn1000 or phages like Muamplac, P1, T5, Aplac etc. Methods for
introducing this phage DNA into the appropriate microorganism are
well known to the skilled worker (see Microbiology, Third Edition,
Eds. Davis, B. D., Dulbecco, R., Eisen, H. N. and Ginsberg, H. S.,
Harper International Edition, 1980). The common procedure of a
transposon mutagenesis is the insertion of a transposable element
within a gene or nearby for example in the promotor or terminator
region and thereby leading to a loss of the gene function.
Procedures to localize the transposon within the genome of the
organisms are well known by a person skilled in the art.
[12248] [0198.0.0.27] Preferably a chemical or biochemical
procedure is used for the mutagenesis of the organisms. A preferred
chemical method is the mutagenesis with
N-methyl-N-nitro-nitrosoguanidine.
[12249] [0199.0.0.27] Other biological method are disclosed by Spee
et al. (Nucleic Acids Research, Vol. 21, No. 3, 1993: 777-778).
Spee et al. teaches a PCR method using dITP for the random
mutagenesis. This method described by Spee et al. was further
improved by Rellos et al. (Protein Expr. Purif., 5, 1994: 270-277).
The use of an in vitro recombination technique for molecular
mutagenesis is described by Stemmer (Proc. Natl. Acad. Sci. USA,
Vol. 91, 1994: 10747-10751). Moore et al. (Nature Biotechnology
Vol. 14, 1996: 458-467) describe the combination of the PCR and
recombination methods for increasing the enzymatic activity of an
esterase toward a para-nitrobenzyl ester. Another route to the
mutagenesis of enzymes is described by Greener et al. in Methods in
Molecular Biology (Vol. 57, 1996: 375-385). Greener et al. use the
specific Escherichia coli strain XL1-Red to generate Escherichia
coli mutants which have increased antibiotic resistance.
[12250] [0200.0.0.27] In one embodiment, the protein according to
the invention or the nucleic acid molecule characterized herein
originates from a eukaryotic or prokaryotic organism such as a
non-human animal, a plant, a microorganism such as a fungi, a
yeast, an alga, a diatom or a bacterium. Nucleic acid molecules,
which advantageously can be used in the process of the invention
originate from yeasts, for example the family Saccharomycetaceae,
in particular the genus Saccharomyces, or yeast genera such as
Candida, Hansenula, Pichia, Yarrowia, Rhodotorula or
Schizosaccharomyces and the especially advantageous from the
species Saccharomyces cerevisiae.
[12251] [0201.0.0.27] In one embodiment, nucleic acid molecules,
which advantageously can be used in the process of the invention
originate from bacteria, for example from Proteobacteria, in
particular from Gammaproteobacteria, more preferred from
Enterobacteriales, e.g. from the family Enterobacteriaceae,
particularly from genera Escherichia, Salmonella, Klebsiella,
advantageously form the species Escherichia coli K12.
[12252] [0202.0.0.27] If, in the process according to the
invention, plants are selected as the donor organism, this plant
may, in principle, be in any phylogenetic relation of the recipient
plant. Donor and recipient plant may belong to the same family,
genus, species, variety or line, resulting in an increasing
homology between the nucleic acids to be integrated and
corresponding parts of the genome of the recipient plant. This also
applies analogously to microorganisms as donor and recipient
organism. It might also be advantageously to use nuclei acids
molecules from very distinct species, since these might exhibit
reduced sensitivity against endogenous regulatory mechanisms and
such sequences might not be recognized by endogenous silencing
mechanisms.
[12253] [0203.0.0.27] Accordingly, one embodiment of the
application relates to the use of nucleic acid molecules in the
process of the invention from plants, e.g. crop plants, e.g. from:
B. napus; Glycine max; sunflower linseed or maize or their
homologues.
[12254] [0204.0.0.27] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule which comprises a nucleic acid
molecule selected from the group consisting of: [12255] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table XII, application no. 27, columns
5 or 7, or a fragment thereof conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof
[12256] b) nucleic acid molecule comprising, preferably at least
the mature form, of a nucleic acid molecule as indicated in Table
XI, application no. 27, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [12257] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [12258] d) nucleic acid molecule encoding a polypeptide
whose sequence has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [12259]
e) nucleic acid molecule which hybridizes with a nucleic acid
molecule of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [12260] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [12261] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [12262]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
primers or primer pairs as indicated in Table XIII, application no.
27, column 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [12263]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from a expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (g), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [12264] j) nucleic acid molecule which
encodes a polypeptide comprising a consensus sequence as indicated
in Table XIV, application no. 27, columns 7, and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [12265] k) nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of a polypeptide as indicated in Table XII, application no. 27,
columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[12266] l) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment of at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (h) or of a
nucleic acid molecule as indicated in Table XI, application no. 27,
columns 5 or 7, or a nucleic acid molecule encoding, preferably at
least the mature form of, the polypeptide as indicated in Table
XII, application no. 27, columns 5 or 7, and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; or which encompasses a sequence which is
complementary thereto; whereby, preferably, the nucleic acid
molecule according to (a) to (l) distinguishes over the sequence
indicated in Table XI, application no. 27, columns 5 or 7, by one
or more nucleotides. In one embodiment, the nucleic acid molecule
does not consist of the sequence shown and indicated in Table XI,
application no. 27, columns 5 or 7: In one embodiment, the nucleic
acid molecule is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table XI, application no. 27,
columns 5 or 7. In another embodiment, the nucleic acid molecule
does not encode a polypeptide of a sequence indicated in Table XII,
application no. 27, columns 5 or 7. In an other embodiment, the
nucleic acid molecule of the present invention is at least 30%,
40%, 50%, or 60% identical and less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI,
application no. 27, columns 5 or 7. In a further embodiment the
nucleic acid molecule does not encode a polypeptide sequence as
indicated in Table XII, application no. 27, columns 5 or 7.
Accordingly, in one embodiment, the nucleic acid molecule of the
differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 27, columns 5 or 7.
Accordingly, in one embodiment, the nucleic acid molecule of the
present invention encodes a polypeptide, which differs at least in
one or more amino acids from a polypeptide indicated in Table XII,
application no. 27, columns 5 or 7. In another embodiment, a
nucleic acid molecule indicated in Table XI, application no. 27,
columns 5 or 7, does not encode a protein of a sequence indicated
in Table XII, application no. 27, columns 5 or 7. Accordingly, in
one embodiment, the protein encoded by a sequences of a nucleic
acid according to (a) to (l) does not consist of a sequence as
indicated in Table XII, application no. 27, columns 5 or 7. In a
further embodiment, the protein of the present invention is at
least 30%, 40%, 50%, or 60% identical to a protein sequence
indicated in Table XII, application no. 27, columns 5 or 7, and
less than 100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 98%, 97%, 96% or 95% identical to a
sequence as indicated in Table XI, application no. 27, columns 5 or
7.
[12267] [0205.0.0.27] The nucleic acid sequences used in the
process are advantageously introduced in a nucleic acid construct,
preferably an expression cassette which makes possible the
expression of the nucleic acid molecules in an organism,
advantageously a plant or a microorganism.
[12268] [0206.0.0.27] Accordingly, the invention also relates to an
nucleic acid construct, preferably to an expression construct,
comprising the nucleic acid molecule of the present invention
functionally linked to one or more regulatory elements or
signals.
[12269] [0207.0.0.27] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes are genes of the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[12270] [0208.0.0.27] In principle, the nucleic acid construct can
comprise the herein described regulator sequences and further
sequences relevant for the expression of the comprised genes. Thus,
the nucleic acid construct of the invention can be used as
expression cassette and thus can be used directly for introduction
into the plant, or else they may be introduced into a vector.
Accordingly in one embodiment the nucleic acid construct is an
expression cassette comprising a microorganism promoter or a
microorganism terminator or both. In another embodiment the
expression cassette encompasses a plant promoter or a plant
terminator or both.
[12271] [0209.0.0.27] Accordingly, in one embodiment, the process
according to the invention comprises the following steps: [12272]
(a) introducing of a nucleic acid construct comprising the nucleic
acid molecule of the invention or used in the process of the
invention or encoding the polypeptide of the present invention or
used in the process of the invention; or [12273] (b) introducing of
a nucleic acid molecule, including regulatory sequences or factors,
which expression increases the expression of the nucleic acid
molecule of the invention or used in the process of the invention
or encoding the polypeptide of the present invention or used in the
process of the invention; [12274] in a cell, or an organism or a
part thereof, preferably in a plant, plant cell or a microorganism,
and [12275] (c) expressing of the gene product encoded by the
nucleic acid construct or the nucleic acid molecule mentioned under
(a) or (b) in the cell or the organism.
[12276] [0210.0.0.27] After the introduction and expression of the
nucleic acid construct the transgenic organism or cell is
advantageously cultured and subsequently harvested. The transgenic
organism or cell may be a prokaryotic or eukaryotic organism such
as a microorganism, a non-human animal and plant for example a
plant or animal cell, a plant or animal tissue, preferably a crop
plant, or a part thereof.
[12277] [0211.0.0.27] To introduce a nucleic acid molecule into a
nucleic acid construct, e.g. as part of an expression cassette, the
codogenic gene segment is advantageously subjected to an
amplification and ligation reaction in the manner known by a
skilled person. It is preferred to follow a procedure similar to
the protocol for the Pfu DNA polymerase or a Pfu/Taq DNA polymerase
mixture. The primers are selected according to the sequence to be
amplified. The primers should expediently be chosen in such a way
that the amplificate comprise the codogenic sequence from the start
to the stop codon. After the amplification, the amplificate is
expediently analyzed. For example, the analysis may consider
quality and quantity and be carried out following separation by gel
electrophoresis. Thereafter, the amplificate can be purified
following a standard protocol (for example Qiagen). An aliquot of
the purified amplificate is then available for the subsequent
cloning step. Suitable cloning vectors are generally known to the
skilled worker.
[12278] [0212.0.0.27] They include, in particular, vectors which
are capable of replication in easy to handle cloning systems like
as bacterial yeast or insect cell based (e.g. baculovirus
expression) systems, that is to say especially vectors which ensure
efficient cloning in E. coli, and which make possible the stable
transformation of plants. Vectors, which must be mentioned in
particular are various binary and cointegrated vector systems which
are suitable for the T-DNA-mediated transformation. Such vector
systems are generally characterized in that they contain at least
the vir genes, which are required for the Agrobacterium-mediated
transformation, and the T-DNA border sequences.
[12279] [0213.0.0.27] In general, vector systems preferably also
comprise further cis-regulatory regions such as promoters and
terminators and/or selection markers by means of which suitably
transformed organisms can be identified. While vir genes and T-DNA
sequences are located on the same vector in the case of
cointegrated vector systems, binary systems are based on at least
two vectors, one of which bears vir genes, but no T-DNA, while a
second one bears T-DNA, but no vir gene. Owing to this fact, the
last-mentioned vectors are relatively small, easy to manipulate and
capable of replication in E. coli and in Agrobacterium. These
binary vectors include vectors from the series pBIB-HYG, pPZP,
pBecks, pGreen. Those which are preferably used in accordance with
the invention are Bin19, pBI101, pBinAR, pGPTV and pCAMBIA. An
overview of binary vectors and their use is given by Hellens et al,
Trends in Plant Science (2000) 5, 446-451.
[12280] [0214.0.0.27] For a vector preparation, vectors may first
be linearized using restriction endonuclease(s) and then be
modified enzymatically in a suitable manner. Thereafter, the vector
is purified, and an aliquot is employed in the cloning step. In the
cloning step, the enzyme-cleaved and, if required, purified
amplificate is cloned together with similarly prepared vector
fragments, using ligase. In this context, a specific nucleic acid
construct, or vector or plasmid construct, may have one or else
more codogenic gene segments. The codogenic gene segments in these
constructs are preferably linked operably to regulatory sequences.
The regulatory sequences include, in particular, plant sequences
like the above-described promoters and terminators. The constructs
can advantageously be propagated stably in microorganisms, in
particular Escherichia coli and/or Agrobacterium tumefaciens, under
selective conditions and enable the transfer of heterologous DNA
into plants or other microorganisms. In accordance with a
particular embodiment, the constructs are based on binary vectors
(overview of a binary vector: Hellens et al., 2000). As a rule,
they contain prokaryotic regulatory sequences, such as replication
origin and selection markers, for the multiplication in
microorganisms such as Escherichia coli and Agrobacterium
tumefaciens. Vectors can further contain agrobacterial T-DNA
sequences for the transfer of DNA into plant genomes or other
eukaryotic regulatory sequences for transfer into other eukaryotic
cells, e.g. Saccharomyces sp. or other prokaryotic regulatory
sequences for the transfer into other prokaryotic cells, e.g.
Corynebacterium sp. or Bacillus sp. For the transformation of
plants, the right border sequence, which comprises approximately 25
base pairs, of the total agrobacterial T-DNA sequence is
advantageously included. Usually, the plant transformation vector
constructs according to the invention contain T-DNA sequences both
from the right and from the left border region, which contain
expedient recognition sites for site-specific acting enzymes which,
in turn, are encoded by some of the vir genes.
[12281] [0215.0.0.27] Suitable host organisms are known to the
skilled worker. Advantageous organisms are described further above
in the present application. They include in particular eukaryotes
or eubacteria, e.g. prokaryotes or archae bacteria. Advantageously
host organisms are microorganisms selected from the group
consisting of Actinomycetaceae, Bacillaceae, Brevibacteriaceae,
Corynebacteriaceae, Enterobacteriacae, Gordoniaceae,
Micrococcaceae, Mycobacteriaceae, Nocardiaceae, Pseudomonaceae,
Rhizobiaceae, Streptomycetaceae, Chaetomiaceae, Choanephoraceae,
Cryptococcaceae, Cunninghamellaceae, Demetiaceae, Moniliaceae,
Mortierellaceae, Mucoraceae, Pythiaceae, Sacharomycetaceae,
Saprolegniaceae, Schizosacharomycetaceae, Sodariaceae,
Sporobolomycetaceae, Tuberculariaceae, Adelotheciaceae,
Dinophyceae, Ditrichaceae and Prasinophyceae. Preferably are
unicellular, microorganisms, e.g. fungi, bacteria or protoza, such
as fungi like the genus Claviceps or Aspergillus or gram-positive
bacteria such as the genera Bacillus, Corynebacterium, Micrococcus,
Brevibacterium, Rhodococcus, Nocardia, Caseobacter or Arthrobacter
or gram-negative bacteria such as the genera Escherichia,
Flavobacterium or Salmonella, or yeasts such as the genera
Rhodotorula, Hansenula, Pichia, Yerrowia, Saccharomyces,
Schizosaccharomyces or Candida.
[12282] [0216.0.0.27] Host organisms which are especially
advantageously selected in the process according to the invention
are microorganisms selected from the group of the genera and
species consisting of Hansenula anomala, Candida utilis, Claviceps
purpurea, Bacillus circulans, Bacillus subtilis, Bacillus sp.,
Brevibacterium albidum, Brevibacterium album, Brevibacterium
cerinum, Brevibacterium flavum, Brevibacterium glutamigenes,
Brevibacterium iodinum, Brevibacterium ketoglutamicum,
Brevibacterium lactofermentum, Brevibacterium linens,
Brevibacterium roseum, Brevibacterium saccharolyticum,
Brevibacterium sp., Corynebacterium acetoacidophilum,
Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes,
Corynebacterium glutamicum (=Micrococcus glutamicum),
Corynebacterium melassecola, Corynebacterium sp. or Escherichia
coli, specifically Escherichia coli K12 and its described
strains.
[12283] [0217.0.0.27] Advantageously preferred in accordance with
the invention are host organisms of the genus Agrobacterium
tumefaciens or plants. Preferred plants are selected from among the
families Aceraceae, Anacardiaceae, Apiaceae, Asteraceae, Apiaceae,
Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Cactaceae,
Caricaceae, Caryophyllaceae, Cannabaceae, Convolvulaceae,
Chenopodiaceae, Elaeagnaceae, Geraniaceae, Gramineae, Juglandaceae,
Lauraceae, Leguminosae, Linaceae, Cucurbitaceae, Cyperaceae,
Euphorbiaceae, Fabaceae, Malvaceae, Nymphaeaceae, Papaveraceae,
Rosaceae, Salicaceae, Solanaceae, Arecaceae, Iridaceae, Liliaceae,
Orchidaceae, Gentianaceae, Labiaceae, Magnoliaceae, Ranunculaceae,
Carifolaceae, Rubiaceae, Scrophulariaceae, Ericaceae, Polygonaceae,
Violaceae, Juncaceae, Poaceae, perennial grass, fodder crops,
vegetables and ornamentals.
[12284] [0218.0.0.27] Especially preferred are plants selected from
the groups of the families Apiaceae, Asteraceae, Brassicaceae,
Cucurbitaceae, Fabaceae, Papaveraceae, Rosaceae, Solanaceae,
Liliaceae or Poaceae. Especially advantageous are, in particular,
crop plants. Accordingly, an advantageous plant preferably belongs
to the group of the genus peanut, oilseed rape, canola, sunflower,
safflower, olive, sesame, hazelnut, almond, avocado, bay,
pumpkin/squash, linseed, soya, pistachio, borage, maize, wheat,
rye, oats, sorghum and millet, triticale, rice, barley, cassava,
potato, sugarbeet, fodder beet, egg plant, and perennial grasses
and forage plants, oil palm, vegetables (brassicas, root
vegetables, tuber vegetables, pod vegetables, fruiting vegetables,
onion vegetables, leafy vegetables and stem vegetables), buckwheat,
Jerusalem artichoke, broad bean, vetches, lentil, alfalfa, dwarf
bean, lupin, clover and lucerne.
[12285] [0219.0.0.27] In order to introduce, into a plant, the
nucleic acid molecule of the invention or used in the process
according to the invention, it has proved advantageous first to
transfer them into an intermediate host, for example a bacterium or
a eukaryotic unicellular cell. The transformation into E. coli,
which can be carried out in a manner known per se, for example by
means of heat shock or electroporation, has proved itself expedient
in this context. Thus, the transformed E. coli colonies can be
analysed for their cloning efficiency. This can be carried out with
the aid of a PCR. Here, not only the identity, but also the
integrity, of the plasmid construct can be verified with the aid of
a defined colony number by subjecting an aliquot of the colonies to
said PCR. As a rule, universal primers which are derived from
vector sequences are used for this purpose, it being possible, for
example, for a forward primer to be arranged upstream of the start
ATG and a reverse primer to be arranged downstream of the stop
codon of the codogenic gene segment. The amplificates are separated
by electrophoresis and assessed with regard to quantity and
quality.
[12286] [0220.0.0.27] The nucleic acid constructs, which are
optionally verified, are subsequently used for the transformation
of the plants or other hosts, e.g. other eukaryotic cells or other
prokaryotic cells. To this end, it may first be necessary to obtain
the constructs from the intermediate host. For example, the
constructs may be obtained as plasmids from bacterial hosts by a
method similar to conventional plasmid isolation.
[12287] [0221.0.0.27] The nucleic acid molecule of the invention or
used in the process according to the invention can also be
introduced into modified viral vectors like baculovirus vectors for
expression in insect cells or plant viral vectors like tobacco
mosaic virus or potato virus X-based vectors. Approaches leading to
the expression of proteins from the modified viral genome including
the the nucleic acid molecule of the invention or used in the
process according to the invention involve for example the
inoculation of tobacco plants with infectious RNA transcribed in
vitro from a cDNA copy of the recombinant viral genome. Another
approach utilizes the transfection of whole plants from wounds
inoculated with Agrobacterium tumefaciens containing cDNA copies of
recombinant plus-sense RNA viruses. Different vectors and virus are
known to the skilled worker for expression in different target eg.
production plants.
[12288] [0222.0.0.27] A large number of methods for the
transformation of plants are known. Since, in accordance with the
invention, a stable Xlntegration of heterologous DNA into the
genome of plants is advantageous, the T-DNA-mediated transformation
has proved expedient in particular. For this purpose, it is first
necessary to transform suitable vehicles, in particular
agrobacteria, with a codogenic gene segment or the corresponding
plasmid construct comprising the nucleic acid molecule of the
invention. This can be carried out in a manner known per se. For
example, said nucleic acid construct of the invention, or said
expression construct or said plasmid construct, which has been
generated in accordance with what has been detailed above, can be
transformed into competent agrobacteria by means of electroporation
or heat shock. In principle, one must differentiate between the
formation of cointegrated vectors on the one hand and the
transformation with binary vectors on the other hand. In the case
of the first alternative, the constructs, which comprise the
codogenic gene segment or the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention have no T-DNA sequences, but the formation of the
cointegrated vectors or constructs takes place in the agrobacteria
by homologous recombination of the construct with T-DNA. The T-DNA
is present in the agrobacteria in the form of Ti or Ri plasmids in
which exogenous DNA has expediently replaced the oncogenes. If
binary vectors are used, they can be transferred to agrobacteria
either by bacterial conjugation or by direct transfer. These
agrobacteria expediently already comprise the vector bearing the
vir genes (currently referred to as helper Ti(Ri) plasmid).
[12289] [0223.0.0.27] One or more markers may expediently also be
used together with the nucleic acid construct, or the vector of the
invention and, if plants or plant cells shall be transformed
together with the T-DNA, with the aid of which the isolation or
selection of transformed organisms, such as agrobacteria or
transformed plant cells, is possible. These marker genes enable the
identification of a successful transfer of the nucleic acid
molecules according to the invention via a series of different
principles, for example via visual identification with the aid of
fluorescence, luminescence or in the wavelength range of light
which is discernible for the human eye, by a resistance to
herbicides or antibiotics, via what are known as nutritive markers
(auxotrophism markers) or antinutritive markers, via enzyme assays
or via phytohormones. Examples of such markers which may be
mentioned are GFP (=green fluorescent protein); the
luciferin/luceferase system, the -galactosidase with its colored
substrates, for example X-Gal, the herbicide resistances to, for
example, imidazolinone, glyphosate, phosphinothricin or
sulfonylurea, the antibiotic resistances to, for example,
bleomycin, hygromycin, streptomycin, kanamycin, tetracyclin,
chloramphenicol, ampicillin, gentamycin, geneticin (G418),
spectinomycin or blasticidin, to mention only a few, nutritive
markers such as the utilization of mannose or xylose, or
antinutritive markers such as the resistance to 2-deoxyglucose.
This list is a small number of possible markers. The skilled worker
is very familiar with such markers. Different markers are
preferred, depending on the organism and the selection method.
[12290] [0224.0.0.27] As a rule, it is desired that the plant
nucleic acid constructs are flanked by T-DNA at one or both sides
of the codogenic gene segment. This is particularly useful when
bacteria of the species Agrobacterium tumefaciens or Agrobacterium
rhizogenes are used for the transformation. A method, which is
preferred in accordance with the invention, is the transformation
with the aid of Agrobacterium tumefaciens. However, biolistic
methods may also be used advantageously for introducing the
sequences in the process according to the invention, and the
introduction by means of PEG is also possible. The transformed
agrobacteria can be grown in the manner known per se and are thus
available for the expedient transformation of the plants. The
plants or plant parts to be transformed are grown or provided in
the customary manner. The transformed agrobacteria are subsequently
allowed to act on the plants or plant parts until a sufficient
transformation rate is reached. Allowing the agrobacteria to act on
the plants or plant parts can take different forms. For example, a
culture of morphogenic plant cells or tissue may be used. After the
T-DNA transfer, the bacteria are, as a rule, eliminated by
antibiotics, and the regeneration of plant tissue is induced. This
is done in particular using suitable plant hormones in order to
initially induce callus formation and then to promote shoot
development.
[12291] [0225.0.0.27] The transfer of foreign genes into the genome
of a plant is called transformation. In doing this the methods
described for the transformation and regeneration of plants from
plant tissues or plant cells are utilized for transient or stable
transformation. An advantageous transformation method is the
transformation in planta. To this end, it is possible, for example,
to allow the agrobacteria to act on plant seeds or to inoculate the
plant meristem with agrobacteria. It has proved particularly
expedient in accordance with the invention to allow a suspension of
transformed agrobacteria to act on the intact plant or at least the
flower primordia. The plant is subsequently grown on until the
seeds of the treated plant are obtained (Clough and Bent, Plant J.
(1998) 16, 735-743). To select transformed plants, the plant
material obtained in the transformation is, as a rule, subjected to
selective conditions so that transformed plants can be
distinguished from untransformed plants. For example, the seeds
obtained in the above-described manner can be planted and, after an
initial growing period, subjected to a suitable selection by
spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into
plants. Further advantageous transformation methods, in particular
for plants, are known to the skilled worker and are described
hereinbelow.
[12292] [0226.0.0.27] Further advantageous and suitable methods are
protoplast transformation by poly(ethylene glycol)-induced DNA
uptake, the "biolistic" method using the gene cannon--referred to
as the particle bombardment method, electroporation, the incubation
of dry embryos in DNA solution, microinjection and gene transfer
mediated by Agrobacterium. Said methods are described by way of
example in B. Jenes et al., Techniques for Gene Transfer, in:
Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D.
Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu.
Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The
nucleic acids or the construct to be expressed is preferably cloned
into a vector, which is suitable for transforming Agrobacterium
tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12
(1984) 8711). Agrobacteria transformed by such a vector can then be
used in known manner for the transformation of plants, in
particular of crop plants such as by way of example tobacco plants,
for example by bathing bruised leaves or chopped leaves in an
agrobacterial solution and then culturing them in suitable media.
The transformation of plants by means of Agrobacterium tumefaciens
is described, for example, by Hofgen and Willmitzer in Nucl. Acid
Res. (1988) 16, 9877 or is known inter alia from F. F. White,
Vectors for Gene Transfer in Higher Plants; in Transgenic Plants,
Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu,
Academic Press, 1993, pp. 15-38.
[12293] [0227.0.0.27] The abovementioned nucleic acid molecules can
be cloned into the nucleic acid constructs or vectors according to
the invention in combination together with further genes, or else
different genes are introduced by transforming several nucleic acid
constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[12294] In addition to a sequence indicated in Table XI,
application no. 27, columns 5 or 7, or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the amino acid biosynthetic pathway such as for
L-lysine, L-threonine and/or L-methionine is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine a sequence as indicated in Table XI,
application no. 27, columns 5 or 7, with genes which generally
support or enhances to growth or yield of the target organisms, for
example genes which lead to faster growth rate of microorganisms or
genes which produces stress-, pathogen, or herbicide resistant
plants.
[12295] [0228.0.0.27] In a further embodiment of the process of the
invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the amino acid
metabolism, in particular in amino acid synthesis.
[12296] [0229.0.0.27] A further advantageous nucleic acid sequence
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes is the
sequence of the ATP/ADP translocator as described in WO 01/20009.
This ATP/ADP translocator leads to an increased synthesis of the
essential amino acids lysine and/or methionine. Furthermore, an
advantageous nucleic acid sequence coexpressed can be threonine
adlolase and/or lysine decarboxylase as described in the state of
the art.
[12297] [0230.0.0.27] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously at least one of the aforementioned
genes or one of the aforementioned nucleic acids is mutated so that
the activity of the corresponding proteins is influenced by
metabolites to a smaller extent compared with the unmutated
proteins, or not at all, and that in particular the production
according to the invention of the respective fine chemical is not
impaired, or so that their specific enzymatic activity is
increased. Less influence means in this connection that the
regulation of the enzymic activity is less by at least 10%,
advantageously at least 20, 30 or 40%, particularly advantageously
by at least 50, 60, 70, 80 or 90%, compared with the starting
organism, and thus the activity of the enzyme is increased by these
figures mentioned compared with the starting organism. An increase
in the enzymatic activity means an enzymatic activity which is
increased by at least 10%, advantageously at least 20, 30, 40 or
50%, particularly advantageously by at least 60, 70, 80, 90, 100,
200, 300, 500 or 1000%, compared with the starting organism. This
leads to an increased productivity of the desired respective fine
chemical or of the desired respective fine chemicals.
[12298] [0231.0.0.27] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a methionine degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[12299] [0232.0.0.27] In another embodiment of the process of the
invention, the organisms used in the process are those in which
simultaneously at least one of the aforementioned nucleic acids or
of the aforementioned genes is mutated in such a way that the
enzymatic activity of the corresponding protein is partially
reduced or completely blocked. A reduction in the enzymatic
activity means an enzymatic activity, which is reduced by at least
10%, advantageously at least 20, 30 or 40%, particularly
advantageously by at least 50, 60 or 70%, preferably more, compared
with the starting organism.
[12300] [0233.0.0.27] If it is intended to transform the host cell,
in particular the plant cell, with several constructs or vectors,
the marker of a preceding transformation must be removed or a
further marker employed in a following transformation. The markers
can be removed from the host cell, in particular the plant cell, as
described hereinbelow via methods with which the skilled worker is
familiar. In particular plants without a marker, in particular
without resistance to antibiotics, are an especially preferred
embodiment of the present invention.
[12301] [0234.0.0.27] In the process according to the invention,
the nucleic acid sequences used in the process according to the
invention are advantageously linked operably to one or more
regulatory signals in order to increase gene expression. These
regulatory sequences are intended to enable the specific expression
of the genes and the expression of protein. Depending on the host
organism for example plant or microorganism, this may mean, for
example, that the gene is expressed and/or overexpressed after
induction only, or that it is expressed and/or overexpressed
constitutively. These regulatory sequences are, for example,
sequences to which the inductors or repressors bind and which thus
regulate the expression of the nucleic acid. In addition to these
novel regulatory sequences, or instead of these sequences, the
natural regulation of these sequences may still be present before
the actual structural genes and, if appropriate, may have been
genetically modified so that the natural regulation has been
switched off and gene expression has been increased. However, the
nucleic acid construct of the invention suitable as expression
cassette (=expression construct=gene construct) can also be simpler
in construction, that is to say no additional regulatory signals
have been inserted before the nucleic acid sequence or its
derivatives, and the natural promoter together with its regulation
has not been removed. Instead, the natural regulatory sequence has
been mutated in such a way that regulation no longer takes place
and/or gene expression is increased. These modified promoters can
also be introduced on their own before the natural gene in the form
of part sequences (=promoter with parts of the nucleic acid
sequences according to the invention) in order to increase the
activity. Moreover, the gene construct can advantageously also
comprise one or more of what are known as enhancer sequences in
operable linkage with the promoter, and these enable an increased
expression of the nucleic acid sequence. Also, it is possible to
insert additional advantageous sequences at the 3' end of the DNA
sequences, such as, for example, further regulatory elements or
terminators.
[12302] [0235.0.0.27] The nucleic acid molecules, which encode
proteins according to the invention and nucleic acid molecules,
which encode other polypeptides may be present in one nucleic acid
construct or vector or in several ones. Advantageously, only one
copy of the nucleic acid molecule of the invention or the nucleic
acid molecule used in the method of the invention or its encoding
genes is present in the nucleic acid construct or vector. Several
vectors or nucleic acid construct or vector can be expressed
together in the host organism. The nucleic acid molecule or the
nucleic acid construct or vectoraccording to the invention can be
inserted in a vector and be present in the cell in a free form. If
a stable transformation is preferred, a vector is used, which is
stably duplicated over several generations or which is else be
inserted into the genome. In the case of plants, integration into
the plastid genome or, in particular, into the nuclear genome may
have taken place. For the insertion of more than one gene in the
host genome the genes to be expressed are present together in one
gene construct, for example in above-described vectors bearing a
plurality of genes.
[12303] [0236.0.0.27] As a rule, regulatory sequences for the
expression rate of a gene are located upstream (5'), within, and/or
downstream (3') relative to to the coding sequence of the nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention or another codogenic gene segment. They
control in particular transcription and/or translation and/or the
transcript stability. The expression level is dependent on the
conjunction of further cellular regulatory systems, such as the
protein biosynthesis and degradation systems of the cell.
[12304] [0237.0.0.27] Regulatory sequences include transcription
and translation regulating sequences or signals, e.g. sequences
located upstream (5'), which concern in particular the regulation
of transcription or translation initiation, such as promoters or
start codons, and sequences located downstream (3'), which concern
in particular the regulation of transcription or translation
termination and transcript stability, such as polyadenylation
signals or stop codons. Regulatory sequences can also be present in
transcribed coding regions as well in transcribed non-coding
regions, e.g. in introns, as for example splicing sites. Promoters
for the regulation of expression of the nucleic acid molecule
according to the invention in a cell and which can be employed are,
in principle, all those which are capable of stimulating the
transcription of genes in the organisms in question, such as
microorganisms or plants. Suitable promoters, which are functional
in these organisms are generally known. They may take the form of
constitutive or inducible promoters. Suitable promoters can enable
the development- and/or tissue-specific expression in multi-celled
eukaryotes; thus, leaf-, root-, flower-, seed-, stomata-, tuber- or
fruit-specific promoters may advantageously be used in plants.
[12305] [0238.0.0.27] The regulatory sequences or factors can, as
described above, have a positive effect on, the expression of the
genes introduced, thus increasing their expression. Thus, an
enhancement of the expression can advantageously take place at the
transcriptional level by using strong transcription signals such as
strong promoters and/or strong enhancers. In addition, enhancement
of expression on the translational level is also possible, for
example by introducing translation enhancer sequences, e.g., the
enhancer e.g. improving the ribosomal binding to the transcript, or
by increasing the stability of the mRNA, e.g. by replacing the
3'UTR coding region by a region encoding a 3'UTR known as
conferring an high stability of the transcript or by stabilization
of the transcript through the elimination of transcript
instability, so that the mRNA molecule is translated more often
than the wild type. For example in plants AU-rich elements (AREs)
and DST (downstream) elements destabilized transcripts. Mutagenesis
studies have demonstrated that residues within two of the conserved
domains, the ATAGAT and the GTA regions, are necessary for
instability function. Therefore removal or mutation of such
elements would obviously lead to more stable transcripts, higher
transcript rates and higher protein activity. Translation enhancers
are also the "overdrive sequence", which comprises the tobacco
mosaic virus 5'-untranslated leader sequence and which increases
the protein/RNA ratio (Gallie et al., 1987, Nucl. Acids Research
15:8693-8711)
[12306] Enhancers are generally defined as cis active elements,
which can stimulate gene transcription independent of position and
orientation. Different enhancers have been identified in plants,
which can either stimulate transcription constitutively or tissue
or stimuli specific. Well known examples for constitutive enhancers
are the enhancer from the 35S promoter (Odell et al., 1985, Nature
313:810-812) or the ocs enhancer (Fromm et al., 1989, Plant Cell 1:
977:984) Another examples are the G-Box motif tetramer which
confers high-level constitutive expression in dicot and monocot
plants (Ishige et al., 1999, Plant Journal, 18, 443-448) or the
petE, a A/T-rich sequence which act as quantitative enhancers of
gene expression in transgenic tobacco and potato plants (Sandhu et
al., 1998; Plant Mol Biol. 37(5):885-96). Beside that, a large
variety of cis-active elements have been described which contribute
to specific expression pattern, like organ specific expression or
induced expression in response to biotic or abiotic stress.
Examples are elements which provide pathogen or wound-induced
expression (Rushton, 2002, Plant Cell, 14, 749-762) or guard
cell-specific expression (Plesch, 2001, Plant Journal 28,
455-464).
[12307] [0239.0.0.27] Advantageous regulatory sequences for the
expression of the nucleic acid molecule according to the invention
in microorganisms are present for example in promoters such as the
cos, tac, rha, trp, tet, trp-tet, lpp, lac, lpp-lac, lacr T7, T5,
T3, gal, trc, ara, SP6, .lamda.-P.sub.R or .lamda.-P.sub.L
promoter, which are advantageously used in Gram-negative bacteria.
Further advantageous regulatory sequences are present for example
in the Gram-positive promoters amy, dnaK, xylS and SPO2, in the
yeast or fungal promoters ADC1, MF.alpha., AC, P-60, UASH, MCB,
PHO, CYC1, GAPDH, TEF, rp28, ADH. Promoters, which are particularly
advantageous, are constitutive, tissue or compartment specific and
inducible promoters. In general, "promoter" is understood as
meaning, in the present context, a regulatory sequence in a nucleic
acid molecule, which mediates the expression of a coding sequence
segment of a nucleic acid molecule. In general, the promoter is
located upstream to the coding sequence segment. Some elements, for
example expression-enhancing elements such as enhancer may,
however, also be located downstream or even in the transcribed
region.
[12308] [0240.0.0.27] In principle, it is possible to use natural
promoters together with their regulatory sequences, such as those
mentioned above, for the novel process. It is also possible
advantageously to use synthetic promoters, either additionally or
alone, in particular when they mediate seed-specific expression
such as described in, for example, WO 99/16890.
[12309] [0241.0.0.27] The expression of the nucleic acid molecules
used in the process may be desired alone or in combination with
other genes or nucleic acids. Multiple nucleic acid molecules
conferring the expression of advantageous genes can be introduced
via the simultaneous transformation of several individual suitable
nucleic acid constructs, i.e. expression constructs, or,
preferably, by combining several expression cassettes on one
construct. It is also possible to transform several vectors with in
each case several expression cassettes stepwise into the recipient
organisms.
[12310] [0242.0.0.27] As described above the transcription of the
genes introduced should advantageously be terminated by suitable
terminators at the 3' end of the biosynthesis genes introduced
(behind the stop codon). A terminator, which may be used for this
purpose is, for example, the OCS1 terminator, the nos3 terminator
or the 35S terminator. As is the case with the promoters, different
terminator sequences should be used for each gene. Terminators,
which are useful in microorganism are for example the fimA
terminator, txn terminator or trp terminator. Such terminators can
be rho-dependent or rho-independent.
[12311] [0243.0.0.27] Different plant promoters such as, for
example, the USP, the LegB4-, the DC3 promoter or the ubiquitin
promoter from parsley or other herein mentioned promoter and
different terminators may advantageously be used in the nucleic
acid construct.
[12312] [0244.0.0.27] In order to ensure the stable Xlntegration,
into the transgenic plant, of nucleic acid molecules used in the
process according to the invention in combination with further
biosynthesis genes over a plurality of generations, each of the
coding regions used in the process should be expressed under the
control of its own, preferably unique, promoter since repeating
sequence motifs may lead to recombination events or to silencing
or, in plants, to instability of the T-DNA.
[12313] [0245.0.0.27] The nucleic acid construct is advantageously
constructed in such a way that a promoter is followed by a suitable
cleavage site for insertion of the nucleic acid to be expressed,
advantageously in a polylinker, followed, if appropriate, by a
terminator located behind the polylinker. If appropriate, this
order is repeated several times so that several genes are combined
in one construct and thus can be introduced into the transgenic
plant in order to be expressed. The sequence is advantageously
repeated up to three times. For the expression, the nucleic acid
sequences are inserted via the suitable cleavage site, for example
in the polylinker behind the promoter. It is advantageous for each
nucleic acid sequence to have its own promoter and, if appropriate,
its own terminator, as mentioned above. However, it is also
possible to insert several nucleic acid sequences behind a promoter
and, if appropriate, before a terminator if a polycistronic
transcription is possible in the host or target cells. In this
context, the insertion site, or the sequence of the nucleic acid
molecules inserted, in the nucleic acid construct is not decisive,
that is to say a nucleic acid molecule can be inserted in the first
or last position in the cassette without this having a substantial
effect on the expression. However, it is also possible to use only
one promoter type in the construct. However, this may lead to
undesired recombination events or silencing effects, as said.
[12314] [0246.0.0.27] Accordingly, in a preferred embodiment, the
nucleic acid construct according to the invention confers
expression of the nucleic acid molecule of the invention or the
nucleic acid molecule used in the method of the invention, and,
optionally further genes, in a plant and comprises one or more
plant regulatory elements. Said nucleic acid construct according to
the invention advantageously encompasses a plant promoter or a
plant terminator or a plant promoter and a plant terminator.
[12315] [0247.0.0.27] A "plant" promoter comprises regulatory
elements, which mediate the expression of a coding sequence segment
in plant cells. Accordingly, a plant promoter need not be of plant
origin, but may originate from viruses or microorganisms, in
particular for example from viruses which attack plant cells.
[12316] [0248.0.0.27] The plant promoter can also originates from a
plant cell, e.g. from the plant, which is transformed with the
nucleic acid construct or vector as described herein.
[12317] This also applies to other "plant" regulatory signals, for
example in "plant" terminators.
[12318] [0249.0.0.27] A nucleic acid construct suitable for plant
expression preferably comprises regulatory elements which are
capable of controlling the expression of genes in plant cells and
which are operably linked so that each sequence can fulfill its
function. Accordingly, the nucleic acid construct can also comprise
transcription terminators. Examples for transcriptional termination
arepolyadenylation signals. Preferred polyadenylation signals are
those which originate from Agrobacterium tumefaciens T-DNA, such as
the gene 3 of the Ti plasmid pTiACH5, which is known as octopine
synthase (Gielen et al., EMBO J. 3 (1984) 835 et seq.) or
functional equivalents thereof, but all the other terminators which
are functionally active in plants are also suitable.
[12319] [0250.0.0.27] The nucleic acid construct suitable for plant
expression preferably also comprises other operably linked
regulatory elements such as translation enhancers, for example the
overdrive sequence, which comprises the tobacco mosaic virus
5'-untranslated leader sequence, which increases the protein/RNA
ratio (Gallie et al., 1987, Nucl. Acids Research 15:8693-8711).
[12320] [0251.0.0.27] Other preferred sequences for use in operable
linkage in gene expression constructs are targeting sequences,
which are required for targeting the gene product into specific
cell compartments (for a review, see Kermode, Crit. Rev. Plant Sci.
15, 4 (1996) 285-423 and references cited therein), for example
into the vacuole, the nucleus, all types of plastids, such as
amyloplasts, chloroplasts, chromoplasts, the extracellular space,
the mitochondria, the endoplasmic reticulum, elaioplasts,
peroxisomes, glycosomes, and other compartments of cells or
extracellular. Sequences, which must be mentioned in this context
are, in particular, the signal-peptide- or transit-peptide-encoding
sequences which are known per se. For example,
plastid-transit-peptide-encoding sequences enable the targeting of
the expression product into the plastids of a plant cellTargeting
sequences are also known for eukaryotic and to a lower extent for
prokaryotic organisms and can advantageously be operable linked
with the nucleic acid molecule of the present invention to achieve
an expression in one of said compartments or extracellular.
[12321] [0252.0.0.27] For expression in plants, the nucleic acid
molecule must, as described above, be linked operably to or
comprise a suitable promoter which expresses the gene at the right
point in time and in a cell- or tissue-specific manner. Usable
promoters are constitutive promoters (Benfey et al., EMBO J. 8
(1989) 2195-2202), such as those which originate from plant
viruses, such as 35S CAMV (Franck et al., Cell 21 (1980) 285-294),
19S CaMV (see also U.S. Pat. No. 5,352,605 and WO 84/02913), 34S
FMV (Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443), the
parsley ubiquitin promoter, or plant promoters such as the Rubisco
small subunit promoter described in U.S. Pat. No. 4,962,028 or the
plant promoters PRP1 [Ward et al., Plant. Mol. Biol. 22 (1993)],
SSU, PGEL1, OCS [Leisner (1988) Proc Natl Acad Sci USA
85(5):2553-2557], lib4, usp, mas [Comai (1990) Plant Mol Biol 15
(3):373-381], STLS1, ScBV (Schenk (1999) Plant Mol Biol
39(6):1221-1230), B33, SAD1 or SAD2 (flax promoters, Jain et al.,
Crop Science, 39 (6), 1999: 1696-1701) or nos [Shaw et al. (1984)
Nucleic Acids Res. 12(20):7831-7846]. Stable, constitutive
expression of the proteins according to the invention a plant can
be advantageous. However, inducible expression of the polypeptide
of the invention or the polypeptide used in the method of the
invention is advantageous, if a late expression before the harvest
is of advantage, as metabolic manipulation may lead to a plant
growth retardation.
[12322] [0253.0.0.27] The expression of plant genes can also be
facilitated as described above via a chemical inducible promoter
(for a review, see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol.
Biol., 48:89-108). Chemically inducible promoters are particularly
suitable when it is desired to express the gene in a time-specific
manner. Examples of such promoters are a salicylic acid inducible
promoter (WO 95/19443), and abscisic acid-inducible promoter (EP
335 528), a tetracyclin-inducible promoter (Gatz et al. (1992)
Plant J. 2, 397-404), a cyclohexanol- or ethanol-inducible promoter
(WO 93/21334) or others as described herein.
[12323] [0254.0.0.27] Other suitable promoters are those which
react to biotic or abiotic stress conditions, for example the
pathogen-induced PRP1 gene promoter (Ward et al., Plant. Mol. Biol.
22 (1993) 361-366), the tomato heat-inducible hsp80 promoter (U.S.
Pat. No. 5,187,267), the potato chill-inducible alpha-amylase
promoter (WO 96/12814) or the wound-inducible pinll promoter
(EP-A-0 375 091) or others as described herein.
[12324] [0255.0.0.27] Preferred promoters are in particular those
which bring about gene expression in tissues and organs in which
the biosynthesis of amino acids takes place, in seed cells, such as
endosperm cells and cells of the developing embryo. Suitable
promoters are the oilseed rape napin gene promoter (U.S. Pat. No.
5,608,152), the Vicia faba USP promoter (Baeumlein et al., Mol Gen
Genet, 1991, 225 (3):459-67), the Arabidopsis oleosin promoter (WO
98/45461), the Phaseolus vulgaris phaseolin promoter (U.S. Pat. No.
5,504,200), the Brassica Bce4 promoter (WO 91/13980), the bean arcs
promoter, the carrot DcG3 promoter, or the Legumin B4 promoter
(LeB4; Baeumlein et al., 1992, Plant Journal, 2 (2):233-9), and
promoters which bring about the seed-specific expression in
monocotyledonous plants such as maize, barley, wheat, rye, rice and
the like. Advantageous seed-specific promoters are the sucrose
binding protein promoter (WO 00/26388), the phaseolin promoter and
the napin promoter. Suitable promoters which must be considered are
the barley Ipt2 or Ipt1 gene promoter (WO 95/15389 and WO
95/23230), and the promoters described in WO 99/16890 (promoters
from the barley hordein gene, the rice glutelin gene, the rice
oryzin gene, the rice prolamin gene, the wheat gliadin gene, the
wheat glutelin gene, the maize zein gene, the oat glutelin gene,
the sorghum kasirin gene and the rye secalin gene). Further
suitable promoters are Amy32b, Amy 6-6 and Aleurain [U.S. Pat. No.
5,677,474], Bce4 (oilseed rape) [U.S. Pat. No. 5,530,149], glycinin
(soya) [EP 571 741], phosphoenolpyruvate carboxylase (soya) [JP
06/62870], ADR12-2 (soya) [WO 98/08962], isocitrate lyase (oilseed
rape) [U.S. Pat. No. 5,689,040] or .alpha.-amylase (barley) [EP 781
849]. Other promoters which are available for the expression of
genes in plants are leaf-specific promoters such as those described
in DE-A 19644478 or light-regulated promoters such as, for example,
the pea petE promoter.
[12325] [0256.0.0.27] Further suitable plant promoters are the
cytosolic FBPase promoter or the potato ST-LSI promoter (Stockhaus
et al., EMBO J. 8, 1989, 2445), the Glycine max
phosphoribosylpyrophosphate amidotransferase promoter (GenBank
Accession No. U87999) or the node-specific promoter described in
EP-A-0 249 676.
[12326] [0257.0.0.27] Other promoters, which are particularly
suitable, are those which bring about plastid-specific expression.
Suitable promoters such as the viral RNA polymerase promoter are
described in WO 95/16783 and WO 97/06250, and the Arabidopsis clpP
promoter, which is described in WO 99/46394.
[12327] [0258.0.0.27] Other promoters, which are used for the
strong expression of heterologous sequences in as many tissues as
possible, in particular also in leaves, are, in addition to several
of the abovementioned viral and bacterial promoters, preferably,
plant promoters of actin or ubiquitin genes such as, for example,
the rice actin1 promoter. Further examples of constitutive plant
promoters are the sugarbeet V-ATPase promoters (WO 01/14572).
Examples of synthetic constitutive promoters are the Super promoter
(WO 95/14098) and promoters derived from G-boxes (WO 94/12015). If
appropriate, chemical inducible promoters may furthermore also be
used, compare EP-A 388186, EP-A 335528, WO 97/06268.
[12328] [0259.0.0.27] As already mentioned herein, further
regulatory sequences, which may be expedient, if appropriate, also
include sequences, which target the transport and/or the
localization of the expression products. Sequences, which must be
mentioned in this context are, in particular, the signal-peptide-
or transit-peptide-encoding sequences which are known per se. For
example, plastid-transit-peptide-encoding sequences enable the
targeting of the expression product into the plastids of a plant
cell.
[12329] [0260.0.0.27] Preferred recipient plants are, as described
above, in particular those plants, which can be transformed in a
suitable manner. These include monocotyledonous and dicotyledonous
plants. Plants which must be mentioned in particular are
agriculturally useful plants such as cereals and grasses, for
example Triticum spp., Zea mays, Hordeum vulgare, oats, Secale
cereale, Oryza sativa, Pennisetum glaucum, Sorghum bicolor,
Triticale, Agrostis spp., Cenchrus ciliaris, Dactylis glomerata,
Festuca arundinacea, Lolium spp., Medicago spp. and Saccharum spp.,
legumes and oil crops, for example Brassica juncea, Brassica napus,
Glycine max, Arachis hypogaea, Gossypium hirsutum, Cicer arietinum,
Helianthus annuus, Lens culinaris, Linum usitatissimum, Sinapis
alba, Trifolium repens and Vicia narbonensis, vegetables and
fruits, for example bananas, grapes, Lycopersicon esculentum,
asparagus, cabbage, watermelons, kiwi fruit, Solanum tuberosum,
Beta vulgaris, cassava and chicory, trees, for example Coffea
species, Citrus spp., Eucalyptus spp., Picea spp., Pinus spp. and
Populus spp., medicinal plants and trees, and flowers.
[12330] [0261.0.0.27] One embodiment of the present invention also
relates to a method for generating a vector, which comprises the
insertion, into a vector, of the nucleic acid molecule
characterized herein, the nucleic acid molecule according to the
invention or the expression cassette according to the invention.
The vector can, for example, be introduced in to a cell, e.g. a
microorganism or a plant cell, as described herein for the nucleic
acid construct, or below under transformation or transfection or
shown in the examples. A transient or stable transformation of the
host or target cell is possible, however, a stable transformation
is preferred. The vector according to the invention is preferably a
vector, which is suitable for expressing the polypeptide according
to the invention in a plant. The method can thus also encompass one
or more steps for integrating regulatory signals into the vector,
in particular signals, which mediate the expression in
microorganisms or plants.
[12331] [0262.0.0.27] Accordingly, the present invention also
relates to a vector comprising the nucleic acid molecule
characterized herein as part of a nucleic acid construct suitable
for plant expression or the nucleic acid molecule according to the
invention.
[12332] [0263.0.0.27] The advantageous vectors of the
inventioncomprise the nucleic acid molecules which encode proteins
according to the invention, nucleic acid molecules which are used
in the process, or nucleic acid construct suitable for plant
expression comprising the nucleic acid molecules used, either alone
or in combination with further genes such as the biosynthesis or
regulatory genes of the respective fine chemical metabolism e.g.
with the genes mentioned herein above. In accordance with the
invention, the term "vector" refers to a nucleic acid molecule,
which is capable of transporting another nucleic acid to which it
is linked. One type of vector is a "plasmid", which means a
circular double-stranded DNA loop into which additional DNA
segments can be ligated. A further type of vector is a viral
vector, it being possible to ligate additional nucleic acids
segments into the viral genome. Certain vectors are capable of
autonomous replication in a host cell into which they have been
introduced (for example bacterial vectors with bacterial
replication origin). Other preferred vectors are advantageously
completely or partly integrated into the genome of a host cell when
they are introduced into the host cell and thus replicate together
with the host genome. Moreover, certain vectors are capable of
controlling the expression of genes with which they are in operable
linkage. In the present context, these vectors are referred to as
"expression vectors". As mentioned above, they are capable of
autonomous replication or may be integrated partly or completely
into the host genome. Expression vectors, which are suitable for
DNA recombination techniques usually take the form of plasmids. In
the present description, "plasmid" and "vector" can be used
interchangeably since the plasmid is the most frequently used form
of a vector. However, the invention is also intended to encompass
these other forms of expression vectors, such as viral vectors,
which exert similar functions. The term vector is furthermore also
to encompass other vectors which are known to the skilled worker,
such as phages, viruses such as SV40, CMV, TMV, transposons, IS
elements, phasmids, phagemids, cosmids, and linear or circular
DNA.
[12333] [0264.0.0.27] The recombinant expression vectors which are
advantageously used in the process comprise the nucleic acid
molecules according to the invention or the nucleic acid construct
according to the invention in a form which is suitable for
expressing, in a host cell, the nucleic acid molecules according to
the invention or described herein. Accordingly, the the recombinant
expression vectors comprise one or more regulatory signals selected
on the basis of the host cells to be used for the expression, in
operable linkage with the nucleic acid sequence to be
expressed.
[12334] [0265.0.0.27] In a recombinant expression vector, "operable
linkage" means that the nucleic acid molecule of interest is linked
to the regulatory signals in such a way that expression of the
nucleic acid molecule is possible: they are linked to one another
in such a way that the two sequences fulfill the predicted function
assigned to the sequence (for example in an in-vitro
transcription/translation system, or in a host cell if the vector
is introduced into the host cell).
[12335] [0266.0.0.27] The term "regulatory sequence" is intended to
comprise promoters, enhancers and other expression control elements
(for example polyadenylation signals These regulatory sequences are
described, for example, in Goeddel: Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990), or see: Gruber and Crosby, in: Methods in Plant Molecular
Biology and Biotechnolgy, CRC Press, Boca Raton, Fla., Ed.: Glick
and Thompson, chapter 7, 89-108, including the references cited
therein. Regulatory sequences encompass those, which control the
constitutive expression of a nucleotide sequence in many types of
host cells and those which control the direct expression of the
nucleotide sequence in specific host cells only, and under specific
conditions. The skilled worker knows that the design of the
expression vector may depend on factors such as the selection of
the host cell to be transformed, the extent to which the desired
protein is expressed, and the like. A preferred selection of
regulatory sequences is described above, for example promoters,
terminators, enhancers and the like. The term regulatory sequence
is to be considered as being encompassed by the term regulatory
signal. Several advantageous regulatory sequences, in particular
promoters and terminators are described above. In general, the
regulatory sequences described as advantageous for nucleic acid
construct suitable for expression are also applicable for
vectors.
[12336] [0267.0.0.27] The recombinant expression vectors used can
be designed specifically for the expression, in prokaryotic and/or
eukaryotic cells, of nucleic acid molecules used in the process.
This is advantageous since intermediate steps of the vector
construction are frequently carried out in microorganisms for the
sake of simplicity. For example, the genes according to the
invention and other genes can be expressed in bacterial cells,
insect cells (using baculovirus expression vectors), yeast cells
and other fungal cells [Romanos (1992), Yeast 8:423-488; van den
Hondel, (1991), in: More Gene Manipulations in Fungi, J. W. Bennet
& L. L. Lasure, Ed., pp. 396-428: Academic Press: San Diego;
and van den Hondel, C. A. M. J. J. (1991), in: Applied Molecular
Genetics of Fungi, Peberdy, J. F., et al., Ed., pp. 1-28, Cambridge
University Press: Cambridge], algae [Falciatore et al., 1999,
Marine Biotechnology. 1, 3:239-251] using vectors and following a
transformation method as described in WO 98/01572, and preferably
in cells of multi-celled plants [see Schmidt, R. and Willmitzer, L.
(1988) Plant Cell Rep.:583-586; Plant Molecular Biology and
Biotechnology, C Press, Boca Raton, Fla., chapter 6/7, pp. 71-119
(1993); F. F. White, in: Transgenic Plants, Bd. 1, Engineering and
Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-43;
Potrykus, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991),
205-225 (and references cited therein)]. Suitable host cells are
furthermore discussed in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). As an alternative, the sequence of the recombinant
expression vector can be transcribed and translated in vitro, for
example using T7 promotor-regulatory sequences and T7
polymerase.
[12337] [0268.0.0.27] Proteins can be expressed in prokaryotes
using vectors comprising constitutive or inducible promoters, which
control the expression of fusion proteins or nonfusion proteins.
Typical fusion expression vectors are, inter alia, pGEX
(Pharmacia
[12338] Biotech Inc; Smith, D. B., and Johnson, K. S. (1988) Gene
67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.), in which glutathione-S-transferase
(GST), maltose-E-binding protein or protein A is fused with the
recombinant target protein. Examples of suitable Xlnducible
nonfusion E. coli expression vectors are, inter alia, pTrc (Amann
et al. (1988) Gene 69:301-315) and pET 11d [Studier et al., Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990) 60-89]. The target gene expression of the
pTrc vector is based on the transcription of a hybrid trp-lac
fusion promoter by the host RNA polymerase. The target gene
expression from the pET 11d vector is based on the transcription of
a T7-gn10-lac fusion promoter, which is mediated by a coexpressed
viral RNA polymerase (T7 gn1). This viral polymerase is provided by
the host strains BL21 (DE3) or HMS174 (DE3) by a resident
A-prophage which harbors a T7 gn1 gene under the transcriptional
control of the lacUV 5 promoter.
[12339] [0269.0.0.27] Other vectors which are suitable Xln
prokaryotic organisms are known to the skilled worker; these
vectors are for example in E. coli pLG338, pACYC184, the pBR
series, such as pBR322, the pUC series such as pUC18 or pUC19, the
M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236, pMBL24, pLG200,
pUR290, pIN-111113-B1, .lamda.gt11 or pBdCI, in Streptomyces
pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or
pBD214, in Corynebacterium pSA77 or pAJ667.
[12340] [0270.0.0.27] In a further embodiment, the expression
vector is a yeast expression vector. Examples of vectors for
expression in the yeasts S. cerevisiae encompass pYeDesaturasec1
(Baldari et al. (1987) Embo J. 6:229-234), pMFa (Kurjan and
Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987)
Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego,
Calif.). Vectors and methods for the construction of vectors which
are suitable for use in other fungi, such as the filamentous fungi,
encompass those which are described in detail in: van den Hondel,
C. A. M. J. J. [(1991), J. F. Peberdy, Ed., pp. 1-28, Cambridge
University Press: Cambridge; or in: More Gene Manipulations in
Fungi; J. W. Bennet & L. L. Lasure, Ed., pp. 396-428: Academic
Press: San Diego]. Examples of other suitable yeast vectors are 2
.mu.M, pAG-1, YEp6, YEp13 or pEMBLYe23.
[12341] [0271.0.0.27] Further vectors, which may be mentioned by
way of example, are pALS1, plL2 or pBB116 in fungi or pLGV23,
pGHlac+, pBIN19, pAK2004 or pDH51 in plants.
[12342] [0272.0.0.27] As an alternative, the nucleic acid sequences
can be expressed in insect cells using baculovirus expression
vectors. Baculovirus vectors, which are available for expressing
proteins in cultured insect cells (for example Sf9 cells) encompass
the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)
and the pVL series (Lucklow and Summers (1989) Virology
170:31-39).
[12343] [0273.0.0.27] The abovementioned vectors are only a small
overview of potentially suitable vectors. Further plasmids are
known to the skilled worker and are described, for example, in:
Cloning Vectors (Ed. Pouwels, P. H., et al., Elsevier,
Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). Further
suitable expression systems for prokaryotic and eukaryotic cells,
see the chapters 16 and 17 by Sambrook, J., Fritsch, E. F., and
Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Edition,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989.
[12344] [0274.0.0.27] Accordingly, one embodiment of the invention
relates to a vector where the nucleic acid molecule according to
the invention is linked operably to regulatory sequences which
permit the expression in a prokaryotic or eukaryotic or in a
prokaryotic and eukaryotic host.
[12345] [0275.0.0.27] Accordingly, one embodiment of the invention
relates to a host cell, which has been transformed stably or
transiently with the vector according to the invention or the
nucleic acid molecule according to the invention or the nucleic
acid construct according to the invention.
[12346] [0276.0.0.27] Depending on the host organism, the organisms
used in the process according to the invention are cultured or
grown in a manner with which the skilled worker is familiar. As a
rule, microorganisms are grown in a liquid medium comprising a
carbon source, usually in the form of sugars, a nitrogen source,
usually in the form of organic nitrogen sources such as yeast
extract or salts such as ammonium sulfate, trace elements such as
iron salts, manganese salts, magnesium salts, and, if appropriate,
vitamins, at temperatures between 0.degree. C. and 100.degree. C.,
preferably between 10.degree. C. and 60.degree. C., while passing
in oxygen. In the event the microorganism is anaerobe, no oxygen is
blown through the culture medium. The pH value of the liquid
nutrient medium may be kept constant, that is to say regulated
during the culturing phase, or not. The organisms may be cultured
batchwise, semibatchwise or continuously. Nutrients may be provided
at the beginning of the fermentation or fed in semicontinuously or
continuously.
[12347] [0277.0.0.27] The amino acids produced can be isolated from
the organism by methods with which the skilled worker is familiar.
For example via extraction, salt precipitation and/or ion-exchange
chromatography. To this end, the organisms may be disrupted
beforehand. The process according to the invention can be conducted
batchwise, semibatchwise or continuously. A summary of known
culture and isolation techniques can be found in the textbook by
Chmiel [Bioproze.beta.technik 1, Einfuhrung in die
Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)],
Demain et al. (Industrial Microbiology and Biotechnology, second
edition, ASM Press, Washington, D.C., 1999, ISBN 1-55581-128-0] or
in the textbook by Storhas (Bioreaktoren and periphere
Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
[12348] [0278.0.0.27] In one embodiment, the present invention
relates to a polypeptide encoded by the nucleic acid molecule
according to the present invention, preferably conferring an
increase in the respective fine chemical content in an organism or
cell after increasing the expression or activity.
[12349] [0279.0.0.27] The present invention also relates to a
process for the production of a polypeptide according to the
present invention, the polypeptide being expressed in a host cell
according to the invention, preferably in a microorganism or a
transgenic plant cell.
[12350] [0280.0.0.27] In one embodiment, the nucleic acid molecule
used in the process for the production of the polypeptide is
derived from a microorganism, preferably from a prokaryotic or
protozoic cell with an eukaryotic organism as host cell. E.g., in
one embodiment the polypeptide is produced in a plant cell or plant
with a nucleic acid molecule derived from a prokaryote or a fungus
or an alga or an other microorganism but not from plant.
[12351] [0281.0.0.27] The skilled worker knows that protein and DNA
expressed in different organisms differ in many respects and
properties, e.g. DNA modulation and imprinting, such as methylation
or post-translational modification, as for example glucosylation,
phosphorylation, acetylation, myristoylation, ADP-ribosylation,
farnesylation, carboxylation, sulfation, ubiquination, etc. though
having the same coding sequence. Preferably, the cellular
expression control of the corresponding protein differs accordingly
in the control mechanisms controlling the activity and expression
of an endogenous protein or another eukaryotic protein. One major
difference between proteins expressed in prokaryotic or eukaryotic
organisms is the amount and pattern of glycosylation. For example
in E. coli there are no glycosylated proteins. Proteins expressed
in yeasts have high mannose content in the glycosylated proteins,
whereas in plants the glycosylation pattern is complex.
[12352] [0282.0.0.27] The polypeptide of the present invention is
preferably produced by recombinant DNA techniques. For example, a
nucleic acid molecule encoding the protein is cloned into a vector
(as described above), the vector is introduced into a host cell (as
described above) and said polypeptide is expressed in the host
cell. Said polypeptide can then be isolated from the cells by an
appropriate purification scheme using standard protein purification
techniques. Alternative to recombinant expression, the polypeptide
or peptide of the present invention can be synthesized chemically
using standard peptide synthesis techniques.
[12353] [0283.0.0.27] Moreover, a native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an antibody against a protein as indicated in
Table XII, application no. 27, column 3. E.g. an antibody against a
polypeptide as indicated in Table XII, application no. 27, columns
5 or 7, or an antigenic part thereof which can be produced by
standard techniques utilizing polypeptides comprising or consisting
of above mentioned sequences, e.g. the polypeptid of the present
invention or fragment thereof. Preferred are monoclonal antibodies
specifically binding to polypeptide as indicated in Table XII,
application no. 27, columns 5 or 7.
[12354] [0284.0.0.27] In one embodiment, the present invention
relates to a polypeptide having the amino acid sequence encoded by
a nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention or obtainable by a
process of the invention. Said polypeptide confers preferably the
aforementioned activity, in particular, the polypeptide confers the
increase of the respective fine chemical in a cell or an organism
or a part thereof after increasing the cellular activity, e.g. by
increasing the expression or the specific activity of the
polypeptide.
[12355] [0285.0.0.27] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 27, columns 5 or 7, or as encoded by a nucleic
acid molecule as indicated in Table XI, application no. 27, columns
5 or 7, or functional homologues thereof.
[12356] [0286.0.0.27] In one advantageous embodiment, in the method
of the present invention the activity of a polypeptide is increased
which comprises or consists of a consensus sequence as indicated in
Table XIV, application no. 27, column 7, and in one another
embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 27, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table XIV, application no. 27, column 7.
[12357] [0287.0.0.27] In one embodiment not more than 15%,
preferably 10%, even more preferred 5%, 4%, 3%, or 2%, most
preferred 1% or 0% of the amino acid position indicated by a letter
are/is replaced another amino acid or, in an other embodiment,
are/is absent and/or replaced. In another embodiment the stretches
of non-conserved amino acids, indicated by (X)n [whereas n
indicates the number of X], vary in their length by 20%, preferably
by 15 or 10%, even more preferred by 5%, 4%, 3%, 2% or most
preferred by only 1%.
[12358] [0288.0.0.27] In one embodiment 20 or less, preferably 15
or 10, preferably 9, 8, 7, or 6, more preferred 5 or 4, even more
preferred 3, even more preferred 2, even more preferred 1, most
preferred 0 amino acids are inserted into the consensus sequence
or, in an other embodiment, are absent and/or replaced.
[12359] [0289.0.0.27] The consensus sequence shown herein was
derived from a multiple alignment of the sequences as listed in
table XII. The consensus sequences of specified domains were
derived from a multiple alignment of all sequences. The letters
represent the one letter amino acid code and indicate that the
amino acids are conserved in all aligned proteins. The letter X
stands for amino acids, which are not conserved in all
sequences.
[12360] In one example, in the cases where only a small selected
subset of amino acids are possible at a certain position these
amino acids are given in brackets. The number of given X indicates
the distances between conserved amino acid residues, e.g.
YX(21-23)F means that conserved tyrosine and phenylalanine residues
are separated from each other by minimum 21 and maximum 23 amino
acid residues in all investigated sequences.
[12361] [0290.0.0.27] The alignment was performed with the Software
AlignX (Sep. 25, 2002) a component of Vector NTI Suite 8.0,
InforMax.TM., Invitrogen.TM. life science software, U.S. Main
Office, 7305 Executive Way, Frederick, Md. 21704, USA with the
following settings: For pairwise alignments: gap opening penalty:
10.0; gap extension penalty 0,1. For multiple alignments: Gap
opening penalty: 10.0; Gap extension penalty: 0.1; Gap separation
penalty range: 8; Residue substitution matrix: blosum62;
Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0,5;
Consensus calculation options: Residue fraction for consensus: 0.9.
Presettings were selected to allow also for the alignment of
conserved amino acids.
[12362] [0291.0.0.27] In one advantageous embodiment, the method of
the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of
plant or microorganism specific consensus sequences.
[12363] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table XII,
application no. 27, columns 5 or 7, by one or more amino acids. In
one embodiment, polypeptide distinguishes form a sequence as
indicated in Table XII, application no. 27, columns 5 or 7, by more
than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, even more preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table XII, application no. 27, columns 5 or 7, by not
more than 80% or 70% of the amino acids, preferably not more than
60% or 50%, more preferred not more than 40% or 30%, even more
preferred not more than 20% or 10%. In an other embodiment, said
polypeptide of the invention does not consist of a sequence as
indicated in Table XII, application no. 27, columns 5 or 7.
[12364] [0292.0.0.27] In one embodiment, the polypeptide of the
invention comprises any one of the sequences not known to the
public before. In one embodiment, the polypeptide of the invention
originates from a non-plant cell, in particular from a
microorganism, and was expressed in a plant cell. In one
embodiment, the present invention relates to a polypeptide encoded
by the nucleic acid molecule of the invention or used in the
process of the invention for which an activity has not been
described yet.
[12365] [0293.0.0.27] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the process
of the invention.
[12366] In one embodiment, the polypeptide of the invention has a
sequence which distinguishes from a sequence as indicated in Table
XII, application no. 27, columns 5 or 7, by one or more amino
acids. In an other embodiment, said polypeptide of the invention
does not consist of the sequence as indicated in Table XII,
application no. 27, columns 5 or 7. In a further embodiment, said
polypeptide of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical. In one embodiment, said polypeptide
does not consist of the sequence encoded by a nucleic acid
molecules as indicated in Table XI, application no. 27, columns 5
or 7.
[12367] [0294.0.0.27] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 27, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 27, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[12368] [0295.0.0.27] The terms "protein" and "polypeptide" used in
this application are interchangeable. "Polypeptide" refers to a
polymer of amino acids (amino acid sequence) and does not refer to
a specific length of the molecule. Thus peptides and oligopeptides
are included within the definition of polypeptide. This term does
also refer to or include post-translational modifications of the
polypeptide, for example, glycosylations, acetylations,
phosphorylations and the like. Included within the definition are,
for example, polypeptides containing one or more analogs of an
amino acid (including, for example, unnatural amino acids, etc.),
polypeptides with substituted linkages, as well as other
modifications known in the art, both naturally occurring and
non-naturally occurring.
[12369] [0296.0.0.27] Preferably, the polypeptide is isolated. An
"isolated" or "purified" protein or nucleic acid molecule or
biologically active portion thereof is substantially free of
cellular material when produced by recombinant DNA techniques or
chemical precursors or other chemicals when chemically
synthesized.
[12370] [0297.0.0.27] The language "substantially free of cellular
material" includes preparations of the polypeptide of the invention
in which the protein is separated from cellular components of the
cells in which it is naturally or recombinantly produced. In one
embodiment, the language "substantially free of cellular material"
includes preparations having less than about 30% (by dry weight) of
"contaminating protein", more preferably less than about 20% of
"contaminating protein", still more preferably less than about 10%
of "contaminating protein", and most preferably less than about 5%
"contaminating protein". The term "Contaminating protein" relates
to polypeptides, which are not polypeptides of the present
invention. When the polypeptide of the present invention or
biologically active portion thereof is recombinantly produced, it
is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less
than about 10%, and most preferably less than about 5% of the
volume of the protein preparation. The language "substantially free
of chemical precursors or other chemicals" includes preparations in
which the polypeptide of the present invention is separated from
chemical precursors or other chemicals, which are involved in the
synthesis of the protein. The language "substantially free of
chemical precursors or other chemicals" includes preparations
having less than about 30% (by dry weight) of chemical precursors
or non-polypeptide of the invention-chemicals, more preferably less
than about 20% chemical precursors or non-polypeptide of the
invention-chemicals, still more preferably less than about 10%
chemical precursors or non-polypeptide of the invention-chemicals,
and most preferably less than about 5% chemical precursors or
non-polypeptide of the invention-chemicals. In preferred
embodiments, isolated proteins or biologically active portions
thereof lack contaminating proteins from the same organism from
which the polypeptide of the present invention is derived.
Typically, such proteins are produced by recombinant
techniques.
[12371] [0297.1.0.27] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity and/or amino acid
sequence of a polypeptide indicated in Table XII, application no.
27, columns 3, 5 or 7.
[12372] [0298.0.0.27] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 27, columns 5 or 7, such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table XII, application no. 27, columns 5 or 7.
[12373] [0299.0.0.27] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 27,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 27, columns 5 or 7, or which is
homologous thereto, as defined above.
[12374] [0300.0.0.27] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table XII,
application no. 27, columns 5 or 7, in amino acid sequence due to
natural variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprise an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence of a sequence as indicated in Table XII,
application no. 27, columns 5 or 7.
[12375] [0301.0.0.27] For the comparison of amino acid sequences
the same algorithms as described above or nucleic acid sequences
can be used. Results of high quality are reached by using the
algorithm of Needleman and Wunsch or Smith and Waterman. Therefore
programs based on said algorithms are preferred. Advantageously the
comparisons of sequences can be done with the program PileUp (J.
Mol. Evolution., 25, 351-360, 1987, Higgins et al., CABIOS, 5 1989:
151-153) or preferably with the programs Gap and BestFit, which are
respectively based on the algorithms of Needleman and Wunsch [J.
Mol. Biol. 48; 443-453 (1970)] and Smith and Waterman [Adv. Appl.
Math. 2; 482-489 (1981)]. Both programs are part of the GCG
software-package [Genetics Computer Group, 575 Science Drive,
Madison, Wis., USA 53711 (1991); Altschul et al. (1997) Nucleic
Acids Res. 25:3389 et seq.]. Therefore preferably the calculations
to determine the percentages of sequence homology are done with the
program Gap over the whole range of the sequences. The following
standard adjustments for the comparison of amino acid sequences
were used: gap weight: 8, length weight: 2, average match: 2.912,
average mismatch: -2.003.
[12376] [0302.0.0.27] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 27, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[12377] [0303.0.0.27] Typically, biologically (or immunologically)
active portions i.e. peptides, e.g., peptides which are, for
example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100 or more
amino acids in length comprise a domain or motif with at least one
activity or epitope of a polypeptide of the present invention or
used in the process of the present invention. Moreover, other
biologically active portions, in which other regions of the
polypeptide are deleted, can be prepared by recombinant techniques
and evaluated for one or more of the activities described
herein.
[12378] [0304.0.0.27] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 27, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[12379] [0305.0.0.27] Any mutagenesis strategies for the
polypeptide of the present invention or the polypeptide used in the
process of the present invention to result in increasing said
activity are not meant to be limiting; variations on these
strategies will be readily apparent to one skilled in the art.
Using such strategies, and incorporating the mechanisms disclosed
herein, the nucleic acid molecule and polypeptide of the invention
or the polypeptide used in the method of the invention may be
utilized to generate plants or parts thereof, expressing one or
more wildtype protein(s) or one or more mutated protein encoding
nucleic acid molecule(s) or polypeptide molecule(s) of the
invention such that the yield, production, and/or efficiency of
production of a desired compound is improved.
[12380] [0306.0.0.27] This desired compound may be any natural
product of plants, which includes the final products of
biosynthesis pathways and intermediates of naturally-occurring
metabolic pathways, as well as molecules which do not naturally
occur in the metabolism of said cells, but which are produced by a
said cells of the invention. Preferably, the compound is a
composition comprising the respective fine chemical or a recovered
respective fine chemical, in particular, the fine chemical, free or
in protein-bound form.
[12381] [0306.1.0.27] Preferably, the compound is a composition
comprising the methionine or a recovered methionine, in particular,
the fine chemical, free or in protein-bound form.
[12382] [0307.0.0.27] The invention also provides chimeric or
fusion proteins.
[12383] [0308.0.0.27] As used herein, an "chimeric protein" or
"fusion protein" comprises an polypeptide operatively linked to a
polypeptide which does not confer above-mentioned activity, in
particular, which does not confer an increase of content of the
respective fine chemical in a cell or an organism or a part
thereof, if its activity is increased.
[12384] [0309.0.0.27] In one embodiment, an reference to a "protein
(=polypeptide) of the invention" or as indicated in Table XII,
application no. 27, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table XII, application no. 27, columns 5 or 7, refers
to a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous a polypeptide of the
invention, preferably which is not substantially homologous to a
polypeptide as indicated in Table XII, application no. 27, columns
5 or 7, e.g., a protein which does not confer the activity
described herein or annotated or known for as indicated in Table
XII, application no. 27, column 3, and which is derived from the
same or a different organism. In one embodiment, a "non-polypeptide
of the invention" or "other polypeptide" not being indicated in
Table XII, application no. 27, columns 5 or 7, does not confer an
increase of the respective fine chemical in an organism or part
thereof.
[12385] [0310.0.0.27] Within the fusion protein, the term
"operatively linked" is intended to indicate that the polypeptide
of the invention or a polypeptide used in the process of the
invention and the "other polypeptide" or a part thereof are fused
to each other so that both sequences fulfil the proposed function
addicted to the sequence used. The "other polypeptide" can be fused
to the N-terminus or C-terminus of the polypeptide of the invention
or used in the process of the invention. For example, in one
embodiment the fusion protein is a GST-LMRP fusion protein in which
the sequences of the polypeptide of the invention or the
polypeptide used in the process of the invention are fused to the
C-terminus of the GST sequences. Such fusion proteins can
facilitate the purification of recombinant polypeptides of the
invention or a polypeptide useful in the process of the
invention.
[12386] [0311.0.0.27] In another embodiment, the fusion protein is
a polypeptide of the invention or a polypeptide used in the process
of the invention containing a heterologous signal sequence at its
N-terminus. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of a polypeptide of the invention or a
polypeptide used in the process of the invention can be increased
through use of a heterologous signal sequence. As already mentioned
above, targeting sequences, are required for targeting the gene
product into specific cell compartment (for a review, see Kermode,
Crit. Rev. Plant Sci. 15, 4 (1996) 285-423 and references cited
therein), for example into the vacuole, the nucleus, all types of
plastids, such as amyloplasts, chloroplasts, chromoplasts, the
extracellular space, the mitochondria, the endoplasmic reticulum,
elaioplasts, peroxisomes, glycosomes, and other compartments of
cells or extracellular. Sequences, which must be mentioned in this
context are, in particular, the signal-peptide- or
transit-peptide-encoding sequences which are known per se. For
example, plastid-transit-peptide-encoding sequences enable the
targeting of the expression product into the plastids of a plant
cell. Targeting sequences are also known for eukaryotic and to a
lower extent for prokaryotic organisms and can advantageously be
operable linked with the nucleic acid molecule of the present
invention to achieve an expression in one of said compartments or
extracellular.
[12387] [0312.0.0.27] Preferably, a chimeric or fusion protein of
the invention is produced by standard recombinant DNA techniques.
For example, DNA fragments coding for the different polypeptide
sequences are ligated together in-frame in accordance with
conventional techniques, for example by employing blunt-ended or
stagger-ended termini for ligation, restriction enzyme digestion to
provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline phosphatase treatment to avoid undesirable
joining, and enzymatic ligation. The fusion gene can be synthesized
by conventional techniques including automated DNA synthesizers.
Alternatively, PCR amplification of gene fragments can be carried
out using anchor primers, which give rise to complementary
overhangs between two consecutive gene fragments which can
subsequently be annealed and reamplified to generate a chimeric
gene sequence (see, for example, Current Protocols in Molecular
Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
Moreover, many expression vectors are commercially available that
already encode a fusion moiety (e.g., a GST polypeptide). The
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention can be cloned into such an
expression vector such that the fusion moiety is linked in-frame to
the encoded protein.
[12388] [0313.0.0.27] Furthermore, folding simulations and computer
redesign of structural motifs of the protein of the invention can
be performed using appropriate computer programs (Olszewski,
Proteins 25 (1996), 286-299; Hoffman, Comput. Appl. Biosci. 11
(1995), 675-679). Computer modelling of protein folding can be used
for the conformational and energetic analysis of detailed peptide
and protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012;
Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45). The appropriate
programs can be used for the identification of interactive sites
the polypeptide of the invention or polypeptides used in the
process of the invention and its substrates or binding factors or
other interacting proteins by computer assistant searches for
complementary peptide sequences (Fassina, Immunomethods (1994),
114-120). Further appropriate computer systems for the design of
protein and peptides are described in the prior art, for example in
Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y.
Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986),
5987-5991. The results obtained from the above-described computer
analysis can be used for, e.g., the preparation of peptidomimetics
of the protein of the invention or fragments thereof. Such
pseudopeptide analogues of the, natural amino acid sequence of the
protein may very efficiently mimic the parent protein (Benkirane,
J. Biol. Chem. 271 (1996), 33218-33224). For example, incorporation
of easily available achiral Q-amino acid residues into a protein of
the invention or a fragment thereof results in the substitution of
amide bonds by polymethylene units of an aliphatic chain, thereby
providing a convenient strategy for constructing a peptidomimetic
(Banerjee, Biopolymers 39 (1996), 769-777).
[12389] [0314.0.0.27] Superactive peptidomimetic analogues of small
peptide hormones in other systems are described in the prior art
(Zhang, Biochem. Biophys. Res. Commun. 224 (1996), 327-331).
Appropriate peptidomimetics of the protein of the present invention
can also be identified by the synthesis of peptidomimetic
combinatorial libraries through successive amide alkylation and
testing the resulting compounds, e.g., for their binding and
immunological properties. Methods for the generation and use of
peptidomimetic combinatorial libraries are described in the prior
art, for example in Ostresh, Methods in Enzymology 267 (1996),
220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.
[12390] [0315.0.0.27] Furthermore, a three-dimensional and/or
crystallographic structure of the protein of the invention can be
used for the design of peptidomimetic inhibitors of the biological
activity of the protein of the invention (Rose, Biochemistry 35
(1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996),
1545-1558).
[12391] [0316.0.0.27] Furthermore, a three-dimensional and/or
crystallographic structure of the protein of the invention and the
identification of interactive sites the polypeptide of the
invention or the polypeptide used in the method of the invention
and its substrates or binding factors can be used for the
identification or design of mutants with modulated binding or turn
over activities. For example, the active centre of the polypeptide
of the present invention can be modelled and amino acid residues
participating in the catalytic reaction can be modulated to
increase or decrease the binding of the substrate to activate or
improve the polypeptide. The identification of the active centre
and the amino acids involved in the catalytic reaction facilitates
the screening for mutants having an increased activity.
[12392] [0317.0.0.27] The sequences shown in column 5 of the Tables
XI to XIV herein have also been described under their Gene/ORF
Locus Name as described in the Table XI, XII, XIII or XIV, column
3.
[12393] [0318.0.0.27] In an especially preferred embodiment, the
polypeptide according to the invention furthermore also does not
have the sequences of those proteins which are encoded by the
sequences shown in the known listed activity of Gene/ORF Locus
Names or as described in the Tables, column 3.
[12394] [0319.0.0.27] One embodiment of the invention also relates
to an antibody, which binds specifically to the polypeptide
according to the invention or parts, i.e. specific fragments or
epitopes of such a protein.
[12395] [0320.0.0.27] The antibodies of the invention can be used
to identify and isolate the polypeptide according to the invention
and encoding genes in any organism, preferably plants, prepared in
plants described herein. These antibodies can be monoclonal
antibodies, polyclonal antibodies or synthetic antibodies as well
as fragments of antibodies, such as Fab, Fv or scFv fragments etc.
Monoclonal antibodies can be prepared, for example, by the
techniques as originally described in KOhler and Milstein, Nature
256 (1975), 495, and Galfr6, Meth. Enzymol. 73 (1981), 3, which
comprise the fusion of mouse myeloma cells to spleen cells derived
from immunized mammals.
[12396] [0321.0.0.27] Furthermore, antibodies or fragments thereof
to the aforementioned peptides can be obtained by using methods,
which are described, e.g., in Harlow and Lane "Antibodies, A
Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. These
antibodies can be used, for example, for the immunoprecipitation
and immunolocalization of proteins according to the invention as
well as for the monitoring of the synthesis of such proteins, for
example, in recombinant organisms, and for the identification of
compounds interacting with the protein according to the invention.
For example, surface plasmon resonance as employed in the BIAcore
system can be used to increase the efficiency of phage antibodies
selections, yielding a high increment of affinity from a single
library of phage antibodies, which bind to an epitope of the
protein of the invention (Schier, Human Antibodies Hybridomas 7
(1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). In
many cases, the binding phenomena of antibodies to antigens are
equivalent to other ligand/anti-ligand binding.
[12397] [0322.0.0.27] In one embodiment, the present invention
relates to an antisense nucleic acid molecule comprising the
complementary sequence of the nucleic acid molecule of the present
invention.
[12398] [0323.0.0.27] Methods to modify the expression levels
and/or the activity are known to persons skilled in the art and
include for instance overexpression, co-suppression, the use of
ribozymes, sense and anti-sense strategies or other gene silencing
approaches like RNA interference (RNAi) or promoter methylation.
"Sense strand" refers to the strand of a double-stranded DNA
molecule that is homologous to an mRNA transcript thereof. The
"anti-sense strand" contains an inverted sequence, which is
complementary to that of the "sense strand".
[12399] In addition the expression levels and/or the activity can
be modified by the introduction of mutations in the regulatory or
coding regions of the nucleic acids of the invention. Furthermore
antibodies can be expressed which specifically binds to a
polypeptide of interest and thereby blocks it activity. The
protein-binding factors can, for example, also be aptamers [Famulok
M and Mayer G (1999) Curr. Top Microbiol. Immunol. 243: 123-36] or
antibodies or antibody fragments or single-chain antibodies.
Obtaining these factors has been described, and the skilled worker
is familiar therewith. For example, a cytoplasmic scFv antibody has
been employed for modulating activity of the phytochrome A protein
in genetically modified tobacco plants [Owen M et al. (1992)
Biotechnology (NY) 10(7): 790-794; Franken E et al. (1997) Curr.
Opin. Biotechnol. 8(4): 411-416; Whitelam (1996) Trend Plant Sci.
1: 286-272].
[12400] [0324.0.0.27] An "antisense" nucleic acid molecule
comprises a nucleotide sequence, which is complementary to a
"sense" nucleic acid molecule encoding a protein, e.g.,
complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an encoding mRNA sequence.
Accordingly, an antisense nucleic acid molecule can bond via
hydrogen bonds to a sense nucleic acid molecule. The antisense
nucleic acid molecule can be complementary to an entire coding
strand of a nucleic acid molecule conferring the expression of the
polypeptide of the invention or used in the process of the present
invention, as the nucleic acid molecule of the invention or the
nucleic acid molecule used in the method of the invention coding
strand, or to only a portion thereof. Accordingly, an antisense
nucleic acid molecule can be antisense to a "coding region" of the
coding strand of a nucleotide sequence of a nucleic acid molecule
of the present invention. The term "coding region" refers to the
region of the nucleotide sequence comprising codons, which are
translated into amino acid residues. Further, the antisense nucleic
acid molecule is antisense to a "noncoding region" of the coding
strand of a nucleotide sequence encoding the polypeptide of the
invention or a polypeptide used in the process of the invention.
The term "noncoding region" refers to 5' and 3' sequences which
flank the coding region that are not translated into a polypeptide,
i.e., also referred to as 5' and 3' untranslated regions (5'-UTR or
3'-UTR).
[12401] [0325.0.0.27] Given the coding strand sequences encoding
the polypeptide of the present invention antisense nucleic acid
molecules of the invention can be designed according to the rules
of Watson and Crick base pairing.
[12402] [0326.0.0.27] The antisense nucleic acid molecule can be
complementary to the entire coding region of the mRNA encoding the
nucleic acid molecule to the invention or used in the process of
the present invention, but can also be an oligonucleotide which is
antisense to only a portion of the coding or noncoding region of
said mRNA. For example, the antisense oligonucleotide can be
complementary to the region surrounding the translation start site
of said mRNA. An antisense oligonucleotide can be, for example,
about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or 200 nucleotides
in length. An antisense nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention can
be constructed using chemical synthesis and enzymatic ligation
reactions using procedures known in the art. For example, an
antisense nucleic acid molecule (e.g., an antisense
oligonucleotide) can be chemically synthesized using naturally
occurring nucleotides or variously modified nucleotides designed to
increase the biological stability of the molecules or to increase
the physical stability of the duplex formed between the antisense
and sense nucleic acids, e.g., phosphorothioate derivatives and
acridine substituted nucleotides can be used. Examples of modified
nucleotides which can be used to generate the antisense nucleic
acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid molecule has been subcloned in an antisense
orientation (i.e., RNA transcribed from the inserted nucleic acid
molecule will be of an antisense orientation to a target nucleic
acid molecule of interest, described further in the following
subsection).
[12403] [0327.0.0.27] The antisense nucleic acid molecules of the
invention are typically administered to a cell or generated in situ
such that they hybridize with or bind to cellular mRNA and/or
genomic DNA encoding a polypeptide of the invention or the
polypeptide used in the method of the invention having
aforementioned the respective fine chemical increasing activity to
thereby inhibit expression of the protein, e.g., by inhibiting
transcription and/or translation.
[12404] [0328.0.0.27] The hybridization can be by conventional
nucleotide complementarity to form a stable duplex, or, for
example, in the case of an antisense nucleic acid molecule which
binds to DNA duplexes, through specific interactions in the major
groove of the double helix. The antisense nucleic acid molecule can
also be delivered to cells using the vectors described herein. To
achieve sufficient intracellular concentrations of the antisense
molecules, vector in which the antisense nucleic acid molecule is
placed under the control of a strong prokaryotic, viral, or
eukaryotic including plant promoters are preferred.
[12405] [0329.0.0.27] In a further embodiment, the antisense
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention can be an--anomeric nucleic
acid molecule. An .alpha.-anomeric nucleic acid molecule forms
specific double-stranded hybrids with complementary RNA in which,
contrary to the usual units, the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res.
[12406] 15:6625-6641). The antisense nucleic acid molecule can also
comprise a 2'-o-methyl-ribonucleotide (Inoue et al. (1987) Nucleic
Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et
al. (1987) FEBS Lett. 215:327-330).
[12407] [0330.0.0.27] Further the antisense nucleic acid molecule
of the invention or the nucleic acid molecule used in the method of
the invention can be also a ribozyme.
[12408] Ribozymes are catalytic RNA molecules with ribonuclease
activity, which are capable of cleaving a single-stranded nucleic
acid, such as an mRNA, to which they have a complementary region.
Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff
and Gerlach (1988) Nature 334:585-591)) can be used to
catalytically cleave mRNA transcripts encoding the polypeptide of
the invention or the polypeptide used in the method of the
invention to thereby inhibit translation of said mRNA. A ribozyme
having specificity for a nucleic acid molecule encoding the
polypeptide of the invention or used in the process of the
invention can be designed based upon the nucleotide sequence of the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention or coding a protein used in the
process of the invention or on the basis of a heterologous sequence
to be isolated according to methods taught in this invention. For
example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved in an
encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071 and
Cech et al. U.S. Pat. No. 5,116,742. Alternatively, mRNA encoding
the polypeptide of the invention or a polypeptide used in the
process of the invention can be used to select a catalytic RNA
having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science
261:1411-1418.
[12409] [0331.0.0.27] The antisense molecule of the present
invention comprises also a nucleic acid molecule comprising a
nucleotide sequences complementary to the regulatory region of an
nucleotide sequence encoding the natural occurring polypeptide of
the invention or the polypeptide used in the method of the
invention, e.g. the polypeptide sequences shown in the sequence
listing, or identified according to the methods described herein,
e.g., its promoter and/or enhancers, e.g. to form triple helical
structures that prevent transcription of the gene in target cells.
See generally, Helene, C. (1991) Anticancer Drug Des. 6(6): 569-84;
Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher,
L. J. (1992) Bioassays 14(12): 807-15.
[12410] [0332.0.0.27] Furthermore the present invention relates to
a double stranded RNA molecule capable for the reduction or
inhibition of the activity of the gene product of a gene encoding
the polypeptide of the invention, a polypeptide used in the process
of the invention, the nucleic acid molecule of the invention or a
nucleic acid molecule used in the process of the invention
encoding.
[12411] [0333.0.0.27] The method of regulating genes by means of
double-stranded RNA ("double-stranded RNA interference"; dsRNAi)
has been described extensively for animal, yeast, fungi and plant
organisms such as Neurospora, zebrafish, Drosophila, mice,
planaria, humans, Trypanosoma, petunia or Arabidopsis (for example
Matzke M A et al. (2000) Plant Mol. Biol. 43: 401-415; Fire A. et
al. (1998) Nature 391: 806-811; WO 99/32619; WO 99/53050; WO
00/68374; WO 00/44914; WO 00/44895; WO 00/49035; WO 00/63364). In
addition RNAi is also documented as an advantageously tool for the
repression of genes in bacteria such as E. coli for example by
Tchurikov et al. [J. Biol. Chem., 2000, 275 (34): 26523-26529].
Fire et al. named the phenomenon RNAi for "RNA interference". The
techniques and methods described in the above references are
expressly referred to. Efficient gene suppression can also be
observed in the case of transient expression or following transient
transformation, for example as the consequence of a biolistic
transformation (Schweizer P et al. (2000) Plant J 2000 24:
895-903). dsRNAi methods are based on the phenomenon that the
simultaneous introduction of complementary strand and counterstrand
of a gene transcript brings about highly effective suppression of
the expression of the gene in question. The resulting phenotype is
very similar to that of an analogous knock-out mutant (Waterhouse P
M et al. (1998) Proc. Natl. Acad. Sci. USA 95: 13959-64).
[12412] [0334.0.0.27] Tuschl et al. [Gens Dev., 1999, 13 (24):
3191-3197] was able to show that the efficiency of the RNAi method
is a function of the length of the duplex, the length of the 3'-end
overhangs, and the sequence in these overhangs. Based on the work
of Tuschl et al. the following guidelines can be given to the
skilled worker: To achieve good results the 5' and 3' untranslated
regions of the used nucleic acid sequence and regions close to the
start codon should be avoided as this regions are richer in
regulatory protein binding sites and interactions between RNAi
sequences and such regulatory proteins might lead to undesired
interactions. Preferably a region of the used mRNA is selected,
which is 50 to 100 nt (=nucleotides or bases) downstream of the AUG
start codon. Only dsRNA (=double-stranded RNA) sequences from exons
are useful for the method, as sequences from introns have no
effect. The G/C content in this region should be greater than 30%
and less than 70% ideally around 50%. A possible secondary
structure of the target mRNA is less important for the effect of
the RNAi method.
[12413] [0335.0.0.27] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XI, application
no. 27, columns 5 or 7, and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived there from),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
application no. 27, columns 5 or 7, and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of an
protein encoded by a nucleic acid sequence of one of the sequences
as indicated in Table XI, application no. 27, columns 5 or 7,
and/or homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[12414] [0336.0.0.27] The term "essentially identical" refers to
the fact that the dsRNA sequence may also include insertions,
deletions and individual point mutations in comparison to the
target sequence while still bringing about an effective reduction
in expression. Preferably, the homology as defined above amounts to
at least 30%, preferably at least 40%, 50%, 60%, 70% or 80%, very
especially preferably at least 90%, most preferably 100%, between
the "sense" strand of an inhibitory dsRNA and a part-segment of a
nucleic acid sequence of the invention (or between the "antisense"
strand and the complementary strand of a nucleic acid sequence,
respectively). The part-segment amounts to at least 10 bases,
preferably at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29 or 30 bases, especially preferably at least 40, 50, 60, 70, 80
or 90 bases, very especially preferably at least 100, 200, 300 or
400 bases, most preferably at least 500, 600, 700, 800, 900 or more
bases or at least 1000 or 2000 bases or more in length. In another
preferred embodiment of the invention the part-segment amounts to
17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 bases, preferably to
20, 21, 22, 23, 24 or 25 bases. These short sequences are preferred
in animals and plants. The longer sequences preferably between 200
and 800 bases are preferred in non-mammalian animals, preferably in
invertebrates, in yeast, fungi or bacteria, but they are also
useable in plants. Long double-stranded RNAs are processed in the
organisms into many siRNAs (=small/short interfering RNAs) for
example by the protein Dicer, which is a ds-specific Rnase III
enzyme. As an alternative, an "essentially identical" dsRNA may
also be defined as a nucleic acid sequence, which is capable of
hybridizing with part of a gene transcript (for example in 400 mM
NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA at 50.degree. C. or 70.degree.
C. for 12 to 16 h).
[12415] [0337.0.0.27] The dsRNA may consist of one or more strands
of polymerized ribonucleotides. Modification of both the
sugar-phosphate backbone and of the nucleosides may furthermore be
present. For example, the phosphodiester bonds of the natural RNA
can be modified in such a way that they encompass at least one
nitrogen or sulfur heteroatom. Bases may undergo modification in
such a way that the activity of, for example, adenosine deaminase
is restricted. These and other modifications are described herein
below in the methods for stabilizing antisense RNA.
[12416] [0338.0.0.27] The dsRNA can be prepared enzymatically; it
may also be synthesized chemically, either in full or in part.
[12417] [0339.0.0.27] The double-stranded structure can be formed
starting from a single, self-complementary strand or starting from
two complementary strands. In a single, self-complementary strand,
"sense" and "antisense" sequence can be linked by a linking
sequence ("linker") and form for example a hairpin structure.
Preferably, the linking sequence may take the form of an intron,
which is spliced out following dsRNA synthesis. The nucleic acid
sequence encoding a dsRNA may contain further elements such as, for
example, transcription termination signals or polyadenylation
signals. If the two strands of the dsRNA are to be combined in a
cell or an organism advantageously in a plant, this can be brought
about in a variety of ways.
[12418] [0340.0.0.27] Formation of the RNA duplex can be initiated
either outside the cell or within the cell. As shown in WO
99/53050, the dsRNA may also encompass a hairpin structure, by
linking the "sense" and "antisense" strands by a "linker" (for
example an intron). The self-complementary dsRNA structures are
preferred since they merely require the expression of a construct
and always encompass the complementary strands in an equimolar
ratio.
[12419] [0341.0.0.27] The expression cassettes encoding the
"antisense" or the "sense" strand of the dsRNA or the
self-complementary strand of the dsRNA are preferably inserted into
a vector and stably inserted into the genome of a plant, using the
methods described herein below (for example using selection
markers), in order to ensure permanent expression of the dsRNA.
[12420] [0342.0.0.27] The dsRNA can be introduced using an amount
which makes possible at least one copy per cell. A larger amount
(for example at least 5, 10, 100, 500 or 1 000 copies per cell) may
bring about more efficient reduction.
[12421] [0343.0.0.27] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 27, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table XI, application no. 27, columns 5 or 7, or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[12422] [0344.0.0.27] Due to the high degree of sequence homology
between sequences from various organisms (e.g. plants), allows the
conclusion that these proteins may be conserved to a high degree
within, for example other, plants, it is optionally possible that
the expression of a dsRNA derived from one of the disclosed
sequences as shown herein or homologs thereof should also have has
an advantageous effect in other plant species. Preferably the
consensus sequences shown herein can be used for the construction
of useful dsRNA molecules.
[12423] [0345.0.0.27] The dsRNA can be synthesized either in vivo
or in vitro. To this end, a DNA sequence encoding a dsRNA can be
introduced into an expression cassette under the control of at
least one genetic control element (such as, for example, promoter,
enhancer, silencer, splice donor or splice acceptor or
polyadenylation signal). Suitable advantageous constructs are
described herein below. Polyadenylation is not required, nor do
elements for initiating translation have to be present.
[12424] [0346.0.0.27] A dsRNA can be synthesized chemically or
enzymatically. Cellular RNA polymerases or bacteriophage RNA
polymerases (such as, for example T3, T7 or SP6 RNA polymerase) can
be used for this purpose. Suitable methods for the in-vitro
expression of RNA are described (WO 97/32016; U.S. Pat. No.
5,593,874; U.S. Pat. No. 5,698,425, U.S. Pat. No. 5,712,135, U.S.
Pat. No. 5,789,214, U.S. Pat. No. 5,804,693). Prior to introduction
into a cell, tissue or organism, a dsRNA which has been synthesized
in vitro either chemically or enzymatically can be isolated to a
higher or lesser degree from the reaction mixture, for example by
extraction, precipitation, electrophoresis, chromatography or
combinations of these methods. The dsRNA can be introduced directly
into the cell or else be applied extracellularly (for example into
the interstitial space).
[12425] [0347.0.0.27] Advantageously the RNAi method leads to only
a partial loss of gene function and therefore enables the skilled
worker to study a gene dose effect in the desired organism and to
fine tune the process of the invention. Furthermore it enables a
person skilled in the art to study multiple functions of a
gene.
[12426] [0348.0.0.27] Stable transformation of the plant with an
expression construct, which brings about the expression of the
dsRNA is preferred, however. Suitable methods are described herein
below.
[12427] [0349.0.0.27] A further embodiment of the invention also
relates to a method for the generation of a transgenic host or host
cell, e.g. a eukaryotic or prokaryotic cell, preferably a
transgenic microorganism, a transgenic plant cell or a transgenic
plant tissue or a transgenic plant, which comprises introducing,
into the plant, the plant cell or the plant tissue, the nucleic
acid construct according to the invention, the vector according to
the invention, or the nucleic acid molecule according to the
invention.
[12428] [0350.0.0.27] A further embodiment of the invention also
relates to a method for the transient generation of a host or host
cell, eukaryotic or prokaryotic cell, preferably a transgenic
microorganism, a transgenic plant cell or a transgenic plant tissue
or a transgenic plant, which comprises introducing, into the plant,
the plant cell or the plant tissue, the nucleic acid construct
according to the invention, the vector according to the invention,
the nucleic acid molecule characterized herein as being contained
in the nucleic acid construct of the invention or the nucleic acid
molecule according to the invention, whereby the introduced nucleic
acid molecules, nucleic acid construct and/or vector is not
integrated into the genome of the host or host cell. Therefore the
transformants are not stable during the propagation of the host in
respect of the introduced nucleic acid molecules, nucleic acid
construct and/or vector.
[12429] [0351.0.0.27] In the process according to the invention,
transgenic organisms are also to be understood as meaning--if they
take the form of plants--plant cells, plant tissues, plant organs
such as root, shoot, stem, seed, flower, tuber or leaf, or intact
plants which are grown for the production of the respective fine
chemical.
[12430] [0352.0.0.27] Growing is to be understood as meaning for
example culturing the transgenic plant cells, plant tissue or plant
organs on or in a nutrient medium or the intact plant on or in a
substrate, for example in hydroponic culture, potting compost or on
a field soil.
[12431] [0353.0.0.27] In a further advantageous embodiment of the
process, the nucleic acid molecules can be expressed in
single-celled plant cells (such as algae), see Falciatore et al.,
1999, Marine Biotechnology 1 (3): 239-251 and references cited
therein, and plant cells from higher plants (for example
spermatophytes such as crops). Examples of plant expression vectors
encompass those which are described in detail herein or in: Becker,
D. [(1992) Plant Mol. Biol. 20:1195-1197] and Bevan, M. W. [(1984),
Nucl. Acids Res. 12:8711-8721; Vectors for Gene Transfer in Higher
Plants; in: Transgenic Plants, Vol. 1, Engineering and Utilization,
Ed.: Kung and R. Wu, Academic Press, 1993, pp. 15-38]. An overview
of binary vectors and their use is also found in Hellens, R.
[(2000), Trends in Plant Science, Vol. 5 No. 10, 446-451.
[12432] [0354.0.0.27] Vector DNA can be introduced into prokaryotic
or eukaryotic cells via conventional transformation or transfection
techniques. The terms "transformation" and "transfection" include
conjugation and transduction and, as used in the present context,
are intended to encompass a multiplicity of prior-art methods for
introducing foreign nucleic acid molecules (for example DNA) into a
host cell, including calcium phosphate coprecipitation or calcium
chloride coprecipitation, DEAE-dextran-mediated transfection,
PEG-mediated transfection, lipofection, natural competence,
chemically mediated transfer, electroporation or particle
bombardment. Suitable methods for the transformation or
transfection of host cells, including plant cells, can be found in
Sambrook et al. (Molecular Cloning: A Laboratory Manual., 2nd Ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989) and in other laboratory handbooks
such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium
protocols, Ed.: Gartland and Davey, Humana Press, Totowa, N.J.
[12433] [0355.0.0.27] The above-described methods for the
transformation and regeneration of plants from plant tissues or
plant cells are exploited for transient or stable transformation of
plants. Suitable methods are the transformation of protoplasts by
polyethylene-glycol-induced DNA uptake, the biolistic method with
the gene gun--known as the particle bombardment method--,
electroporation, the incubation of dry embryos in DNA-containing
solution, microinjection and the Agrobacterium-mediated gene
transfer. The abovementioned methods are described for example in
B. Jenes, Techniques for Gene Transfer, in: Transgenic Plants, Vol.
1, Engineering and Utilization, edited by S. D. Kung and R. Wu,
Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant
Physiol. Plant Molec. Biol. 42 (1991) 205-225. The construct to be
expressed is preferably cloned into a vector, which is suitable for
transforming Agrobacterium tumefaciens, for example pBin19 (Bevan,
Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed with
such a vector can then be used in the known manner for the
transformation of plants, in particular crop plants, such as, for
example, tobacco plants, for example by bathing scarified leaves or
leaf segments in an agrobacterial solution and subsequently
culturing them in suitable media. The transformation of plants with
Agrobacterium tumefaciens is described for example by Hofgen and
Willmitzer in Nucl. Acid Res. (1988) 16,9877 or known from, inter
alia, F. F. White, Vectors for Gene Transfer in Higher Plants; in
Transgenic Plants, Vol. 1, Engineering and Utilization, edited by
S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
[12434] [0356.0.0.27] To select for the successful transfer of the
nucleic acid molecule, vector or nucleic acid construct of the
invention according to the invention into a host organism, it is
advantageous to use marker genes as have already been described
above in detail. It is known of the stable or transient integration
of nucleic acids into plant cells that only a minority of the cells
takes up the foreign DNA and, if desired, integrates it into its
genome, depending on the expression vector used and the
transfection technique used. To identify and select these
integrants, a gene encoding for a selectable marker (as described
above, for example resistance to antibiotics) is usually introduced
into the host cells together with the gene of interest. Preferred
selectable markers in plants comprise those, which confer
resistance to an herbicide such as glyphosate or gluphosinate.
Other suitable markers are, for example, markers, which encode
genes involved in biosynthetic pathways of, for example, sugars or
amino acids, such as .beta.-galactosidase, ura3 or ilv2. Markers,
which encode genes such as luciferase, gfp or other fluorescence
genes, are likewise suitable. These markers and the aforementioned
markers can be used in mutants in whom these genes are not
functional since, for example, they have been deleted by
conventional methods. Furthermore, nucleic acid molecules, which
encode a selectable marker, can be introduced into a host cell on
the same vector as those, which encode the polypeptides of the
invention or used in the process or else in a separate vector.
Cells which have been transfected stably with the nucleic acid
introduced can be identified for example by selection (for example,
cells which have integrated the selectable marker survive whereas
the other cells die).
[12435] [0357.0.0.27] Since the marker genes, as a rule
specifically the gene for resistance to antibiotics and herbicides,
are no longer required or are undesired in the transgenic host cell
once the nucleic acids have been introduced successfully, the
process according to the invention for introducing the nucleic
acids advantageously employs techniques which enable the removal,
or excision, of these marker genes. One such a method is what is
known as cotransformation. The cotransformation method employs two
vectors simultaneously for the transformation, one vector bearing
the nucleic acid according to the invention and a second bearing
the marker gene(s). A large proportion of transformants receives
or, in the case of plants, comprises (up to 40% of the
transformants and above), both vectors. In case of transformation
with Agrobacteria, the transformants usually receive only a part of
the vector, the sequence flanked by the T-DNA, which usually
represents the expression cassette. The marker genes can
subsequently be removed from the transformed plant by performing
crosses. In another method, marker genes integrated into a
transposon are used for the transformation together with desired
nucleic acid (known as the Ac/Ds technology). The transformants can
be crossed with a transposase resource or the transformants are
transformed with a nucleic acid construct conferring expression of
a transposase, transiently or stable. In some cases (approx. 10%),
the transposon jumps out of the genome of the host cell once
transformation has taken place successfully and is lost. In a
further number of cases, the transposon jumps to a different
location. In these cases, the marker gene must be eliminated by
performing crosses. In microbiology, techniques were developed
which make possible, or facilitate, the detection of such events. A
further advantageous method relies on what are known as
recombination systems, whose advantage is that elimination by
crossing can be dispensed with. The best-known system of this type
is what is known as the Cre/lox system. Cre1 is a recombinase,
which removes the sequences located between the loxP sequences. If
the marker gene is integrated between the loxP sequences, it is
removed, once transformation has taken place successfully, by
expression of the recombinase. Further recombination systems are
the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., J. Biol.
Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol.,
149, 2000: 553-566). A site-specific integration into the plant
genome of the nucleic acid sequences according to the invention is
possible. Naturally, these methods can also be applied to
microorganisms such as yeast, fungi or bacteria.
[12436] [0358.0.0.27] Agrobacteria transformed with an expression
vector according to the invention may also be used in the manner
known per se for the transformation of plants such as experimental
plants like Arabidopsis or crop plants, such as, for example,
cereals, maize, oats, rye, barley, wheat, soya, rice, cotton,
sugarbeet, canola, sunflower, flax, hemp, potato, tobacco, tomato,
carrot, bell peppers, oilseed rape, tapioca, cassava, arrow root,
tagetes, alfalfa, lettuce and the various tree, nut, and grapevine
species, in particular oil-containing crop plants such as soya,
peanut, castor-oil plant, sunflower, maize, cotton, flax, oilseed
rape, coconut, oil palm, safflower (Carthamus tinctorius) or cocoa
beans, for example by bathing scarified leaves or leaf segments in
an agrobacterial solution and subsequently growing them in suitable
media.
[12437] [0359.0.0.27] In addition to the transformation of somatic
cells, which then has to be regenerated into intact plants, it is
also possible to transform the cells of plant meristems and in
particular those cells which develop into gametes. In this case,
the transformed gametes follow the natural plant development,
giving rise to transgenic plants. Thus, for example, seeds of
Arabidopsis are treated with agrobacteria and seeds are obtained
from the developing plants of which a certain proportion is
transformed and thus transgenic (Feldman, K A and Marks M D (1987).
Mol Gen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua
and J Shell, eds, Methods in Arabidopsis Research. Word Scientific,
Singapore, pp. 274-289). Alternative methods are based on the
repeated removal of the influorescences and incubation of the
excision site in the center of the rosette with transformed
agrobacteria, whereby transformed seeds can likewise be obtained at
a later point in time (Chang (1994). Plant J. 5: 551-558; Katavic
(1994). Mol Gen Genet, 245: 363-370). However, an especially
effective method is the vacuum infiltration method with its
modifications such as the "floral dip" method. In the case of
vacuum infiltration of Arabidopsis, intact plants under reduced
pressure are treated with an agrobacterial suspension (Bechthold, N
(1993). C R Acad Sci Paris Life Sci, 316: 1194-1199), while in the
case of the "floral dip" method the developing floral tissue is
incubated briefly with a surfactant-treated agrobacterial
suspension (Clough, S J and Bent, A F (1998). The Plant J. 16,
735-743). A certain proportion of transgenic seeds are harvested in
both cases, and these seeds can be distinguished from nontransgenic
seeds by growing under the above-described selective conditions. In
addition the stable transformation of plastids is of advantages
because plastids are inherited maternally is most crops reducing or
eliminating the risk of transgene flow through pollen. The
transformation of the chloroplast genome is generally achieved by a
process, which has been schematically displayed in Klaus et al.,
2004 (Nature Biotechnology 22(2), 225-229). Briefly the sequences
to be transformed are cloned together with a selectable marker gene
between flanking sequences homologous to the chloroplast genome.
These homologous flanking sequences direct site specific
integration into the plastome. Plastidal transformation has been
described for many different plant species and an overview can be
taken from Bock (2001) Transgenic plastids in basic research and
plant biotechnology. J Mol Biol. 2001 Sep. 21; 312 (3): 425-38 or
Maliga, P (2003) Progress towards commercialization of plastid
transformation technology. Trends Biotechnol. 21, 20-28. Further
biotechnological progress has recently been reported in form of
marker free plastid transformants, which can be produced by a
transient cointegrated maker gene (Klaus et al., 2004, Nature
Biotechnology 22 (2), 225-229).
[12438] [0360.0.0.27] The genetically modified plant cells can be
regenerated via all methods with which the skilled worker is
familiar. Suitable methods can be found in the abovementioned
publications by S. D. Kung and R. Wu, Potrykus or Hofgen and
Willmitzer.
[12439] [0361.0.0.27] Accordingly, the present invention thus also
relates to a plant cell comprising the nucleic acid construct
according to the invention, the nucleic acid molecule according to
the invention or the vector according to the invention.
[12440] [0362.0.0.27] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, the nucleic acid construct of
the invention, the antisense molecule of the invention, the vector
of the invention or a nucleic acid molecule encoding the
polypeptide of the invention or the polypeptide used in the method
of the invention, e.g. the polypeptide as indicated in Table XII,
application no. 27, columns 5 or 7 cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention or the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention is increased, e.g. due to an increased
expression or specific activity of the subject matters of the
invention in a cell or an organism or a part thereof. In one
embodiment, transgenic for a polypeptide having an activity of a
polypeptide as indicated in Table XII, application no. 27, columns
5 or 7, means herein that due to modulation or manipulation of the
genome, an activity as annotated for a polypeptide as indicated in
Table XII, application no. 27, column 3, e.g. having a sequence as
indicated in Table XII, application no. 27, columns 5 or 7, is
increased in a cell or an organism or a part thereof. Examples are
described above in context with the process of the invention
[12441] [0363.0.0.27] "Transgenic", for example regarding a nucleic
acid molecule, an nucleic acid construct or a vector comprising
said nucleic acid molecule or an organism transformed with said
nucleic acid molecule, nucleic acid construct or vector, refers to
all those subjects originating by recombinant methods in which
either [12442] a) the nucleic acid sequence, or [12443] b) a
genetic control sequence linked operably to the nucleic acid
sequence, for example a promoter, or [12444] c) (a) and (b) are not
located in their natural genetic environment or have been modified
by recombinant methods, an example of a modification being a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide residues. Natural genetic environment refers to the
natural chromosomal locus in the organism of origin, or to the
presence in a genomic library. In the case of a genomic library,
the natural genetic environment of the nucleic acid sequence is
preferably retained, at least in part. The environment flanks the
nucleic acid sequence at least at one side and has a sequence of at
least 50 bp, preferably at least 500 bp, especially preferably at
least 1000 bp, very especially preferably at least 5000 bp, in
length.
[12445] [0364.0.0.27] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a polypeptide of the invention with the corresponding
protein-encoding sequence--becomes a transgenic expression cassette
when it is modified by non-natural, synthetic "artificial" methods
such as, for example, mutagenization. Such methods have been
described (U.S. Pat. No. 5,565,350; WO 00/15815; also see
above).
[12446] [0365.0.0.27] Further, the plant cell, plant tissue or
plant can also be transformed such that further enzymes and
proteins are (over)expressed which expression supports an increase
of the respective fine chemical.
[12447] [0366.0.0.27] However, transgenic also means that the
nucleic acids according to the invention are located at their
natural position in the genome of an organism, but that the
sequence has been modified in comparison with the natural sequence
and/or that the regulatory sequences of the natural sequences have
been modified. Preferably, transgenic/recombinant is to be
understood as meaning the transcription of the nucleic acids used
in the process according to the invention occurs at a non-natural
position in the genome, that is to say the expression of the
nucleic acids is homologous or, preferably, heterologous. This
expression can be transiently or of a sequence integrated stably
into the genome.
[12448] [0367.0.0.27] The term "transgenic plants" used in
accordance with the invention also refers to the progeny of a
transgenic plant, for example the T.sub.1, T.sub.2, T.sub.3 and
subsequent plant generations or the BC.sub.1, BC.sub.2, BC.sub.3
and subsequent plant generations. Thus, the transgenic plants
according to the invention can be raised and selfed or crossed with
other individuals in order to obtain further transgenic plants
according to the invention. Transgenic plants may also be obtained
by propagating transgenic plant cells vegetatively. The present
invention also relates to transgenic plant material, which can be
derived from a transgenic plant population according to the
invention. Such material includes plant cells and certain tissues,
organs and parts of plants in all their manifestations, such as
seeds, leaves, anthers, fibers, tubers, roots, root hairs, stems,
embryo, calli, cotelydons, petioles, harvested material, plant
tissue, reproductive tissue and cell cultures, which are derived
from the actual transgenic plant and/or can be used for bringing
about the transgenic plant.
[12449] [0368.0.0.27] Any transformed plant obtained according to
the invention can be used in a conventional breeding scheme or in
in vitro plant propagation to produce more transformed plants with
the same characteristics and/or can be used to introduce the same
characteristic in other varieties of the same or related species.
Such plants are also part of the invention. Seeds obtained from the
transformed plants genetically also contain the same characteristic
and are part of the invention. As mentioned before, the present
invention is in principle applicable to any plant and crop that can
be transformed with any of the transformation method known to those
skilled in the art.
[12450] [0369.0.0.27] In an especially preferred embodiment, the
organism, the host cell, plant cell, plant, microorganism or plant
tissue according to the invention is transgenic.
[12451] [0370.0.0.27] Accordingly, the invention therefore relates
to transgenic organisms transformed with at least one nucleic acid
molecule, nucleic acid construct or vector according to the
invention, and to cells, cell cultures, tissues, parts--such as,
for example, in the case of plant organisms, plant tissue, for
example leaves, roots and the like--or propagation material derived
from such organisms, or intact plants. The terms "recombinant
(host)", and "transgenic (host)" are used interchangeably in this
context. Naturally, these terms refer not only to the host organism
or target cell in question, but also to the progeny, or potential
progeny, of these organisms or cells. Since certain modifications
may occur in subsequent generations owing to mutation or
environmental effects, such progeny is not necessarily identical
with the parental cell, but still comes within the scope of the
term as used herein.
[12452] [0371.0.0.27] Suitable organisms for the process according
to the invention or as hosts are all these eukaryotic or
prokaryotic organisms, which are capable of synthesizing the
respective fine chemical. The organisms used as hosts are
microorganisms, such as bacteria, fungi, yeasts or algae, non-human
animals, or plants, such as dictotyledonous or monocotyledonous
plants.
[12453] [0372.0.0.27] In principle all plants can be used as host
organism, especially the plants mentioned above as source organism.
Preferred transgenic plants are, for example, selected from the
families Aceraceae, Anacardiaceae, Apiaceae, Asteraceae,
Brassicaceae, Cactaceae, Cucurbitaceae, Euphorbiaceae, Fabaceae,
Malvaceae, Nymphaeaceae, Papaveraceae, Rosaceae, Salicaceae,
Solanaceae, Arecaceae, Bromeliaceae, Cyperaceae, Iridaceae,
Liliaceae, Orchidaceae, Gentianaceae, Labia-ceae, Magnoliaceae,
Ranunculaceae, Carifolaceae, Rubiaceae, Scrophulariaceae,
Caryophyllaceae, Ericaceae, Polygonaceae, Violaceae, Juncaceae or
Poaceae and preferably from a plant selected from the group of the
families Apiaceae, Asteraceae, Brassicaceae, Cucurbitaceae,
Fabaceae, Papaveraceae, Rosaceae, Solanaceae, Liliaceae or Poaceae.
Preferred are crop plants such as plants advantageously selected
from the group of the genus peanut, oilseed rape, canola,
sunflower, safflower, olive, sesame, hazelnut, almond, avocado,
bay, pumpkin/squash, linseed, soya, pistachio, borage, maize,
wheat, rye, oats, sorghum and millet, triticale, rice, barley,
cassava, potato, sugarbeet, egg plant, alfalfa, and perennial
grasses and forage plants, oil palm, vegetables (brassicas, root
vegetables, tuber vegetables, pod vegetables, fruiting vegetables,
onion vegetables, leafy vegetables and stem vegetables), buckwheat,
Jerusalem artichoke, broad bean, vetches, lentil, dwarf bean,
lupin, clover and Lucerne for mentioning only some of them.
[12454] [0373.0.0.27] Preferred plant cells, plant organs, plant
tissues or parts of plants originate from the under source organism
mentioned plant families, preferably from the abovementioned plant
genus, more preferred from abovementioned plants species.
[12455] [0374.0.0.27] Transgenic plants comprising the amino acids
synthesized in the process according to the invention can be
marketed directly without isolation of the compounds synthesized.
In the process according to the invention, plants are understood as
meaning all plant parts, plant organs such as leaf, stalk, root,
tubers or seeds or propagation material or harvested material or
the intact plant. In this context, the seed encompasses all parts
of the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue. The amino acids produced in the
process according to the invention may, however, also be isolated
from the plant in the form of their free amino acids or bound in
proteins. Amino acids produced by this process can be harvested by
harvesting the organisms either from the culture in which they grow
or from the field. This can be done via expressing, grinding and/or
extraction, salt precipitation and/or ion-exchange chromatography
of the plant parts, preferably the plant seeds, plant fruits, plant
tubers and the like.
[12456] [0375.0.0.27] In a further embodiment, the present
invention relates to a process for the generation of a
microorganism, comprising the introduction, into the microorganism
or parts thereof, of the nucleic acid construct of the invention,
or the vector of the invention or the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention.
[12457] [0376.0.0.27] In another embodiment, the present invention
relates also to a transgenic microorganism comprising the nucleic
acid molecule of the invention or the nucleic acid molecule used in
the method of the invention, the nucleic acid construct of the
invention or the vector as of the invention. Appropriate
microorganisms have been described herein before under source
organism, preferred are in particular aforementioned strains
suitable for the production of fine chemicals.
[12458] [0377.0.0.27] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced amino acid composition or the produced respective fine
chemical is isolated.
[12459] [0378.0.0.27] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the fine
chemicals produced in the process can be isolated. The resulting
fine chemicals can, if appropriate, subsequently be further
purified, if desired mixed with other active ingredients such as
vitamins, amino acids, carbohydrates, antibiotics and the like,
and, if appropriate, formulated.
[12460] [0379.0.0.27] In one embodiment, the fatty acid is the fine
chemical.
[12461] [0380.0.0.27] The amino acids obtained in the process are
suitable as starting material for the synthesis of further products
of value. For example, they can be used in combination with each
other or alone for the production of pharmaceuticals, foodstuffs,
animal feeds or cosmetics. Accordingly, the present invention
relates a method for the production of a pharmaceuticals, food
stuff, animal feeds, nutrients or cosmetics comprising the steps of
the process according to the invention, including the isolation of
the amino acid composition produced or the fine chemical produced
if desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the amino acids produced in the
process or of the transgenic organisms in animal feeds, foodstuffs,
medicines, food supplements, cosmetics or pharmaceuticals.
[12462] [0381.0.0.27] In principle all microorganisms can be used
as host organism especially the ones mentioned under source
organism above. It is advantageous to use in the process of the
invention transgenic microorganisms such as fungi such as the genus
Claviceps or Aspergillus or Gram-positive bacteria such as the
genera Bacillus, Corynebacterium, Micrococcus, Brevibacterium,
Rhodococcus, Nocardia, Caseobacter or Arthrobacter or Gram-negative
bacteria such as the genera Escherichia, Flavobacterium or
Salmonella or yeasts such as the genera Rhodotorula, Hansenula or
Candida. Particularly advantageous organisms are selected from the
group of genera Corynebacterium, Brevibacterium, Escherichia,
Bacillus, Rhodotorula, Hansenula, Candida, Claviceps or
Flavobacterium. It is very particularly advantageous to use in the
process of the invention microorganisms selected from the group of
genera and species consisting of Hansenula anomala, Candida utilis,
Claviceps purpurea, Bacillus circulans, Bacillus subtilis, Bacillus
sp., Brevibacterium albidum, Brevibacterium album, Brevibacterium
cerinum, Brevibacterium flavum, Brevibacterium glutamigenes,
Brevibacterium iodinum, Brevibacterium ketoglutamicum,
Brevibacterium lactofermentum, Brevibacterium linens,
Brevibacterium roseum, Brevibacterium saccharolyticum,
Brevibacterium sp., Corynebacterium acetoacidophilum,
Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes,
Corynebacterium glutamicum (=Micrococcus glutamicum),
Corynebacterium melassecola, Corynebacterium sp. or Escherichia
coli, specifically Escherichia coli K12 and its described
strains.
[12463] [0382.0.0.27] The process of the invention is, when the
host organisms are microorganisms, advantageously carried out at a
temperature between 0.degree. C. and 95.degree. C., preferably
between 10.degree. C. and 85.degree. C., particularly preferably
between 15.degree. C. and 75.degree. C., very particularly
preferably between 15.degree. C. and 45.degree. C. The pH is
advantageously kept at between pH 4 and 12, preferably between pH 6
and 9, particularly preferably between pH 7 and 8, during this. The
process of the invention can be operated batchwise, semibatchwise
or continuously. A summary of known cultivation methods is to be
found in the textbook by Chmiel (Bioproze.beta.technik 1.
Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag,
Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and
periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden,
1994)). The culture medium to be used must meet the requirements of
the respective strains in a suitable manner. Descriptions of
culture media for various microorganisms are present in the
handbook "Manual of Methods for General Bacteriology" of the
American Society for Bacteriology (Washington D. C., USA, 1981).
These media, which can be employed according to the invention
include, as described above, usually one or more carbon sources,
nitrogen sources, inorganic salts, vitamins and/or trace elements.
Preferred carbon sources are sugars such as mono-, di- or
polysaccharides. Examples of very good carbon sources are glucose,
fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,
maltose, sucrose, raffinose, starch or cellulose. Sugars can also
be added to the media via complex compounds such as molasses, or
other byproducts of sugar refining. It may also be advantageous to
add mixtures of various carbon sources. Other possible carbon
sources are oils and fats such as, for example, soybean oil,
sunflower oil, peanut oil and/or coconut fat, fatty acids such as,
for example, palmitic acid, stearic acid and/or linoleic acid,
alcohols and/or polyalcohols such as, for example, glycerol,
methanol and/or ethanol and/or organic acids such as, for example,
acetic acid and/or lactic acid. Nitrogen sources are usually
organic or inorganic nitrogen compounds or materials, which contain
these compounds. Examples of nitrogen sources include ammonia in
liquid or gaseous form or ammonium salts such as ammonium sulfate,
ammonium chloride, ammonium phosphate, ammonium carbonate or
ammonium nitrate, nitrates, urea, amino acids or complex nitrogen
sources such as corn steep liquor, soybean meal, soybean protein,
yeast extract, meat extract and others. The nitrogen sources may be
used singly or as a mixture. Inorganic salt compounds, which may be
present in the media include the chloride, phosphorus or sulfate
salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium,
manganese, zinc, copper and iron.
[12464] [0383.0.0.27] For preparing sulfur-containing fine
chemicals, in particular the respective fine chemical, e.g. amino
acids containing sulfur it is possible to use as sulfur source
inorganic sulfur-containing compounds such as, for example,
sulfates, sulfites, dithionites, tetrathionates, thiosulfates,
sulfides or else organic sulfur compounds such as mercaptans and
thiols.
[12465] [0384.0.0.27] It is possible to use as phosphorus source
phosphoric acid, potassium dihydrogenphosphate or dipotassium
hydrogenphosphate or the corresponding sodium-containing salts.
Chelating agents can be added to the medium in order to keep the
metal ions in solution. Particularly suitable chelating agents
include dihydroxyphenols such as catechol or protocatechuate, or
organic acids such as citric acid. The fermentation media employed
according to the invention for cultivating microorganisms normally
also contain other growth factors such as vitamins or growth
promoters, which include, for example, biotin, riboflavin,
thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine.
Growth factors and salts are often derived from complex media
components such as yeast extract, molasses, corn steep liquor and
the like. Suitable precursors can moreover be added to the culture
medium. The exact composition of the media compounds depends
greatly on the particular experiment and is chosen individually for
each specific case. Information about media optimization is
obtainable from the textbook "Applied Microbiol. Physiology, A
Practical Approach" (editors P. M. Rhodes, P. F. Stanbury, IRL
Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also
be purchased from commercial suppliers such as Standard 1 (Merck)
or BHI (Brain heart infusion, DIEGO) and the like. All media
components are sterilized either by heat (1.5 bar and 121.degree.
C. for 20 min) or by sterilizing filtration. The components can be
sterilized either together or, if necessary, separately. All media
components can be present at the start of the cultivation or
optionally be added continuously or batchwise. The temperature of
the culture is normally between 15.degree. C. and 45.degree. C.,
preferably at 25.degree. C. to 40.degree. C., and can be kept
constant or changed during the experiment. The pH of the medium
should be in the range from 5 to 8.5, preferably around 7. The pH
for the cultivation can be controlled during the cultivation by
adding basic compounds such as sodium hydroxide, potassium
hydroxide, ammonia or aqueous ammonia or acidic compounds such as
phosphoric acid or sulfuric acid. Foaming can be controlled by
employing antifoams such as, for example, fatty acid polyglycol
esters. The stability of plasmids can be maintained by adding to
the medium suitable substances having a selective effect, for
example antibiotics. Aerobic conditions are maintained by
introducing oxygen or oxygen-containing gas mixtures such as, for
example, ambient air into the culture. The temperature of the
culture is normally from 20.degree. C. to 45.degree. C. and
preferably from 25.degree. C. to 40.degree. C. The culture is
continued until formation of the desired product is at a maximum.
This aim is normally achieved within 10 hours to 160 hours.
[12466] [0385.0.0.27] The fermentation broths obtained in this way,
containing in particular L-methionine, L-threonine and/or L-lysine,
normally have a dry matter content of from 7.5 to 25% by weight.
Sugar-limited fermentation is additionally advantageous, at least
at the end, but especially over at least 30% of the fermentation
time. This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 3 g/l during
this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed entirely or
partly by separation methods, such as, for example, centrifugation,
filtration, decantation or a combination of these methods, from the
fermentation broth or left completely in it. The fermentation broth
can then be thickened or concentrated by known methods, such as,
for example, with the aid of a rotary evaporator, thin-film
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. This concentrated fermentation broth can then be
worked up by freeze-drying, spray drying, spray granulation or by
other processes.
[12467] [0386.0.0.27] However, it is also possible to purify the
amino acid produced further. For this purpose, the
product-containing composition is subjected to a chromatography on
a suitable resin, in which case the desired product or the
impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use. The purified product can be
concentrated by filtration or ultrafiltration and stored at a
temperature at which the stability of the product is a maximum.
[12468] [0387.0.0.27] The identity and purity of the isolated
compound(s) can be determined by prior art techniques. These
include high performance liquid chromatography (HPLC),
spectroscopic methods, mass spectrometry (MS), staining methods,
thin-layer chromatography, NIRS, enzyme assay or microbiological
assays. These analytical methods are summarized in: Patek et al.
(1994) Appl. Environ. Microbiol. 60:133-140; Malakhova et al.
(1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540,
pp. 540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, Vol. 17.
[12469] [0388.0.0.27] In yet another aspect, the invention also
relates to harvestable parts and to propagation material of the
transgenic plants according to the invention which either contain
transgenic plant cells expressing a nucleic acid molecule according
to the invention or which contains cells which show an increased
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention, e.g. an increased
expression level or higher activity of the described protein.
[12470] [0389.0.0.27] Harvestable parts can be in principle any
useful parts of a plant, for example, flowers, pollen, seedlings,
tubers, leaves, stems, fruit, seeds, roots etc. Propagation
material includes, for example, seeds, fruits, cuttings, seedlings,
tubers, rootstocks etc. Preferred are seeds, fruits, seedlings or
tubers as harvestable or propagation material.
[12471] [0390.0.0.27] The invention furthermore relates to the use
of the transgenic organisms according to the invention and of the
cells, cell cultures, parts--such as, for example, roots, leaves
and the like as mentioned above in the case of transgenic plant
organisms--derived from them, and to transgenic propagation
material such as seeds or fruits and the like as mentioned above,
for the production of foodstuffs or feeding stuffs, pharmaceuticals
or fine chemicals.
[12472] [0391.0.0.27] Accordingly in another embodiment, the
present invention relates to the use of the nucleic acid molecule,
the organism, e.g. the microorganism, the plant, plant cell or
plant tissue, the vector, or the polypeptide of the present
invention for making fatty acids, carotenoids, isoprenoids,
vitamins, lipids, wax esters, (poly)saccharides and/or
polyhydroxyalkanoates, and/or its metabolism products, in
particular, steroid hormones, cholesterol, prostaglandin,
triacylglycerols, bile acids and/or ketone bodies producing cells,
tissues and/or plants. There are a number of mechanisms by which
the yield, production, and/or efficiency of production of fatty
acids, carotenoids, isoprenoids, vitamins, wax esters, lipids,
(poly)saccharides and/or polyhydroxyalkanoates, and/or its
metabolism products, in particular, steroid hormones, cholesterol,
triacylglycerols, prostaglandin, bile acids and/or ketone bodies or
further of above defined fine chemicals incorporating such an
altered protein can be affected. In the case of plants, by e.g.
increasing the expression of acetyl-CoA which is the basis for many
products, e.g., fatty acids, carotenoids, isoprenoids, vitamines,
lipids, (poly)saccharides, wax esters, and/or
polyhydroxyalkanoates, and/or its metabolism products, in
particular, prostaglandin, steroid hormones, cholesterol,
triacylglycerols, bile acids and/or ketone bodies in a cell, it may
be possible to increase the amount of the produced said compounds
thus permitting greater ease of harvesting and purification or in
case of plants more efficient partitioning. Further, one or more of
said metabolism products, increased amounts of the cofactors,
precursor molecules, and intermediate compounds for the appropriate
biosynthetic pathways maybe required. Therefore, by increasing the
number and/or activity of transporter proteins involved in the
import of nutrients, such as carbon sources (i.e., sugars),
nitrogen sources (i.e., amino acids, ammonium salts), phosphate,
and sulfur, it may be possible to improve the production of acetyl
CoA and its metabolism products as mentioned above, due to the
removal of any nutrient supply limitations on the biosynthetic
process. In particular, it may be possible to increase the yield,
production, and/or efficiency of production of said compounds, e.g.
fatty acids, carotenoids, isoprenoids, vitamins, was esters,
lipids, (poly)saccharides, and/or polyhydroxyalkanoates, and/or its
metabolism products, in particular, steroid hormones, cholesterol,
prostaglandin, triacylglycerols, bile acids and/or ketone bodies
molecules etc. in plants.
[12473] [0392.0.0.27] Furthermore preferred is a method for the
recombinant production of pharmaceuticals or fine chemicals in host
organisms, wherein a host organism is transformed with one of the
above-described nucleic acid constructs comprising one or more
structural genes which encode the desired fine chemical or catalyze
the biosynthesis of the desired fine chemical, the transformed host
organism is cultured, and the desired fine chemical is isolated
from the culture medium. This method can be applied widely to fine
chemicals such as enzymes, vitamins, amino acids, sugars, fatty
acids, and natural and synthetic flavourings, aroma substances and
colorants or compositions comprising these. Especially preferred is
the additional production of further amino acids, tocopherols and
tocotrienols and carotenoids or compositions comprising said
compounds. The transformed host organisms are cultured and the
products are recovered from the host organisms or the culture
medium by methods known to the skilled worker or the organism
itself servers as food or feed supplement. The production of
pharmaceuticals such as, for example, antibodies or vaccines, is
described by Hood E E, Jilka J M. Curr Opin Biotechnol. 1999
August; 10(4):382-6; Ma J K, Vine N D. Curr Top Microbiol Immunol.
1999; 236:275-92.
[12474] [0393.0.0.27] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps:
(a) contacting, e.g. hybridising, the nucleic acid molecules of a
sample, e.g. cells, tissues, plants or microorganisms or a nucleic
acid library, which can contain a candidate gene encoding a gene
product conferring an increase in the respective fine chemical
after expression, with the nucleic acid molecule of the present
invention; (b) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to a nucleic acid
molecule sequence as indicated in Table XI, application no. 27,
columns 5 or 7, and, optionally, isolating the full length cDNA
clone or complete genomic clone; (c) introducing the candidate
nucleic acid molecules in host cells, preferably in a plant cell or
a microorganism, appropriate for producing the respective fine
chemical; (d) expressing the identified nucleic acid molecules in
the host cells; (e) assaying the respective fine chemical level in
the host cells; and (f) identifying the nucleic acid molecule and
its gene product which expression confers an increase in the the
respective fine chemical level in the host cell after expression
compared to the wild type.
[12475] [0394.0.0.27] Relaxed hybridisation conditions are: After
standard hybridisation procedures washing steps can be performed at
low to medium stringency conditions usually with washing conditions
of 40.degree.-55.degree. C. and salt conditions between 2.times.SSC
and 0,2.times.SSC with 0.1% SDS in comparison to stringent washing
conditions as e.g. 60.degree.-68.degree. C. with 0.1% SDS. Further
examples can be found in the references listed above for the
stringent hybridization conditions. Usually washing steps are
repeated with increasing stringency and length until a useful
signal to noise ratio is detected and depend on many factors as the
target, e.g. its purity, GC-content, size etc, the probe, e.g. its
length, is it a RNA or a DNA probe, salt conditions, washing or
hybridisation temperature, washing or hybridisation time etc.
[12476] [0395.0.0.27] In an other embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [12477] (a) identifying
nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the respective fine chemical after expression, which are at least
20%, preferably 25%, more preferably 30%, even more preferred are
35%. 40% or 50%, even more preferred are 60%, 70% or 80%, most
preferred are 90% or 95% or more homology to the nucleic acid
molecule of the present invention, for example via homology search
in a data bank; [12478] (b) introducing the candidate nucleic acid
molecules in host cells, preferably in a plant cells or
microorganisms, appropriate for producing the respective fine
chemical; [12479] (c) expressing the identified nucleic acid
molecules in the host cells; [12480] (d) assaying the the
respective fine chemical level in the host cells; and [12481] (e)
identifying the nucleic acid molecule and its gene product which
expression confers an increase in the the respective fine chemical
level in the host cell after expression compared to the wild type.
[12482] Eventually gene products conferring the increase in the
respective fine chemical production can also be identify according
to a identical or similar 3D structure in step (a) and by the above
described method.
[12483] [0396.0.0.27] The nucleic acid molecules identified can
then be used for the production of the respective fine chemical in
the same way as the nucleic acid molecule of the present invention.
Accordingly, in one embodiment, the present invention relates to a
process for the production of the respective fine chemical,
comprising (a) identifying a nucleic acid molecule according to
aforementioned steps [12484] (a) to (f) or (a) to (e) and
recovering the free or bound fine chemical from a organism having
an increased cellular activity of a polypeptide encoded by the
isolated nucleic acid molecule compared to a wild type.
[12485] [0397.0.0.27] Furthermore, in one embodiment, the present
invention relates to a method for the identification of a compound
stimulating production of the respective fine chemical to said
plant comprising: [12486] a) contacting cells which express the
polypeptide of the present invention or its mRNA with a candidate
compound under cell cultivation conditions; [12487] b) assaying an
increase in expression of said polypeptide or said mRNA; [12488] c)
comparing the expression level to a standard response made in the
absence of said candidate compound; whereby, an increased
expression over the standard indicates that the compound is
stimulating production of the respective fine chemical.
[12489] [0398.0.0.27] Furthermore, in one embodiment, the present
invention relates to a method for the screening for agonists or an
antagonist of the activity of the polypeptide of the present
invention or used in the process of the present invention, e.g. a
polypeptide conferring an increase of the respective fine chemical
in an organism or a part thereof after increasing the activity in
an organism or a part thereof, comprising: [12490] (a) contacting
cells, tissues, plants or microorganisms which express the
polypeptide according to the invention with a candidate compound or
a sample comprising a plurality of compounds under conditions which
permit the expression the polypeptide of the present invention or
used in the process of the present invention; [12491] (b) assaying
the respective fine chemical level or the polypeptide expression
level in the cell, tissue, plant or microorganism or the media the
cell, tissue, plant or microorganisms is cultured or maintained in;
and [12492] (c) identifying a agonist or antagonist by comparing
the measured the respective fine chemical level or polypeptide of
the invention or used in the invention expression level with a
standard the respective fine chemical or polypeptide expression
level measured in the absence of said candidate compound or a
sample comprising said plurality of compounds, whereby an increased
level over the standard indicates that the compound or the sample
comprising said plurality of compounds is an agonist and a
decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an
antagonist.
[12493] [0399.0.0.27] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increased the respective fine chemical production in a
plant or microorganism, comprising the steps: [12494] (j) culturing
a cell or tissue or microorganism or maintaining a plant expressing
the polypeptide according to the invention or a nucleic acid
molecule encoding said polypeptide and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and the
polypeptide of the present invention or used in the process of the
invention; and [12495] (k) identifying if the compound is an
effective agonist by detecting the presence or absence or increase
of a signal produced by said readout system.
[12496] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in fine chemical production.
[12497] [0399.1.0.27] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table XII,
columns 5 or 7, or a homolog thereof, e.g. comparing the phenotype
of nearly identical organisms with low and high activity of a
protein as indicated in Table XII, columns 5 or 7, after incubation
with the drug.
[12498] [0400.0.0.27] Said compound may be chemically synthesized
or microbiologically produced and/or comprised in, for example,
samples, e.g., cell extracts from, e.g., plants, animals or
microorganisms, e.g. pathogens. Furthermore, said compound(s) may
be known in the art but hitherto not known to be capable of
suppressing or activating the polypeptide of the present invention.
The reaction mixture may be a cell free extract or may comprise a
cell or tissue culture. Suitable set ups for the method of the
invention are known to the person skilled in the art and are, for
example, generally described in Alberts et al., Molecular Biology
of the Cell, third edition (1994), in particular Chapter 17. The
compounds may be, e.g., added to the reaction mixture, culture
medium, injected into the cell or sprayed onto the plant.
[12499] [0401.0.0.27] If a sample containing a compound is
identified in the method of the invention, then it is either
possible to isolate the compound from the original sample
identified as containing the compound capable of activating or
increasing the content of the respective fine chemical in an
organism or part thereof, or one can further subdivide the original
sample, for example, if it consists of a plurality of different
compounds, so as to reduce the number of different substances per
sample and repeat the method with the subdivisions of the original
sample. Depending on the complexity of the samples, the steps
described above can be performed several times, preferably until
the sample identified according to the method of the invention only
comprises a limited number of or only one substance(s). Preferably
said sample comprises substances of similar chemical and/or
physical properties, and most preferably said substances are
identical. Preferably, the compound identified according to the
above described method or its derivative is further formulated in a
form suitable for the application in plant breeding or plant cell
and tissue culture.
[12500] [0402.0.0.27] The compounds which can be tested and
identified according to a method of the invention may be expression
libraries, e.g., cDNA expression libraries, peptides, proteins,
nucleic acids, antibodies, small organic compounds, hormones,
peptidomimetics, PNAs or the like (Milner, Nature Medicine 1
(1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79
(1994), 193-198 and references cited supra). Said compounds can
also be functional derivatives or analogues of known inhibitors or
activators. Methods for the preparation of chemical derivatives and
analogues are well known to those skilled in the art and are
described in, for example, Beilstein, Handbook of Organic
Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New
York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York,
USA. Furthermore, said derivatives and analogues can be tested for
their effects according to methods known in the art. Furthermore,
peptidomimetics and/or computer aided design of appropriate
derivatives and analogues can be used, for example, according to
the methods described above. The cell or tissue that may be
employed in the method of the invention preferably is a host cell,
plant cell or plant tissue of the invention described in the
embodiments hereinbefore.
[12501] [0403.0.0.27] Thus, in a further embodiment the invention
relates to a compound obtained or identified according to the
method for identifying an agonist of the invention said compound
being an agonist of the polypeptide of the present invention or
used in the process of the present invention.
[12502] [0404.0.0.27] Accordingly, in one embodiment, the present
invention further relates to a compound identified by the method
for identifying a compound of the present invention.
[12503] [0405.0.0.27] Said compound is, for example, a homologous
of the polypeptide of the present invention. Homologues of the
polypeptid of the present invention can be generated by
mutagenesis, e.g., discrete point mutation or truncation of the
polypeptide of the present invention. As used herein, the term
"homologue" refers to a variant form of the protein, which acts as
an agonist of the activity of the polypeptide of the present
invention. An agonist of said protein can retain substantially the
same, or a subset, of the biological activities of the polypeptide
of the present invention. In particular, said agonist confers the
increase of the expression level of the polypeptide of the present
invention and/or the expression of said agonist in an organisms or
part thereof confers the increase of free and/or bound the
respective fine chemical in the organism or part thereof.
[12504] [0406.0.0.27] In one embodiment, the invention relates to
an antibody specifically recognizing the compound or agonist of the
present invention.
[12505] [0407.0.0.27] The invention also relates to a diagnostic
composition comprising at least one of the aforementioned nucleic
acid molecules, vectors, proteins, antibodies or compounds of the
invention and optionally suitable means for detection.
[12506] [0408.0.0.27] The diagnostic composition of the present
invention is suitable for the isolation of mRNA from a cell and
contacting the mRNA so obtained with a probe comprising a nucleic
acid probe as described above under hybridizing conditions,
detecting the presence of mRNA hybridized to the probe, and thereby
detecting the expression of the protein in the cell. Further
methods of detecting the presence of a protein according to the
present invention comprise immunotechniques well known in the art,
for example enzyme linked immunosorbent assay. Furthermore, it is
possible to use the nucleic acid molecules according to the
invention as molecular markers or primer in plant breeding.
Suitable means for detection are well known to a person skilled in
the arm, e.g. buffers and solutions for hydridization assays, e.g.
the aforementioned solutions and buffers, further and means for
Southern-, Western-, Northern--etc.--blots, as e.g. described in
Sambrook et al. are known.
[12507] [0409.0.0.27] In another embodiment, the present invention
relates to a kit comprising the nucleic acid molecule, the vector,
the host cell, the polypeptide, the antisense nucleic acid, the
antibody, plant cell, the plant or plant tissue, the harvestable
part, the propagation material and/or the compound or agonist or
antagonists identified according to the method of the
invention.
[12508] [0410.0.0.27] The compounds of the kit of the present
invention may be packaged in containers such as vials, optionally
with/in buffers and/or solution. If appropriate, one or more of
said components might be packaged in one and the same container.
Additionally or alternatively, one or more of said components might
be adsorbed to a solid support as, e.g. a nitrocellulose filter, a
glass plate, a chip, or a nylon membrane or to the well of a micro
titerplate. The kit can be used for any of the herein described
methods and embodiments, e.g. for the production of the host cells,
transgenic plants, pharmaceutical compositions, detection of
homologous sequences, identification of antagonists or agonists, as
food or feed or as a supplement thereof, as supplement for the
treating of plants, etc.
[12509] [0411.0.0.27] Further, the kit can comprise instructions
for the use of the kit for any of said embodiments, in particular
for the use for producing organisms or part thereof having an
increased free or bound the respective fine chemical content.
[12510] [0412.0.0.27] In one embodiment said kit comprises further
a nucleic acid molecule encoding one or more of the aforementioned
protein, and/or an antibody, a vector, a host cell, an antisense
nucleic acid, a plant cell or plant tissue or a plant.
[12511] [0413.0.0.27] In a further embodiment, the present
invention relates to a method for the production of a agricultural
composition providing the nucleic acid molecule, the vector or the
polypeptide of the invention or the polypeptide used in the method
of the invention or comprising the steps of the method according to
the invention for the identification of said compound, agonist or
antagonist; and formulating the nucleic acid molecule, the vector
or the polypeptide of the invention or the polypeptide used in the
method of the invention or the agonist, or compound identified
according to the methods or processes of the present invention or
with use of the subject matters of the present invention in a form
applicable as plant agricultural composition.
[12512] [0414.0.0.27] In another embodiment, the present invention
relates to a method for the production of a "the respective fine
chemical"-production supporting plant culture composition
comprising the steps of the method for of the present invention;
and formulating the compound identified in a form acceptable as
agricultural composition.
[12513] [0415.0.0.27] Under "acceptable as agricultural
composition" is understood, that such a composition is in agreement
with the laws regulating the content of fungicides, plant
nutrients, herbicides, etc. Preferably such a composition is
without any harm for the protected plants and the animals (humans
included) fed therewith.
[12514] [0416.0.0.27] The present invention also pertains to
several embodiments relating to further uses and methods. The
nucleic acid molecule, polypeptide, protein homologues, fusion
proteins, primers, vectors, host cells, described herein can be
used in one or more of the following methods: identification of
plants useful for the respective fine chemical production as
mentioned and related organisms; mapping of genomes; identification
and localization of sequences of interest; evolutionary studies;
determination of regions required for function; modulation of an
activity.
[12515] [0417.0.0.27] The nucleic acid molecule of the invention or
the nucleic acid molecule used in the method of the invention, the
vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the amino acid production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect plants against herbicides, which
block the amino acid, in particular the respective fine chemical,
synthesis in said plant. Inhibitors may inhibit one or more of the
steps for the synthesis of methionine. The first committed step for
the synthesis of Lys, Met and Thr is the first step, in which
aspartate is phosphorylated to aspartyl-b-phosphate, catalyzed by
aspartokinase: E. coli has 3 isozymes of aspartokinase that respond
differently to each of the 3 amino acids, with regard to enzyme
inhibition and feedback inhibition. The biosynthesis of lysine,
methionine and threonine are not, then, controlled as a group. The
pathway from aspartate to lysine has 10 steps. The pathway from
aspartate to threonine has 5 steps. The pathway from aspartate to
methionine has 7 steps. Regulation of the three pathways also
occurs at the two branch points: [12516] b-Aspartate-semialdehyde
(homoserine and lysine) [12517] Homoserine (threonine and
methionine)
[12518] The regulation results from feedback inhibition by the
amino acid products of the branches, indicated in the brackets
above. One important step in the synthesis of this group of 3 amino
acids is the step in which homocysteine is converted to methionine,
catalyzed by the enzyme methionine synthase:
##STR00003##
[12519] In this reaction, homocysteine is methylated to methionine,
and the Cl donor is N5-methyl-THF. Thus, inhibition of one or more
of the methionine synthesis enzymes, including also the provision
of donor molecules, can inhibit the synthesis of methionine.
[12520] Examples of herbicides blocking the amino acid synthesis in
plants are for example sulfonylurea and imidazolinone herbicides,
which catalyze the first step in branched-chain amino acid
biosynthesis. Inhibitors of the methionine synthesis may for
example described in Danishpajooh 10, 2001 Nitric oxide inhibits
methionine synthase activity in vivo and disrupts carbon flow
through the folate pathway. J. Biol. Chem. 276: 27296-27303; Datko
A H, 1982 Methionine biosynthesis in Lemna-inhibitor studies. Plant
Physiol. 69: 1070-1076; Lavrador K, 1998 A new series of cyclic
amino acids as inhibitors of S-adenosyl L-methionine synthetase.
Bioorg. Med. Chem. Lett. 8: 1629-1634; Thompson G A, 1982
Methionine synthesis in Lemna-inhibition of cystathionine
gamma-synthase by propargylglycine. Plant Physiol. 70: 1347-1352.
In some organisms the methionine synthesis is inhibited by ethanol,
lead, mercury, aluminium, thimerosal, cupper, N20, as e.g.
discussed in M. Waly, H. Oleteanu et al., 2004, Molecular
Psychiatry, 1-13.
[12521] Interestingly, Arabidopsis seed germination was strongly
delayed in the presence of DL-propargylglycine, a specific
inhibitor of methionine synthesis. Furthermore, this compound
totally inhibited seedling growth. These phenotypic effects were
largely alleviated upon methionine supplementation in the
germination medium. The results indicated that methionine synthase
and S-adenosylmethionine synthetase are fundamental components
controlling metabolism in the transition from a quiescent to a
highly active state during seed germination. Moreover, the observed
temporal patterns of accumulation of these proteins are consistent
with an essential role of endogenous ethylene in Arabidopsis only
after radicle protrusion; s. Gallarado, K., 2002, Importance of
methionine biosynthesis for Arabidopsis seed germination and
seedling growth, Physiolgia Plantarum, 116(2), pp 238-247.
Accordingly, the overexpression of a polypeptide of the present
invention in a plant may protect the plant against a herbicide
inhibiting methionine synthesis.
[12522] [0418.0.0.27] Accordingly, the nucleic acid molecules of
the present invention have a variety of uses. First, they may be
used to identify an organism or a close relative thereof. Also,
they may be used to identify the presence thereof or a relative
thereof in a mixed population of microorganisms or plants. By
probing the extracted genomic
[12523] DNA of a culture of a unique or mixed population of plants
under stringent conditions with a probe spanning a region of the
gene of the present invention which is unique to this, one can
ascertain whether the present invention has been used or whether it
or a close relative is present.
[12524] [0419.0.0.27] Further, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention may be sufficiently homologous to the sequences of
related species such that these nucleic acid molecules may serve as
markers for the construction of a genomic map in related
organism.
[12525] [0420.0.0.27] Accordingly, the present invention relates to
a method for breeding plants for the production of the respective
fine chemical, comprising [12526] (a) providing a first plant
variety produced according to the process of the invention
preferably (over)expressing the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention; [12527] (b) crossing the first plant variety with a
second plant variety; and [12528] (c) selecting the offspring
plants which overproduce the respective fine chemical by means of
analysis the distribution of a molecular marker in the offspring
representing the first plant variety and its capability to
(over)produce the respective fine chemical.
[12529] Details about the use of molecular markers in breeding can
be found in Kumar et al., 1999 (Biotech Adv., 17:143-182) and
Peleman and van der Voort 2003 (Trends Plant Sci. 2003 July;
8(7):330-334)
[12530] The molecular marker can e.g. relate to the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention and/or its expression level. Accordingly,
the molecular marker can be a probe or a PCR primer set useful for
identification of the genomic existence or genomic localisation of
the nucleic acid molecule of the invention or the nucleic acid
molecule used in the method of the invention, e.g. in a Southern
blot analysis or a PCR or its expression level, i.g. in a Northern
Blot analysis or a quantitative PCR.
[12531] Accordingly, in one embodiment, the present invention
relates to the use of the nucleic acid molecule of the present
invention or encoding the polypeptide of the present invention as
molecular marker for breeding, especially for breeding for a high
or low respective fine chemical production.
[12532] [0421.0.0.27] The nucleic acid molecules of the invention
are also useful for evolutionary and protein structural studies. By
comparing the sequences of the invention or used in the process of
the invention to those encoding similar enzymes from other
organisms, the evolutionary relatedness of the organisms can be
assessed. Similarly, such a comparison permits an assessment of
which regions of the sequence are conserved and which are not,
which may aid in determining those regions of the protein which are
essential for the functioning of the enzyme. This type of
determination is of value for protein engineering studies and may
give an indication of what the protein can tolerate in terms of
mutagenesis without losing function.
[12533] [0422.0.0.27] Accordingly, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention can be used for the identification of other nucleic acids
conferring an increase of the respective fine chemical after
expression.
[12534] [0423.0.0.27] Further, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention or a fragment of a gene conferring the expression of the
polypeptide of the invention or the polypeptide used in the method
of the invention, preferably comprising the nucleic acid molecule
of the invention, can be used for marker assisted breeding or
association mapping of the respective fine chemical derived
traits
[12535] [0424.0.0.27] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention or the polypeptide used
in the method of the invention, the nucleic acid construct of the
invention, the organisms, the host cell, the microorganisms, the
plant, plant tissue, plant cell, or the part thereof of the
invention, the vector of the invention, the agonist identified with
the method of the invention, the nucleic acid molecule identified
with the method of the present invention, can be used for the
production of the respective fine chemical or of the fine chemical
and one or more other amino acids, in particular Threoinine,
Alanine, Glutamin, Glutamic acid, Valine, Asparagine,
Phenylalanine, Leucine, Proline, Tryptophan Tyrosine, Valine,
Isoleucine and Arginine. Accordingly, the nucleic acid of the
invention, or the nucleic acid molecule identified with the method
of the present invention or the complement sequences thereof, the
polypeptide of the invention or the polypeptide used in the method
of the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the respective fine chemical in a organism or part
thereof, e.g. in a cell.
[12536] [0425.0.0.27] Further, the nucleic acid of the invention,
the polypeptide of the invention or the polypeptide used in the
method of the invention, the nucleic acid construct of the
invention, the organisms, the host cell, the microorganisms, the
plant, plant tissue, plant cell, or the part thereof of the
invention, the vector of the invention, the antagonist or the
agonist identified with the method of the invention, the antibody
of the present invention, the antisense molecule of the present
invention or the nucleic acid molecule identified with the method
of the present invention, can be used for the preparation of an
agricultural composition.
[12537] [0426.0.0.27] Furthermore, the nucleic acid of the
invention, the polypeptide of the invention or the polypeptide used
in the method of the invention, the nucleic acid construct of the
invention, the organisms, the host cell, the microorganisms, the
plant, plant tissue, plant cell, or the part thereof of the
invention, the vector of the invention, antagonist or the agonist
identified with the method of the invention, the antibody of the
present invention, the antisense molecule of the present invention
or the nucleic acid molecule identified with the method of the
present invention, can be used for the identification and
production of compounds capable of conferring a modulation of the
respective fine chemical levels in an organism or parts thereof,
preferably to identify and produce compounds conferring an increase
of the respective fine chemical levels in an organism or parts
thereof, if said identified compound is applied to the organism or
part thereof, i.e. as part of its food, or in the growing or
culture media.
[12538] [0427.0.0.27] These and other embodiments are disclosed and
encompassed by the description and examples of the present
invention. Further literature concerning any one of the methods,
uses and compounds to be employed in accordance with the present
invention may be retrieved from public libraries, using for example
electronic devices. For example the public database "Medline" may
be utilized which is available on the Internet, for example under
hftp://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases
and addresses, such as hftp://www.ncbi.nlm.nih.gov/,
hftp://www.infobiogen.fr/,
hftp://www.fmi.ch/biology/research-tools.html,
hftp://www.tigr.org/, are known to the person skilled in the art
and can also be obtained using, e.g., hftp://www.lycos.com. An
overview of patent information in biotechnology and a survey of
relevant sources of patent information useful for retrospective
searching and for current awareness is given in Berks, TIBTECH 12
(1994), 352-364.
[12539] [0428.0.0.27]
[12540] [0429.0.0.27] The present invention is illustrated by the
examples, which follow. The present examples illustrate the basic
invention without being intended as limiting the subject of the
invention. The content of all of the references, patent
applications, patents and published patent applications cited in
the present patent application is herewith incorporated by
reference.
[0430.0.0.27] EXAMPLES
[0431.0.0.27] Example 1
Cloning into in Escherichia coli
[12541] [0432.0.0.27] A DNA polynucleotide with a sequence as
indicated in Table XI, application no. 27, column 5 and encoding a
polypeptide as listed in Table 1 below, was cloned into the
plasmids pBR322 (Sutcliffe, J. G. (1979) Proc. Natl Acad. Sci. USA,
75: 3737-3741); pACYC177 (Change & Cohen (1978) J. Bacteriol.
134: 1141-1156); plasmids of the pBS series (pBSSK+, pBSSK- and
others; Stratagene, LaJolla, USA) or cosmids such as SuperCosi
(Stratagene, LaJolla, USA) or Lorist6 (Gibson, T. J. Rosenthal, A.,
and Waterson, R. H. (1987) Gene 53: 283-286) for expression in E.
coli using known, well-established procedures (see, for example,
Sambrook, J. et al. (1989) "Molecular Cloning: A Laboratory
Manual". Cold Spring Harbor Laboratory Press or Ausubel, F. M. et
al. (1994) "Current Protocols in Molecular Biology", John Wiley
& Sons).
[0433.0.0.27] Example 2
DNA Sequencing and Computerized Functional Analysis
[12542] [0434.0.0.27] The DNA was sequenced by standard procedures,
in particular the chain determination method, using ABI377
sequencers (see, for example, Fleischman, R. D. et al. (1995)
"Whole-genome Random Sequencing and Assembly of Haemophilus
Influenzae Rd., Science 269; 496-512)".
[0435.0.0.27] Example 3
In-Vivo and In-Vitro Mutagenesis
[12543] [0436.0.0.27] An in vivo mutagenesis of Corynebacterium
glutamicum for the production of the respective fine chemical can
be carried out by passing a plasmid DNA (or another vector DNA)
through E. coli and other microorganisms (for example Bacillus spp.
or yeasts such as Saccharomyces cerevisiae), which are not capable
of maintaining the integrity of its genetic information. Usual
mutator strains have mutations in the genes for the DNA repair
system [for example mutHLS, mutD, mutT and the like; for
comparison, see Rupp, W. D. (1996) DNA repair mechanisms in
Escherichia coli and Salmonella, pp. 2277-2294, ASM: Washington].
The skilled worker knows these strains. The use of these strains is
illustrated for example in Greener, A. and Callahan, M. (1994)
Strategies 7; 32-34.
[12544] [0436.1.0.27] In-vitro mutation methods such as increasing
the spontaneous mutation rates by chemical or physical treatment
are well known to the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagenesis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[12545] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[12546] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.0.27] Example 4
DNA Transfer Between Escherichia coli and Corynebacterium
glutamicum
[12547] [0438.0.0.27] Several Corynebacterium and Brevibacterium
species comprise endogenous plasmids (such as, for example, pHM1519
or pBL1) which replicate autonomously (for a review, see, for
example, Martin, J. F. et al. (1987) Biotechnology 5: 137-146).
Shuttle vectors for Escherichia coli and Corynebacterium glutamicum
can be constructed easily using standard vectors for E. coli
(Sambrook, J. et al., (1989), "Molecular Cloning: A Laboratory
Manual", Cold Spring Harbor Laboratory Press or Ausubel, F. M. et
al. (1994) "Current Protocols in Molecular Biology", John Wiley
& Sons), which have a replication origin for, and suitable
marker from, Corynebacterium glutamicum added. Such replication
origins are preferably taken from endogenous plasmids, which have
been isolated from Corynebacterium and Brevibacterium species.
Genes, which are used in particular as transformation markers for
these species are genes for kanamycin resistance (such as those
which originate from the Tn5 or Tn-903 transposon) or for
chloramphenicol resistance (Winnacker, E. L. (1987) "From Genes to
Clones--Introduction to Gene Technology, VCH, Weinheim). There are
many examples in the literature of the preparation of a large
multiplicity of shuttle vectors which are replicated in E. coli and
C. glutamicum and which can be used for various purposes including
the overexpression of genes (see, for example, Yoshihama, M. et al.
(1985) J. Bacteriol. 162: 591-597, Martin, J. F. et al., (1987)
Biotechnology, 5: 137-146 and Eikmanns, B. J. et al. (1992) Gene
102: 93-98). Suitable vectors, which replicate in coryneform
bacteria are, for example, pZ1 (Menke) et al., Appl. Environ.
Microbiol., 64, 1989: 549-554) pEkEx1 (Eikmanns et al., Gene 102,
1991: 93-98) or pHS2-1 (Sonnen et al, Gene 107, 1991: 69-74). These
vectors are based on the cryptic plasmids pHM1519, pBL1 or pGA1.
Other plasmid vectors such as, for example, those based on pCG4
(U.S. Pat. No. 4,489,160), pNG2 (Serwold-Davis et al., FEMS
Microbiol. Lett., 66, 1990: 119-124) or pAG1 (U.S. Pat. No.
5,158,891) can be used in the same manner.
[12548] [0439.0.0.27] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into Corynebacterium
glutamicum strains. The transformation of C. glutamicum can be
achieved by protoplast transformation (Kastsumata, R. et al.,
(1984) J. Bacteriol. 159, 306-311), electroporation (Liebl, E. et
al., (1989) FEMS Microbiol. Letters, 53: 399-303) and in those
cases where specific vectors are used also by conjugation (such as,
for example, described in Schafer, A., et al. (1990) J. Bacteriol.
172: 1663-1666). Likewise, it is possible to transfer the shuttle
vectors for C. glutamicum to E. coli by preparing plasmid DNA from
C. glutamicum (using standard methods known in the art) and
transforming it into E. coli. This transformation step can be
carried out using standard methods, but preferably using an
Mcr-deficient E. coli strain, such as NM522 (Gough & Murray
(1983) J. Mol. Biol. 166: 1-19).
[12549] [0440.0.0.27] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the coryneform
bacteria, standard techniques known to the skilled worker also
exist for this purpose. Examples, which are used for this purpose
are plasmid vectors as they have been described by Remscheid et al.
(App). Environ. Microbiol., 60, 1994: 126-132) for the duplication
and amplification of the hom-thrB operon. In this method, the
complete gene is cloned into a plasmid vector which is capable of
replication in a host such as E. coli, but not in C. glutamicum.
Suitable vectors are, for example, pSUP301 (Simon et al.,
Bio/Technology 1, 1983: 784-791), pK1Bmob or pK19mob (Schafer et
al., Gene 145, 1994: 69-73), pGEM-T (Promega Corp., Madison, Wis.,
USA), pCR2.1-TOPO (Schuman, J. Biol. Chem., 269, 1994: 32678-32684,
U.S. Pat. No. 5,487,993), pCR.RTM.Blunt (Invitrogen, Groningen, the
Netherlands) or pEM1 (Schrumpf et al., J. Bacteriol., 173, 1991:
4510-4516).
[0441.0.0.27] Example 5
Determining the Expression of the Mutant/Transgenic Protein
[12550] [0442.0.0.27] The observations of the activity of a
mutated, or transgenic, protein in a transformed host cell are
based on the fact that the protein is expressed in a similar manner
and in a similar quantity as the wild-type protein. A suitable
method for determining the transcription quantity of the mutant, or
transgenic, gene (a sign for the amount of mRNA which is available
for the translation of the gene product) is to carry out a Northern
blot (see, for example, Ausubel et al., (1988) Current Protocols in
Molecular Biology, Wiley: New York), where a primer which is
designed in such a way that it binds to the gene of interest is
provided with a detectable marker (usually a radioactive or
chemiluminescent marker) so that, when the total RNA of a culture
of the organism is extracted, separated on a gel, applied to a
stable matrix and incubated with this probe, the binding and
quantity of the binding of the probe indicates the presence and
also the amount of mRNA for this gene. Another method is a
quantitative PCR. This information detects the extent to which the
gene has been transcribed. Total cell RNA can be isolated from
Corynebacterium glutamicum or other microorganisms by a variety of
methods, which are known in the art, e.g. as described in Bormann,
E. R. et al., (1992) Mol. Microbiol. 6: 317-326.
[12551] [0443.0.0.27] Standard techniques, such as Western blot,
may be employed to determine the presence or relative amount of
protein translated from this mRNA (see, for example, Ausubel et al.
(1988) "Current Protocols in Molecular Biology", Wiley, New York).
In this method, total cell proteins are extracted, separated by gel
electrophoresis, transferred to a matrix such as nitrocellulose and
incubated with a probe, such as an antibody, which binds
specifically to the desired protein. This probe is usually provided
directly or indirectly with a chemiluminescent or colorimetric
marker, which can be detected readily. The presence and the
observed amount of marker indicates the presence and the amount of
the sought mutant protein in the cell. However, other methods are
also known.
[0444.0.0.27] Example 6
Growth of Genetically Modified Corynebacterium glutamicum: Media
and Culture Conditions
[12552] [0445.0.0.27] Genetically modified Corynebacteria are grown
in synthetic or natural growth media. A number of different growth
media for Corynebacteria are known and widely available (Lieb et
al. (1989) Appl. Microbiol. Biotechnol. 32: 205-210; von der Osten
et al. (1998) Biotechnology Letters 11: 11-16; Patent DE 4 120 867;
Liebl (1992) "The Genus Corynebacterium", in: The Procaryotes, Vol.
II, Balows, A., et al., Ed. Springer-Verlag).
[12553] [0446.0.0.27] Said media, which can be used according to
the invention usually consist of one or more carbon sources,
nitrogen sources, inorganic salts, vitamins and trace elements.
Preferred carbon sources are sugars such as mono-, di- or
polysaccharides. Examples of very good carbon sources are glucose,
fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,
maltose, sucrose, raffinose, starch or cellulose. Sugars may also
be added to the media via complex compounds such as molasses or
other by-products of sugar refining. It may also be advantageous to
add mixtures of various carbon sources. Other possible carbon
sources are alcohols and/or organic acids such as methanol,
ethanol, acetic acid or lactic acid. Nitrogen sources are usually
organic or inorganic nitrogen compounds or materials containing
said compounds. Examples of nitrogen sources include ammonia gas,
aqueous ammonia solutions or ammonium salts such as NH.sub.4Cl, or
(NH.sub.4).sub.2SO.sub.4, NH.sub.4OH, nitrates, urea, amino acids
or complex nitrogen sources such as cornsteep liquor, soybean
flour, soybean protein, yeast extract, meat extract and others.
Mixtures of the above nitrogen sources may be used
advantageously.
[12554] [0447.0.0.27] Inorganic salt compounds, which may be
included in the media comprise the chloride, phosphorus or sulfate
salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium,
manganese, zinc, copper and iron. Chelating agents may be added to
the medium in order to keep the metal ions in solution.
Particularly suitable chelating agents include dihydroxyphenols
such as catechol or protocatechulate or organic acids such as
citric acid. The media usually also contain other growth factors
such as vitamins or growth promoters, which include, for example,
biotin, riboflavin, thiamine, folic acid, nicotinic acid,
panthothenate and pyridoxine. Growth factors and salts are
frequently derived from complex media components such as yeast
extract, molasses, cornsteep liquor and the like. The exact
composition of the compounds used in the media depends heavily on
the particular experiment and is decided upon individually for each
specific case. Information on the optimization of media can be
found in the textbook "Applied Microbiol. Physiology, A Practical
Approach" (Ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) S.
53-73, ISBN 0 19 963577 3). Growth media can also be obtained from
commercial suppliers, for example Standard 1 (Merck) or BHI (Brain
heart infusion, DIEGO) and the like.
[12555] [0448.0.0.27] All media components are sterilized, either
by heat (20 min at 1.5 bar and 121.degree. C.) or by filter
sterilization. The components may be sterilized either together or,
if required, separately. All media components may be present at the
start of the cultivation or added continuously or batchwise, as
desired.
[12556] [0449.0.0.27] The culture conditions are defined separately
for each experiment. The temperature is normally between 15.degree.
C. and 45.degree. C. and may be kept constant or may be altered
during the experiment. The pH of the medium should be in the range
from 5 to 8.5, preferably around 7.0, and can be maintained by
adding buffers to the media. An example of a buffer for this
purpose is a potassium phosphate buffer. Synthetic buffers such as
MOPS, HEPES, ACES and the like may be used as an alternative or
simultaneously. The culture pH value may also be kept constant
during the culture period by addition of, for example, NaOH or
NH.sub.4OH. If complex media components such as yeast extract are
used, additional buffers are required less since many complex
compounds have a high buffer capacity. When using a fermenter for
the culture of microorganisms, the pH value can also be regulated
using gaseous ammonia.
[12557] [0450.0.0.27] The incubation period is generally in a range
of from several hours to several days. This time period is selected
in such a way that the maximum amount of product accumulates in the
fermentation broth. The growth experiments, which are disclosed can
be carried out in a multiplicity of containers such as microtiter
plates, glass tubes, glass flasks or glass or metal fermenters of
various sizes. To screen a large number of clones, the
microorganisms should be grown in microtiter plates, glass tubes or
shake flasks, either using simple flasks or baffle flasks. 100 ml
shake flasks filled with 10% (based on the volume) of the growth
medium required are preferably used. The flasks should be shaken on
an orbital shaker (amplitude 25 mm) at a rate ranging from 100 to
300 rpm. Evaporation losses can be reduced by maintaining a humid
atmosphere; as an alternative, a mathematical correction should be
carried out for the evaporation losses.
[12558] [0451.0.0.27] If genetically modified clones are examined,
an unmodified control clone, or a control clone, which contains the
basic plasmid without insertion, should also be included in the
tests. If a transgenic sequence is expressed, a control clone
should advantageously again be included in these tests. The medium
is advantageously inoculated to an OD600 of 0.5 to 1.5 using cells
which have been grown on agar plates, such as CM plates (10 g/l
glucose, 2.5 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5 g/l yeast
extract, 5 g/l meat extract, 22 g/l agar, pH value 6.8 established
with 2M NaOH), which have been incubated at 30.degree. C. The media
are inoculated for example by introducing of a preculture of seed
organisms.
[12559] [0451.1.0.27] For example, the media are inoculated by
introducing of a saline solution of C. glutamicum cells from CM
plates or by addition of a liquid preculture of this bacterium.
[0452.0.0.27] Example 7
In-Vitro Analysis of the Function of the Proteins Encoded by the
Transformed Sequences
[12560] [0453.0.0.27] The determination of the activities and
kinetic parameters of enzymes is well known in the art. Experiments
for determining the activity of a specific modified enzyme must be
adapted to the specific activity of the wild-enzyme type, which is
well within the capabilities of the skilled worker. Overviews of
enzymes in general and specific details regarding the structure,
kinetics, principles, methods, applications and examples for the
determination of many enzyme activities can be found for example in
the following literature: Dixon, M., and Webb, E. C: (1979)
Enzymes, Longmans, London; Fersht (1985) Enzyme Structure and
Mechanism, Freeman, New York; Walsh (1979) Enzymatic Reaction
Mechanisms. Freeman, San Francisco; Price, N. C., Stevens, L.
(1982) Fundamentals of Enzymology. Oxford Univ. Press: Oxford;
Boyer, P. D: Ed. (1983) The Enzymes, 3rd Ed. Academic Press, New
York; Bisswanger, H. (1994) Enzymkinetik, 2nd Ed. VCH, Weinheim
(ISBN 3527300325); Bergmeyer, H. U., Bergmeyer, J., GraBI, M. Ed.
(1983-1986) Methods of Enzymatic Analysis, 3rd Ed. Vol. I-XII,
Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of Industrial
Chemistry (1987) Vol. A9, "Enzymes", VCH, Weinheim, pp.
352-363.
[0454.0.0.27] Example 8
Analysis of the Effect of the Nucleic Acid Molecule on the
Production of the Amino Acids
[12561] [0455.0.0.27] The effect of the genetic modification in C.
glutamicum on the production of an amino acid can be determined by
growing the modified microorganisms under suitable conditions (such
as those described above) and analyzing the medium and/or the
cellular components for the increased production of the amino acid.
Such analytical techniques are well known to the skilled worker and
encompass spectroscopy, thin-layer chromatography, various types of
staining methods, enzymatic and microbiological methods and
analytical chromatography such as high-performance liquid
chromatography (see, for example, Ullman, Encyclopedia of
Industrial Chemistry, Vol. A2, pp. 89-90 and pp. 443-613, VCH:
Weinheim (1985); Fallon, A., et al., (1987) "Applications of HPLC
in Biochemistry" in: Laboratory Techniques in Biochemistry and
Molecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol.
3, Chapter III: "Product recovery and purification", pp. 469-714,
VCH: Weinheim; Better, P. A. et al. (1988) Bioseparations:
downstream processing for Biotechnology, John Wiley and Sons;
Kennedy, J. F. and Cabral, J. M. S. (1992) Recovery processes for
biological Materials, John Wiley and Sons; Shaeiwitz, J. A. and
Henry, J. D. (1988) Biochemical Separations, in Ullmann's
Encyclopedia of Industrial Chemistry, Vol. B3; chapter 11, pp.
1-27, VCH: Weinheim; and Dechow, F. J. (1989) Separation and
purification techniques in biotechnology, Noyes Publications).
[12562] [0456.0.0.27] In addition to the determination of the
fermentation end product, other components of the metabolic
pathways which are used for the production of the desired compound,
such as intermediates and by-products, may also be analyzed in
order to determine the total productivity of the organism, the
yield and/or production efficiency of the compound. The analytical
methods encompass determining the amounts of nutrients in the
medium (for example sugars, hydrocarbons, nitrogen sources,
phosphate and other ions), determining biomass composition and
growth, analyzing the production of ordinary metabolites from
biosynthetic pathways and measuring gases generated during the
fermentation. Standard methods for these are described in Applied
Microbial Physiology; A Practical Approach, P. M. Rhodes and P. F.
Stanbury, Ed. IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN:
0199635773) and the references cited therein.
[0457.0.0.27] Example 9
Purification of the Amino Acid
[12563] [0458.0.0.27] The amino acid can be recovered from cells or
from the supernatant of the above-described culture by a variety of
methods known in the art. For example, the culture supernatant is
recovered first. To this end, the cells are harvested from the
culture by slow centrifugation. Cells can generally be disrupted or
lysed by standard techniques such as mechanical force or
sonication. The cell debris is removed by centrifugation and the
supernatant fraction, if appropriate together with the culture
supernatant, is used for the further purification of the amino
acid. However, it is also possible to process the supernatant alone
if the amino acid is present in the supernatant in sufficiently
high a concentration. In this case, the amino acid, or the amino
acid mixture, can be purified further for example via extraction
and/or salt precipitation or via ion-exchange chromatography.
[12564] [0459.0.0.27] If required and desired, further
chromatography steps with a suitable resin may follow, the amino
acid, but not many contaminants in the sample, being retained on
the chromatography resin or the contaminants, but not the sample
with the product (amino acid), being retained on the resin. If
necessary, these chromatography steps may be repeated, using
identical or other chromatography resins. The skilled worker is
familiar with the selection of suitable chromatography resin and
the most effective use for a particular molecule to be purified.
The purified product can be concentrated by filtration or
ultrafiltration and stored at a temperature at which maximum
product stability is ensured. Many purification methods, which are
not limited to the above purification method are known in the art.
They are described, for example, in Bailey, J. E. & 011 is, D.
F. Biochemical Engineering Fundamentals, McGraw-Hill: New York
(1986).
[12565] [0460.0.0.27] Identity and purity of the amino acid
isolated can be determined by standard techniques of the art. They
encompass high-performance liquid chromatography (HPLC),
spectroscopic methods, mass spectrometry (MS), staining methods,
thin-layer chromatography, NIRS, enzyme assay or microbiological
assays. These analytical methods are compiled in: Patek et al.
(1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al.
(1996) Biotekhnologiya 11:27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19: 67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540,
pp. 540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, Vol. 17.
[0461.0.0.27] Example 10
Cloning SEQ ID NO: 108199 for the Expression in Plants
[12566] [0462.0.0.27] Unless otherwise specified, standard methods
as described in Sambrook et al., Molecular Cloning: A laboratory
manual, Cold Spring Harbor 1989, Cold Spring Harbor Laboratory
Press are used.
[12567] [0463.0.0.27] SEQ ID NO: 108199 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[12568] [0464.0.0.27] The composition for the protocol of the Pfu
Turbo DNA polymerase was as follows: 1.times.PCR buffer
(Stratagene), 0.2 mM of each dNTP, 100 ng genomic DNA of
Saccharomyces cerevisiae (strain S288C; Research Genetics, Inc.,
now Invitrogen) or Escherichia coli (strain MG1655; E. coli Genetic
Stock Center), 50 .mu.mol forward primer, 50 .mu.mol reverse
primer, 2.5 u Pfu Turbo DNA polymerase. The amplification cycles
were as follows:
[12569] [0465.0.0.27] 1 cycle of 3 minutes at 94-95.degree. C.,
followed by 25-36 cycles of in each case 1 minute at 95.degree. C.
or 30 seconds at 94.degree. C., 45 seconds at 50.degree. C., 30
seconds at 50.degree. C. or 30 seconds at 55.degree. C. and 210-480
seconds at 72.degree. C., followed by 1 cycle of 8 minutes at
72.degree. C., then 4.degree. C. The composition for the protocol
of the Herculase polymerase was as follows: 1.times.PCR buffer
(Stratagene), 0.2 mM of each dNTP, 100 ng genomic DNA of
Saccharomyces cerevisiae (strain S288C; Research Genetics, Inc.,
now Invitrogen) or Escherichia coli (strain MG1655; E. coli Genetic
Stock Center), 50 .mu.mol forward primer, 50 .mu.mol reverse
primer, 2.5 u Herculase polymerase. The amplification cycles were
as follows:
[12570] [0466.0.0.27] 1 cycle of 2-3 minutes at 94.degree. C.,
followed by 25-30 cycles of in each case 30 seconds at 94.degree.
C., 30 seconds at 55-60.degree. C. and 5-10 minutes at 72.degree.
C., followed by 1 cycle of 10 minutes at 72.degree. C., then
4.degree. C.
[12571] [0467.0.0.27] The following primer sequences were selected
for the gene SEQ ID NO: 108199: [12572] i) forward primer SEQ ID
NO: 108249 [12573] ii) reverse primer SEQ ID NO: 108250
[12574] [0468.0.0.27] Thereafter, the amplificate was purified over
QIAquick columns following the standard protocol (Qiagen).
[12575] [0469.0.0.27] For the cloning of PCR-products, produced by
Pfu Turbo DNA polymerase, the vector DNA (30 ng) was restricted
with SmaI following the standard protocol (MBI Fermentas) and
stopped by addition of high-salt buffer. The restricted vector
fragments were purified via Nucleobond columns using the standard
protocol (Macherey-Nagel). Thereafter, the linearized vector was
dephosphorylated following the standard protocol (MBI
Fermentas).
[12576] [0470.0.0.27] The PCR-products, produced by Pfu Turbo DNA
polymerase, were directly cloned into the processed binary vector.
The PCR-products, produced by Pfu Turbo DNA polymerase, were
phosphorylated using a T4 DNA polymerase using a standard protocol
(e.g. MBI Fermentas) and cloned into the processed binary
vector.
[12577] [0471.0.0.27] The DNA termini of the PCR-products, produced
by Herculase DNA polymerase, were blunted in a second synthesis
reaction using Pfu Turbo DNA polymerase. The composition for the
protocol of the blunting the DNA-termini was as follows: 0.2 mM
blunting dTTP and 1.25 u Pfu Turbo DNA polymerase. The reaction was
incubated at 72.degree. C. for 30 minutes. Then the PCR-products
were cloned into the processed vector as well. The DNA termini of
the PCR-products, produced by Herculase DNA polymerase, were
blunted in a second synthesis reaction using Pfu Turbo DNA
polymerase. The composition for the protocol of the blunting the
DNA-termini was as follows: 0.2 mM blunting dTTP and 1.25 u Pfu
Turbo DNA polymerase. The reaction was incubated at 72.degree. C.
for 30 minutes. Then the PCR-products were phosphorylated using a
T4 DNA polymerase using a standard protocol (e.g. MBI Fermentas)
and cloned into the processed vector as well.
[12578] [0472.0.0.27] A binary vector comprising a selection
cassette (promoter, selection marker, terminator) and an expression
cassette with promoter, cloning cassette and terminator sequence
between the T-DNA border sequences was used. In addition to those
within the cloning cassette, the binary vector has no SmaI cleavage
site. Binary vectors which can be used are known to the skilled
worker; an overview of binary vectors and their use can be found in
Hellens, R., Mullineaux, P. and Klee H., [(2000) "A guide to
Agrobacterium binary vectors", Trends in Plant Science, Vol. 5 No.
10, 446-451. Depending on the vector used, cloning may
advantageously also be carried out via other restriction enzymes.
Suitable advantageous cleavage sites can be added to the ORF by
using suitable primers for the PCR amplification.
[12579] [0473.0.0.27] Approximately 30 ng of prepared vector and a
defined amount of prepared amplificate were mixed and ligated by
addition of ligase.
[12580] [0474.0.0.27] The ligated vectors were transformed in the
same reaction vessel by addition of competent E. coli cells (strain
DHSalpha) and incubation for 20 minutes at 1.degree. C. followed by
a heat shock for 90 seconds at 42.degree. C. and cooling to
4.degree. C. Then, complete medium (SOC) was added and the mixture
was incubated for 45 minutes at 37.degree. C. The entire mixture
was subsequently plated onto an agar plate with antibiotics
(selected as a function of the binary vector used) and incubated
overnight at 37.degree. C.
[12581] [0475.0.0.27] The outcome of the cloning step was verified
by amplification with the aid of primers which bind upstream and
downstream of the integration site, thus allowing the amplification
of the insertion. In addition combinations of the above mentioned
gene specific primers and upstream and downstream primers were used
in PCR reactions to identify clones with the correct insert
orientation. The amplifications were carried as described in the
protocol of Taq DNA polymerase (Gibco-BRL).
[12582] [0476.0.0.27] The amplification cycles were as follows: 1
cycle of 5 minutes at 94.degree. C., followed by 35 cycles of in
each case 15 seconds at 94.degree. C., 15 seconds at 50-66.degree.
C. and 5 minutes at 72.degree. C., followed by 1 cycle of 10
minutes at 72.degree. C., then 4.degree. C.
[12583] [0477.0.0.27] Several colonies were checked, but only one
colony for which a PCR product of the expected size was detected
was used in the following steps.
[12584] [0478.0.0.27] A portion of this positive colony was
transferred into a reaction vessel filled with complete medium (LB)
and incubated overnight at 37.degree. C. The LB medium contained an
antibiotic chosen to suit the binary vector (see above) used and
the resistance gene present therein in order to select the
clone.
[12585] [0479.0.0.27] The plasmid preparation was carried out as
specified in the Qiaprep standard protocol (Qiagen).
[0480.0.0.27] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 108199
[12586] [0481.0.0.27] 1 ng of the plasmid DNA isolated was
transformed by electroporation into competent cells of
Agrobacterium tumefaciens, of strain GV 3101 pMP90 (Koncz and
Schell, Mol. Gen. Gent. 204, 383-396, 1986). The choice of the
agrobacterial strain depends on the choice of the binary vector. An
overview of possible strains and their properties is found in
Hellens, R., Mullineaux, P. and Klee H., (2000) "A guide to
Agrobacterium binary vectors, Trends in Plant Science, Vol. 5 No.
10, 446-451. Thereafter, complete medium (YEP) was added and the
mixture was transferred into a fresh reaction vessel for 3 hours at
28.degree. C. Thereafter, all of the reaction mixture was plated
onto YEP agar plates supplemented with the respective antibiotics,
for example rifampicin and gentamycin for GV3101 pMP90, and a
further antibiotic for the selection onto the binary vector, was
plated, and incubated for 48 hours at 28.degree. C.
[12587] [0482.0.0.27] The agrobacteria generated in Example 10,
which contains the plasmid construct were then used for the
transformation of plants.
[12588] [0483.0.0.27] A colony was picked from the agar plate with
the aid of a pipette tip and taken up in 3 ml of liquid TB medium,
which also contained suitable antibiotics, depending on the
agrobacterial strain and the binary plasmid. The preculture was
grown for 48 hours at 28.degree. C. and 120 rpm.
[12589] [0484.0.0.27] 400 ml of LB medium containing the same
antibiotics as above were used for the main culture. The preculture
was transferred into the main culture. It was grown for 18 hours at
28.degree. C. and 120 rpm. After centrifugation at 4 000 rpm, the
pellet was resuspended in infiltration medium (MS medium, 10%
sucrose).
[12590] [0485.0.0.27] In order to grow the plants for the
transformation, dishes (Piki Saat 80, green, provided with a screen
bottom, 30.times.20.times.4.5 cm, from Wiesauplast,
Kunststofftechnik, Germany) were half-filled with a GS 90 substrate
(standard soil, Werkverband E. V., Germany). The dishes were
watered overnight with 0.05% Proplant solution (Chimac-Apriphar,
Belgium). Arabidopsis thaliana C24 seeds (Nottingham Arabidopsis
Stock Centre, UK; NASC Stock N906) were scattered over the dish,
approximately 1 000 seeds per dish. The dishes were covered with a
hood and placed in the stratification facility (8 h, 110
.mu.mol/m2/s-1, 22.degree. C.; 16 h, dark, 6.degree. C.). After 5
days, the dishes were placed into the short-day controlled
environment chamber (8 h 130 .mu.mol/m2/s-1, 22.degree. C.; 16 h,
dark 20.degree. C.), where they remained for approximately 10 days
until the first true leaves had formed.
[12591] [0486.0.0.27] The seedlings were transferred into pots
containing the same substrate (Teku pots, 7 cm, LC series,
manufactured by POppelmann GmbH & Co, Germany). Five plants
were pricked out into each pot. The pots were then returned into
the short-day controlled environment chamber for the plant to
continue growing.
[12592] [0487.0.0.27] After 10 days, the plants were transferred
into the greenhouse cabinet (supplementary illumination, 16 h, 340
E, 22.degree. C.; 8 h, dark, 20.degree. C.), where they were
allowed to grow for further 17 days.
[12593] [0488.0.0.27] For the transformation, 6-week-old
Arabidopsis plants which had just started flowering were immersed
for 10 seconds into the above-described agrobacterial suspension
which had previously been treated with 10 l Silwett L77 (Crompton
S. A., Osi Specialties, Switzerland). The method in question is
described in Clough and Bent, 1998 (Clough, J C and Bent, A F. 1998
Floral dip: a simplified method for Agrobacterium-mediated
transformation of Arabidopsis thaliana, Plant J. 16:735-743.
[12594] [0489.0.0.27] The plants were subsequently placed for 18
hours into a humid chamber. Thereafter, the pots were returned to
the greenhouse for the plants to continue growing. The plants
remained in the greenhouse for another 10 weeks until the seeds
were ready for harvesting.
[12595] [0490.0.0.27] Depending on the resistance marker used for
the selection of the transformed plants the harvested seeds were
planted in the greenhouse and subjected to a spray selection or
else first sterilized and then grown on agar plates supplemented
with the respective selection agent. In case of
BASTA.RTM.-resistance, plantlets were sprayed four times at an
interval of 2 to 3 days with 0.02% BASTA.RTM. and transformed
plants were allowed to set seeds. The seeds of the transgenic A.
thaliana plants were stored in the freezer (at -20.degree. C.).
[0491.0.0.27] Example 12
Plant Culture for Bioanalytical Analyses
[12596] [0492.0.0.27] For the bioanalytical analyses of the
transgenic plants, the latter were grown uniformly a specific
culture facility. To this end the GS-90 substrate as the compost
mixture was introduced into the potting machine (Laible System
GmbH, Singen, Germany) and filled into the pots. Thereafter, 35
pots were combined in one dish and treated with Previcur. For the
treatment, 25 ml of Previcur were taken up in 10 l of tap water.
This amount was sufficient for the treatment of approximately 200
pots. The pots were placed into the Previcur solution and
additionally irrigated overhead with tap water without Previcur.
They were used within four days.
[12597] [0493.0.0.27] For the sowing, the seeds, which had been
stored in the refrigerator (at -20.degree. C.), were removed from
the Eppendorf tubes with the aid of a toothpick and transferred
into the pots with the compost. In total, approximately 5 to 12
seeds were distributed in the middle of the pot.
[12598] [0494.0.0.27] After the seeds had been sown, the dishes
with the pots were covered with matching plastic hood and placed
into the stratification chamber for 4 days in the dark at 4.degree.
C. The humidity was approximately 90%. After the stratification,
the test plants were grown for 22 to 23 days at a 16-h-light,
8-h-dark rhythm at 20.degree. C., an atmospheric humidity of 60%
and a CO.sub.2 concentration of approximately 400 ppm. The light
sources used were Powerstar HQI-T 250 W/D Daylight lamps from
Osram, which generate a light resembling the solar color spectrum
with a light intensity of approximately 220 E/m2/s-1.
[12599] [0495.0.0.27] When the plants were 8, 9 and 10 days old,
they were subjected to selection for the resistance marker
Approximately 1400 pots with transgenic plants were treated with 11
0,015% vol/vol of Basta (Glufosinate-ammonium) solution in water
(Aventis Cropsience, Germany). After a further 3 to 4 days, the
transgenic, resistant seedlings (plantlets in the 4-leaf stage)
could be distinguished clearly from the untransformed plantlets.
The nontransgenic seedlings were bleached or dead. The transgenic
resistance plants were thinned when they had reached the age of 14
days. The plants, which had grown best in the center of the pot
were considered the target plants. All the remaining plants were
removed carefully with the aid of metal tweezers and discarded.
[12600] [0496.0.0.27] During their growth, the plants received
overhead irrigation with distilled water (onto the compost) and
bottom irrigation into the placement grooves. Once the grown plants
had reached the age of 23 days, they were harvested.
[0497.0.0.27] Example 13
Metabolic Analysis of Transformed Plants
[12601] [0498.0.0.27] The modifications identified in accordance
with the invention, in the content of above-described metabolites,
were identified by the following procedure.
[12602] a) Sampling and Storage of the Samples
[12603] [0499.0.0.27] Sampling was performed directly in the
controlled-environment chamber. The plants were cut using small
laboratory scissors, rapidly weighed on laboratory scales,
transferred into a pre-cooled extraction sleeve and placed into an
aluminum rack cooled by liquid nitrogen. If required, the
extraction sleeves can be stored in the freezer at -80.degree. C.
The time elapsing between cutting the plant to freezing it in
liquid nitrogen amounted to not more than 10 to 20 seconds.
[12604] b) Lyophilization
[12605] [0500.0.0.27] During the experiment, care was taken that
the plants either remained in the deep-frozen state (temperatures
<-40.degree. C.) or were freed from water by lyophilization
until the first contact with solvents.
[12606] [0501.0.0.27] The aluminum rack with the plant samples in
the extraction sleeves was placed into the pre-cooled (-40.degree.
C.) lyophilization facility. The initial temperature during the
main drying phase was -35.degree. C. and the pressure was 0.120
mbar. During the drying phase, the parameters were altered
following a pressure and temperature program. The final temperature
after 12 hours was +30.degree. C. and the final pressure was 0.001
to 0.004 mbar. After the vacuum pump and the refrigerating machine
had been switched off, the system was flushed with air (dried via a
drying tube) or argon.
[12607] c) Extraction
[12608] [0502.0.0.27] Immediately after the lyophilization
apparatus had been flushed, the extraction sleeves with the
lyophilized plant material were transferred into the 5 ml
extraction cartridges of the ASE device (Accelerated Solvent
Extractor ASE 200 with Solvent Controller and AutoASE software
(DIONEX)).
[12609] [0503.0.0.27] The 24 sample positions of an ASE device
(Accelerated Solvent Extractor ASE 200 with Solvent Controller and
AutoASE software (DIONEX)) were filled with plant samples,
including some samples for testing quality control.
[12610] [0504.0.0.27] The polar substances were extracted with
approximately 10 ml of methanol/water (80/20, v/v) at T=70.degree.
C. and p=140 bar, 5 minutes heating-up phase, 1 minute static
extraction. The more lipophilic substances were extracted with
approximately 10 ml of methanol/dichloromethane (40/60, v/v) at
T=70.degree. C. and p=140 bar, 5 minute heating-up phase, 1 minute
static extraction. The two solvent mixtures were extracted into the
same glass tubes (centrifuge tubes, 50 ml, equipped with screw cap
and pierceable septum for the ASE (DIONEX)).
[12611] [0505.0.0.27] The solution was treated with internal
standards: ribitol, L-glycine-2,2-d.sub.2,
L-alanine-2,3,3,3-d.sub.4, methionine-methyl-d.sub.3, and
.alpha.-methylglucopyranoside and methyl nonadecanoate, methyl
undecanoate, methyl tridecanoate, methyl pentadecanoate, methyl
nonacosanoate.
[12612] [0506.0.0.27] The total extract was treated with 8 ml of
water. The solid residue of the plant sample and the extraction
sleeve were discarded.
[12613] [0507.0.0.27] The extract was shaken and then centrifuged
for 5 to 10 minutes at least at 1 400 g in order to accelerate
phase separation. 1 ml of the supernatant methanol/water phase
("polar phase", colorless) was removed for the further GC analysis,
and 1 ml was removed for the LC analysis. The remainder of the
methanol/water phase was discarded. 0.5 ml of the organic phase
("lipid phase", dark green) was removed for the further GC analysis
and 0.5 ml was removed for the LC analysis. All the portions
removed were evaporated to dryness using the IR Dancer infrared
vacuum evaporator (Hettich). The maximum temperature during the
evaporation process did not exceed 40.degree. C. Pressure in the
apparatus was not less than 10 mbar.
[12614] d) Processing the Lipid Phase for the LC/MS or LC/MS/MS
Analysis
[12615] [0508.0.0.27] The lipid extract, which had been evaporated
to dryness was taken up in mobile phase. The polar extract, which
had been evaporated to dryness was taken up in mobile phase.
[12616] [0509.0.0.27] e) LC-MS Analyisis
[12617] The LC part was carried out on a commercially available
LCMS system from Agilent
[12618] Technologies, USA. For polar extracts 10 .mu.l are injected
into the system at a flow rate of 200 .mu.l/min. The separation
column (Reversed Phase C18) was maintained at 15.degree. C. during
chromatography. For lipid extracts 5 .mu.l are injected into the
system at a flow rate of 200 .mu.l/min. The separation column
(Reversed Phase C18) was maintained at 30.degree. C. HPLC was
performed with gradient elution
[12619] The mass spectrometric analysis was performed on a Applied
Biosystems API 4000 triple quadrupole instrument with turbo ion
spray source. For polar extracts the instrument measures in
negative ion mode in fullscan mode from 100-1000 amu. For lipid
extracts the instrument measures in positive ion mode in fullscan
mode from 100-1000 amu.
[12620] f) Derivatization of the Lipid Phase for the GC/MS
Analysis
[12621] [0510.0.0.27] For the transmethanolysis, a mixture of 140
.mu.l of chloroform, 37 .mu.l of hydrochloric acid (37% by weight
HCl in water), 320 .mu.l of methanol and 20 .mu.l of toluene was
added to the evaporated extract. The vessel was sealed tightly and
heated for 2 hours at 100.degree. C., with shaking. The solution
was subsequently evaporated to dryness. The residue was dried
completely. The methoximation of the carbonyl groups was carried
out by reaction with methoxyamine hydrochloride (5 mg/ml in
pyridine, 100 l for 1.5 hours at 60.degree. C.) in a tightly sealed
vessel. 20 .mu.l of a solution of odd-numbered, straight-chain
fatty acids (solution of each 0.3 mg/mL of fatty acids from 7 to 25
carbon atoms and each 0.6 mg/mL of fatty acids with 27, 29 and 31
carbon atoms in 3/7 (v/v) pyridine/toluene) were added as time
standards. Finally, the derivatization with 100 .mu.l of
N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was
carried out for 30 minutes at 60.degree. C., again in the tightly
sealed vessel. The final volume before injection into the GC was
220 .mu.l.
[12622] g) Derivatization of the Polar Phase for GC/MS Analysis
[12623] [0511.0.0.27] The methoximation of the carbonyl groups was
carried out by reaction with methoxyamine hydrochloride (5 mg/ml in
pyridine, 50 l for 1.5 hours at 60.degree. C.) in a tightly sealed
vessel. 10 .mu.l of a solution of odd-numbered, straight-chain
fatty acids (solution of each 0.3 mg/mL of fatty acids from 7 to 25
carbon atoms and each 0.6 mg/mL of fatty acids with 27, 29 and 31
carbon atoms in 3/7 (v/v) pyridine/toluene) were added as time
standards. Finally, the derivatization with 50 .mu.l of
N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was
carried out for 30 minutes at 60.degree. C., again in the tightly
sealed vessel. The final volume before injection into the GC was
110 .mu.l. .mu.l.
[12624] h) GC-MS Analysis
[12625] [0512.0.0.27] The GC-MS systems consist of an Agilent 6890
GC coupled to an Agilent 5973 MSD. The autosamplers are CompiPal or
GCPal from CTC. For the analysis usual commercial capillary
separation columns (30 m.times.0.25 mm.times.0.25 .mu.m) with
different poly-methyl-siloxane stationary phases containing 0% up
to 35% of aromatic moieties, depending on the analysed sample
materials and fractions from the phase separation step, are used
(for example: DB-1 ms, HP-5 ms, DB-XLB, DB-35 ms, Agilent
Technologies). Up to 1 .mu.L of the final volume is injected
splitless and the oven temperature program is started at 70.degree.
C. and ended at 340.degree. C. with different heating rates
depending on the sample material and fraction from the phase
separation step in order to achieve a sufficient chromatographic
separation and number of scans within each analyte peak. Usual
GC-MS standard conditions, for example constant flow with nominal 1
to 1.7 ml/min. and helium as the mobile phase gas are used.
Ionisation is done by electron impact with 70 eV, scanning within a
m/z range from 15 to 600 with scan rates from 2.5 to 3 scans/sec
and standard tune conditions.
[12626] i) Analysis of the Various Plant Samples
[12627] [0513.0.0.27] The samples were measured in individual
series of 20 plant samples each (also referred to as sequences),
each sequence containing at least 5 wild-type plants as controls.
The peak area of each analyte was divided by the peak area of the
respective internal standard. The data were standardized for the
fresh weight established for the plant. The values calculated thus
were related to the wild-type control group by being divided by the
mean of the corresponding data of the wild-type control group of
the same sequence. The values obtained were referred to as
ratio_by_WT, they are comparable between sequences and indicate how
much the analyte concentration in the mutant differs in relation to
the wild-type control. Appropriate controls were done before to
proof that the vector and transformation procedure itself has no
significant influence on the metabolic composition of the plants.
Therefore the described changes in comparison with wildtypes were
caused by the introduced genes.
[12628] [0514.0.0.27] As an alternative, the amino acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55).
[12629] [0515.0.0.27]
[12630] [0516.0.0.27] When the analyses were repeated
independently, all results proved to be significant.
[0517.0.0.27] Example 14a
Engineering Ryegrass Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, e.g. Derived from Plants or an
Other Organism
[12631] [0518.0.0.27] Seeds of several different ryegrass varieties
can be used as explant sources for transformation, including the
commercial variety Gunne available from Svalof Weibull seed company
or the variety Affinity. Seeds are surface-sterilized sequentially
with 1% Tween-20 for 1 minute, 100% bleach for 60 minutes, 3 rinses
with 5 minutes each with de-ionized and distilled H2O, and then
germinated for 3-4 days on moist, sterile filter paper in the dark.
Seedlings are further sterilized for 1 minute with 1% Tween-20, 5
minutes with 75% bleach, and rinsed 3 times with ddH2O, 5 min
each.
[12632] [0519.0.0.27] Surface-sterilized seeds are placed on the
callus induction medium containing Murashige and Skoog basal salts
and vitamins, 20 g/l sucrose, 150 mg/l asparagine, 500 mg/l casein
hydrolysate, 3 g/l Phytagel, 10 mg/l BAP, and 5 mg/l dicamba.
Plates are incubated in the dark at 25.degree. C. for 4 weeks for
seed germination and embryogenic callus induction.
[12633] [0520.0.0.27] After 4 weeks on the callus induction medium,
the shoots and roots of the seedlings are trimmed away, the callus
is transferred to fresh media, is maintained in culture for another
4 weeks, and is then transferred to MSO medium in light for 2
weeks. Several pieces of callus (11-17 weeks old) are either
strained through a 10 mesh sieve and put onto callus induction
medium, or are cultured in 100 ml of liquid ryegrass callus
induction media (same medium as for callus induction with agar) in
a 250 ml flask. The flask is wrapped in foil and shaken at 175 rpm
in the dark at 23.degree. C. for 1 week. Sieving the liquid culture
with a 40-mesh sieve is collected the cells. The fraction collected
on the sieve is plated and is cultured on solid ryegrass callus
induction medium for 1 week in the dark at 25.degree. C. The callus
is then transferred to and is cultured on MS medium containing 1%
sucrose for 2 weeks.
[12634] [0521.0.0.27] Transformation can be accomplished with
either Agrobacterium or with particle bombardment methods. An
expression vector is created containing a constitutive plant
promoter and the cDNA of the gene in a pUC vector. The plasmid DNA
is prepared from E. coli cells using with Qiagen kit according to
manufacturer's instruction. Approximately 2 g of embryogenic callus
is spread in the center of a sterile filter paper in a Petri dish.
An aliquot of liquid MSO with 10 g/l sucrose is added to the filter
paper. Gold particles (1.0 .mu.m in size) are coated with plasmid
DNA according to method of Sanford et al., 1993 and are delivered
to the embryogenic callus with the following parameters: 500 .mu.g
particles and 2 .mu.g DNA per shot, 1300 psi and a target distance
of 8.5 cm from stopping plate to plate of callus and 1 shot per
plate of callus.
[12635] [0522.0.0.27] After the bombardment, calli are transferred
back to the fresh callus development medium and maintained in the
dark at room temperature for a 1-week period. The callus is then
transferred to growth conditions in the light at 25.degree. C. to
initiate embryo differentiation with the appropriate selection
agent, e.g. 250 nM Arsenal, 5 mg/l PPT or 50 mg/L Kanamycin. Shoots
resistant to the selection agent are appearing and once rooted are
transferred to soil.
[12636] [0523.0.0.27] Samples of the primary transgenic plants (TO)
are analyzed by PCR to confirm the presence of T-DNA. These results
are confirmed by Southern hybridization in which DNA is
electrophoresed on a 1% agarose gel and transferred to a positively
charged nylon membrane (Roche Diagnostics). The PCR DIG Probe
Synthesis Kit (Roche Diagnostics) is used to prepare a
digoxigenin-labelled probe by PCR, and used as recommended by the
manufacturer.
[12637] [0524.0.0.27] Transgenic TO ryegrass plants are propagated
vegetatively by excising tillers. The transplanted tillers are
maintained in the greenhouse for 2 months until well established.
The shoots are defoliated and allowed to grow for 2 weeks.
[0525.0.0.27] Example 14b
Engineering Soybean Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, e.g. Derived from Plants or Another
Organism
[12638] [0526.0.0.27] Soybean can be transformed according to the
following modification of the method described in the Texas A&M
patent U.S. Pat. No. 5,164,310. Several commercial soybean
varieties are amenable to transformation by this method. The
cultivar Jack (available from the Illinois Seed Foundation) is
commonly used for transformation. Seeds are sterilized by immersion
in 70% (v/v) ethanol for 6 min and in 25 commercial bleach (NaOCI)
supplemented with 0.1% (v/v) Tween for 20 min, followed by rinsing
4 times with sterile double distilled water. Removing the radicle,
hypocotyl and one cotyledon from each seedling propagates seven-day
seedlings. Then, the epicotyl with one cotyledon is transferred to
fresh germination media in petri dishes and incubated at 25.degree.
C. under a 16-hr photoperiod (approx. 100 E-m-2s-1) for three
weeks. Axillary nodes (approx. 4 mm in length) are cut from 3-4
week-old plants. Axillary nodes are excised and incubated in
Agrobacterium LBA4404 culture.
[12639] [0527.0.0.27] Many different binary vector systems have
been described for plant transformation (e.g. An, G. in
Agrobacterium Protocols. Methods in Molecular Biology vol 44, pp
47-62, Gartland KMA and MR Davey eds. Humana Press, Totowa, N.J.
Many are based on the vector pBIN19 described by Bevan (Nucleic
Acid Research. 1984. 12:8711-8721) that includes a plant gene
expression cassette flanked by the left and right border sequences
from the Ti plasmid of Agrobacterium tumefaciens. A plant gene
expression cassette consists of at least two genes--a selection
marker gene and a plant promoter regulating the transcription of
the cDNA or genomic DNA of the trait gene. Various selection marker
genes can be used as described above, including the Arabidopsis
gene encoding a mutated acetohydroxy acid synthase (AHAS) enzyme
(US patents 57673666 and 6225105). Similarly, various promoters can
be used to regulate the trait gene to provide constitutive,
developmental, tissue or environmental regulation of gene
transcription as described above. In this example, the 34S promoter
(GenBank Accession numbers M59930 and X16673) is used to provide
constitutive expression of the trait gene.
[12640] [0528.0.0.27] After the co-cultivation treatment, the
explants are washed and transferred to selection media supplemented
with 500 mg/L timentin. Shoots are excised and placed on a shoot
elongation medium. Shoots longer than 1 cm are placed on rooting
medium for two to four weeks prior to transplanting to soil.
[12641] [0529.0.0.27] The primary transgenic plants (TO) are
analyzed by PCR to confirm the presence of T-DNA. These results are
confirmed by Southern hybridization in which DNA is electrophoresed
on a 1% agarose gel and transferred to a positively charged nylon
membrane (Roche Diagnostics). The PCR DIG Probe Synthesis Kit
(Roche Diagnostics) is used to prepare a digoxigenin-labelled probe
by PCR, and is used as recommended by the manufacturer.
[0530.0.0.27] Example 14c
Engineering Corn Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, e.g. Derived from Plants or Another
Organism
[12642] [0530.1.0.27] Amplification of for example SEQ ID NO:
108199 was achieved as described in example 10 except that the
upstream primer SEQ ID NO: 108249 and the reverse primer SEQ ID NO:
108250 contained the following 5''extensions:
TABLE-US-00147 i) forward primer: SEQ ID NO: 108488
5'-GGGTCGCTCCTACGCG-3' ii) reverse primer SEQ ID NO: 108491
5'-CTCGGGCTCGGCGTCC-3'
[12643] [0530.2.0.27]: Vector Construction
[12644] The maize transformation vector for constitutive expression
was constructed as follows.
[12645] As base vectors, the vectors EG073qcz (SEQ ID NO: 108485)
and EG065qcz (SEQ ID NO: 108486) were chosen. The MCS from EG065qcz
was deleted by digestion of the vector with Asp718 and Pstl,
followed by blunting of the vector using T4 DNA polymerase. The
blunted vector was religated. The vector generated was called
EG065-MCS. The LIC cassette was cloned in the vector EG065-MCS by
hybridizing the following oligos, generating a DNA fragment with
ends able to ligate into a SmaI and SacI digested vector. This
fragment was ligated into the vector EG065-MCS that had been
digested with SmaI and SacI. The generated vector was called
EG065-LIC. The complete expression cassette comprising ScBV (Schenk
(1999) Plant Mol Biol
[12646] 39(6):1221-1230) promoter, LIC cassette and terminator was
cut out of EG065-LIC with Ascl and Pacl and ligated into the vector
EG073qcz that had previously been digested with Ascl and Pacl. The
resulting binary vector for corn transformation was called pMME0607
(SEQ ID NO: 108487).
TABLE-US-00148 Oligo POCCLicMluISacIIfw: (SEQ ID NO: 108489)
gggtcgctcctacgcgtcaatgatccgcggacgccgagcccgagct Oligo
POCCLicMluISacIrev: (SEQ ID NO: 108490)
cgggctcggcgtccgcggatcattgacgcgtaggagcgaccc
[12647] For cloning of a polynucleotide of the invention, for
example the ORF of SEQ ID NO: 108199, from Linum usitatissimum the
vector DNA was treated with the restriction enzyme Mlul and SacII.
The reaction was stopped by inactivation at 70.degree. C. for 20
minutes and purified over QIAquick columns following the standard
protocol (Qiagen).
[12648] Then the PCR-product representing the amplified ORF and the
vector DNA were treated with T4 DNA polymerase according to the
standard protocol (MBI Fermentas) to produce single stranded
overhangs with the parameters 1 unit T4 DNA polymerase at
37.degree. C. for 2-10 minutes for the vector and 1 u T4 DNA
polymerase at 15.degree. C. for 10-60 minutes for the PCR product
representing SEQ ID NO: 108199.
[12649] The reaction was stopped by addition of high-salt buffer
and purified over QIAquick columns following the standard protocol
(Qiagen).
[12650] Approximately 30 ng of prepared vector and a defined amount
of prepared amplificate were mixed and hybridized at 65.degree. C.
for 15 minutes followed by 37.degree. C. 0,1.degree. C./1 seconds,
followed by 37.degree. C. 10 minutes, followed by 0,1.degree. C./1
seconds, then 4.degree. C.
[12651] The ligated constructs were transformed in the same
reaction vessel by addition of competent E. coli cells (strain
DHSalpha) and incubation for 20 minutes at 1.degree. C. followed by
a heat shock for 90 seconds at 42.degree. C. and cooling to
4.degree. C. Then, complete medium (SOC) was added and the mixture
was incubated for 45 minutes at 37.degree. C. The entire mixture
was subsequently plated onto an agar plate with 0.05 mg/ml
kanamycine and incubated overnight at 37.degree. C.
[12652] The outcome of the cloning step was verified by
amplification with the aid of primers which bind upstream and
downstream of the integration site, thus allowing the amplification
of the insertion. The amplifications were carried as described in
the protocol of Taq DNA polymerase (Gibco-BRL).
[12653] The amplification cycles were as follows: 1 cycle of 5
minutes at 94.degree. C., followed by 35 cycles of in each case 15
seconds at 94.degree. C., 15 seconds at 50-66.degree. C. and 5
minutes at 72.degree. C., followed by 1 cycle of 10 minutes at
72.degree. C., then 4.degree. C.
[12654] Several colonies were checked, but only one colony for
which a PCR product of the expected size was detected was used in
the following steps.
[12655] A portion of this positive colony was transferred into a
reaction vessel filled with complete medium (LB) supplemented with
kanamycin 0 and incubated overnight at 37.degree. C.
[12656] The plasmid preparation was carried out as specified in the
Qiaprep standard protocol (Qiagen).
[0530.3.0.27] Example 14 c.a.
Corn Transformation
[12657] The preparation of the immature embryos and Agrobacterium
were basically as stated in U.S. Pat. No. 5,591,616. In brief, the
Agrobacterium strain LBA4404 transformed with the plasmid by a
standard method, such as the triple cross method or the
electroporation, was grown on LB plates for 2 days prior to
cocultivation. A loop of cells was resuspended in liquid infection
media at an O.D. of approximately 1.0. Immature Embryos of about
1.5 mm in size were incubated in the soln of agrobacterium for
around 30 minutes. Excised embryos were removed from liquid and
then co-cultivated in the dark at 22.degree. C. with Agrobacterium
tumefaciens on solid MS-based callus induction medium containing 2
mg/l 2,4-D, 10 um AgNO3, and 200 um Acetosyringone. After several
days of co-cultivation, embryos were transferred to MS-based media
containing 2 mg/l 2,4, 10 um AgNO3 and 200 mg/l Timentin the dark
at 27.degree. C. for 1 week. Embryos were transferred to MS-based
selection media containing imidazoline herbicide (500 nM Pursuit)
as a selection agent in the dark for 3 weeks. After 3 weeks
putative transgenic events were transferred to an MS-based media
containing 2 mg/L Kinetin 500 nM Pursuit, 200 mg/l Timentin and
incubated under cool white fluorescent light (100 uE/m2/s-1 with
photoperiod of 16 hrs) at 25.degree. C. for 2-3 weeks, or until
shoots develop. The shoots were transferred to MS-based rooting
medium and incubated under light at 25.degree. C. for 2 weeks. The
rooted shoots were transplanted to 4 inch pots containing
artificial soil mix. Metro-Mix.RTM. 360 in and grown in an
environmental chamber for 1-2 weeks. The environmental chamber
maintained 16-h-light, 8-h-dark cycles at 27.degree. C. day and
22.degree. C. respectively. Light was supplied by a mixture of
incandescent and cool white fluorescent bulbs with an intensity of
.about.400 uE/m2/s-1. After plants were grown to 4-6 leaf stage
they were moved to 14 inch pots containing Metro-Mix.RTM. 360.
Supplemental metal-halide lamps were used to maintain >800
uE/m2/s-1 with a 16-h-light, 8-h-dark cycles at 28.degree. C. day
and 22.degree. C. Transplantation occurs weekly on Tuesday. Peters
20-20-20 plus micronutrients (200 ppm) is used to fertilize plants
2.times. weekly on Monday and Thursday after sampling of TO's is
performed. T1 seeds were produced from plants that exhibit
tolerance to the imidazolinone herbicides and which are PCR
positive for the transgenes. T0 plants with single locus insertions
of the T-DNA (self-pollinated) produced T1 generation that
segregated for the transgene in a 3:1 ratio. Progeny containing
copies of the transgene were tolerant of imidazolinone herbicides
and could be detected by PCR analysis.
[0530.4.0.27] Example 14 c.b.
Growth of T0 corn plants for metabolic analysis
[12658] Plants were grown under the following standardized
conditions to properly stage them for TO sampling. T0 plantlets
were transferred to 14" pots in the greenhouse after they grow to
4-6 leaf stage (1-3 weeks). pBSMM232 containing plants were
produced carried along with each experiment to serve as controls
for TO samples. Plantlets were moved to 14'' pots on Tuesday of
each week. Plants were grown for 9 days until the 7-13 leaf stage
is reached. On Thursday between 10 am and 2 .mu.m leaf sampling was
performed on the 3rd youngest (1.sup.st fully elongated). Within 30
seconds 250-500 mg of leaf material (without midrib), were removed
weighed and placed into pre-extracted glass thimbles in liquid
nitrogen. A second sample (opposite side of the midrib) from each
plant was sampled as described above for qPCR analysis.
[0530.5.0.27] Example 14 c.c.
Growth of T1 Corn Plant for Metabolic Analysis
[12659] For the bioanalytical analyses of the transgenic plants,
the latter were grown uniformly in a specific culture facility. To
this end the GS-90 substrate as the compost mixture was introduced
into the potting machine (Laible System GmbH, Singen, Germany) and
filled into the pots. Thereafter, 26 pots were combined in one dish
and treated with Previcur. For the treatment, 25 ml of Previcur
were taken up in 10 l of tap water. This amount was sufficient for
the treatment of approximately 150 pots. The pots were placed into
the Previcur solution and additionally irrigated overhead with tap
water without Previcur. They were used within four days.
[12660] For the sowing, the seeds, which had been stored at room
temperature were removed from the paper-bag and transferred into
the pots with the soil. In total, approximately 1 to 3 seeds were
distributed in the middle of the pot.
[12661] After the seeds had been sown, the dishes with the pots
were covered with matching plastic hood and placed into growth
chambers for 2 days. After this time the plastic hood was removed
and plants were placed on the growth table and cultivated for 22 to
24 days under following growth conditions: 16-h-light, 8-h-dark
rhythm at 20.degree. C., an atmospheric humidity of 60% and a
CO.sub.2 concentration of approximately 400 ppm. The light sources
used were Powerstar HQI-T 250 W/D Daylight lamps from Osram, which
generate a light resembling the solar color spectrum with a light
intensity of approximately 220 pE/m2/s-1.
[12662] When the plants were 7 days old, they were subjected to
select transgenic plants. For this purposes pieces of plant leaves
were sampled and a PCR reaction with the respective primers for the
transgene were performed. Plants exhibiting the transgene were used
for the metabolic analysis. The nontransgenic seedlings were
removed. The transgenic plants were thinned when they had reached
the age of 18 days. The transgenic plants, which had grown best in
the center of the pot were considered the target plants. All the
remaining plants were removed carefully with the aid of metal
tweezers and discarded.
[12663] During their growth, the plants received overhead
irrigation with distilled water (onto the compost) and bottom
irrigation into the placement grooves. Once the grown plants had
reached the age of 24 days, they were harvested.
[0530.6.0.27] Example 14 c.d.
Metabolic Analysis of Maize Leaves
[12664] The modifications identified in accordance with the
invention, in the content of above-described metabolites, were
identified by the following procedure.
a) Sampling and Storage of the Samples
[12665] Sampling was performed in corridor next to the green house.
The leaves were incised twice using small laboratory scissors and
this part of the leave was removed manually from the middle rib.
The sample was rapidly weighed on laboratory scales, transferred
into a pre-cooled extraction sleeve and placed into kryo-box cooled
by liquid nitrogen. The time elapsing between cutting the leave to
freezing it in liquid nitrogen amounted to not more than 30
seconds. The boxes were stored in a freezer at -80 C, an shipped on
dry ice.
b) Lyophilization
[12666] During the experiment, care was taken that the plants
either remained in the deep-frozen state (temperatures
<-40.degree. C.) or were freed from water by lyophilization
until the first contact with solvents. Before entering the
analytical process the extraction sleeves with the samples were
transferred to a pre-cooled aluminium rack.
[12667] The aluminum rack with the plant samples in the extraction
sleeves was placed into the pre-cooled (-40.degree. C.)
lyophilization facility. The initial temperature during the main
drying phase was -35.degree. C. and the pressure was 0.120 mbar.
During the drying phase, the parameters were altered following a
pressure and temperature program. The final temperature after 12
hours was +30.degree. C. and the final pressure was 0.001 to 0.004
mbar. After the vacuum pump and the refrigerating machine had been
switched off, the system was flushed with air (dried via a drying
tube) or argon.
c) Extraction
[12668] Immediately after the lyophilization apparatus had been
flushed, the extraction sleeves with the lyophilized plant material
were transferred into the 5 ml extraction cartridges of the ASE
device (Accelerated Solvent Extractor ASE 200 with Solvent
Controller and AutoASE software (DIONEX)).
[12669] Immediately after the lyophilization apparatus had been
flushed, the extraction sleeves with the lyophilized plant material
were transferred into the 5 ml extraction cartridges of the ASE
device (Accelerated Solvent Extractor ASE 200 with Solvent
Controller and AutoASE software (DIONEX)).
[12670] The 24 sample positions of an ASE device (Accelerated
Solvent Extractor ASE 200 with Solvent Controller and AutoASE
software (DIONEX)) were filled with plant samples, including some
samples for testing quality control.
[12671] The polar substances were extracted with approximately 10
ml of methanol/water (80/20, v/v) at T=70.degree. C. and p=140 bar,
5 minutes heating-up phase, 1 minute static extraction. The more
lipophilic substances were extracted with approximately 10 ml of
methanol/dichloromethane (40/60, v/v) at T=70.degree. C. and p=140
bar, 5 minute heating-up phase, 1 minute static extraction. The two
solvent mixtures were extracted into the same glass tubes
(centrifuge tubes, 50 ml, equipped with screw cap and pierceable
septum for the ASE (DIONEX)).
[12672] The solution was treated with internal standards: ribitol,
L-glycine-2,2-d.sub.2, L-alanine-2,3,3,3-d.sub.4,
methionine-methyl-d.sub.3, and .alpha.-methylglucopyranoside and
methyl nona-decanoate, methyl undecanoate, methyl tridecanoate,
methyl pentadecanoate, methyl nonacosanoate.
[12673] The total extract was treated with 8 ml of water. The solid
residue of the plant sample and the extraction sleeve were
discarded.
[12674] The extract was shaken and then centrifuged for 5 to 10
minutes at least at 1 400 g in order to accelerate phase
separation. 0.5 ml of the supernatant methanol/water phase ("polar
phase", colorless) was removed for the further GC analysis, and 0.5
ml was removed for the LC analysis. The remainder of the
methanol/water phase of all samples was used for additional quality
controls. 0.5 ml of the organic phase ("lipid phase", dark green)
was removed for the further GC analysis and 0.5 ml was removed for
the LC analysis. All the portions removed were evaporated to
dryness using the IR Dancer infrared vacuum evaporator (Hettich).
The maximum temperature during the evaporation process did not
exceed 40.degree. C. Pressure in the apparatus was not less than 10
mbar.
d) Processing the Lipid Phase for the LC/MS or LC/MS/MS
Analysis
[12675] The lipid extract, which had been evaporated to dryness was
taken up in mobile phase. The HPLC was run with gradient
elution.
[12676] The polar extract, which had been evaporated to dryness was
taken up in mobile phase. The HPLC was run with gradient
elution.
e) LC/MSMS Analysis
[12677] The LC part was carried out on a commercially available
LCMS system from Agilent Technologies, USA. For polar extracts 10
.mu.l are injected into the system at a flow rate of 200 .mu.l/min.
The separation column (Reversed Phase C18) was maintained at
15.degree. C. during chromatography. For lipid extracts 5 .mu.l are
injected into the system at a flow rate of 200 .mu.l/min. The
separation column (Reversed Phase C18) was maintained at 30.degree.
C. HPLC was performed with gradient elution
[12678] The mass spectrometric analysis was performed on a Applied
Biosystems API 4000 triple quadrupole instrument with turbo ion
spray source. For polar extracts the instrument measures in
negative ion mode in fullscan mode from 100-1000 amu. For lipid
extracts the instrument measures in positive ion mode in fullscan
mode from 100-1000 amu.
f) Derivatization of the Lipid Phase for the GC/MS Analysis
[12679] For the transmethanolysis, a mixture of 140 .mu.l of
chloroform, 37 .mu.l of hydrochloric acid (37% by weight HCl in
water), 320 .mu.l of methanol and 20 .mu.l of toluene was added to
the evaporated extract. The vessel was sealed tightly and heated
for 2 hours at 100.degree. C., with shaking. The solution was
subsequently evaporated to dryness. The residue was dried
completely.
[12680] The methoximation of the carbonyl groups was carried out by
reaction with methoxyamine hydrochloride (20 mg/ml in pyridine, 100
.mu.l for 1.5 hours at 60.degree. C.) in a tightly sealed vessel.
20 .mu.l of a solution of odd-numbered, straight-chain fatty acids
(solution of each 0.3 mg/mL of fatty acids from 7 to 25 carbon
atoms and each 0.6 mg/mL of fatty acids with 27, 29 and 31 carbon
atoms in 3/7 (v/v) pyridine/toluene) were added as time standards.
Finally, the derivatization with 100 .mu.l of
N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was
carried out for 30 minutes at 60.degree. C., again in the tightly
sealed vessel. The final volume before injection into the GC was
220 .mu.l.
g) Derivatization of the Polar Phase for the GC/MS Analysis
[12681] The methoximation of the carbonyl groups was carried out by
reaction with methoxyamine hydrochloride (20 mg/ml in pyridine, 50
.mu.l for 1.5 hours at 60.degree. C.) in a tightly sealed vessel.
10 .mu.l of a solution of odd-numbered, straight-chain fatty acids
(solution of each 0.3 mg/mL of fatty acids from 7 to 25 carbon
atoms and each 0.6 mg/mL of fatty acids with 27, 29 and 31 carbon
atoms in 3/7 (v/v) pyridine/toluene) were added as time standards.
Finally, the derivatization with 50 .mu.l of
N-methyl-N-(trimethylsilyl)-2,2,2-trifluoroacetamide (MSTFA) was
carried out for 30 minutes at 60.degree. C., again in the tightly
sealed vessel. The final volume before injection into the GC was
110 .mu.l.
[12682] GC/MS Analysis
[12683] The GC-MS systems consist of an Agilent 6890 GC coupled to
an Agilent 5973 MSD. The autosamplers are CompiPal or GCPal from
CTC. For the analysis usual commercial capillary separation columns
(30 m.times.0.25 mm.times.0.25 .mu.m) with different
poly-methyl-siloxane stationary phases containing 0% up to 35% of
aromatic moieties, depending on the analysed sample materials and
fractions from the phase separation step, are used (for example:
DB-1 ms, HP-5 ms, DB-XLB, DB-35 ms, Agilent Technologies). Up to 1
.mu.L of the final volume is injected splitless and the oven
temperature program is started at 70.degree. C. and ended at
340.degree. C. with different heating rates depending on the sample
material and fraction from the phase separation step in order to
achieve a sufficient chromatographic separation and number of scans
within each analyte peak. Usual GC-MS standard conditions, for
example constant flow with nominal 1 to 1.7 ml/min. and helium as
the mobile phase gas are used. Ionisation is done by electron
impact with 70 eV, scanning within a m/z range from 15 to 600 with
scan rates from 2.5 to 3 scans/sec and standard tune
conditions.
i) Analysis of the Various Plant Samples
[12684] The samples were measured in individual series of 20 plant
(leaf) samples each (also referred to as sequences), each sequence
containing at least 5 samples from individual control plants
containing GUS. The peak area of each analyte was divided by the
peak area of the respective internal standard. The data were
standardized for the fresh weight established for the respective
harvested sample. The values calculated were then related to the
GUS-containing control group by being divided by the mean of the
corresponding data of the control group of the same sequence. The
values obtained were referred to as ratio_by_WT, they are
comparable between sequences and indicate how much the analyte
concentration in the mutant differs in relation to the control. The
GUS-containing plants were chosen in order to assure that the
vector and transformation procedure itself has no significant
influence on the metabolic composition of the plants. Therefore the
described changes in comparison with the controls were caused by
the introduced genes.
[12685] [0531.0.0.27] Transformation of maize (Zea Mays L.) can
also be performed with a modification of the method described by
Ishida et al. (1996. Nature Biotech 14745-50). Transformation is
genotype-dependent in corn and only specific genotypes are amenable
to transformation and regeneration. The inbred line A188
(University of Minnesota) or hybrids with A188 as a parent are good
sources of donor material for transformation (Fromm et al. 1990
Biotech 8:833-839), but other genotypes can be used successfully as
well. Ears are harvested from corn plants at approximately 11 days
after pollination (DAP) when the length of immature embryos is
about 1 to 1.2 mm. Immature embryos are co-cultivated with
Agrobacterium tumefaciens that carry "super binary" vectors and
transgenic plants are recovered through organogenesis. The super
binary vector system of Japan Tobacco is described in WO patents
WO94/00977 and WO95/06722. Vectors can be constructed as described.
Various selection marker genes can be used including the maize gene
encoding a mutated acetohydroxy acid synthase (AHAS) enzyme (U.S.
Pat. No. 6,025,541). Similarly, various promoters can be used to
regulate the trait gene to provide constitutive, developmental,
tissue or environmental regulation of gene transcription. In this
example, the 34S promoter (GenBank Accession numbers M59930 and
X16673 can be used to provide constitutive expression of the trait
gene.
[12686] [0532.0.0.27] Excised embryos can be grown on callus
induction medium, then maize regeneration medium, containing
imidazolinone as a selection agent. The Petri plates can be
incubated in the light at 25.degree. C. for 2-3 weeks, or until
shoots develop. The green shoots can be transferred from each
embryo to maize rooting medium and incubated at 25.degree. C. for
2-3 weeks, until roots develop. The rooted shoots can be
transplanted to soil in the greenhouse. T1 seeds can be produced
from plants that exhibit tolerance to the imidazolinone herbicides
and which can be PCR positive for the transgenes.
[12687] [0533.0.0.27] The T1 generation of single locus insertions
of the T-DNA can segregate for the transgene in a 3:1 ratio. Those
progeny containing one or two copies of the transgene can be
tolerant of the imidazolinone herbicide. Homozygous T2 plants can
exhibited similar phenotypes as the T1 plants. Hybrid plants (F1
progeny) of homozygous transgenic plants and non-transgenic plants
can also exhibit increased similar phenotypes.
[0534.0.0.27] Example 14d
Engineering Wheat Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, e.g. Derived from Plants or Another
Organism
[12688] [0535.0.0.27] Transformation of wheat can be performed with
the method described by Ishida et al. (1996 Nature Biotech.
14745-50). The cultivar Bobwhite (available from CYMMIT, Mexico)
can commonly be used in transformation. Immature embryos can be
co-cultivated with Agrobacterium tumefaciens that carry "super
binary" vectors, and transgenic plants are recovered through
organogenesis. The super binary vector system of Japan Tobacco is
described in WO patents WO94/00977 and WO95/06722. Vectors can be
constructed as described. Various selection marker genes can be
used including the maize gene encoding a mutated acetohydroxy acid
synthase (AHAS) enzyme (U.S. Pat. No. 6,025,541). Similarly,
various promoters can be used to regulate the trait gene to provide
constitutive, developmental, tissue or environmental regulation of
gene transcription. The 34S promoter (GenBank Accession numbers
M59930 and X16673) can be used to provide constitutive expression
of the trait gene.
[12689] [0536.0.0.27] After incubation with Agrobacterium, the
embryos can be grown on callus induction medium, then regeneration
medium, containing imidazolinone as a selection agent. The Petri
plates can be incubated in the light at 25.degree. C. for 2-3
weeks, or until shoots develop. The green shoots can be transferred
from each embryo to rooting medium and incubated at 25.degree. C.
for 2-3 weeks, until roots develop. The rooted shoots can be
transplanted to soil in the greenhouse. T1 seeds can be produced
from plants that exhibit tolerance to the imidazolinone herbicides
and which are PCR positive for the transgenes.
[12690] [0537.0.0.27] The T1 generation of single locus insertions
of the T-DNA can segregate for the transgene in a 3:1 ratio. Those
progeny containing one or two copies of the transgene can be
tolerant of the imidazolinone herbicide. Homozygous T2 plants
exhibited similar phenotypes.
[0538.0.0.27] Example 14e
Engineering Rapeseed/Canola Plants by Over-Expressing the
Polynucleotide Characterized in the Invention, e.g. Derived from
Plants or Another Organism
[12691] [0539.0.0.27] Cotyledonary petioles and hypocotyls of 5-6
day-old young seedlings can be used as explants for tissue culture
and transformed according to Babic et al. (1998, Plant Cell Rep 17:
183-188). The commercial cultivar Westar (Agriculture Canada) can
be the standard variety used for transformation, but other
varieties can be used.
[12692] [0540.0.0.27] Agrobacterium tumefaciens LBA4404 containing
a binary vector can be used for canola transformation. Many
different binary vector systems have been described for plant
transformation (e.g. An, G. in Agrobacterium Protocols. Methods
in
[12693] Molecular Biology vol 44, pp 47-62, Gartland KMA and MR
Davey eds. Humana Press, Totowa, N.J.). Many are based on the
vector pBIN19 described by Bevan (Nucleic Acid Research. 1984.
12:8711-8721) that includes a plant gene expression cassette
flanked by the left and right border sequences from the Ti plasmid
of Agrobacterium tumefaciens. A plant gene expression cassette can
consist of at least two genes--a selection marker gene and a plant
promoter regulating the transcription of the cDNA or genomic DNA of
the trait gene. Various selection marker genes can be used
including the Arabidopsis gene encoding a mutated acetohydroxy acid
synthase (AHAS) enzyme (US patents 57673666 and 6225105).
Similarly, various promoters can be used to regulate the trait gene
to provide constitutive, developmental, tissue or environmental
regulation of gene transcription. The 34S promoter (GenBank
Accession numbers M59930 and X16673) can be used to provide
constitutive expression of the trait gene.
[12694] [0541.0.0.27] Canola seeds can be surface-sterilized in 70%
ethanol for 2 min., and then in 30% Clorox with a drop of Tween-20
for 10 min, followed by three rinses with sterilized distilled
water. Seeds can be then germinated in vitro 5 days on half
strength MS medium without hormones, 1% sucrose, 0.7% Phytagar at
23.degree. C., 16 hr. light. The cotyledon petiole explants with
the cotyledon attached can be excised from the in vitro seedlings,
and can be inoculated with Agrobacterium by dipping the cut end of
the petiole explant into the bacterial suspension. The explants can
be then cultured for 2 days on MSBAP-3 medium containing 3 mg/l
BAP, 3% sucrose, 0.7% Phytagar at 23.degree. C., 16 hr light. After
two days of co-cultivation with Agrobacterium, the petiole explants
can be transferred to MSBAP-3 medium containing 3 mg/l BAP,
cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and
can then be cultured on MSBAP-3 medium with cefotaxime,
carbenicillin, or timentin and selection agent until shoot
regeneration. When the shoots are 5-10 mm in length, they can be
cut and transferred to shoot elongation medium (MSBAP-0.5,
containing 0.5 mg/l BAP). Shoots of about 2 cm in length can be
transferred to the rooting medium (MSO) for root induction.
[12695] [0542.0.0.27] Samples of the primary transgenic plants (TO)
can be analyzed by PCR to confirm the presence of T-DNA. These
results can be confirmed by Southern hybridization in which DNA is
electrophoresed on a 1% agarose gel and are transferred to a
positively charged nylon membrane (Roche Diagnostics). The PCR DIG
Probe Synthesis Kit (Roche Diagnostics) can be used to prepare a
digoxigenin-labelled probe by PCR, and used as recommended by the
manufacturer.
[0543.0.0.27] Example 14f
Engineering Alfalfa Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, e.g. Derived from Plants or Another
Organism
[12696] [0544.0.0.27] A regenerating clone of alfalfa (Medicago
sativa) can be transformed using the method of (McKersie et al.,
1999 Plant Physiol 119: 839-847). Regeneration and transformation
of alfalfa can be genotype dependent and therefore a regenerating
plant is required. Methods to obtain regenerating plants have been
described. For example, these can be selected from the cultivar
Rangelander (Agriculture Canada) or any other commercial alfalfa
variety as described by Brown D C W and A Atanassov (1985. Plant
Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3
variety (University of Wisconsin) can be selected for use in tissue
culture (Walker et al., 1978 Am J Bot 65:654-659).
[12697] [0545.0.0.27] Petiole explants can be cocultivated with an
overnight culture of Agrobacterium tumefaciens C58C1 pMP90
(McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404
containing a binary vector. Many different binary vector systems
have been described for plant transformation (e.g. An, G. in
Agrobacterium Protocols. Methods in Molecular Biology vol 44, pp
47-62, Gartland KMA and MR Davey eds. Humana Press, Totowa, N.J.).
Many are based on the vector pBIN19 described by Bevan (Nucleic
Acid Research. 1984. 12:8711-8721) that includes a plant gene
expression cassette flanked by the left and right border sequences
from the Ti plasmid of Agrobacterium tumefaciens. A plant gene
expression cassette can consist of at least two genes--a selection
marker gene and a plant promoter regulating the transcription of
the cDNA or genomic DNA of the trait gene. Various selection marker
genes can be used including the Arabidopsis gene encoding a mutated
acetohydroxy acid synthase (AHAS) enzyme (US patents 57673666 and
6225105). Similarly, various promoters can be used to regulate the
trait gene that provides constitutive, developmental, tissue or
environmental regulation of gene transcription. The 34S promoter
(GenBank Accession numbers M59930 and X16673) can be used to
provide constitutive expression of the trait gene.
[12698] [0546.0.0.27] The explants can be cocultivated for 3 d in
the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L
thioproline, 4.35 g/L K2SO4, and 100 .mu.m acetosyringinone. The
explants can be washed in half-strength Murashige-Skoog medium
(Murashige and Skoog, 1962) and plated on the same SH induction
medium without acetosyringinone but with a suitable selection agent
and suitable antibiotic to inhibit Agrobacterium growth. After
several weeks, somatic embryos can be transferred to BOi2Y
development medium containing no growth regulators, no antibiotics,
and 50 g/L sucrose. Somatic embryos are subsequently germinated on
half-strength Murashige-Skoog medium. Rooted seedlings can be
transplanted into pots and grown in a greenhouse.
[12699] [0547.0.0.27] The T0 transgenic plants are propagated by
node cuttings and rooted in Turface growth medium. The plants are
defoliated and grown to a height of about 10 cm (approximately 2
weeks after defoliation).
[0548.0.0.27] Example 14 g
Engineering Alfalfa Plants by Over-Expressing the Polynucleotide
Characterized in the Invention, Derived e.g. from Saccharomyces
cerevisiae, E. Coli or Plants or Another Organism
[12700] [0549.0.0.27] A regenerating clone of alfalfa (Medicago
sativa) can be transformed using the method of (McKersie et al.,
1999 Plant Physiol 119: 839-847). Regeneration and transformation
of alfalfa can be genotype dependent and therefore a regenerating
plant is required. Methods to obtain regenerating plants have been
described. For example, these can be selected from the cultivar
Rangelander (Agriculture Canada) or any other commercial alfalfa
variety as described by Brown D C W and A Atanassov (1985. Plant
Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3
variety (University of Wisconsin) has been selected for use in
tissue culture (Walker et al., 1978 Am J Bot 65:654-659).
[12701] [0550.0.0.27] Petiole explants can be cocultivated with an
overnight culture of Agrobacterium tumefaciens C58C1 pMP90
(McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404
containing a binary vector. Many different binary vector systems
have been described for plant transformation (e.g. An, G. in
Agrobacterium Protocols. Methods in Molecular Biology vol 44, pp
47-62, Gartland KMA and MR Davey eds. Humana Press, Totowa, N.J.).
Many are based on the vector pBIN19 described by Bevan (Nucleic
Acid Research. 1984. 12:8711-8721) that includes a plant gene
expression cassette flanked by the left and right border sequences
from the Ti plasmid of Agrobacterium tumefaciens. A plant gene
expression cassette consists of at least two genes--a selection
marker gene and a plant promoter regulating the transcription of
the cDNA or genomic DNA of the trait gene. Various selection marker
genes can be used including the Arabidopsis gene encoding a mutated
acetohydroxy acid synthase (AHAS) enzyme (US patents 57673666 and
6225105). Similarly, various promoters can be used to regulate the
trait gene that provides constitutive, developmental, tissue or
environmental regulation of gene transcription. In this example,
the 34S promoter (GenBank Accession numbers M59930 and X16673) can
be used to provide constitutive expression of the trait gene.
[12702] [0551.0.0.27] The explants are cocultivated for 3 d in the
dark on SH induction medium containing 288 mg/L Pro, 53 mg/L
thioproline, 4.35 g/L K2SO4, and 100 .mu.m acetosyringinone. The
explants are washed in half-strength Murashige-Skoog medium
(Murashige and Skoog, 1962) and plated on the same SH induction
medium without acetosyringinone but with a suitable selection agent
and suitable antibiotic to inhibit
[12703] Agrobacterium growth. After several weeks, somatic embryos
are transferred to BOi2Y development medium containing no growth
regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are
subsequently germinated on half-strength Murashige-Skoog medium.
Rooted seedlings are transplanted into pots and grown in a
greenhouse.
[12704] [0552.0.0.27] The T0 transgenic plants are propagated by
node cuttings and rooted in Turface growth medium. The plants are
defoliated and grown to a height of about 10 cm (approximately 2
weeks after defoliation).
[12705] [0552.1.0.27] ./.
[0552.2.0.27] Example 16
Preparation of Homologous Sequences from Plants
[12706] Different plants can be grown under standard or varying
conditions in the greenhouse. RNA can be extracted following the
protocol of Jones, Dunsmuir and Bedbrook (1985) EMBO J. 4:
2411-2418. Approx. 1 gram of tissue material from various organs is
ground in liquid nitrogen. The powder is transferred to a 13 ml
Falcon tube containing 4.5 ml NTES buffer (100 mM NaCl, 10 mM
Tris/HCl pH 7.5, 1 mM EDTA, 1% SDS; in RNase-free water) and 3 ml
phenol/chloroform/isoamylalcohol (25/24/1), immediately mixed and
stored on ice. The mixture is spun for 10 minutes at 7000 rpm using
a centrifuge (Sorval; SM24 or SS34 rotor). The supernatant is
transferred to a new tube, 1/10th volume of 3 M NaAcetate (pH 5.2;
in RNase-free water) and 1 volume of isopropanol is added, mixed at
stored for 1 hour or overnight at -20.degree. C. The mixture is
spun for 10 minutes at 7000 rpm. The supernatant is discarded and
the pellet washed with 70% ethanol (v/v). The mixture is spun for 5
minutes at 7000 rpm, the supernatant is discarded and the pellet is
air-dried. 1 ml RNase-free water is added and allow the DNA/RNA
pellet to dissolve on ice at 4 C. The nucleic acid solution is
transferred to a 2 ml Eppendorf tube and 1 ml of 4 M LiAcetate is
added. After mixing the solution is kept for at least 3 hours, or
overnight, at 4 C. The mixture is spun for 10 minutes at 14000 rpm,
the supernatant discarded, the pellet washed with 70% Ethanol,
air-dried and dissolved in 200 .mu.l of RNase-free water.
[12707] Total RNA can be used to construct a cDNA-library according
to the manufacturer's protocol (for example using the ZAP-cDNA
synthesis and cloning kit of Stratagene, La Jolla, USA). Basically,
messenger RNA (mRNA) is primed in the first strand synthesis with a
oligo(dT) linker-primer and is reverse-transcribed using reverse
transcriptase. After second strand cDNA synthesis, the
double-stranded cDNA is ligated into the Uni-ZAP XR vector. The
Uni-ZAP XR vector allows in vivo excision of the pBluescript
phagemid. The polylinker of the pBluescript phagemid has 21 unique
cloning sites flanked by T3 and T7 promoters and a choice of 6
different primer sites for DNA sequencing. Systematic single run
sequencing of the expected 5 prime end of the clones can allow
preliminary annotation of the sequences for example with the help
of the pedant pro Software package (Biomax, Munchen). Clones for
the nucleic acids of the invention or used in the process according
to the invention can be identified based on homology search with
standard algorithms like blastp or gap. Identified putative full
length clones with identity or high homology can be subjected to
further sequencing in order to obtain the complete sequence.
[12708] Additional new homologous sequences can be identified in a
similar manner by preparing respective cDNA libraries from various
plant sources as described above. Libraries can then be screened
with available sequences of the invention under low stringency
conditions for example as described in Sambrook et al., Molecular
Cloning: A laboratory manual, Cold Spring Harbor 1989, Cold Spring
Harbor Laboratory Press. Purified positive clones can be subjected
to the in vivo excision and complete sequencing. A pairwise
sequence alignment of the original and the new sequence using the
blastp or gap program allows the identification of orthologs,
meaning homologous sequences from different organisms, which should
have a sequence identity of at least 30%. Furthermore the
conservation of functionally important amino acid residues or
domains, which can be identified by the alignment of several
already available paralogs, can identify a new sequence as an new
orthologs.
[12709] Alternatively libraries can be subjected to mass sequencing
and obtained sequences can be stored in a sequence database, which
then can be screened for putative orthologs by different search
algorithms, for example the tbastn algorithm to search the obtained
nucleic acid sequences with a amino acid sequence of the invention.
Clones with the highest sequence identity are used for a complete
sequence determination and orthologs can be identified as described
above.
[12710] [0553.0.0.27] [12711] 1. A process for the production of
methionine, which comprises (a) increasing or generating the
activity of a protein as indicated in Table XII, application no.
27, columns 5 or 7, or a functional equivalent thereof in a
non-human organism, or in one or more parts thereof; and (b)
growing the organism under conditions which permit the production
of methionine in said organism. [12712] 2. A process for the
production of methionine, comprising the increasing or generating
in an organism or a part thereof the expression of at least one
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: [12713] a) nucleic acid molecule
encoding of a polypeptide as indicated in Table XII, application
no. 27, columns 5 or 7, or a fragment thereof, which confers an
increase in the amount of methionine in an organism or a part
thereof; [12714] b) nucleic acid molecule comprising of a nucleic
acid molecule as indicated in Table XI, application no. 27, columns
5 or 7; [12715] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of methionine in an
organism or a part thereof; [12716] d) nucleic acid molecule which
encodes a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
methionine in an organism or a part thereof; [12717] e) nucleic
acid molecule which hybridizes with a nucleic acid molecule of (a)
to (c) under stringent hybridisation conditions and conferring an
increase in the amount of methionine in an organism or a part
thereof; [12718] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table XIII, application no.
27, columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [12719]
g) nucleic acid molecule encoding a polypeptide which is isolated
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (f) and conferring
an increase in the amount of methionine in an organism or a part
thereof; [12720] h) nucleic acid molecule encoding a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 27, columns 7, and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; and [12721] i) nucleic acid molecule which is obtainable
by screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof. [12722] or comprising a sequence which is complementary
thereto. [12723] 3. The process of claim 1 or 2, comprising
recovering of the free or bound methionine. [12724] 4. The process
of any one of claims 1 to 3, comprising the following steps:
[12725] (a) selecting an organism or a part thereof expressing a
polypeptide encoded by the nucleic acid molecule characterized in
claim 2; [12726] (b) mutagenizing the selected organism or the part
thereof; [12727] (c) comparing the activity or the expression level
of said polypeptide in the mutagenized organism or the part thereof
with the activity or the expression of said polypeptide of the
selected organisms or the part thereof; [12728] (d) selecting the
mutated organisms or parts thereof, which comprise an increased
activity or expression level of said polypeptide compared to the
selected organism or the part thereof; [12729] (e) optionally,
growing and cultivating the organisms or the parts thereof; and
[12730] (f) recovering, and optionally isolating, the free or bound
methionine produced by the selected mutated organisms or parts
thereof. [12731] 5. The process of any one of claims 1 to 4,
wherein the activity of said protein or the expression of said
nucleic acid molecule is increased or generated transiently or
stably. [12732] 6. An isolated nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[12733] a) nucleic acid molecule encoding of a polypeptide as
indicated in Table XII, application no. 27, columns 5 or 7, or a
fragment thereof, which confers an increase in the amount of
methionine in an organism or a part thereof; [12734] b) nucleic
acid molecule comprising of a nucleic acid molecule as indicated in
Table XI, application no. 27, columns 5 or 7; [12735] c) nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as a
result of the degeneracy of the genetic code and conferring an
increase in the amount of methionine in an organism or a part
thereof; [12736] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of methionine
in an organism or a part thereof; [12737] e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of methionine in an organism or a part thereof; [12738]
f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table XIII, application no. 27, columns 7, and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [12739] g) nucleic acid
molecule encoding a polypeptide which is isolated with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (f) and conferring an increase in
the amount of methionine in an organism or a part thereof; [12740]
h) nucleic acid molecule encoding a polypeptide comprising a
consensus sequence as indicated in Table XIV, application no. 27,
columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[12741] i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof. [12742] whereby the
nucleic acid molecule distinguishes over the sequence as indicated
in Table XI, application no. 27, columns 5 or 7, by one or more
nucleotides. [12743] 7. A nucleic acid construct which confers the
expression of the nucleic acid molecule of claim 6, comprising one
or more regulatory elements. [12744] 8. A vector comprising the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7. [12745] 9. The vector as claimed in claim 8,
wherein the nucleic acid molecule is in operable linkage with
regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic, host. [12746] 10. A
host cell, which has been transformed stably or transiently with
the vector as claimed in claim 8 or 9 or the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 2 to 5. [12747]
11. The host cell of claim 10, which is a transgenic host cell.
[12748] 12. The host cell of claim 10 or 11, which is a plant cell,
an animal cell, a microorganism, or a yeast cell, a fungus cell, a
prokaryotic cell, an eukaryotic cell or an archaebacterium. [12749]
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. [12750] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table XII, application no. 27, columns 5 or 7, by one
or more amino acids. [12751] 15. An antibody, which binds
specifically to the polypeptide as claimed in claim 14. [12752] 16.
A plant tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [12753] 17. A method for screening for
agonists and antagonists of the activity of a polypeptide encoded
by the nucleic acid molecule of claim 6 conferring an increase in
the amount of methionine in an organism or a part thereof
comprising: (a) contacting cells, tissues, plants or microorganisms
which express the a polypeptide encoded by the nucleic acid
molecule of claim 5 conferring an increase in the amount of
methionine in an organism or a part thereof with a candidate
compound or a sample comprising a plurality of compounds under
conditions which permit the expression the polypeptide; (b)
assaying the methionine level or the polypeptide expression level
in the cell, tissue, plant or microorganism or the media the cell,
tissue, plant or microorganisms is cultured or maintained in; and
(c) identifying a agonist or antagonist by comparing the measured
methionine level or polypeptide expression level with a standard
methionine or polypeptide expression level measured in the absence
of said candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. [12754] 18. A process for the
identification of a compound conferring increased methionine
production in a plant or microorganism, comprising the steps: (a)
culturing a plant cell or tissue or microorganism or maintaining a
plant expressing the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
methionine in an organism or a part thereof and a readout system
capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
dais readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
methionine in an organism or a part thereof; (b) identifying if the
compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout system.
[12755] 19. A method for the identification of a gene product
conferring an increase in methionine production in a cell,
comprising the following steps: (a) contacting the nucleic acid
molecules of a sample, which can contain a candidate gene encoding
a gene product conferring an increase in methionine after
expression with the nucleic acid molecule of claim 6; (b)
identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; (c) introducing the candidate nucleic acid molecules in
host cells appropriate for producing methionine; (d) expressing the
identified nucleic acid molecules in the host cells; (e) assaying
the methionine level in the host cells; and (f) identifying nucleic
acid molecule and its gene product which expression confers an
increase in the methionine level in the host cell in the host cell
after expression compared to the wild type. [12756] 20. A method
for the identification of a gene product conferring an increase in
methionine production in a cell, comprising the following steps:
(a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the methionine amount or level in
an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing methionine; (c) expressing the identified
nucleic acid molecules in the host cells; (d) assaying the
methionine level in the host cells; and (e) identifying nucleic
acid molecule and its gene product which expression confers an
increase in the methionine level in the host cell after expression
compared to the wild type. [12757] 21. A method for the production
of an agricultural composition comprising the steps of the method
of any one of claims 17 to 20 and formulating the compound
identified in any one of claims 17 to 20 in a form acceptable for
an application in agriculture. [12758] 22. A composition comprising
the nucleic acid molecule of claim 6, the polypeptide of claim 14,
the nucleic acid construct of claim 7, the vector of any one of
claim 8 or 9, an antagonist or agonist identified according to
claim 17, the compound of claim 18, the gene product of claim 19 or
20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. [12759] 23. Use of the nucleic acid molecule as
claimed in claim 6 for the identification of a nucleic acid
molecule conferring an increase of methionine after expression.
[12760] 24. Use of the polypeptide of claim 14 or the nucleic acid
construct claim 7 or the gene product identified according to the
method of claim 19 or 20 for identifying compounds capable of
conferring a modulation of methionine levels in an organism.
[12761] 25. Food or feed composition comprising the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the harvested material of
claim 16, the host cell of claim 10 to 12 or the gene product
identified according to the method of claim 19 or 20.
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[12762] [0000.0.28.28] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and corresponding embodiments as
described herein as follows.
[12763] [0001.0.0.28] to [0007.0.0.28]: see [0001.0.0.27] to
[0007.0.0.27]
[12764] [0007.1.28.28] Following the approach of deregulating
specific enzymes in the amino acid biosynthetic pathway an increase
of the levels of free threonine is disclosed in U.S. Pat. No.
5,942,660 which is achieved by overexpression of either a wild-type
or deregulated aspartate kinase, homoserine dehydrogenase or
threonine synthase.
[12765] [0008.0.0.28] see [0008.0.0.27]
[12766] [0009.0.28.28] As described above, the essential amino
acids are necessary for humans and many mammals, for example for
livestock. Threonine is an important constituent in many body
proteins and is necessary for the formation of tooth enamel
protein, collagen and elastin, which both needed for healthy skin
and wound healing. It is a precursor to the amino acids glycine and
serine. It acts as a lipotropic in controlling fat build-up in the
liver. Threonine is an immune stimulant because it promotes thymus
growth and activity. It is a component of digestive enzymes and
immune secretions from the gut, particularly mucins. It has been
used as a supplement to help alleviate anxiety and some cases of
depression. In animal production, as an important essential amino
acid, threonine is normally the second limiting amino acid for pigs
and the third limiting amino acid for chicken (Gallus gallus f.
domestica, e.g. laying hen or broiler).
[12767] [0010.0.0.28] see [0010.0.0.27]
[12768] [0011.0.0.28] see [0011.0.0.27]
[12769] [0012.0.28.28] It is an object of the present invention to
develop an inexpensive process for the synthesis of threonine,
preferably L-threonine. Threonine is together with lysine and
methionine (depending on the organism) one of the amino acids which
are most frequently limiting.
[12770] [0013.0.0.28] see [0013.0.0.27]
[12771] [0014.0.28.28] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is threonine, preferably
L-threonine. Accordingly, in the present invention, the term "the
fine chemical" as used herein relates to "threonine". Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising threonine.
[12772] [0015.0.28.28] In one embodiment, the term "the fine
chemical" means threonine, preferably L-threonine. Throughout the
specification the term "the fine chemical" means threonine,
preferably L-threonine, its salts, ester or amids in free form or
bound to proteins. In a preferred embodiment, the term "the fine
chemical" means threonine, preferably L-threonine, in free form or
its salts or bound to proteins.
[12773] [0016.0.28.28] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [12774] (a) increasing or generating the activity of one
or more of a protein as shown in table XII, application no. 28,
column 3 encoded by the nucleic acid sequences as shown in table
XI, application no. 28, column 5, in a non-human organism or in one
or more parts thereof or [12775] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
amino acid of the invention or fine chemicals comprising amino
acids of the invention, in said organism or in the culture medium
surrounding the organism.
[12776] [0017.0.0.28] see [0017.0.0.27]
[12777] [0018.0.0.28] to [0019.0.0.28] see [0018.0.0.27] to
[0019.0.0.27]
[12778] [0020.0.28.28] Surprisingly it was found, that the
transgenic expression of the Zea mays protein as indicated in Table
XII, application no. 28, column 5, line 3 in a plant conferred an
increase in Threonine content of the transformed plants. Thus, in
one embodiment, said protein or its homologs are used for the
production of Threonine. Surprisingly it was found, that the
transgenic expression of the Zea mays protein as indicated in Table
XII, application no. 28, column 5, line 4 in thaliana plant
conferred an increase in Threonine content of the transformed
plants. Thus, in one embodiment, said protein or its homologs are
used for the production of Threonine.
[12779] [0021.0.0.28] see [0021.0.0.27]
[12780] [0022.0.28.28] The sequence of YKR057W from Saccharomyces
cerevisiae has been published in Dujon et al., Nature 369 (6479),
371-378, 1994 and Goffeau et al., Science 274 (5287), 546-547, 1996
and its activity is being defined as an ribosomal protein, similar
to S21 ribosomal proteins, involved in ribosome biogenesis and
translation. Accordingly, in one embodiment, the process of the
present invention comprises the use of a ribosomal protein, similar
to S21 ribosomal proteins, involved in ribosome biogenesis and
translation from Saccaromyces cerevisiae or a plant or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of threonine, in particular for increasing the amount of
threonine, preferably L-threonine in free or bound form in an
organism or a part thereof, as mentioned. The sequence of YOR245C
from Saccharomyces cerevisiae has been published in Dujon, B. et
al., Nature 387 (6632 Suppl), 98-102 (1997) and its activity is
defined as a acyl-CoA:diacylglycerol acyltransferase. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product defined as a acyl-CoA:diacylglycerol
acyltransferase from Saccaromyces cerevisiae or a plant or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning threonine, in particular for increasing the
amount of threonine, preferably L-threonine in free or bound form
in an organism or a part thereof, as mentioned. In one embodiment,
in the process of the present invention the activity of a
acyl-CoA:diacylglycerol acyltransferase is increased or generated,
e.g. from Saccharomyces cerevisiae or a homolog thereof.
[12781] [0022.1.0.28] to [0023.0.0.28] see [0022.1.0.27] to
[0023.0.0.27]
[12782] [0023.1.28.28] Homologs of the polypeptide disclosed in
table XII, application no. 28, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 28, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 28, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 28,
column 7, resp.
[12783] [0024.0.0.28]: see [0024.0.0.27]
[12784] [0025.0.28.28] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 28, column 3 "if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 28, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[12785] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 28,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 28,
column 3 of a a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[12786] [0025.1.28.28] In one embodiment, the polypeptide of the
invention confers said activity, e.g. the increase of the fine
chemical in an organism or a part thereof, if it is derived from an
organism, which is evolutionary distant to the organism in which it
is expressed. For example origin and expressing organism are
derived from different families, orders, classes or phylums.
[12787] [0026.0.0.28] to [0033.0.0.28]: see [0026.0.0.27] to
[0033.0.0.27]
[12788] [0034.0.28.28] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 28, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[12789] [0035.0.0.28] to [0044.0.0.28]: see [0035.0.0.27] to
[0044.0.0.27]
[12790] [0045.0.28.28] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
28, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 28, column 6 of
the respective line, between 10% and 50%, 10% and 100%, 10% and
200%, 10% and 300%, 10% and 400%, 10% and 500%, 20% and 50%, 20%
and 250%, 20% and 500%, 50% and 100%, 50% and 250%, 50% and 500%,
500% and 1000% or more
[12791] [0046.0.28.28] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
28, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 28, column 6 of
the respective line confers an increase of the respective fine
chemical and of further amino acid or their precursors.
[12792] [0047.0.0.28] see [0047.0.0.27]
[12793] [0048.0.0.28] see [0048.0.0.27]
[12794] [0049.0.28.28] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 28, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 28, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 28, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[12795] [0050.0.28.28] For the purposes of the present invention,
the term "threonine" and "L-threonine" also encompass the
corresponding salts, such as, for example, threonine hydrochloride
or threonine sulfate. Preferably the term threonine is intended to
encompass the term L-threonine.
[12796] [0051.0.0.28] see [0051.0.0.27]
[12797] [0052.0.0.28] see [0052.0.0.27]
[12798] [0053.0.28.28] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[12799] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 28, columns 5 and 7 or its homologs activity
having herein-mentioned amino acid of the invention increasing
activity; and/or [12800] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no.
[12801] 28, columns 5 and 7, e.g. a nucleic acid sequence encoding
a polypeptide having the activity of a protein as indicated in
table XII, application no. 28, columns 5 and 7 or its homologs
activity or of a mRNA encoding the polypeptide of the present
invention having herein-mentioned amino acid of the invention
increasing activity; and/or [12802] c) increasing the specific
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the invention or of
the polypeptide of the present invention having herein-mentioned
amino acid increasing activity, e.g. of a polypeptide having the
activity of a protein as indicated in table XII, application no.
28, columns 5 and 7 or its homologs activity, or decreasing the
inhibiitory regulation of the polypeptide of the invention; and/or
[12803] d) generating or increasing the expression of an endogenous
or artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned amino acid of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 28, columns 5
and 7 or its homologs activity; and/or [12804] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned amino acid of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 28, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [12805] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned amino acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 28, columns 5 and 7 or its
homologs activity, and/or [12806] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned amino acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 28, columns 5 and 7 or its
homologs activity; and/or [12807] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 28, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[12808] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [12809] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[12810] [0054.0.28.28] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 28 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 28, column 3 or its homologs.
[12811] [0055.0.0.28] to [0064.0.0.28] see [0055.0.0.27] to
[0064.0.0.27]
[12812] [0065.0.28.28] The activation of an endogenous polypeptide
having above-mentioned activity, of the polypeptide of the
invention, e.g. conferring the increase of the fine chemical after
increase of expression or activity can also be increased by
introducing a synthetic transcription factor, which binds close to
the coding region of an endogenous polypeptide of the invention- or
its endogenous homolog-encoding gene and activates its
transcription. A chimeric zinc finger protein can be construed,
which comprises a specific DNA-binding domain and an activation
domain as e.g. the VP16 domain of Herpes Simplex virus. The
specific binding domain can bind to the regulatory region of the
endogenous protein-coding region. The expression of the chimeric
transcription factor in a organism, in particular in a plant, leads
to a specific expression of an endogenous polypeptid of the
invention, in particular a plant homolog thereof, see e.g. in
WO01/52620, Oriz, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, 13290
or Guan, Proc. Natl. Acad. Sci. USA, 2002, Vol. 99, 13296.
[12813] [0066.0.0.28] to [0069.0.0.28]: see [0066.0.0.27] to
[0069.0.0.27]
[12814] [0070.0.28.28] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, into an
organism alone or in combination with other genes, it is possible
not only to increase the biosynthetic flux towards the end product,
but also to increase, modify or create de novo an advantageous,
preferably novel metabolites composition in the organism, e.g. an
advantageous amino acid composition comprising a higher content of
(from a viewpoint of nutritional physiology limited) fine
chemicals, in particular amino acids, likewise the fine
chemical.
[12815] [0071.0.0.28] see [0071.0.0.27]
[12816] [0072.0.28.28] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous hydroxy containing compounds. Examples of such
compounds are, in addition to threonine, serine, homoserine,
phosphohomoserine or hydroxyproline or methionine.
[12817] [0073.0.28.28] Accordingly, in one embodiment, the process
according to the invention relates to a process which comprises:
[12818] (a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [12819] (b) increasing an activity of
a polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table XII, application no. 28, columns 5 or 7, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the fine chemical in the organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [12820] (c) growing the organism,
preferably a microorganism, a non-human animal, a plant or animal
cell, a plant or animal tissue or a plant under conditions which
permit the production of the fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and [12821] (d) if desired, revovering, optionally
isolating, the free and/or bound the fine chemical and, optionally
further free and/or bound amino acids synthetized by the organism,
the microorganism, the non-human animal, the plant or animal cell,
the plant or animal tissue or the plant.
[12822] [0074.0.0.28] to [0084.0.0.28]: see [0074.0.0.27] to
[0084.0.0.27]
[12823] [0085.0.28.28] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [12824] (a) a nucleic acid sequence as
indicated in Table XI, application no. 28, columns 5 or 7, or
[12825] (b) a genetic regulatory element, for example a promoter,
which is functionally linked to the nucleic acid sequence as
indicated in Table XI, application no. 28, columns 5 or 7, or a
derivative thereof, or [12826] (c) (a) and (b) is/are not present
in its/their natural genetic environment or has/have been modified
by means of genetic manipulation methods, it being possible for the
modification to be, by way of example, a substitution, addition,
deletion, inversion or insertion of one or more nucleotide
radicals. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[12827] [0086.0.0.28]: see [0086.0.0.27]
[12828] [0087.0.0.28]: see [0087.0.0.27]
[12829] [0088.0.28.28] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose amino acid
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for monogastric animals is limited by a few essential amino acids
such as lysine, threonine or methionine.
[12830] [0088.1.28.28] In one embodiment, after an activity of a
polypeptide of the present invention has been increased or
generated, or after the expression of a nucleic acid molecule or
polypeptide according to the invention has been generated or
increased, the transgenic plant generated can be grown on or in a
nutrient medium or else in the soil and subsequently harvested.
[12831] [0089.0.0.28] to [0097.0.0.28]: see [0089.0.0.27] to
[0097.0.0.27]
[12832] [0098.0.28.28] In a further embodiment, the fine chemical
threonine is produced in accordance with the invention and, if
desired, is isolated. The production of further amino acids such as
methionine, lysine and/or mixtures of amino acid by the process
according to the invention is advantageous.
[12833] [0099.0.0.28] to [0102.0.0.28]: see [0099.0.0.27] to
[0102.0.0.27]
[12834] [0103.0.28.28] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical threonine comprising or generating in an organism or a
part thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [12835] (a) nucleic acid molecule encoding,
preferably at least the mature form, of a polypeptide having a
sequence as indicated in Table XII, application no. 28, columns 5
or 7; [12836] (b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table XI, application no. 28, columns 5 or 7;
[12837] (c) nucleic acid molecule whose sequence can be deduced
from a polypeptide sequence encoded by a nucleic acid molecule of
(a) or (b) as result of the degeneracy of the genetic code and
conferring an increase in the amount of the fine chemical threonine
in an organism or a part thereof; [12838] (d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the fine chemical threonine in an organism or a part thereof;
[12839] (e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical threonine in an organism or a part thereof; [12840] (f)
nucleic acid molecule encoding a polypeptide, the polypeptide being
derived by substituting, deleting and/or adding one or more amino
acids of the amino acid sequence of the polypeptide encoded by the
nucleic acid molecules (a) to (d), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical threonine
in an organism or a part thereof; [12841] (g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and and conferring an increase in the amount of the fine
chemical threonine in an organism or a part thereof; [12842] (h)
nucleic acid molecule comprising a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers pairs having a sequence as
indicated in Table XIII, application no. 28, columns 7, and
conferring an increase in the amount of the fine chemical threonine
in an organism or a part thereof; [12843] (i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from an expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and and conferring an increase in
the amount of the fine chemical threonine in an organism or a part
thereof; [12844] (j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence having a sequences as
indicated in Table XIV, application no. 28, column 7, and
conferring an increase in the amount of the fine chemical threonine
in an organism or a part thereof; [12845] (k) nucleic acid molecule
comprising one or more of the nucleic acid molecule encoding the
amino acid sequence of a polypeptide encoding a domain of a
polypeptide indicated in Table XII, application no. 28, columns 5
or 7, and conferring an increase in the amount of the fine chemical
threonine in an organism or a part thereof; and [12846] (l) nucleic
acid molecule which is obtainable by screening a suitable library
under stringent conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k), preferably to
(a) to (c), or with a fragment of at least 15 nt, preferably 20 nt,
30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k), preferably to (a) to (c), and
conferring an increase in the amount of the fine chemical threonine
in an organism or a part thereof; or which comprises a sequence
which is complementary thereto.
[12847] [0104.0.28.28] In one embodiment, the nucleic acid molecule
of the invention distinguishes over the sequence indicated in Table
XI, application no. 28, columns 5 or 7, by one or more nucleotides.
In one embodiment, the nucleic acid molecule of the present
invention does not consist of the sequence shown in indicated in
Table XI, application no. 28, columns 5 or 7. In another
embodiment, the nucleic acid molecule does not encode a polypeptide
of a sequence indicated in Table XI, application no.
[12848] 28, columns 5 or 7.
[12849] [0105.0.0.28] to [0107.0.0.28]: see [0105.0.0.27] to
[0107.0.0.27]
[12850] [0108.0.28.28] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 28, columns 5 or 7, nucleic
acid molecules which are derived from an amino acid sequences as
indicated in Table XII, application no. 28, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 28, column 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of a polypeptide as indicated in Table XII application no.
28, column 3, 5 or 7, or e.g. conferring a increase of the fine
chemical threonine after increasing its expression or activity are
advantageously increased in the process according to the
invention.
[12851] [0109.0.0.28] see [0109.0.0.27]
[12852] [0110.0.28.28] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide of the
invention can be determined from generally accessible
databases.
[12853] [0111.0.0.28] see [0111.0.0.27]
[12854] [0112.0.28.28] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XIIa or XII, application no.
28, column 3, or having the sequence of a polypeptide as indicated
in Table XII, application no. 28, columns 5 and 7, and conferring
an increase of the fine chemical threonine.
[12855] [0113.0.0.28] to [0120.0.0.28]: see [0113.0.0.27] to
[0120.0.0.27]
[12856] [0121.0.28.28] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 28, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a increase of the fine chemical threonine
after increasing its activity
[12857] [0122.0.0.28] to [127.0.0.28]: see [0122.0.0.27] to
[0127.0.0.27]
[12858] [0128.0.28.28] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 28, column 7, by means of polymerase chain reaction
can be generated on the basis of a sequence as indicated in Table
XI, application no. 28, columns 5 or 7, or the sequences derived
from sequences as indicated in Table XII, application no. 28,
columns 5 or 7.
[12859] [0129.0.28.28] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequences indicated in Table XIV,
application no. 28, column 7, are derived from said alignments.
[12860] [0130.0.28.28] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of the fine
chemical after increasing its expression or activity or further
functional homologs of the polypeptide of the invention from other
organisms.
[12861] [0131.0.0.28] to [0138.0.0.28]: see [0131.0.0.27] to
[0138.0.0.27]
[12862] [0139.0.28.28] Polypeptides having above-mentioned
activity, i.e. conferring a threonine increase, derived from other
organisms, can be encoded by other DNA sequences which hybridize to
a sequences indicated in Table XI, application no. 28, columns 5 or
7, under relaxed hybridization conditions and which code on
expression for peptides having the threonine increasing
activity.
[12863] [0140.0.0.28] to [0146.0.0.28]: see [0140.0.0.27] to
[0146.0.0.27]
[12864] [0147.0.28.28] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 28, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences such
that it can hybridize to one of said nucleotide sequences thereby
forming a stable duplex. Preferably, the hybridisation is performed
under stringent hybridization conditions. However, a complement of
one of the herein disclosed sequences is preferably a sequence
complement thereto according to the base pairing of nucleic acid
molecules well known to the skilled person. For example, the bases
A and G undergo base pairing with the bases T and U or C, resp. and
visa versa. Modifications of the bases can influence the
base-pairing partner.
[12865] [0148.0.28.28] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 28,
columns 5 or 7, or a functional portion thereof and preferably has
above mentioned activity, in particular has
the--fine-chemical-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homog's.
[12866] [0149.0.28.28] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 28,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity, e.g. conferring an increase of the fine
chemical.
[12867] [00149.1.28.28] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table
XI, application no. 28, columns 5 or 7, has further one or more of
the activities annotated or known for the a protein as indicated in
Table XII, application no. 28, column 3.
[12868] [0150.0.28.28] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table XI, application no. 28, columns
5 or 7, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of fine chemical threonine if
its activity is increased. The nucleotide sequences determined from
the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences indicated in Table
[12869] XI, application no. 28, columns 5 or 7, an anti-sense
sequence of one of the sequences indicated in Table XI, application
no. 28, columns 5 or 7, or naturally occurring mutants thereof.
Primers based on a nucleotide of invention can be used in PCR
reactions to clone homologues of the polypeptide of the invention
or of the polypeptide used in the process of the invention, e.g. as
the primers described in the examples of the present invention,
e.g. as shown in the examples. A PCR with the primer pairs
indicated in Table XIII, application no. 28, column 7, will result
in a fragment of a polynucleotide sequence as indicated in Table
XI, application no. 28, columns 5 or 7.
[12870] [0151.0.0.28] see [0151.0.0.27]
[12871] [0152.0.28.28] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 28, columns 5
or 7 such that the protein or portion thereof maintains the ability
to participate in threonine production, in particular a threonine
increasing activity as mentioned above or as described in the
examples in plants or microorganisms is comprised.
[12872] [0153.0.28.28] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 28,
columns 5 or 7 such that the protein or portion thereof is able to
participate in the increase of threonine production. In one
embodiment, a protein or portion thereof as indicated in Table XII,
application no. 28, columns 5 or 7, has for example an activity of
a polypeptide indicated in Table XII, application no. 28, column
3.
[12873] [0154.0.28.28] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 28, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the fine chemical.
[12874] [0155.0.0.28] see [0155.0.0.27]
[12875] [0156.0.0.28] see [0156.0.0.27]
[12876] [0157.0.28.28] The invention further relates to nucleic
acid molecules that differ from one of a nucleotide sequences as
indicated in Table XI, application no. 28, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in threonine in an organism, e.g. as that polypeptides
comprising the consensus sequences as indicated in Table XIV,
application no. 28, columns 7, or of the polypeptide as indicated
in Table XII, application no. 28, columns 5 or 7, or their
functional homologues. Advantageously, the nucleic acid molecule of
the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in another
embodiment having, a consensus sequences as indicated in Table XIV,
application no. 28, columns 7, or of the polypeptide as as
indicated in Table XII, application no. 28, columns 5 or 7, or the
functional homologues. In a still further embodiment, the nucleic
acid molecule of the invention encodes a full length protein which
is substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table XIV, application no. 28,
column 7, or of a polypeptide as indicated in Table XII,
application no. 28, columns 5 or 7, or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 28, columns 5 or 7.
[12877] [0158.0.0.28] to [0160.0.0.28]: see [0158.0.0.27] to
[0160.0.0.27]
[12878] [0161.0.28.28] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no.
[12879] 28, columns 5 or 7. The nucleic acid molecule is preferably
at least 20, 30, 50, 100, 250 or more nucleotides in length.
[12880] [0162.0.0.28]: see [0162.0.0.27]
[12881] [0163.0.28.28] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 28, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the fine chemical
increase after increasing the expression or activity thereof or the
activity of a protein of the invention or used in the process of
the invention.
[12882] [0164.0.0.28]: see [0164.0.0.27]
[12883] [0165.0.28.28] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, columns 5 or 7.
[12884] [0166.0.0.28]: see [0166.0.0.27]
[12885] [0167.0.0.28] see [0167.0.0.27]
[12886] [0168.0.28.28] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table XII, application no.
28, columns 5 or 7, yet retain said activity described herein. The
nucleic acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table XII, application no. 28, columns 5 or 7, and is
capable of participation in the increase of production of the fine
chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table XII,
application no. 28, columns 5 or 7, more preferably at least about
70% identical to one of the sequences as indicated in Table XII,
application no. 28, columns 5 or 7, even more preferably at least
about 80%, 90% or 95% homologous to a sequence as indicated in
Table XII, application no. 28, columns 5 or 7, and most preferably
at least about 96%, 97%, 98%, or 99% identical to the sequence as
indicated in Table XII, application no. 28, columns 5 or 7.
[12887] [0169.0.0.28] to [0172.0.0.28]: see [0169.0.0.27] to
[0172.0.0.27]
[12888] [0173.0.28.28] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108442 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108442 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[12889] [0174.0.0.28]: see [0174.0.0.27]
[12890] [0175.0.28.28] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108443 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108443 by the above program algorithm with the
above parameter set, has a 80% homology.
[12891] [0176.0.28.28] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 28,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least least 30%, 35%, 40%, 45% or
50%, preferably at least 55%, 60%, 65% or 70% by preference at
least 80%, especially preferably at least 85% or 90%, 91%, 92%, 93%
or 94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 28, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 28, columns
5 or 7.
[12892] [0177.0.28.28] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 28,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptides as indicated in Table XII,
application no. 28, columns 5 or 7, according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 28, columns
5 or 7.
[12893] [0178.0.0.28] see [0178.0.0.27]
[12894] [0179.0.28.28] A nucleic acid molecule encoding an
homologous to a protein sequence of as indicated in Table XII,
application no. 28, columns 5 or 7, can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table XI, application no.
28, columns 5 or 7, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences of
sequences as indicated in Table XI, application no. 28, columns 5
or 7, by standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[12895] [0180.0.0.28] to [0183.0.0.28]: see [0180.0.0.27] to
[0183.0.0.27]
[12896] [0184.0.28.28] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 28,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 28, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 28,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[12897] [0185.0.28.28] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one more sequence as indicated in Table
XI, application no. 28, columns 5 or 7. In one embodiment it is
preferred that the nucleic acid molecule comprises as little as
possible other nucleotide sequences not shown in any one of
sequences as indicated in Table XI, application no. 28, columns 5
or 7. In one embodiment, the nucleic acid molecule comprises less
than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or 40 further
nucleotides. In a further embodiment, the nucleic acid molecule
comprises less than 30, 20 or 10 further nucleotides. In one
embodiment, a nucleic acid molecule use in the process of the
invention is identical to a sequence as indicated in Table XI,
application no. 28, columns 5 or 7.
[12898] [0186.0.28.28] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table XII,
application no. 28, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polynucleotide used in
the process of the invention is identical to the sequences as
indicated in Table XII, application no. 28, columns 5 or 7.
[12899] [0187.0.28.28] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table XII, application no.
28, columns 5 or 7, comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence
encoding a sequences as indicated in Table XII, application no. 28,
columns 5 or 7.
[12900] [0188.0.28.28] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide as indicated in Table XII, application no. 28,
columns 5 or 7, preferably compared to a sequence as indicated in
Table XII, application no. 28, column 5, and expressed under
identical conditions.
[12901] [0189.0.28.28] Homologues of sequences as indicated in
Table XI, application no. 28, columns 5 or 7, or of derived
sequences as indicated in Table XII, application no. 28, columns 5
or 7, also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives which comprise
noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[12902] [0190.0.0.28] to [0203.0.0.28]: see [0190.0.0.27] to
[0203.0.0.27]
[12903] [0204.0.28.28] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule which comprises a
nucleic acid molecule selected from the group consisting of:
[12904] (a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 28, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical threonine in an organism or a part thereof [12905] (b)
nucleic acid molecule comprising, preferably at least the mature
form, of a nucleic acid molecule as indicated in Table XI,
application no. 28, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical threonine in an organism or a part thereof; [12906] (c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical threonine
in an organism or a part thereof; [12907] (d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the respective fine chemical threonine in an organism
or a part thereof; [12908] (e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the respective fine chemical threonine in an organism
or a part thereof; [12909] (f) nucleic acid molecule encoding a
polypeptide, the polypeptide being derived by substituting,
deleting and/or adding one or more amino acids of the amino acid
sequence of the polypeptide encoded by the nucleic acid molecules
(a) to (d), preferably to (a) to (c), and conferring an increase in
the amount of the respective fine chemical threonine in an organism
or a part thereof; [12910] (g) nucleic acid molecule encoding a
fragment or an epitope of a polypeptide which is encoded by one of
the nucleic acid molecules of (a) to (e), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical threonine in an organism or a part thereof; [12911] (h)
nucleic acid molecule comprising a nucleic acid molecule which is
obtained by amplifying a cDNA library or a genomic library using
primers or primer pairs as indicated in Table XIII, application no.
28, column 7, and conferring an increase in the amount of the
respective fine chemical threonine in an organism or a part
thereof; [12912] (i) nucleic acid molecule encoding a polypeptide
which is isolated, e.g. from a expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (g), preferably to (a) to (c) and
conferring an increase in the amount of the respective fine
chemical threonine in an organism or a part thereof; [12913] (j)
nucleic acid molecule which encodes a polypeptide comprising the
consensus sequence as indicated in Table XIV, application no. 28,
column 7, and conferring an increase in the amount of the
respective fine chemical threonine in an organism or a part
thereof; [12914] (k) nucleic acid molecule encoding the amino acid
sequence of a polypeptide encoding a domaine of a polypeptide as
indicated in Table XII, application no. 28, columns 5 or 7, and
conferring an increase in the amount of the respective fine
chemical threonine in an organism or a part thereof; and [12915]
(l) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment of at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (h) or of a
nucleic acid molecule as indicated in Table XI, application no. 28,
columns 5 or 7, or a nucleic acid molecule encoding, preferably at
least the mature form of, the polypeptide as indicated in Table
XII, application no. 28, columns 5 or 7, and conferring an increase
in the amount of the respective fine chemical threonine in an
organism or a part thereof; or which encompasses a sequence which
is complementary thereto; whereby, preferably, the nucleic acid
molecule according to (a) to (l) distinguishes over a sequence
depicted in as indicated in Table XI, columns 5 or 7, by one or
more nucleotides. In one embodiment, the nucleic acid molecule of
the invention does not consist of a sequence as indicated in Table
XI, application no. 28, columns 5 or 7. In one embodiment, the
nucleic acid molecule is less than 100%, 99.999%, 99.99%, 99.9% or
99% identical to a sequence indicated in Table XI, application no.
28, columns 5 or 7. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table XII, application no. 28, columns 5 or 7. In an other
embodiment, the nucleic acid molecule of the present invention is
at least 30%, 40%, 50%, or 60% identical and less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence indicated in
Table XI, application no. 28, columns 5 or 7. In a further
embodiment the nucleic acid molecule does not encode a polypeptide
sequence as indicated in Table XII application no. 28, columns 5 or
7. Accordingly, in one embodiment, the nucleic acid molecule of the
differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 28, columns 5 or 7.
Accordingly, in one embodiment, the nucleic acid molecule of the
present invention encodes a polypeptide, which differs at least in
one or more amino acids from a polypeptide indicated in Table XII,
columns 5 or 7. In another embodiment, a nucleic acid molecule
indicated in Table XI, columns 5 or 7, does not encode a protein of
a sequence indicated in Table XII, application no. 28, columns 5 or
7. Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to [12916] (l) does
not consist of a sequence as indicated in Table XII, application
no. 28, columns 5 or 7. In a further embodiment, the protein of the
present invention is at least 30%, 40%, 50%, or 60% identical to a
protein sequence indicated in Table XII, application no. 28,
columns 5 or 7, and less than 100%, preferably less than 99.999%,
99.99% or 99.9%, more preferably less than 99%, 985, 97%, 96% or
95% identical to a sequence as indicated in Table XI, application
no. 28, columns 5 or 7.
[12917] [0205.0.0.28] to [0226.0.0.28]: see [0205.0.0.27] to
[0226.0.0.27]
[12918] [0227.0.28.28] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[12919] In addition to a sequence as indicated in Table XI,
application no. 28, columns 5 or 7, or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the amino acid biosynthetic pathway such as for
Llysine, L-methionine and/or L-threonine is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acid
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine sequences as indicated in Table XI,
application no. 28, columns 5 or 7, with genes which generally
support or enhances to growth or yield of the target organisms, for
example genes which lead to faster growth rate of microorganisms or
genes which produces stress-, pathogen, or herbicide resistant
plants.
[12920] [0228.0.0.28] to [0230.0.0.28]: see [0228.0.0.27] to
[0230.0.0.27]
[12921] [0231.0.28.28] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a threonine degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[12922] [0232.0.0.28] to [0282.0.0.28]: see [0232.0.0.27] to
[0282.0.0.27]
[12923] [0283.0.28.28] Moreover, a native polypeptide conferring
the increase of the fine chemical threonine in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an antibody against a protein as indicated in
Table XII, application no. 28, column 3. E.g. an antibody against a
polypeptide as indicated in Table XII, application no. 28, columns
5 or 7, which can be produced by standard techniques utilizing
polypeptides comprising or consisting of above mentioned sequences,
e.g. the polypeptid of the present invention or fragment thereof.
Preferred are monoclonal antibodies.
[12924] [0284.0.0.28]: see [0284.0.0.27]
[12925] [0285.0.28.28] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 28, columns 5 or 7, or as coded by a nucleic
acid molecule as indicated in Table XII, application no. 28,
columns 5 or 7, or functional homologues thereof.
[12926] [0286.0.28.28] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 28, column 7 and in one
other embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 28, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid.
[12927] [0287.0.0.28] to [0290.0.0.28]: see [0287.0.0.27] to
[0290.0.0.27]
[12928] [0291.0.28.28] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[12929] Accordingly, in one embodiment, the present invention
relates to a polypeptide comprising or consisting of plant or
microorganism specific consensus sequences. In one embodiment, said
polypeptide of the invention distinguishes over a sequence as
indicated in Table XII, application no. 28, columns 5 or 7, by one
or more amino acids. In one embodiment, the polypeptide
distinguishes from a sequence as indicated in Table XII,
application no. 28, columns 5 or 7, by more than1, 2, 3, 4, 5, 6,
7, 8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or 30
amino acids, even more preferred are more than 40, 50, or 60 amino
acids and, preferably, the sequence of the polypeptide of the
invention distinguishes from a sequence as indicated in Table XII,
application no. 28, columns 5 or 7, by not more than 80% or 70% of
the amino acids, preferably not more than 60% or 50%, more
preferred not more than 40% or 30%, even more preferred not more
than 20% or 10%. In another embodiment, said polypeptide of the
invention does not consist of a sequence as indicated in Table XII,
application no. 28, columns 5 or 7.
[12930] [0292.0.0.28]: see [0292.0.0.27]
[12931] [0293.0.28.28] In one embodiment, the invention relates to
a polypeptide conferring an increase in the fine chemical threonine
in an organism or part being encoded by the nucleic acid molecule
of the invention or by the nucleic acid molecule of the invention
used in the process of the invention.
[12932] In one embodiment, the poylpeptide of the invention is
having a sequence which distinguishes from a sequence as indicated
in Table XII, application no. 28, columns 5 or 7, by one or more
amino acids. In another embodiment, said polypeptide of the
invention does not consist of the sequence as indicated in Table
XII, application no. 28, columns 5 or 7. In a further embodiment,
said polypeptide of the present invention is less than 100%,
99.999%, 99.99%, 99.9% or 99% identical. In one embodiment, said
polypeptide does not consist of the sequence encoded by a nucleic
acid molecules as indicated in Table XI, application no. 28,
columns 5 or 7.
[12933] [0294.0.28.28] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 28, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 28, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[12934] [0295.0.0.28] to [0297.0.0.28]: see [0295.0.0.27] to
[0297.0.0.27]
[12935] [0297.1.28.28] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table XII, application no. 28, columns 3, 5 or 7.
[12936] [0298.0.28.28] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 28, columns 5 or 7, such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table XII, application no. 28, columns 5 or 7.
[12937] [0299.0.28.28] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 28,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 28, columns 5 or 7, or which is
homologous thereto, as defined above.
[12938] [0300.0.28.28] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table XII,
application no. 28, columns 5 or 7, in the amino acid sequence due
to natural variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprises an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence of a sequence as indicated in Table XII
application no. 28, columns 5 or 7.
[12939] [0301.0.0.28] see [0301.0.0.27]
[12940] [0302.0.28.28] Biologically active portions of a
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 28, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to a polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of a polypeptide of the
present invention or used in the process of the present
invention.
[12941] [0303.0.0.28]: see [0303.0.0.27]
[12942] [0304.0.28.28] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 28, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[12943] [0305.0.0.28] to [0308.0.0.28]: see [0305.0.0.27] to
[0308.0.0.27]
[12944] [0309.0.28.28] In one embodiment, an reference to a
"protein (=polypeptide)" of the invention or as indicated in Table
XII, application no. 28, columns 5 or 7, refers to a polypeptide
having an amino acid sequence corresponding to the polypeptide of
the invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table XII, application no. 28, columns 5 or 7, refers
to a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous to a polypeptide of
the invention, preferably which is not substantially homologous to
a polypeptide as indicated in Table XII, application no. 28,
columns 5 or 7, e.g., a protein which does not confer the activity
described herein or annotated or known for as indicated in Table
XII, application no. 28, column 3, and which is derived from the
same or a different organism. In one embodiment a "non-polypeptide
of the invention" or "other polypeptide" not being indicate in
Table XII, application no. 28, columns 5 or 7, does not confer an
increase of the fine chemical in an organism or part thereof.
[12945] [0310.0.0.28] to [0334.0.0.28]: see [0310.0.0.27] to
[0334.0.0.27]
[12946] [0335.0.28.28] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in
[12947] Table XII, application no. 28, columns 5 or 7, and/or
homologs thereof. As described inter alia in WO 99/32619, dsRNAi
approaches are clearly superior to traditional antisense
approaches. The invention therefore furthermore relates to
double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived therefrom), bring about altered
metabolic activity by the reduction in the expression of a nucleic
acid sequences as indicated in Table XI, application no. 28,
columns 5 or 7, and/or homologs thereof. In a double-stranded RNA
molecule for reducing the expression of an protein encoded by a
nucleic acid sequence of one of the sequences as indicated in Table
XII, application no. 28, columns 5 or 7, and/or homologs thereof,
one of the two RNA strands is essentially identical to at least
part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[12948] [0336.0.0.28] to [0342.0.0.28]: see [0336.0.0.27] to
[0342.0.0.27]
[12949] [0343.0.28.28] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 28, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table XI, application no. 28, columns 5 or 7, or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[12950] [0344.0.0.28] to [0361.0.0.28]: see [0344.0.0.27] to
[0361.0.0.27]
[12951] [0362.0.28.28] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the fine chemical
threonine in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 28, columns 5 or 7. Due to the above
mentioned activity the fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. In one embodiment transgenic for a polypeptide having an
activity of a polypeptide as indicated in Table XII, application
no. 28, columns 5 or 7, means herein that due to modulation or
manipulation of the genome, an activity as annotated for a
polypeptide as indicated in Table XII, application no. 28, columns
3, e.g. having a sequence as indicated in Table XII, application
no. 28, columns 5 or 7, is increased in a cell or an organism or a
part thereof. Examples are described above in context with the
process of the invention.
[12952] [0363.0.0.28]: see [0363.0.0.27]
[12953] [0364.0.28.28] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a polypeptide of the invention with the corresponding
protein-encoding-sequence--becomes a transgenic expression cassette
when it is modified by non-natural, synthetic "artificial" methods
such as, for example, mutagenization. Such methods have been
described (U.S. Pat. No. 5,565,350; WO 00/15815; also see
above).
[12954] [0365.0.0.28] to [0382.0.0.28]: see [0365.0.0.27] to
[0382.0.0.27]
[12955] [0383.0.28.28] For preparing hydroxy containing fine
chemicals, in particular the fine chemical threonine, it is
possible to use as hydroxy source organic hydroxy-containing
compounds such as, for example, alcohols, hydroxy-containing
organic acids, acetals, or compounds containing carbonyl or
carboxyl groups to be reduced by known methods of the art.
[12956] [0384.0.0.28] to [0392.0.0.28]: see [0384.0.0.27] to
[0392.0.0.27]
[12957] [0393.0.28.28] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [12958] (a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical threonine after expression, with the
nucleic acid molecule of the present invention; [12959] (b)
identifying the nucleic acid molecules, which hybridize under
relaxed stringent conditions with the nucleic acid molecule of the
present invention in particular to the nucleic acid molecule
sequence as indicated in Table XI, application no. 28, columns 5 or
7, and, optionally, isolating the full length cDNA clone or
complete genomic clone; [12960] (c) introducing the candidate
nucleic acid molecules in host cells, preferably in a plant cell or
a microorganism, appropriate for producing the fine chemical
threonine; [12961] (d) expressing the identified nucleic acid
molecules in the host cells; [12962] (e) assaying the the fine
chemical level in the host cells; and [12963] (f) identifying the
nucleic acid molecule and its gene product which expression confers
an increase in the the fine chemical level in the host cell after
expression compared to the wild type.
[12964] [0394.0.0.28] to [0399.0.0.28]: see [0394.0.0.27] to
[0399.0.0.27]
[12965] [0399.1.28.28] One can think to screen for increased
production of the fine chemical threonine by for example searching
for a resistance to a drug blocking the synthesis of the fine
chemical threonine and looking whether this effect is dependent on
the activity or expression of a polypeptide as indicated in Table
XII, application no. 28, columns 5 or 7, or a homolog thereof, e.g.
comparing the phenotyp of nearly identical organisms with low and
high activity of a protein as indicated in Table XII, application
no. 28, columns 5 or 7, after incubation with the drug.
[12966] [0400.0.0.28] to [0430.0.0.28]: see [0400.0.0.27] to
[0430.0.0.27]
[12967] [0431.0.0.28] to [0460.0.0.28]: see [0431.0.0.27] to
[0460.0.0.27]
[0461.0.28.28] Example 10
Cloning SEQ ID NO: 108442 for the Expression in Plants
[12968] [0462.0.0.28]: see [0462.0.0.27]
[12969] [0463.0.28.28] SEQ ID NO: 108442 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[12970] [0464.0.0.28] to [0466.0.0.28]: see [0464.0.0.27] to
[0466.0.0.27]
[12971] [0467.0.28.28] The following primer sequences were selected
for the gene SEQ ID NO: 108442: [12972] i) forward primer SEQ ID
NO: 108448: [12973] ii) reverse primer SEQ ID NO: 108449:
[12974] [0468.0.0.28] to [0479.0.0.28]: see [0468.0.0.27] to
[0479.0.0.27]
[0480.0.28.28] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 108442
[12975] [0481.0.0.28] to [0513.0.0.28]: see [0481.0.0.27] to
[0513.0.0.27]
[12976] [0514.0.28.28] As an alternative, the amino acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55).
[12977] Arabidopsis thaliana plants were engineered as described in
Example 11.
[12978] [0515.0.0.28] to [0552.0.0.28]: see [0515.0.0.27] to
[0552.0.0.27]
[12979] [0552.1.28.28]: ./.
[12980] [0553.0.28.28] [12981] 1. A process for the production of
threonine, which comprises [12982] (a) increasing or generating the
activity of a protein as indicated in Table XII, application no.
28, columns 5 or 7, or a functional equivalent thereof in a
non-human organism, or in one or more parts thereof; and [12983]
(b) growing the organism under conditions which permit the
production of threonine in said organism. [12984] 2. A process for
the production of threonine, comprising the increasing or
generating in an organism or a part thereof the expression of at
least one nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [12985] (a) nucleic acid
molecule encoding of a polypeptide as indicated in Table XII,
application no. 28, columns 5 or 7, or a fragment thereof, which
confers an increase in the amount of threonine in an organism or a
part thereof; [12986] (b) nucleic acid molecule comprising of the
nucleic acid molecule as indicated in Table XI, application no. 28,
columns 5 or 7; [12987] (c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as a result of the degeneracy of the
genetic code and conferring an increase in the amount of threonine
in an organism or a part thereof; [12988] (d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of threonine in an organism or a part thereof; [12989] (e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under under stringent hybridisation conditions and
conferring an increase in the amount of threonine in an organism or
a part thereof; [12990] (f) nucleic acid molecule which encompasses
a nucleic acid molecule which is obtained by amplifying nucleic
acid molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table XIII, columns 5 or 7,
lines 6 to 15, 339 to 355 and conferring an increase in the amount
of the fine chemical threonine in an organism or a part thereof;
[12991] (g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of threonine in an
organism or a part thereof; [12992] (h) nucleic acid molecule
encoding a polypeptide comprising a consensus sequence as indicated
in Table XIV, application no. 28, columns 5 or 7, and conferring an
increase in the amount of the fine chemical threonine in an
organism or a part thereof; and [12993] (i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical threonine in an organism or a part
thereof. [12994] or comprising a sequence which is complementary
thereto. [12995] 3. The process of claim 1 or 2, comprising
recovering of the free or bound threonine. [12996] 4. The process
of any one of claims 1 to 3, comprising the following steps:
[12997] (a) selecting an organism or a part thereof expressing a
polypeptide encoded by the nucleic acid molecule characterized in
claim 2; [12998] (b) mutagenizing the selected organism or the part
thereof; [12999] (c) comparing the activity or the expression level
of said polypeptide in the mutagenized organism or the part thereof
with the activity or the expression of said polypeptide of the
selected organisms or the part thereof; [13000] (d) selecting the
mutated organisms or parts thereof, which comprise an increased
activity or expression level of said polypeptide compared to the
selected organism or the part thereof; [13001] (e) optionally,
growing and cultivating the organisms or the parts thereof; and
[13002] (f) recovering, and optionally isolating, the free or bound
threonine produced by the selected mutated organisms or parts
thereof. [13003] 5. The process of any one of claims 1 to 4,
wherein the activity of said protein or the expression of said
nucleic acid molecule is increased or generated transiently or
stably. [13004] 6. An isolated nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[13005] (a) nucleic acid molecule encoding a polypeptide as
indicated in Table XII, application no. 28, columns 5 or 7, or a
fragment thereof, which confers an increase in the amount of
threonine in an organism or a part thereof; [13006] (b) nucleic
acid molecule comprising a nucleic acid as indicated in Table XI,
application no. 28, columns 5 or 7; [13007] (c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of threonine in an organism or a part thereof; [13008] (d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of threonine in an organism or a part
thereof; [13009] (e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of threonine in an organism or a part thereof; [13010] (f) nucleic
acid molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers in Table XIII, application
no. 28, column 8, and conferring an increase in the amount of the
fine chemical threonine in an organism or a part thereof; [13011]
(g) nucleic acid molecule encoding a polypeptide which is isolated
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (f) and conferring
an increase in the amount of threonine in an organism or a part
thereof; [13012] (h) nucleic acid molecule encoding a polypeptide
comprising the consensus sequence shown in Table XIV, application
no. 28, column 8, and conferring an increase in the amount of the
fine chemical in an organism or a part thereof; and [13013] (i)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment thereof having at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (k) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof. [13014] whereby the nucleic acid
molecule distinguishes over the sequence as indicated in Table XI,
application no. 28, columns 5 or 7, by one or more nucleotides.
[13015] 7. A nucleic acid construct which confers the expression of
the nucleic acid molecule of claim 6, comprising one or more
regulatory elements. [13016] 8. A vector comprising the nucleic
acid molecule as claimed in claim 6 or the nucleic acid construct
of claim 7. [13017] 9. The vector as claimed in claim 8, wherein
the nucleic acid molecule is in operable linkage with regulatory
sequences for the expression in a prokaryotic or eukaryotic, or in
a prokaryotic and eukaryotic, host. [13018] 10. A host cell, which
has been transformed stably or transiently with the vector as
claimed in claim 9 or 10 or the nucleic acid molecule as claimed in
claim 6 or the nucleic acid construct of claim 7 or produced as
described in claim any one of claims 2 to 5. [13019] 11. The host
cell of claim 10, which is a transgenic host cell. [13020] 12. The
host cell of claim 10 or 11, which is a plant cell, an animal cell,
a microorganism, or a yeast cell, a fungus cell, a prokaryotic
cell, an eukaryotic cell or an archaebacterium. [13021] 13. A
process for producing a polypeptide, wherein the polypeptide is
expressed in a host cell as claimed in any one of claims 10 to 12.
[13022] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a Sequence as
indicated in Table XII, application no. 28, columns 5 or 7, by one
or more amino acids [13023] 15. An antibody, which binds
specifically to the polypeptide as claimed in claim 14. [13024] 16.
A plant tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [13025] 17. A method for screening for
agonists and antagonists of the activity of a polypeptide encoded
by the nucleic acid molecule of claim 6 conferring an increase in
the amount of threonine in an organism or a part thereof
comprising: [13026] (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of threonine in an organism or a part thereof with a
candidate compound or a sample comprising a plurality of compounds
under conditions which permit the expression the polypeptide;
[13027] (b) assaying the threonine level or the polypeptide
expression level in the cell, tissue, plant or microorganism or the
media the cell, tissue, plant or microorganisms is cultured or
maintained in; and [13028] (c) identifying a agonist or antagonist
by comparing the measured threonine level or polypeptide expression
level with a standard threonine or polypeptide expression level
measured in the absence of said candidate compound or a sample
comprising said plurality of compounds, whereby an increased level
over the standard indicates that the compound or the sample
comprising said plurality of compounds is an agonist and a
decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[13029] 18. A process for the identification of a compound
conferring increased threonine production in a plant or
microorganism, comprising the steps: [13030] (a) culturing a plant
cell or tissue or microorganism or maintaining a plant expressing
the polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of threonine in an organism or
a part thereof and a readout system capable of interacting with the
polypeptide under suitable conditions which permit the interaction
of the polypeptide with said readout system in the presence of a
compound or a sample comprising a plurality of compounds and
capable of providing a detectable signal in response to the binding
of a compound to said polypeptide under conditions which permit the
expression of said readout system and of the polypeptide encoded by
the nucleic acid molecule of claim 6 conferring an increase in the
amount of threonine in an organism or a part thereof; [13031] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [13032] 19. A method for the identification of a
gene product conferring an increase in threonine production in a
cell, comprising the following steps: [13033] (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in threonine
after expression with the nucleic acid molecule of claim 6; [13034]
(b) identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; [13035] (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing threonine;
[13036] (d) expressing the identified nucleic acid molecules in the
host cells; [13037] (e) assaying the threonine level in the host
cells; and [13038] (f) identifying nucleic acid molecule and its
gene product which expression confers an increase in the threonine
level in the host cell after expression compared to the wild type.
[13039] 20. A method for the identification of a gene product
conferring an increase in threonine production in a cell,
comprising the following steps: [13040] (a) identifying in a data
bank nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the threonine amount or level in an organism or a part thereof
after expression, and which are at least 20% homolog to the nucleic
acid molecule of claim 6; [13041] (b) introducing the candidate
nucleic acid molecules in host cells appropriate for producing
threonine; [13042] (c) expressing the identified nucleic acid
molecules in the host cells; [13043] (d) assaying the threonine
level in the host cells; and [13044] (e) identifying the nucleic
acid molecule and its gene product which expression confers an
increase in the threonine level in the host cell after expression
compared to the wild type. [13045] 21. A method for the production
of an agricultural composition comprising the steps of the method
of any one of claims 17 to 20 and formulating the compound
identified in any one of claims 17 to 20 in a form acceptable for
an application in agriculture. [13046] 22. A composition comprising
the nucleic acid molecule of claim 6, the polypeptide of claim 14,
the nucleic acid construct of claim 7, the vector of any one of
claim 8 or 9, an antagonist or agonist identified according to
claim 17, the compound of claim 18, the gene product of claim 19 or
20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. [13047] 23. Use of the nucleic acid molecule as
claimed in claim 6 for the identification of a nucleic acid
molecule conferring an increase of threonine after expression.
[13048] 24. Use of the polypeptide of claim 14 or the nucleic acid
construct claim 7 or the gene product identified according to the
method of claim 19 or 20 for identifying compounds capable of
conferring a modulation of threonine levels in an organism. [13049]
25. Food or feed composition comprising the nucleic acid molecule
of claim 6, the polypeptide of claim 14, the nucleic acid construct
of claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the harvested material of claim
16, the host cell of claim 10 to 12 or the gene product identified
according to the method of claim 19 or 20.
[13050] [0554.0.0.28] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[13051] [0000.0.29.29]: In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and corresponding embodiments as
described herein as follows.
[13052] [0001.0.0.29] to [0008.0.0.29]: see [0.0.01.0.0.27] to
[0008.0.0.27]
[13053] [0009.0.29.29] As described above, the essential amino
acids are necessary for humans and many mammals, for example for
livestock. Tryptophane (L-tryptophane) is one of the most reactive
amino acids. At pH 4.0-6.0 tryptophane amino group reacts with
aldehydes producing Schiff-bases. On the other hand if the amino
group is blocked by acetylation, tryptophane reacts with aldehydes
yielding carboline derivatives (carboline
1,2,3,4-tetrahydro-carboline-3-carboxylic acid). Tryptophane plays
a unique role in defense against infection because of its relative
scarcity compared to other amino acids. During infection, the body
induces tryptophane-catabolizing enzymes which increase
tryptophane's scarcity in an attempt to starve the infecting
organisms [R. R. Brown, Y. Ozaki, S. P. Datta, et al., Implications
of interferon-induced tryptophane catabolism in cancer, auto-immune
diseases and AIDS. In: Kynurenine and Serotonin Pathways, R.
Schwarcz, et al., (Eds.), Plenum Press, New York, 1991]. In most
proteins, tryptophane is the least abundant essential amino acid,
comprising approximately 1% of plant proteins and 1.5% of animal
proteins. Although the minimum daily requirement for tryptophane is
160 mg for women and 250 mg for men, 500-700 mg are recommended to
ensure high-quality protein intake. Actual tryptophane utilization
is substantially higher. Men use approximately 3.5 grams of
tryptophane to make one days's worth of protein [J. C. Peters,
Tryptophane Nutrition and Metabolism: an Overview. In: Kynurenine
and Serotonin Pathways, R. Schwarcz, et al., (Eds.), Plenum Press,
New York, 1991]. The balance is obtained by hepatic recycling of
tryptophane from used (catabolized) proteins.
[13054] Dietary tryptophane is well absorbed intestinally. About
10% of the tryptophane circulating in the bloodstream is free, and
90% is bound to the protein albumin. The tryptophane binding site
on albumin also has affinity for free fatty acids (FFAs), so
tryptophane is displaced when FFAs rise, as when fasting.
[13055] Although tryptophane is not usually the limiting amino acid
in protein synthesis, tryptophane may become insufficient for the
normal functioning of other tryptophane-dependent pathways.
Numerous lines of research point to tryptophane's central role in
regulation of feeding and other behaviors. Tryptophane is not only
typically the least abundant amino acid in the livers free amino
acid pool, but liver tryptophane-tRNA levels fall faster during
food deprivation than other indispensable amino acids [Q. R.
Rogers, The nutritional and metabolic effects of amino acid
imbalances. In: Protein Metabolism and Nutrition, D. J. A. Cole
(Ed.), Butterworths, London, 1976]. Under fasting conditions, and
possibly in wasting syndromes, tryptophane may become the
rate-limiting amino acid for protein synthesis [Peters, 1991].
[13056] [0010.0.0.29] and [0011.0.0.29]: see [0010.0.0.27] and
[0011.0.0.27]
[13057] [0012.0.29.29] It is an object of the present invention to
develop an inexpensive process for the synthesis of tryptophane,
preferably L-tryptophane. Tryptophane is together with methionine,
lysine and threonine (depending on the organism) one of the amino
acids which are most frequently limiting.
[13058] [0013.0.0.29]: see [0013.0.0.27]
[13059] [0014.0.29.29] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is tryptophane, preferably
L-tryptophane. Accordingly, in the present invention, the term "the
fine chemical" as used herein relates to "tryptophane". Further,
the term "the fine chemicals" as used herein also relates to fine
chemicals comprising tryptophane.
[13060] [0015.0.29.29] In one embodiment, the term "the fine
chemical" means tryptophane, preferably L-tryptophane. Throughout
the specification the term "the fine chemical" means tryptophane,
preferably L-tryptophane, its salts, ester or amids in free form or
bound to proteins. In a preferred embodiment, the term "the fine
chemical" means tryptophane, preferably L-tryptophane, in free form
or its salts or bound to proteins.
[13061] In one embodiment, the term "the fine chemical" and the
term "the respective fine chemical" mean at least one chemical
compound with an activity of the above mentioned fine chemical.
[13062] [0016.0.29.29] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [13063] (a) increasing or generating the activity of one
or more [13064] of a protein as shown in table XII, application no.
29, column 3 encoded by the nucleic acid sequences as shown in
table XI, application no. 29, column 5, in a non-human organism or
in one or more parts thereof or [13065] (b) growing the organism
under conditions which permit the production of the fine chemical,
thus amino acid of the invention or fine chemicals comprising amino
acids of the invention, in said organism or in the culture medium
surrounding the organism.
[13066] [0017.0.0.29] to [0019.0.0.29]: see [0017.0.0.27] and
[0019.0.0.27]
[13067] [0020.0.29.29] Surprisingly it was found, that the
transgenic expression of the Zea mays protein as indicated in Table
XII, application no. 29, column 5, line 5 in a plant conferred an
increase in tryptophane content of the transformed plants. Thus, in
one embodiment, said protein or its homologs are used for the
production of tryptophane.
[13068] [0021.0.0.29]: see [0021.0.0.27]
[13069] [0022.0.29.29] The sequence of b3983 from Escherichia coli
K12 has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as a 50S
ribosomal subunit protein L12. Accordingly, in one embodiment, the
process of the present invention comprises the use of a gene
product with the activity of the Escherichia coli ribosomal protein
L11 superfamily, preferably a protein with a 50S ribosomal subunit
protein L12 activity from E. coli or a plant or its homolog, e.g.
as shown herein, for the production of the fine chemical, meaning
of tryptophane, in particular for increasing the amount of
tryptophane in free or bound form in an organism or a part thereof,
as mentioned.
[13070] [0022.1.0.29]: to [0023.0.0.29]: see [0022.1.0.27] to
[0023.0.0.27]
[13071] [0023.1.29.29] Homologs of the polypeptide disclosed in
table XII, application no. 29, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 29, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 29, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 29,
column 7, resp.
[13072] [0024.0.0.29]: see [0024.0.0.27]
[13073] [0025.0.29.29] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 29, column 3 "if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 29, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[13074] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 29,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 29,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[13075] [0025.1.0.29]: see [0025.1.0.27]
[13076] [0025.2.0.29]: see [0025.2.0.27]
[13077] [0026.0.0.29] to [0033.0.0.29]: see [0026.0.0.27] to
[0033.0.0.27]
[13078] [0034.0.29.29] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 29, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[13079] [0035.0.0.29] to [0044.0.0.29]: see [0035.0.0.27] to
[0044.0.0.27]
[13080] [0045.0.29.29] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
29, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 29, column 6 of
the respective line, between 10% and 50%, 10% and 100%, 10% and
200%, 10% and 300%, 10% and 400%, 10% and 500%, 20% and 50%, 20%
and 250%, 20% and 500%, 50% and 100%, 50% and 250%, 50% and 500%,
500% and 1000% or more
[13081] [0046.0.29.29] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
29, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 29, column 6 of
the respective line confers an increase of the respective fine
chemical and of further amino acid or their precursors.
[13082] [0047.0.0.29] and [0048.0.0.29]: see [0047.0.0.27] and
[0048.0.0.27]
[13083] [0049.0.29.29] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 29, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 29, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 29, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[13084] [0050.0.29.29] For the purposes of the present invention,
the term "tryptophane" and "L-tryptophane" also encompass the
corresponding salts, such as, for example, tryptophane
hydrochloride or tryptophane sulfate. Preferably the term
tryptophane is intended to encompass the term L-tryptophane.
[13085] [0051.0.0.29] and [0052.0.0.29]: see [0051.0.0.27] and
[0052.0.0.27]
[13086] [0053.0.29.29] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[13087] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 29, columns 5 and 7 or its homologs activity
having herein-mentioned amino acid of the invention increasing
activity; and/or [13088] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 29,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 29, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned amino acid of the invention increasing
activity; and/or [13089] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned amino acid increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 29, columns 5 and 7 or its
homologs activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [13090] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned amino acid of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 29, columns 5
and 7 or its homologs activity; and/or [13091] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned amino acid of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 29, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [13092] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned amino acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 29, columns 5 and 7 or its
homologs activity, and/or [13093] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned amino acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 29, columns 5 and 7 or its
homologs activity; and/or [13094] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 29, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[13095] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [13096] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[13097] [0054.0.29.29] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 29 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 29, column 3 or its homologs.
[13098] [0055.0.0.29] to [0071.0.0.29]: see [0055.0.0.27] to
[0071.0.0.27]
[13099] [0072.0.29.29] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to tryptophane chorismic acid, anthralinic acid,
N-5'-Phosphoribosyl-anthranilate,
1-(o-Carboxyphenylamino)-1-deoxyribulose 5-phosphate.,
1-(Indol-3-yl)-glycerin-3-phosphate, and 5-hydroxytrytophane.
[13100] [0073.0.29.29] Accordingly, in one embodiment, the process
according to the invention relates to a process which comprises:
[13101] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [13102] b) increasing an activity of a
polypeptide of the invention or the polypeptide used in the method
of the invention or a homolog thereof, e.g. as indicated in Table
XII, application no. 29, columns 5 or 7, or of a polypeptide being
encoded by the nucleic acid molecule of the present invention and
described below, e.g. conferring an increase of the respective fine
chemical in the organism, preferably in a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant, [13103] c) growing the organism, preferably the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue or the plant under conditions which permit
the production of the respective fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and [13104] d) if desired, recovering, optionally
isolating, the free and/or bound the fine chemical and, optionally
further free and/or bound amino acids synthetized by the organism,
the microorganism, the non-human animal, the plant or animal cell,
the plant or animal tissue or the plant.
[13105] [0074.0.0.29] to [0084.0.0.29]: see [0074.0.0.27] to
[0084.0.0.27]
[13106] [0085.0.29.29] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [13107] a) a nucleic acid sequence as
indicated in Table XI, application no. 29, columns 5 or 7, a
derivative thereof, or [13108] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 29, columns
5 or 7, or a derivative thereof, or [13109] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[13110] [0086.0.0.29] and [0087.0.0.29]: see [0086.0.0.27] and
[0087.0.0.27]
[13111] [0088.0.29.29] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose amino acid
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for monogastric animals is limited by a few essential amino acids
such as lysine, threonine or methionine or tryptophane.
[13112] [0088.1.0.29] to [0097.0.0.29]: see [0088.1.0.27] to
[0097.0.0.27]
[13113] [0098.0.29.29] In a preferred embodiment, the fine chemical
(tryptophane) is produced in accordance with the invention and, if
desired, is isolated. The production of further amino acids such as
methionine, lysine and/or threonine mixtures of amino acid by the
process according to the invention is advantageous.
[13114] [0099.0.0.29] to [0102.0.0.29]: see [0099.0.0.27] to
[0102.0.0.27]
[13115] [0103.0.29.29] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[13116] (a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide having a sequence as indicated in
Table XII, application no. 29, columns 5 or 7; [13117] (b) nucleic
acid molecule comprising, preferably at least the mature form, of a
nucleic acid molecule having a sequence as indicated in Table XI,
application no. 29, columns 5 or 7; [13118] (c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[13119] (d) nucleic acid molecule encoding a polypeptide which has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13120] (e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under r
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[13121] (f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c) and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [13122] (g)
nucleic acid molecule encoding a fragment or an epitope of a
polypeptide which is encoded by one of the nucleic acid molecules
of (a) to (e), preferably to (a) to (c) and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; [13123] (h) nucleic acid molecule comprising a nucleic
acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers pairs having a sequence as indicated in Table XIII,
application no. 29, columns 7, and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[13124] (i) nucleic acid molecule encoding a polypeptide which is
isolated, e.g. from an expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (h), preferably to (a) to (c), and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13125] (j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence having a
sequences as indicated in Table XIV, application no. 29, column 7,
and conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13126] (k) nucleic acid molecule
comprising one or more of the nucleic acid molecule encoding the
amino acid sequence of a polypeptide encoding a domain of a
polypeptide indicated in Table XII, application no. 29, columns 5
or 7, and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; and [13127] (l) nucleic acid
molecule which is obtainable by screening a suitable library under
stringent conditions with a probe comprising one of the sequences
of the nucleic acid molecule of (a) to (k), preferably to (a) to
(c), or with a fragment of at least 15 nt, preferably 20 nt, 30 nt,
50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k), preferably to (a) to (c), and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; or which comprises a sequence which is
complementary thereto.
[13128] [00103.1.29.29.] In one embodiment, the nucleic acid
molecule used in the process of the invention distinguishes over
the sequence indicated in Table XI, application no. 29, columns 5
or 7, by one or more nucleotides. In one embodiment, the nucleic
acid molecule used in the process of the invention does not consist
of the sequence indicated in Table XI, application no. 29, columns
5 or 7: In one embodiment, the nucleic acid molecule used in the
process of the invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence indicated in Table XI, application
no. 29, columns 5 or 7. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table XII, application no. 29, columns 5 or 7.
[13129] [00103.2.29.29] ./.
[13130] [0104.0.29.29] In one embodiment, the nucleic acid molecule
of the invention or used in the process of the invention
distinguishes over the sequence indicated in Table XI, application
no. 29, columns 5 or 7, by one or more nucleotides. In one
embodiment, the nucleic acid molecule of the present invention or
used in the process of the invention does not consist of the
sequence n indicated in Table XI, application no. 29, columns 5 or
7: In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to a sequence indicated in Table XI, application no. 29,
columns 5 or 7. In another embodiment, the nucleic acid molecule
does not encode a polypeptide of a sequence indicated in Table XI,
application no. 29, columns 5 or 7.
[13131] [0105.0.0.29] to [0107.0.0.29]: see [0105.0.0.27] to
[0107.0.0.27]
[13132] [0108.0.29.29] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 29, columns 5 or 7, nucleic
acid molecules which are derived from a amino acid sequences as
indicated in Table XII, application no. 29, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 29, column 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of a polypeptide as indicated in Table XII, application
no. 29, column 3, 5 or 7, or e.g. conferring a increase of the fine
chemical after increasing its expression or activity are
advantageously increased in the process according to the
invention.
[13133] [0109.0.0.29]: see [0109.0.0.27]
[13134] [0110.0.29.29] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
used in the process of the invention, e.g. of a protein as
indicated in Table XII, application no. 29, column 5, or being
encoded by a nucleic acid molecule indicated in Table XI,
application no. 29, column 5, or of its homologs, e.g. as indicated
in Table XII, application no. 29, column 7, can be determined from
generally accessible databases.
[13135] [0111.0.0.29]: see [0111.0.0.27]
[13136] [0112.0.29.29] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XII, application no. 29,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 29, columns 5 and 7, and conferring an
tryptophane increase.
[13137] [0113.0.0.29] to [120.0.0.29]: see [0113.0.0.27] to
[0120.0.0.27]
[13138] [0121.0.29.29] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 29, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a increase of the fine chemical after
increasing its activity.
[13139] [0122.0.0.29] to [0127.0.0.29]: see [0122.0.0.27] to
[0127.0.0.27]
[13140] [0128.0.29.29] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 29, column 7, by means of polymerase chain reaction
can be generated on the basis of a sequence as indicated in Table
XI, application no. 29, columns 5 or 7, or the sequences derived
from sequences as indicated in Table XII, application no. 29,
columns 5 or 7.
[13141] [0129.0.29.29] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention or the polypeptide used in the method of the invention,
from which conserved regions, and in turn, degenerate primers can
be derived. Conserved region for the polypeptide of the invention
or the polypeptide used in the method of the invention are
indicated in the alignments shown in the figures. Conserved regions
are those, which show a very little variation in the amino acid in
one particular position of several homologs from different origin.
The consensus sequences indicated in Table XIV, application no. 29,
column 7, are derived from said alignments.
[13142] [0130.0.0.29] to [0138.0.0.29]: see [0130.0.0.27] to
[0138.0.0.27]
[13143] [0139.0.29.29] Polypeptides having above-mentioned
activity, i.e. conferring a tryptophane increase, derived from
other organisms, can be encoded by other DNA sequences which
hybridize to a sequences indicated in Table XI, application no. 29,
columns 5 or 7, under relaxed hybridization conditions and which
code on expression for peptides having the tryptophane increasing
activity.
[13144] [0140.0.0.29] to [0146.0.0.29]: see [0140.0.0.27] to
[0146.0.0.27]
[13145] [0147.0.29.29] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 29, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences such
that it can hybridize to one of said nucleotide sequences thereby
forming a stable duplex. Preferably, the hybridisation is performed
under stringent hybridization conditions. However, a complement of
one of the herein disclosed sequences is preferably a sequence
complement thereto according to the base pairing of nucleic acid
molecules well known to the skilled person. For example, the bases
A and G undergo base pairing with the bases T and U or C, resp. and
visa versa. Modifications of the bases can influence the
base-pairing partner.
[13146] [0148.0.29.29] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 29,
columns 5 or 7, or a functional portion thereof and preferably has
above mentioned activity, in particular has
the--fine-chemical-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homologs.
[13147] [0149.0.29.29] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 29,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity and as indicated in indicated in Table
XII, application no. 29, columns 5 or 7, e.g. conferring an
increase of the fine chemical.
[13148] [0149.1.29.29] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table
XI, application no. 29, columns 5 or 7, has further one or more of
the activities annotated or known for a protein as indicated in
Table XII, application no. 29, column 3.
[13149] [0150.0.29.29] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table XI, application no. 29, columns 5 or 7, for example a
fragment which can be used as a probe or primer or a fragment
encoding a biologically active portion of the polypeptide of the
present invention or of a polypeptide used in the process of the
present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of tryptophane if its activity is increased.
The nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences indicated in Table XI, application no. 29, columns
5 or 7, an anti-sense sequence of one of the sequences indicated in
Table XI, application no. 29, columns 5 or 7, or naturally
occurring mutants thereof. Primers based on a nucleotide sequence
of the invention can be used in PCR reactions to clone homologues
of the polypeptide of the invention or of the polypeptide used in
the process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primer pairs indicated in Table XIII, application no.
29, column 7, will result in a fragment of a polynucleotide
sequence as indicated in Table XI, application no. 29, columns 5 or
7.
[13150] [0151.0.0.29]: [see 0151.0.0.27]
[13151] [0152.0.29.29] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 29, columns 5
or 7, such that the protein or portion thereof maintains the
ability to participate in tryptophane production, in particular a
tryptophane increasing activity as mentioned above or as described
in the examples in plants or microorganisms is comprised.
[13152] [0153.0.29.29] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 29,
columns 5 or 7, such that the protein or portion thereof is able to
participate in the increase of tryptophane production. In one
embodiment, a protein or portion thereof as indicated in Table XII,
application no. 29, columns 5 or 7, has for example an activity of
a polypeptide indicated in Table XII, application no. 29, column
3.
[13153] [0154.0.29.29] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 29, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the fine chemical.
[13154] [0155.0.0.29] and [0156.0.0.29]: see [0155.0.0.27] to
[0156.0.0.27]
[13155] [0157.0.29.29] The invention further relates to nucleic
acid molecules that differ from one of a nucleotide sequences as
indicated in Table XI, application no. 29, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in tryptophane in a organism, e.g. as that polypeptides
comprising the consensus sequences as indicated in Table XIV,
application no. 29, columns 7, or of the polypeptide as indicated
in Table XII, application no. 29, columns 5 or 7, or their
functional homologues. Advantageously, the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, a consensus sequences as indicated in Table XIV,
application no. 29, columns 7, or of the polypeptide as indicated
in Table XII, application no. 29, columns 5 or 7, or the functional
homologues. In a still further embodiment, the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention encodes a full length protein which is
substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table XIV, application no. 29,
column 7, or of a polypeptide as indicated in Table XII,
application no. 29, columns 5 or 7, or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 29, columns 5 or 7.
Preferably the nucleic acid molecule of the invention is a
functional homologue or identical to a nucleic acid molecule
indicated in Table XI, application no. 29, columns 5 or 7.
[13156] [0158.0.0.29] to [0160.0.0.29]: see [0158.0.0.27] to
[0160.0.0.27]
[13157] [0161.0.29.29] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 29, columns 5 or
7. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[13158] [0162.0.0.29]: see [0162.0.0.27]
[13159] [0163.0.29.29] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 29, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the fine chemical
increase after increasing the expression or activity thereof or the
activity of a protein of the invention or used in the process of
the invention.
[13160] [0164.0.0.29]: see [0164.0.0.27]
[13161] [0165.0.29.29] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, columns 5 or 7, lines 16 to 18 and/or lines
356 to 362.
[13162] [0166.0.0.29] and [0167.0.0.29]: see [0166.0.0.27] and
[0167.0.0.27]
[13163] [0168.0.29.29] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the fine
chemical in an organisms or parts thereof that contain changes in
amino acid residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in as sequence as indicated in Table XII, application no.
29, columns 5 or 7, yet retain said activity described herein. The
nucleic acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table XII, application no. 29, columns 5 or 7, and is
capable of participation in the increase of production of the fine
chemical after increasing its activity, e.g. its expression.
Preferably, the protein encoded by the nucleic acid molecule is at
least about 60% identical to a sequence as indicated in Table XII,
application no. 29, columns 5 or 7, more preferably at least about
70% identical to one of the sequences as indicated in Table XII,
application no. 29, columns 5 or 7, even more preferably at least
about 80%, 90% or 95% homologous to a sequence as indicated in
Table XII, application no. 29, columns 5 or 7, and most preferably
at least about 96%, 97%, 98%, or 99% identical to the sequence as
indicated in Table XII, application no. 29, columns 5 or 7.
[13164] [0169.0.0.29] to [0172.0.0.29]: see [0169.0.0.27] to
[0172.0.0.27]
[13165] [0173.0.29.29] For example a sequence which has a 80%
homology with sequence SEQ ID No: 108346 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID No: 108346 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[13166] [0174.0.0.29]: see [0174.0.0.27]
[13167] [0175.0.29.29] For example a sequence which has a 80%
homology with sequence SEQ ID No: 108347 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID No: 108347 by the above program algorithm with the
above parameter set, has a 80% homology.
[13168] [0176.0.29.29] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 29,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 29, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 29, columns
5 or 7.
[13169] [0177.0.29.29] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 29,
columns 5 or 7 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptides as indicated in Table XII,
application no. 29, columns 5 or 7, according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 29, columns
5 or 7.
[13170] [0178.0.0.29]: see [0178.0.0.27]
[13171] [0179.0.29.29] A nucleic acid molecule encoding an
homologous to a protein sequence of as indicated in Table XII,
application no. 29, columns 5 or 7, can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table XI, application no.
29, columns 5 or 7, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences of a
sequences as indicated in Table XI, application no. 29, columns 5
or 7, by standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[13172] [0180.0.0.29] to [0183.0.0.29]: see [0180.0.0.27] to
[0183.0.0.27]
[13173] [0184.0.29.29] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 29,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 29, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 29,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[13174] [0185.0.29.29] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 29, columns 5 or 7. In one embodiment it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotide sequences not shown in any one of
sequences as indicated in Table XI, application no. 29, columns 5
or 7. In one embodiment, the nucleic acid molecule comprises less
than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or 40 further
nucleotides. In a further embodiment, the nucleic acid molecule
comprises less than 30, 20 or 10 further nucleotides. In one
embodiment, a nucleic acid molecule use in the process of the
invention is identical to a sequences as indicated in Table XI,
application no. 29, columns 5 or 7.
[13175] [0186.0.29.29] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table XII,
application no. 29, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 29, columns 5 or 7.
[13176] [0187.0.29.29] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table XII, application no.
29, columns 5 or 7, and comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a sequences as indicated in Table XII,
application no. 29, columns 5 or 7.
[13177] [0188.0.29.29] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide as indicated in Table XII, application no. 29,
columns 5 or 7, preferably compared to a sequence as indicated in
Table XII, application no. 29, column 5, and expressed under
identical conditions.
[13178] [0189.0.29.29] Homologues of a sequences as indicated in
Table XI, application no. 29, columns 5 or 7, or of a derived
sequences as indicated in Table XII, application no. 29, columns 5
or 7, also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives which comprise
noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[13179] [0190.0.0.29] to [0203.0.0.29]: see [0190.0.0.27] to
[0203.0.0.27]
[13180] [0204.0.29.29] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule which comprises a
nucleic acid molecule selected from the group consisting of:
[13181] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 29, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the fine chemical in an
organism or a part thereof [13182] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule as indicated in Table XII, application no. 29, columns 5
or 7, or a fragment thereof conferring an increase in the amount of
the fine chemical in an organism or a part thereof; [13183] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [13184] d) nucleic acid molecule encoding a
polypeptide whose sequence has at least 50% identity with the amino
acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the fine chemical in an organism or a part thereof; [13185] e)
nucleic acid molecule which hybridizes with a nucleic acid molecule
of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13186] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [13187] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13188] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table XIII, application no. 29, column 7, and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13189] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from a expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[13190] j) nucleic acid molecule which encodes a polypeptide
comprising the consensus sequence as indicated in Table XIV,
application no. 29, column 7, and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[13191] k) nucleic acid molecule encoding the amino acid sequence
of a polypeptide encoding a domain of a polypeptide as indicated in
Table XII, application no. 29, columns 5 or 7, and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; and [13192] l) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of a nucleic acid molecule as indicated in Table XI,
application no. 29, columns 5 or 7, or a nucleic acid molecule
encoding, preferably at least the mature form of, the polypeptide
as indicated in Table XII, application no. 29, columns 5 or 7, and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; or which encompasses a sequence which
is complementary thereto; whereby, preferably, the nucleic acid
molecule according to (a) to (l) distinguishes over the sequence
indicated in Table XI, application no. 29, columns 5 or 7, by one
or more nucleotides. In one embodiment, the nucleic acid molecule
does not consist of the sequence shown and in indicated in Table
XI, application no. 29, columns 5 or 7: In one embodiment, the
nucleic acid molecule is less than 100%, 99.999%, 99.99%, 99.9% or
99% identical to a sequence indicated in Table XI, application no.
29, columns 5 or 7. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table XII, application no. 29, columns 5 or 7. In an other
embodiment, the nucleic acid molecule of the present invention is
at least 30%, 40%, 50%, or 60% identical and less than 100%,
99.999%, 99.99%, 99.9% or 99% identical to a sequence indicated in
Table XI, application no. 29, columns 5 or 7. In a further
embodiment the nucleic acid molecule does not encode a polypeptide
sequence as indicated in Table XII, application no. 29, columns 5
or 7. Accordingly, in one embodiment, the nucleic acid molecule of
the differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 29, columns 5 or 7.
Accordingly, in one embodiment, the nucleic acid molecule of the
present invention encodes a polypeptide, which differs at least in
one or more amino acids from a polypeptide indicated in Table XII,
application no. 29, columns 5 or 7. In another embodiment, a
nucleic acid molecule indicated in Table XI, application no. 29,
columns 5 or 7, does not encode a protein of a sequence indicated
in Table XII, application no. 29, columns 5 or 7. Accordingly, in
one embodiment, the protein encoded by a sequences of a nucleic
acid according to (a) to (l) does not consist of a sequence as
indicated in Table XII, application no. 29, columns 5 or 7. In a
further embodiment, the protein of the present invention is at
least 30%, 40%, 50%, or 60% identical to a protein sequence
indicated in Table XII, application no. 29, columns 5 or 7, and
less than 100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 985, 97%, 96% or 95% identical to a
sequence as indicated in Table XI, application no. 29, columns 5 or
7.
[13193] [0205.0.0.29] to [0226.0.0.29]: see [0205.0.0.27] to
[0226.0.0.27]
[13194] [0227.0.29.29] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorgansim.
[13195] In addition to a sequence as indicated in Table XI,
application no. 29, columns 5 or 7, or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the amino acid biosynthetic pathway such as for
Llysine, L-threonine and/or L-methionine or L-tryptophane is
expressed in the organisms such as plants or microorganisms. It is
also possible that the regulation of the natural genes has been
modified advantageously so that the gene and/or its gene product is
no longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all.
[13196] In addition it might be advantageously to combine a
sequences as indicated in Table
[13197] XI, application no. 29, columns 5 or 7, with genes which
generally support or enhances to growth or yield of the target
organismn, for example genes which lead to faster growth rate of
microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[13198] [0228.0.0.29] to [0230.0.0.29]: see [0228.0.0.27] to
[0230.0.0.27]
[13199] [0231.0.0.29] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a tryptophane degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[13200] [0232.0.0.29] to [0282.0.0.29]: see [0232.0.0.27] to
[0282.0.0.27]
[13201] [0283.0.29.29] Moreover, a native polypeptide conferring
the increase of the fine chemical in an organism or part thereof
can be isolated from cells (e.g., endothelial cells), for example
using the antibody of the present invention as described below, in
particular, an antibody against a protein as indicated in Table
XII, application no. 29, column 3, E.g. an antibody against a
polypeptide as indicated in Table XII, application no. 29, columns
5 or 7, which can be produced by standard techniques utilizing
polypeptides comprising or consisting of above mentioned sequences,
e.g. the polypeptide of the present invention or fragment thereof,
Preferred are monoclonal antibodies, specifically binding to
polypeptide as indicated in Table XII, application no. 29, columns
5 or 7.
[13202] [0284.0.0.29] see [0284.0.0.27]
[13203] [0285.0.29.29] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 29, columns 5 or 7, or as encoded by a nucleic
acid molecule as indicated in Table XI, application no. 29, columns
5 or 7, or functional homologues thereof.
[13204] [0286.0.29.29] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 29, column 7, and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 29, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences as indicated in Table XIV, application no.
29, column 7
amino acidamino acid
[13205] [0287.0.0.29] to [290.0.0.29]: see [0287.0.0.27] to
[0290.0.0.27]
[13206] [0291.0.29.29] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[13207] Accordingly, in one embodiment, the present invention
relates to a polypeptide comprising or consisting of plant or
microorganism specific consensus sequences. In one embodiment, said
polypeptide of the invention distinguishes over a sequence as
indicated in Table XII, application no. 29, columns 5 or 7, by one
or more amino acids. In one embodiment, polypeptide distinguishes
form a sequence as indicated in Table XII, application no. 29,
columns 5 or 7 by more than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino
acids, preferably by more than 10, 15, 20, 25 or 30 amino acids,
even more preferred are more than 40, 50, or 60 amino acids and,
preferably, the sequence of the polypeptide of the invention
distinguishes from a sequence as indicated in Table XII,
application no. 29, columns 5 or 7, by not more than 80% or 70% of
the amino acids, preferably not more than 60% or 50%, more
preferred not more than 40% or 30%, even more preferred not more
than 20% or 10%. In an other embodiment, said polypeptide of the
invention does not consist of a sequence as indicated in Table XII,
application no. 29, columns 5 or 7.
[13208] [0292.0.0.29]: see [0292.0.0.27]
[13209] [0293.0.29.29] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the process
of the invention.
[13210] In one embodiment, the polypeptide of the invention has a
sequence which distinguishes from a sequence as indicated in Table
XII, application no. 29, columns 5 or 7, by one or more amino
acids. In an other embodiment, said polypeptide of the invention
does not consist of the sequence as indicated in Table XII,
application no. 29, columns 5 or 7. In a further embodiment, said
polypeptide of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical. In one embodiment, said polypeptide
does not consist of the sequence encoded by a nucleic acid
molecules as indicated in Table XI, application no. 29, columns 5
or 7.
[13211] [0294.0.29.29] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 29, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 29, columns 5 or 7, to 362 by one or more amino
acids, preferably by more than 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, even more
preferred are more than 40, 50, or 60 amino acids but even more
preferred by less than 70% of the amino acids, more preferred by
less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[13212] [0295.0.0.29] to [0297.0.0.29]: see [0295.0.0.27] to
[0297.0.0.27]
[13213] [0297.1.29.29] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity and/or amino acid
sequence of a polypeptide indicated in Table XII, application no.
29, columns 3, 5 or 7.
[13214] [0298.0.29.29] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 29, columns 5 or 7, such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table XII, application no. 29, columns 5 or 7.
[13215] [0299.0.29.29] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 29,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 29, columns 5 or 7, or which is
homologous thereto, as defined above.
[13216] [0300.0.29.29] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table XII,
application no. 29, columns 5 or 7, in amino acid sequence due to
natural variation or mutagenesis, as described in detail
herein.
[13217] Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of a sequence as indicated in Table
XII, application no. 29, columns 5 or 7.
[13218] [0301.0.0.29]: see [0301.0.0.27]
[13219] [0302.0.29.29] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 29, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[13220] [0303.0.0.29]: see [0303.0.0.27]
[13221] [0304.0.29.29] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 29, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[13222] [0305.0.0.29] to [0306.0.0.29]: see [0305.0.0.27] to
[0306.0.0.27]
[13223] [00306.1.29.29] Preferrably, the compound is a composition
comrising the tryptophane or a recovered tryptophane, in
particular, the fine chemical, free or in protein-bound form.
[13224] [0307.0.0.29]: to [0308.0.0.29]: see [0307.0.0.27] to
[0308.0.0.27]
[13225] [0309.0.29.29] In one embodiment, a reference to a "protein
(=polypeptide)" of the invention or as indicated in Table XII,
application no. 29, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide"--not
being indicated in Table XII, application no. 29, columns 5 or 7,
refers to a polypeptide having an amino acid sequence corresponding
to a protein which is not substantially homologous a polypeptide of
the invention, preferably which is not substantially homologous to
a as indicated in Table XII, application no. 29, columns 5 or 7,
e.g., a protein which does not confer the activity described herein
or annotated or known for as indicated in Table XII, application
no.
[13226] 29, column 3, and which is derived from the same or a
different organism. In one embodiment a "non-polypeptide of the
invention" or "other polypeptide" not being indicate in Table XII,
application no. 29, columns 5 or 7, does not confer an increase of
the fine chemical in an organism or part thereof.
[13227] [0310.0.0.29] to [334.0.0.29]: see [0310.0.0.27] to
[0334.0.0.27]
[13228] [0335.0.29.29] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XII, application
no. 29, columns 5 or 7, and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
application no. 29, columns 5 or 7, and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of an
protein encoded by a nucleic acid sequence of one of the sequences
as indicated in Table XI, application no. 29, columns 5 or 7,
and/or homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[13229] [0336.0.0.29] to [0342.0.0.29]: see [0336.0.0.27] to
[0342.0.0.27]
[13230] [0343.0.29.29] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 29, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table XI, application no. 29, columns 5 or 7, or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[13231] [0344.0.0.29] to [0361.0.0.29]: see [0344.0.0.27] to
[0361.0.0.27]
[13232] [0362.0.29.29] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the fine chemical in
a cell or an organism or a part thereof, e.g. the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention, the nucleic acid construct of the
invention, the antisense molecule of the invention, the vector of
the invention or a nucleic acid molecule encoding the polypeptide
of the invention, e.g. the polypeptide as indicated in Table XII,
application no. 29, columns 5 or 7. Due to the above mentioned
activity the fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity of the polypeptide of the invention or the
polypeptide used in the method of the invention or nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention is increased, e.g. due to an increased
expression or specific activity of the subject matters of the
invention in a cell or an organism or a part thereof. In one
embodiment transgenic for a polypeptide having an activity of a
polypeptide as indicated in Table XII, application no. 29, columns
5 or 7, means herein that due to modulation or manipulation of the
genome, an activity as annotated for a polypeptide as indicated in
Table XII, application no. 29, columns 3, e.g. having a sequence as
indicated in Table XII, application no. 29, columns 5 or 7, is
increased in a cell or an organism or a part thereof. Examples are
described above in context with the process of the invention.
[13233] [0363.0.0.29] to [0382.0.0.29]: see [0363.0.0.27] to
[0382.0.0.27]
[13234] [0383.0.29.29] For preparing aromatic compound-containing
fine chemicals, in particular the fine chemical, it is possible to
use as aromat source organic aromatic-containing compounds such as,
for example, benzene, naphthaline, indole, pyrrole, furen, oxazole,
imidazole, thiophene, pyrridin, pyrrimidine or else organic
aromatic compounds such as benzoic acid and chorismic, shikimic,
aminobenzoic, kynurenic acids or pyridoxidal.
[13235] [0384.0.0.29]: see [0384.0.0.27]
[13236] [0385.0.29.29] The fermentation broths obtained in this
way, containing in particular
[13237] L-tryptophane, L-methionine, L-threonine and/or L-lysine,
normally have a dry matter content of from 7.5 to 25% by weight.
Sugar-limited fermentation is additionally advantageous, at least
at the end, but especially over at least 30% of the fermentation
time. This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 3 g/l during
this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed entirely or
partly by separation methods, such as, for example, centrifugation,
filtration, decantation or a combination of these methods, from the
fermentation broth or left completely in it. The fermentation broth
can then be thickened or concentrated by known methods, such as,
for example, with the aid of a rotary evaporator, thin-film
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. This concentrated fermentation broth can then be
worked up by freeze-drying, spray drying, spray granulation or by
other processes.
[13238] [0386.0.0.29] to [392.0.0.29]: see [0386.0.0.27] to
[0392.0.0.27]
[13239] [0393.0.29.29] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [13240] (a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [13241] (b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table XI, application no. 29, columns 5 or 7, and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[13242] (c) introducing the candidate nucleic acid molecules in
host cells, preferably in a plant cell or a microorganism,
appropriate for producing the fine chemical; [13243] (d) expressing
the identified nucleic acid molecules in the host cells; [13244]
(e) assaying the fine chemical level in the host cells; and [13245]
(f) identifying the nucleic acid molecule and its gene product
which expression confers an increase in the fine chemical level in
the host cell after expression compared to the wild type.
[13246] [0394.0.0.29] to [0399.0.0.29]: see [0394.0.0.27] to
[0399.0.0.27]
[13247] [0399.1.29.29] One can think to screen for increased
production of the fine chemical by for example searching for a
resistance to a drug blocking the synthesis of the fine chemical
and looking whether this effect is dependent on the activity or
expression of a polypeptide as indicated in Table XII, application
no. 29, columns 5 or 7, or a homolog thereof, e.g. comparing the
phenotype of nearly identical organisms with low and high activity
of a protein as indicated in Table XII, application no. 29, columns
5 or 7, after incubation with the drug.
[13248] [0400.0.0.29] to [0416.0.0.29]: see [0400.0.0.27] to
[0416.0.0.27]
[13249] [0417.0.29.29] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the amino acid production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect plants against herbicides, which
block the amino acid, in particular the fine chemical, synthesis in
said plant. Examples of herbicides blocking the amino acid
synthesis in plants are for example sulfonylurea and imidazolinone
herbicides which catalyze the first step in branched-chain amino
acid biosynthesis.
[13250] [0418.0.0.29] to [0423.0.0.29]: see [0418.0.0.27] to
[0423.0.0.27]
[13251] [0424.0.29.29] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the fine chemical or of the fine
chemical and one or more other amino acids, in particular
methionine, threonine, alanine, glutamine, glutamic acid, valine,
asparagine, phenylalanine, leucine, proline, Tryptophan tyrosine,
isoleucine and arginine.
[13252] Accordingly, the nucleic acid of the invention, or the
nucleic acid molecule identified with the method of the present
invention or the complement sequences thereof, the polypeptide of
the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the fine chemical in a organism or part thereof, e.g.
in a cell.
[13253] [0425.0.0.29] to [0430.0.0.29]: see [0425.0.0.27] to
[0430.0.0.27]
[13254] [0431.0.0.29] to [0460.0.0.29]: see [0431.0.0.27] to
[0460.0.0.27]
[0461.0.29.29] Example 10
Cloning SEQ ID NO: 108346 for the Expression in Plants
[13255] [0462.0.0.29] see [0462.0.0.27]
[13256] [0463.0.29.29] SEQ ID NO: 108346 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[13257] [0464.0.0.29] to [0466.0.0.29]: see [0464.0.0.27] to
[0466.0.0.27]
[13258] [0467.0.29.29] The following primer sequences were selected
for the gene SEQ ID No: 108346:
[13259] i) forward primer SEQ ID No: 108348
[13260] ii) reverse primer SEQ ID No: 108349
[13261] [0468.0.0.29] to [0479.0.0.29]: see [0468.0.0.27] to
[0479.0.0.27]
[0480.0.29.29] Example 11
Generation of Transgenic Plants which Express SEQ ID No: 108346
[13262] [0481.0.0.29] to [0513.0.0.29]: see [0481.0.0.27] to
[0513.0.0.27]
[13263] [0514.0.29.29] As an alternative, the amino acids can be
detected advantageously via HPLC separation in ethanolic extract as
described by Geigenberger et al. (Plant Cell & Environ, 19,
1996: 43-55).
[13264] [0515.0.0.29] to [0552.0.0.29]: see [0515.0.0.27] to
[0552.0.0.27]
[13265] [0553.0.29.29]
1. A process for the production of tryptophane, which comprises
[13266] (a) increasing or generating the activity of a protein as
indicated in Table XII, application no. 29, columns 5 or 7, or a
functional equivalent thereof in a non-human organism, or in one or
more parts thereof; and [13267] (b) growing the organism under
conditions which permit the production of tryptophane in said
organism. 2. A process for the production of tryptophane,
comprising the increasing or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [13268] (a) nucleic acid molecule encoding of a
polypeptide as indicated in Table XII, application no. 29, columns
5 or 7, or a fragment thereof, which confers an increase in the
amount of tryptophane in an organism or a part thereof; [13269] (b)
nucleic acid molecule comprising of the nucleic acid molecule as
indicated in Table XI, application no. 29, columns 5 or 7; [13270]
(c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of tryptophane in an organism
or a part thereof; [13271] (d) nucleic acid molecule which encodes
a polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of tryptophane
in an organism or a part thereof; [13272] (e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of tryptophane in an organism or a part thereof; [13273]
(f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table XIII, application no. 29, column 7, and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13274] (g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
tryptophane in an organism or a part thereof; [13275] (h) nucleic
acid molecule encoding a polypeptide comprising a consensus
sequence as indicated in Table XIV, application no. 29, column 7,
and conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13276] (i) nucleic acid molecule which
is obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof. or
comprising a sequence which is complementary thereto. 3. The
process of claim 1 or 2, comprising recovering of the free or bound
tryptophane. 4. The process of any one of claims 1 to 3, comprising
the following steps: [13277] (a) selecting an organism or a part
thereof expressing a polypeptide encoded by the nucleic acid
molecule characterized in claim 2; [13278] (b) mutagenizing the
selected organism or the part thereof; [13279] (c) comparing the
activity or the expression level of said polypeptide in the
mutagenized organism or the part thereof with the activity or the
expression of said polypeptide of the selected organisms or the
part thereof; [13280] (d) selecting the mutated organisms or parts
thereof, which comprise an increased activity or expression level
of said polypeptide compared to the selected organism or the part
thereof; [13281] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [13282] (f) recovering, and
optionally isolating, the free or bound tryptophane produced by the
selected mutated organisms or parts thereof. 5. The process of any
one of claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [13283] (a) nucleic acid molecule encoding of a
polypeptide as indicated in Table XII, application no. 29, columns
5 or 7, or a fragment thereof, which confers an increase in the
amount of tryptophane in an organism or a part thereof; [13284] (b)
nucleic acid molecule comprising of a nucleic acid as indicated in
Table XI, application no. 29, columns 5 or 7; [13285] (c) nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as a
result of the degeneracy of the genetic code and conferring an
increase in the amount of tryptophane in an organism or a part
thereof; [13286] (d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of tryptophane
in an organism or a part thereof; [13287] (e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
under stringent hybridisation conditions and conferring an increase
in the amount of tryptophane in an organism or a part thereof;
[13288] (f) nucleic acid molecule which encompasses a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers or
primer pairs as indicated in Table XIII, application no. 29, column
7, and conferring an increase in the amount of tryptophane in an
organism or a part thereof; [13289] (g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
tryptophane in an organism or a part thereof; [13290] (h) nucleic
acid molecule encoding a polypeptide comprising the consensus
sequence as indicated in Table XIV, application no. 29, column 7,
and conferring an increase in the amount of the fine chemical in an
organism or a part thereof; and [13291] (i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof.
whereby the nucleic acid molecule distinguishes over the sequence
as indicated in Table XI, application no. 29, columns 5 or 7, by
one or more nucleotides. 7. A nucleic acid construct which confers
the expression of the nucleic acid molecule of claim 6, comprising
one or more regulatory elements. 8. A vector comprising the nucleic
acid molecule as claimed in claim 6 or the nucleic acid construct
of claim 7. 9. The vector as claimed in claim 8, wherein the
nucleic acid molecule is in operable linkage with regulatory
sequences for the expression in a prokaryotic or eukaryotic, or in
a prokaryotic and eukaryotic host. 10. A host cell, which has been
transformed stably or transiently with the vector as claimed in
claim 9 or 10 or the nucleic acid molecule as claimed in claim 6 or
the nucleic acid construct of claim 7 or produced as described in
claim any one of claims 2 to 5. 11. The host cell of claim 10,
which is a transgenic host cell. 12. The host cell of claim 10 or
11, which is a plant cell, an animal cell, a microorganism, or a
yeast cell, a fungus cell, a prokaryotic cell, an eukaryotic cell
or an archaebacterium. 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. 14. A polypeptide produced by the
process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table XII,
application no. 29, columns 5 or 7, by one or more amino acids. 15.
An antibody, which binds specifically to the polypeptide as claimed
in claim 14. 16. A plant tissue, propagation material, harvested
material or a plant comprising the host cell as claimed in claim 12
which is plant cell or an Agrobacterium. 17. A method for screening
for agonists and antagonists of the activity of a polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of tryptophane in an organism or a part
thereof comprising: [13292] (a) contacting cells, tissues, plants
or microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of tryptophane in an organism or a part thereof with a
candidate compound or a sample comprising a plurality of compounds
under conditions which permit the expression the polypeptide;
[13293] (b) assaying the tryptophane level or the polypeptide
expression level in the cell, tissue, plant or microorganism or the
media the cell, tissue, plant or microorganisms is cultured or
maintained in; and [13294] (c) identifying a agonist or antagonist
by comparing the measured tryptophane level or polypeptide
expression level with a standard tryptophane or polypeptide
expression level measured in the absence of said candidate compound
or a sample comprising said plurality of compounds, whereby an
increased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an agonist and
a decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
18. A method for the identification of a compound conferring
increased tryptophane production in a plant or microorganism,
comprising the steps: [13295] a) culturing a plant cell or tissue
or microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of tryptophane in an organism or a part
thereof and a readout system capable of interacting with the
polypeptide under suitable conditions which permit the interaction
of the polypeptide with dais readout system in the presence of a
compound or a sample comprising a plurality of compounds and
capable of providing a detectable signal in response to the binding
of a compound to said polypeptide under conditions which permit the
expression of said readout system and of the polypeptide encoded by
the nucleic acid molecule of claim 6 conferring an increase in the
amount of tryptophane in an organism or a part thereof; [13296] b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. 19. A method for the identification of a gene
product conferring an increase in tryptophane production in a cell,
comprising the following steps: [13297] a) contacting the nucleic
acid molecules of a sample, which can contain a candidate gene
encoding a gene product conferring an increase in tryptophane after
expression with the nucleic acid molecule of claim 6; [13298] b)
identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; [13299] c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing tryptophane;
[13300] d) expressing the identified nucleic acid molecules in the
host cells; [13301] e) assaying the tryptophane level in the host
cells; and [13302] f) identifying nucleic acid molecule and its
gene product which expression confers an increase in the
tryptophane level in the host cell in the host cell after
expression compared to the wild type. 20. A method for the
identification of a gene product conferring an increase in
tryptophane production in a cell, comprising the following steps:
[13303] a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the tryptophane amount or level
in an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; [13304]
b) introducing the candidate nucleic acid molecules in host cells
appropriate for producing tryptophane; [13305] c) expressing the
identified nucleic acid molecules in the host cells; [13306] d)
assaying the tryptophane level in the host cells; and [13307] e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the tryptophane level in the host
cell after expression compared to the wild type. 21. A method for
the production of an agricultural composition comprising the steps
of the method of any one of claims 17 to 20 and formulating the
compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. 22. A composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of any
one of claim 8 or 9, an antagonist or agonist identified according
to claim 17, the compound of claim 18, the gene product of claim 19
or 20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. 23. Use of the nucleic acid molecule as claimed
in claim 6 for the identification of a nucleic acid molecule
conferring an increase of tryptophane after expression. 24. Use of
the polypeptide of claim 14 or the nucleic acid construct claim 7
or the gene product identified according to the method of claim 19
or 20 for identifying compounds capable of conferring a modulation
of tryptophane levels in an organism. 25. Food or feed composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 17, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20. 26. Use of the nucleic acid molecule of
claim 6, the polypeptide of claim 14, the nucleic acid construct of
claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the host cell of claim 10 to 12
or the gene product identified according to the method of claim 19
or 20 for the protection of a plant against a tryptophane synthesis
inhibiting herbicide.
[13308] [0554.0.0.29] Abstract: see [0554.0.0.27]:
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[13309] [0000.0.30.30] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and corresponding embodiments as
described herein as follows.
[13310] [0001.0.0.30] to [0008.0.0.30]: see [0001.0.0.27] to
[0008.0.0.27]
[13311] [0009.0.30.30] As described above, the essential amino
acids are necessary for humans and many mammals, for example for
livestock. The branched-chain amino acids (BCAA) leucine and valine
are among the nine dietary indispensable amino acids for humans.
BCAA accounts for 35-40% of the dietary indispensable amino acids
in body protein and 14% of the total amino acids in skeletal muscle
(Ferrando et al., (1995) Oral branched chain amino acids decrease
whole-body proteolysis. J. Parenter. Enteral Nutr. 19: 47-54.13).
They share a common membrane transport system and enzymes for their
transamination and irreversible oxidation (Block, K. P. (1989)
Interactions among leucine and valine with special reference to the
branched chain amino acid antagonism. In: Absorption and
Utilization of Amino Acids (Friedman, M., ed.), pp. 229-244, CRC
Press, Boca Raton, Fla. and Champe, P. C. & Harvey, R. A.
(1987) Amino acids: metabolism of carbon atoms. In: Biochemistry
(Champ, P. C. & Harvery, P. A., eds.), pp. 242-252, J. B.
Lippincott, Philadelphia, Pa.). Further, for patient suffering from
Maple Syrup Urine Disease (MSUD) a reduced uptake of those
branched-chain amino acids is essential.
[13312] Dietary sources of the branched-chain amino acids are
principally derived from animal and vegetable proteins. The
branched-chain amino acids (BCAA) leucine and valine are limiting
for the growth of many mammals. Therefore the branched-chain amino
acids are supplemented in the feed of broiler, leg hens, turkey,
swine or cattle diets.
[13313] [0010.0.0.30] see [0010.0.0.27]
[13314] [0011.0.0.30] see [0011.0.0.27]
[13315] [0012.0.30.30] It is an object of the present invention to
develop an inexpensive process for the synthesis of leucine and/or
valine, preferably L-leucine and/or L-valine.
[13316] [0013.0.0.30] see [0013.0.0.27]
[13317] [0014.0.30.30] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is leucine and/or valine,
preferably L-leucine and/or L-valine. Accordingly, in the present
invention, the term "the fine chemical" as used herein relates to
"leucine and/or valine". Further, the term "the fine chemicals" as
used herein also relates to fine chemicals comprising leucine
and/or valine.
[13318] [0015.0.30.30] In one embodiment, the term "the fine
chemical" means leucine and/or valine, preferably L-leucine and/or
L-valine. Throughout the specification the term "the fine chemical"
means leucine and/or valine, preferably L-leucine and/or L-valine,
its salts, ester or amids in free form or bound to proteins. In a
preferred embodiment, the term "the fine chemical" means leucine
and/or valine, preferably L-leucine and/or L-valine, in free form
or its salts or bound to proteins.
[13319] [0016.0.30.30] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [13320] (a) increasing or generating the activity of one
or more of a protein as shown in table XII, application no. 30,
column 3 encoded by the nucleic acid sequences as shown in table
XI, application no. 30, column 5, in a non-human organism or in one
or more parts thereof or [13321] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
amino acids of the invention or fine chemicals comprising amino
acids of the invention, in said organism or in the culture medium
surrounding the organism.
[13322] [0016.1.30.30] Accordingly, the term "the fine chemical"
means in one embodiment "valine" in relation to all sequences
listed in Tables XI to XIV, line 6 and/or 8 and/or 9 or homologs
thereof and means in one embodiment "leucine" in relation to all
sequences listed in Table XI to XIV, line 7 or homologs
thereof.
[13323] [0017.0.0.30] to [0019.0.0.30] see [0017.0.0.27] to
[0019.0.0.27]
[13324] [0020.0.30.30] Surprisingly it was found, that the
transgenic expression of the Zea mays protein as indicated in Table
XII, column 5, line 6 in a plant conferred an increase in valine
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of valine.
[13325] Surprisingly it was found, that the transgenic expression
of the Zea mays protein as indicated in Table XII, column 5, line 7
in a plant conferred an increase in leucine content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of leucine.
[13326] Surprisingly it was found, that the transgenic expression
of the Linum usitatissimum protein as indicated in Table XII,
column 5, line 8 in a plant conferred an increase in valine content
of the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of valine.
[13327] Surprisingly it was found, that the transgenic expression
of the Hordeum vulgare protein as indicated in Table XII, column 5,
line 9 in a plant conferred an increase in valine content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of valine.
[13328] [0021.0.0.30] see [0021.0.0.27]
[13329] [0022.0.30.30] The sequence of YKR057W from Saccharomyces
cerevisiae has been published in Dujon et al., Nature 369 (6479),
371-378, 1994 and Goffeau et al., Science 274 (5287), 546-547, 1996
and its activity is beeing defined as a ribosomal protein, similar
to S21A, S26A and/or YS25 ribosomal proteins, involved in ribosome
biogenesis and translation. Accordingly, in one embodiment, the
process of the present invention comprises the use of a ribosomal
protein, similar to S21 A, S26A and/or YS25 ribosomal proteins,
involved in ribosome biogenesis and translation from Saccaromyces
cerevisiae or a plant or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a ribosomal
protein, similar to S21 A, S26A and/or YS25 ribosomal proteins,
involved in ribosome biogenesis and translation is increased or
generated, e.g. from Saccharomyces cerevisiae or a plant or a
homolog thereof. The sequence of YNL135C from Saccharomyces
cerevisiae has been published in Philippsen, P., Nature 387 (6632
Suppl), 93-98, 1997 and Goffeau, A., Science 274 (5287), 546-547,
1996, and its activity is beeing defined as a peptidylprolyl
isomerase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a peptidylprolyl isomerase
or its homolog, for the production of the fine chemical, meaning of
leucine and/or isoleucine and/or valine, in particular for
increasing the amount of leucine and/or isoleucine and/or valine,
preferably leucine and/or isoleucine and/or valine in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a peptidylprolyl isomerase is increased or generated, e.g. from
Saccharomyces cerevisiae or a plant or a homolog thereof.
[13330] The sequence of b1343 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as an ATP-dependent RNA helicase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein with an ATP-dependent RNA
helicase from E. coli or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of an ATP-dependent
RNA helicase is increased or generated, e.g. from E. coli or a
plant or a homolog thereof.
[13331] The sequence of b3938 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a transcriptional repressor
for methionine biosynthesis. Accordingly, in one embodiment, the
process of the present invention comprises the use of a
transcriptional repressor activity for methionine biosynthesis from
E. coli or a plant or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of leucine and/or
isoleucine and/or valine, in particular for increasing the amount
of leucine and/or isoleucine and/or valine, preferably leucine
and/or isoleucine and/or valine in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a transcriptional
repressor for methionine biosynthesis is increased or generated,
e.g. from E. coli or a plant or a homolog thereof.
[13332] [0022.1.0.30] see [0022.1.0.27]
[13333] [0023.0.0.30] for the disclosure of the paragraph
[0023.0.0.30] see paragraph [0023.0.0.27] above.
[13334] [0023.1.30.30] Homologs of the polypeptide disclosed in
table XII, application no. 30, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 30, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 30, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 30,
column 7, resp.
[13335] [0024.0.0.30] see [0024.0.0.07]
[13336] [0025.0.30.30] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 30, column 3" if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 30, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[13337] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 30,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 30,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[13338] [0025.1.30.30] In one embodiment, the polypeptide of the
invention or the polypeptide used in the method of the invention
confers said activity, e.g. the increase of the fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary distant to the organism in which it is
expressed. For example origin and expressing organism are derived
from different families, orders, classes or phylums.
[13339] [0025.2.30.30] In one embodiment, the polypeptide of the
invention or the polypeptide used in the method of the invention
confers said activity, e.g. the increase of the fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table XI,
application no. 30, column 4 and is expressed in an organism, which
is evolutionary distant to the origin organism. For example origin
and expressing organism are derived from different families,
orders, classes or phylums whereas origin and the organsim
indicated in Table XI, application no. 30, column 4 are derived
from the same families, orders, classes or phylums.
[13340] [0026.0.0.30] to [0033.0.0.30]: see [0026.0.0.27] to
[0033.0.0.27]
[13341] [0034.0.30.30] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 30, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[13342] [0035.0.0.30] to [0044.0.0.30]: see [0035.0.0.27] to
[0044.0.0.27]
[13343] [0045.0.30.30] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
30, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line,
between 10% and 50%, 10% and 100%, 10% and 200%, 10% and 300%, 10%
and 400%, 10% and 500%, 20% and 50%, 20% and 250%, 20% and 500%,
50% and 100%, 50% and 250%, 50% and 500%, 500% and 1000% or
more.
[13344] [0046.0.30.30] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
30, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line
confers an increase of the respective fine chemical and of further
amino acids or their precursors.
[13345] [0047.0.0.30] see [0047.0.0.27]
[13346] [0048.0.0.30] see [0048.0.0.27]
[13347] [0049.0.30.30] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 30, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 30, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 30, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[13348] [0050.0.30.30] For the purposes of the present invention,
the term "leucine" and/or "valine" and "L-leucine" and/or
"L-valine" also encompass the corresponding salts, such as, for
example, leucine- and/or valine-hydrochloride or leucine and/or
valine sulfate. Preferably the term leucine and/or valine is
intended to encompass the term L-leucine and/or L-valine.
[13349] [0051.0.0.30] see [0051.0.0.27]
[13350] [0052.0.0.30] see [0052.0.0.27]
[13351] [0053.0.30.30] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[13352] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 30, columns 5 and 7 or its homologs activity
having herein-mentioned amino acids of the invention increasing
activity; and/or [13353] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 30,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 30, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned amino acids of the invention increasing
activity; and/or [13354] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned amino acid increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 30, columns 5 and 7 or its
homologs activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [13355] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned amino acids of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 30, columns 5
and 7 or its homologs activity; and/or [13356] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned amino acids of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 30, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [13357] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned amino acids of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 30, columns 5 and 7 or its
homologs activity, and/or [13358] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned amino acids of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 30, columns 5 and 7 or its
homologs activity; and/or [13359] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 30, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[13360] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [13361] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[13362] [0054.0.30.30] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 30 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 30, column 3 or its homologs.
[13363] [0055.0.0.30] to [0064.0.0.30] see [0055.0.0.27] to
[0064.0.0.27]
[13364] [0065.0.30.30] The activation of an endogenous polypeptide
having above-mentioned activity, e.g. having the activity of a
YKR057W, YNL135C, b1343 and/or b3983 protein or of the polypeptide
of the invention, e.g. conferring the increase of leucine and/or
valine after increase of expression or activity can also be
increased by introducing a synthetic transcription factor, which
binds close to the coding region of YKR057W, YNL135C, b1343 and/or
b3983 protein encoding gene and activates its transcription. A
chimeric zinc finger protein can be construed, which comprises a
specific DNA-binding domain and an activation domain as e.g. the
VP16 domain of Herpes Simplex virus. The specific binding domain
can bind to the regulatory region of the YKR057W, YNL135C, b1343
and/or b3983 protein encoding gene. The expression of the chimeric
transcription factor in a organism, in particular in a plant, leads
to a specific expression of YKR057W, YNL135C, b1343 and/or b3983
protein, see e.g. in WO01/52620, Oriz, Proc. Natl. Acad. Sci. USA,
2002, Vol. 99, 13290 or Guan, Proc. Natl. Acad. Sci. USA, 2002,
Vol. 99, 13296.
[13365] [0066.0.0.30] to [0069.0.0.30]: see [0066.0.0.27] to
[0069.0.0.27]
[13366] [0070.0.30.30] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding the
YKR057W, YNL135C, b1343 and/or b3983 protein into an organism alone
or in combination with other genes, it is possible not only to
increase the biosynthetic flux towards the end product, but also to
increase, modify or create de novo an advantageous, preferably
novel metabolites composition in the organism, e.g. an advantageous
amino acid composition comprising a higher content of (from a
viewpoint of nutritional physiology limited) amino acids, like
tryptophane, methionine, lysine and/or threonine.
[13367] [0071.0.0.30] see [0071.0.0.27]
[13368] [0072.0.30.30] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to leucine and/or valine 2-Acetolactate,
2-3-Dihydroxyisovalerate, 2-Oxoisovalerate, 3-Hydroxyisobutyrate,
3-Hydroxy-lsobutyryl-CoA, Methylacrylyl-CoA, Isobutyryl-CoA,
2-Aceto-2-hydroxybutyrate, 2:3-Di-OH-3-methylvalerate,
2-Oxo-3-methylvalerate, 2-Methylacetoacetyl-CoA,
2-Methyl-3-hydroxybutyryl-CoA, Tiglyl-CoA, 2 Methylbutyryl-CoA,
22-Isopropyl malate, 3-Isopropylmalate, Oxoleucine, Isovaleryl-CoA,
3-Methylcrotonyl-CoA and/or 3-Methylglutaconyl-CoA.
[13369] [0073.0.30.30] Accordingly, in one embodiment, the process
according to the invention relates to a process which comprises:
[13370] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [13371] b) increasing the YKR057W,
YNL135C, b1343 and/or b3983 protein activity or of a polypeptide
being encoded by the nucleic acid molecule of the present invention
and described below, e.g. conferring an increase of the fine
chemical in the organism, preferably in the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant, [13372] c) growing the organism, preferably
the microorganism, the non-human animal, the plant or animal cell,
the plant or animal tissue or the plant under conditions which
permit the production of the fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and [13373] d) if desired, revovering, optionally
isolating, the free and/or bound the fine chemical and, optionally
further free and/or bound amino acids synthetized by the organism,
the microorganism, the non-human animal, the plant or animal cell,
the plant or animal tissue or the plant.
[13374] [0074.0.0.30] to [0084.0.0.30]: see [0074.0.0.27] to
[0084.0.0.27]
[13375] [0085.0.30.30] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [13376] a) the nucleic acid sequence as
depicted in Table XI, application no. 30, columns 5 and 7, or a
derivative thereof, or [13377] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as depicted in Table XI, application no.
[13378] 30, columns 5 and 7, or a derivative thereof, or [13379] c)
(a) and (b) is/are not present in its/their natural genetic
environment or has/have been modified by means of genetic
manipulation methods, it being possible for the modification to be,
by way of example, a substitution, addition, deletion, inversion or
insertion of one or more nucleotide. "Natural genetic environment"
means the natural chromosomal locus in the organism of origin or
the presence in a genomic library. In the case of a genomic
library, the natural, genetic environment of the nucleic acid
sequence is preferably at least partially still preserved. The
environment flanks the nucleic acid sequence at least on one side
and has a sequence length of at least 50 bp, preferably at least
500 bp, particularly preferably at least 1000 bp, very particularly
preferably at least 5000 bp.
[13380] [0086.0.0.30]: see [0086.0.0.27]
[13381] [0087.0.0.30]: see [0087.0.0.27]
[13382] [0088.0.30.30] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose amino acid
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for monogastric animals is limited by a few essential amino acids
such as lysine, threonine or methionine or tryptophane. After the
YKR057W, YNL135C, b1343 and/or b3983 protein activity has been
increased or generated, or after the expression of nucleic acid
molecule or polypeptide according to the invention has been
generated or increased, the transgenic plant generated thus is
grown on or in a nutrient medium or else in the soil and
subsequently harvested.
[13383] [0089.0.0.30] to [0097.0.0.30]: see [0089.0.0.27] to
[0097.0.0.27]
[13384] [0098.0.30.30] In a preferred embodiment, the fine chemical
(leucine and/or valine) is produced in accordance with the
invention and, if desired, is isolated. The production of further
amino acids such as methionine, lysine and/or threonine mixtures of
amino acid by the process according to the invention is
advantageous.
[13385] [0099.0.0.30] to [0102.0.0.30]: see [0099.0.0.27] to
[0102.0.0.27]
[13386] [0103.0.30.30] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[13387] a) nucleic acid molecule encoding, preferably at least the
mature form, of the polypeptide shown in Table XII, application no.
30, columns 5 and 7, or a fragment thereof, which confers an
increase in the amount of the fine chemical in an organism or a
part thereof; [13388] b) nucleic acid molecule comprising,
preferably at least the mature form, of the nucleic acid molecule
shown in Table XI, application no. 30, columns 5 and 7, [13389] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [13390] d) nucleic acid molecule encoding a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [13391] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [13392] f) nucleic acid molecule encoding a
polypeptide, the polypeptide being derived by substituting,
deleting and/or adding one or more amino acids of the amino acid
sequence of the polypeptide encoded by the nucleic acid molecules
(a) to (d), preferably to (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[13393] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13394] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers in Table XIII, application no. 30, column 7, and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13395] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from an expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[13396] j) nucleic acid molecule which encodes a polypeptide
comprising the consensus sequence shown in Table XIV, application
no. 30, column 7, and conferring an increase in the amount of the
fine chemical in an organism or a part thereof; [13397] k) nucleic
acid molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of the polypeptide shown in Table XI, application no. 30, columns 5
and 7, and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; and [13398] l) nucleic
acid molecule which is obtainable by screening a suitable library
under stringent conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k), preferably to
(a) to (c), or with a fragment of at least 15 nt, preferably 20 nt,
30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k), preferably to (a) to (c), and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; or which comprises a sequence which is
complementary thereto.
[13399] [0103.1.30.30] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table XI, application no. 30, columns 5 or 7
by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence shown in indicated in Table XI, application no. 30,
columns 5 or 7. In one embodiment, the nucleic acid molecule used
in the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI,
application no. 30, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table XII, application no. 30, columns 5 or 7.
[13400] [0104.0.30.30] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence depicted in
Table XI, application no. 30, columns 5 and 7, by one or more
nucleotides or does not consist of the sequence shown in Table XI,
application no. 30, columns 5 and 7. In one embodiment, the nucleic
acid molecule of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to the sequence shown in Table XI.
In another embodiment, the nucleic acid molecule does not encode a
polypeptide of the sequence shown in Table XII, application no. 30,
preferably of sequences as shown in Table XI, application no. 30,
columns 5 or 7.
[13401] [0105.0.0.30] to [0107.0.0.30]: see [0105.0.0.27] to
[0107.0.0.27]
[13402] [0108.0.30.30] Nucleic acid molecules with the sequence
shown in Table XI, acid molecules which are derived from the amino
acid sequences shown in Table XII, application no. 30, columns 5 or
7, or from polypeptides comprising the consensus sequence shown in
Table XIV, application no. 30, columns 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of an YKR057W, YNL135C, b1343 and/or b3983 protein or
conferring a leucine and/or valine increase after increasing its
expression or activity are advantageously increased in the process
according to the invention.
[13403] [0109.0.0.30] see [0109.0.0.27]
[13404] [0110.0.30.30] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with YKR057W, YNL135C, b1343 and/or b3983
protein activity can be determined from generally accessible
databases.
[13405] [0111.0.0.30] see [0111.0.0.27]
[13406] [0112.0.30.30] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with YKR057W,
YNL135C, b1343 and/or b3983 protein activity and conferring a
leucine and/or valine increase.
[13407] [0113.0.0.30] to [0120.0.0.30]: see [0113.0.0.27] to
[0120.0.0.27]
[13408] [0121.0.30.30] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences shown in Table XII,
application no. 30, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a leucine and/or valine increase after
increasing its activity, e.g. having the activity of an YKR057W,
YNL135C, b1343 and/or b3983 protein.
[13409] [0122.0.0.30] to [127.0.0.30]: see [0122.0.0.27] to
[0127.0.0.27]
[13410] [0128.0.30.30] Synthetic oligonucleotide primers for the
amplification, e.g. as shown in Table XIII, application no. 30,
column 7, by means of polymerase chain reaction can be generated on
the basis of a sequence shown herein, for example the sequence
shown in Table XI, application no. 30, columns 5 or 7, or the
sequences derived from Table XII, application no. 30.
[13411] [0129.0.30.30] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence shown in Table XIV,
application no. 30, column 7, is derived from said alignments.
[13412] [0130.0.30.30] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of leucine
and/or valine after increasing the expression or activity or having
an YKR057W, YNL135C, b1343 and/or b3983 activity or further
functional homologs of the polypeptide of the invention from other
organisms.
[13413] [0131.0.0.30] to [0138.0.0.30]: see [0131.0.0.27] to
[0138.0.0.27]
[13414] [0139.0.30.30] Polypeptides having above-mentioned
activity, i.e. conferring the fine chemical increase, derived from
other organisms, can be encoded by other DNA sequences which
hybridize to the sequences shown in Table XI, application no. 30,
columns 5 or 7, under relaxed hybridization conditions and which
code on expression for peptides having the leucine and/or valine
increasing activity.
[13415] [0140.0.0.30] to [0146.0.0.30]: see [0140.0.0.27] to
[0146.0.0.27]
[13416] [0147.0.30.30] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 30, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences s
such that it can hybridize to one of said nucleotide sequences
thereby forming a stable duplex. Preferably, the hybridisation is
performed under stringent hybridization conditions. However, a
complement of one of the herein disclosed sequences is preferably a
sequence complement thereto according to the base pairing of
nucleic acid molecules well known to the skilled person. For
example, the bases A and G undergo base pairing with the bases T
and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[13417] [0148.0.30.30] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 30,
columns 5 or 7 or a portion thereof and preferably has above
mentioned activity, in particular, of valine and/or leucine
increasing activity after increasing the activity or an activity of
a product of a gene encoding said sequences or their homologs.
[13418] [0149.0.30.30] The nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 30,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity and as indicated in indicated in Table
XII.
[13419] [0150.0.30.30] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table XI, application no. 30, columns
5 or 7, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of valine and/or leucine,
resp., if its activity is increased. The nucleotide sequences
determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table XI,
application no. 30, columns 5 or 7, an anti-sense sequence of one
of the sequences, e.g., as indicated in Table XI, application no.
30, columns 5 or 7, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table XIII, application no. 30, column 7, will result in a fragment
of a polynucleotide sequence as indicated in Table XI, application
no. 30, columns 5 or 7 or its gene product.
[13420] [0151.0.0.30] see [0151.0.0.27]
[13421] [0152.0.30.30] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to the amino acid
sequence of Table XII, application no. 30, columns 5 or 7, such
that the protein or portion thereof maintains the ability to
participate in the fine chemical production, in particular a
leucine and/or valine increasing the activity as mentioned above or
as described in the examples in plants or microorganisms is
comprised.
[13422] [0153.0.30.30] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence shown in Table XII, application no. 30, columns 5 or
7, such that the protein or portion thereof is able to participate
in the increase of the fine chemical production. For examples
having an YKR057W, YNL135C, b1343 and/or b3983 activity are
described herein.
[13423] [0154.0.30.30] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence of Table XII, application no. 30, columns 5 or 7, and
having above-mentioned activity, e.g. conferring preferably the
increase of the fine chemical.
[13424] [0155.0.0.30] and [0156.0.0.30]: see [0155.0.0.27] and
[0156.0.0.27]
[13425] [0157.0.30.30] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences
shown in Table XI, application no. 30, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the fine chemical in a organism, e.g. comprising the
consensus sequences as indicated in Table XIV, application no. 30,
column 7, or of the polypeptide as indicated in Table XII,
application no. 30, columns 5 or 7, or their functional homologues.
Advantageously, the nucleic acid molecule of the invention or the
nucleic acid molecule used in the method of the invention
comprises, or in an other embodiment has, a nucleotide sequence
encoding a protein comprising, or in an other embodiment having, a
consensus sequences as indicated in Table XIV, application no. 30,
column 7, or of the polypeptide as indicated in Table XII,
application no. 30, columns 5 or 7, or the functional homologues.
In a still further embodiment, the nucleic acid molecule of the
invention or the nucleic acid molecule used in the method of the
invention encodes a full length protein which is substantially
homologous to an amino acid sequence comprising a consensus
sequence as indicated in Table XIV, application no. 30, column 7,
or of a polypeptide as indicated in Table XII, application no. 30,
columns 5 or 7, or the functional homologues thereof. However, in a
preferred embodiment, the nucleic acid molecule of the present
invention does not consist of a sequence as indicated in Table XI,
application no. 30, columns 5 or 7,
[13426] [0158.0.0.30] to [0160.0.0.30]: see [0158.0.0.27] to
[0160.0.0.27]
[13427] [0161.0.30.30] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising the
sequence shown in Table XI, application no. 30, columns 5 or 7. The
nucleic acid molecule is preferably at least 20, 30, 50, 100, 250
or more nucleotides in length.
[13428] [0162.0.0.30]: see [0162.0.0.27]
[13429] [0163.0.30.30] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
of Table XI, application no. 30, columns 5 or 7, corresponds to a
naturally-occurring nucleic acid molecule of the invention. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein). Preferably, the nucleic
acid molecule encodes a natural protein having above-mentioned
activity, e.g. conferring the fine chemical increase after
increasing the expression or activity thereof or the activity of a
protein of the invention or used in the process of the
invention.
[13430] [0164.0.0.30]: see [0164.0.0.27]
[13431] [0165.0.30.30] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. in
Table XI, application no. 30, columns 5 or 7.
[13432] [0166.0.0.30] and [0167.0.0.30]: see [0166.0.0.27] and
[0167.0.0.27]
[13433] [0168.0.30.30] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 30, columns 5 or 7, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 30,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table XII, application no. 30, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 30, columns 5
or 7, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table XII, application no.
30, columns 5 or 7, and most preferably at least about 96%, 97%,
98%, or 99% identical to the sequence as indicated in Table XII,
application no. 30, columns 5 or 7.
[13434] [0169.0.0.30] to [0172.0.0.30]: see [0169.0.0.27] to
[0172.0.0.27]
[13435] [0173.0.30.30] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108442 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108442 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[13436] [0174.0.0.30]: see [0174.0.0.27]
[13437] [0175.0.30.30] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108443 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108443 by the above program algorithm with the
above parameter set, has a 80% homology.
[13438] [0176.0.30.30] Functional equivalents derived from one of
the polypeptides as shown in Table XII, application no. 30, columns
5 or 7, according to the invention by substitution, insertion or
deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at
least 55%, 60%, 65% or 70% by preference at least 80%, especially
preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very
especially preferably at least 95%, 97%, 98% or 99% homology with
one of the polypeptides as shown in Table XII, application no. 30,
columns 5 or 7, according to the invention and are distinguished by
essentially the same properties as the polypeptide as shown in
Table XII, application no. 30, columns 5 or 7.
[13439] [0177.0.30.30] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 30,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptide as indicated in Table XII,
application no. 30, columns 5 or according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 30, columns
5 or 7.
[13440] [0178.0.0.30] see [0178.0.0.27]
[13441] [0179.0.30.30] A nucleic acid molecule encoding an
homologous to a protein sequence of Table XI, application no. 30,
columns 5 or 7, preferably of Table XII, application no. 30, column
7, can be created by introducing one or more nucleotide
substitutions, additions or deletions into a nucleotide sequence of
the nucleic acid molecule of the present invention, in particular
of Table XI, application no. 30, columns 5 or 7, such that one or
more amino acid substitutions, additions or deletions are
introduced into the encoded protein. Mutations can be introduced
into the encoding sequences of Table XI, application no. 30,
columns 5 or 7, by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis.
[13442] [0180.0.0.30] to [0183.0.0.30]: see [0180.0.0.27] to
[0183.0.0.27]
[13443] [0184.0.30.30] Homologues of the nucleic acid sequences
used, with the sequence shown in Table XI, application no. 30,
columns 5 or 7, or of the nucleic acid sequences derived from the
sequences Table XI, application no. 30, columns 5 or 7, comprise
also allelic variants with at least approximately 30%, 35%, 40% or
45% homology, by preference at least approximately 50%, 60% or 70%,
more preferably at least approximately 90%, 91%, 92%, 93%, 94% or
95% and even more preferably at least approximately 96%, 97%, 98%,
99% or more homology with one of the nucleotide sequences shown or
the abovementioned derived nucleic acid sequences or their
homologues, derivatives or analogues or parts of these. Allelic
variants encompass in particular functional variants which can be
obtained by deletion, insertion or substitution of nucleotides from
the sequences shown, preferably from Table XI, application no. 30,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[13444] [0185.0.30.30] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 30, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table XI, application no. 30, columns 5 or 7. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequences as indicated in Table XI, application no. 30, columns 5
or 7.
[13445] [0186.0.30.30] Also preferred is that the nucleic acid
molecule used in the process of the invention encodes a polypeptide
comprising the sequence shown in Table XII, application no. 30,
columns 5 or 7. In one embodiment, the nucleic acid molecule
encodes less than 150, 130, 100, 80, 60, 50, 40 or 30 further amino
acids. In a further embodiment, the encoded polypeptide comprises
less than 20, 15, 10, 9, 8, 7, 6 or 5 further amino acids. In one
embodiment used in the inventive process, the encoded polypeptide
is identical to the sequences shown in Table XII.
[13446] [0187.0.30.30] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence shown in Table XII, application no. 30,
columns 5 or 7, comprises less than 100 further nucleotides. In a
further embodiment, said nucleic acid molecule comprises less than
30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence of
the sequences shown in Table XI, application no. 30, columns 5 or
7.
[13447] [0188.0.30.30] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 30, columns 5 or 7 and is expressed under
identical conditions.
[13448] [0189.0.30.30] Homologues of a sequences as indicated in
Table XI, application no. 30, columns 5 or 7, or of a derived
sequences as indicated in Table XII, application no. 30, columns 5
or 7 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[13449] [0190.0.0.30] to [0203.0.0.30]: see [0190.0.0.27] to
[0203.0.0.27]
[13450] [0204.0.0.30] Accordingly, in one embodiment, the invention
relates to a nucleic acid molecule which comprises a nucleic acid
molecule selected from the group consisting of: [13451] a) nucleic
acid molecule encoding, preferably at least the mature form, of a
polypeptide as indicated in Table XII, application no. 30, columns
5 or 7, or a fragment thereof conferring an increase in the amount
of the respective fine chemical, in particular according to table
XII, application no. 30, column 6 in an organism or a part thereof
[13452] b) nucleic acid molecule comprising, preferably at least
the mature form, of a nucleic acid molecule as indicated in Table
XI, application no. 30, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [13453] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[13454] d) nucleic acid molecule encoding a polypeptide whose
sequence has at least 50% identity with the amino acid sequence of
the polypeptide encoded by the nucleic acid molecule of (a) to (c)
and conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13455] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[13456] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [13457] g)
nucleic acid molecule encoding a fragment or an epitope of a
polypeptide which is encoded by one of the nucleic acid molecules
of (a) to (e), preferably to (a) to (c) and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; [13458] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying a cDNA library or a
genomic library using primers or primer pairs as indicated in Table
XIII, application no. 30, column 7, and conferring an increase in
the amount of the respective fine chemical, in particular according
to table XII, application no. 30, column 6 in an organism or a part
thereof; [13459] i) nucleic acid molecule encoding a polypeptide
which is isolated, e.g. from a expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (g), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13460] j) nucleic acid molecule which
encodes a polypeptide comprising a consensus sequence as indicated
in Table XIV, application no. 30, columns 5 or 7, and conferring an
increase in the amount of the respective fine chemical, in
particular according to table XII, application no. 30, column 6 in
an organism or a part thereof; [13461] k) nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a
domaine of a polypeptide as indicated in Table XII, application no.
30, columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical, in particular accccording to table XII,
application no. 30, column 6 in an organism or a part thereof; and
[13462] l) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment of at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (h) or of a
nucleic acid molecule as indicated in Table XI, application no. 30,
columns 5 or 7, or a nucleic acid molecule encoding, preferably at
least the mature form of, a polypeptide as indicated in Table XII,
application no. 30, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical according to table XII,
application no. 30, column 6 in an organism or a part thereof;
[13463] or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over the sequence indicated in Table
XI, application no. 30, columns 5 or 7, by one or more nucleotides.
In one embodiment, the nucleic acid molecule does not consist of
the sequence shown and indicated in Table XI, application no. 30,
columns 5 or 7,
[13464] In one embodiment, the nucleic acid molecule is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table XI, application no. 30, columns 5 or 7.
[13465] In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table XII,
application no. 30, columns 5 or 7.
[13466] In an other embodiment, the nucleic acid molecule of the
present invention is at least 30%, 40%, 50%, or 60% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 30, columns 5 or
7.
[13467] In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table XII,
application no. 30, columns 5 or 7.
[13468] Accordingly, in one embodiment, the nucleic acid molecule
of the differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 30, columns 5 or
7.
[13469] Accordingly, in one embodiment, the nucleic acid molecule
of the present invention encodes a polypeptide, which differs at
least in one or more amino acids from a polypeptide indicated in
Table XII, application no. 30, columns 5 or 7.
[13470] In another embodiment, a nucleic acid molecule indicated in
Table XI, application no. 30, columns 5 or 7.
[13471] Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table XII, application no.
30, columns 5 or 7.
[13472] In a further embodiment, the protein of the present
invention is at least 30%, 40%, 50%, or 60% identical to a protein
sequence indicated in Table XII, application no. 30, columns 5 or
7, and less than 100%, preferably less than 99.999%, 99.99% or
99.9%, more preferably less than 99%, 985, 97%, 96% or 95%
identical to a sequence as indicated in Table XI, columns 5 or
7.
[13473] [0205.0.0.30] to [0226.0.0.30]: see [0205.0.0.27] to
[0226.0.0.27]
[13474] [0227.0.30.30] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[13475] In addition to the sequence mentioned in Table XI,
application no. 30, columns 5 or 7, or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the amino acid biosynthetic pathway such as for
Llysine, L-threonine and/or L-methionine or L-leucine and/or valine
is expressed in the organisms such as plants or microorganisms. It
is also possible that the regulation of the natural genes has been
modified advantageously so that the gene and/or its gene product is
no longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine the sequences shown in Table XI,
application no. 30, columns 5 or 7, with genes which generally
support or enhances to growth or yield of the target organisms, for
example genes which lead to faster growth rate of microorganisms or
genes which produces stress-, pathogen, or herbicide resistant
plants.
[13476] [0228.0.0.30] to [0230.0.0.30]: see [0228.0.0.27] to
[0230.0.0.27]
[13477] [0231.0.30.30] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a leucine and/or valine degrading
protein is attenuated, in particular by reducing the rate of
expression of the corresponding gene.
[13478] [0232.0.0.30] to [0282.0.0.30]: see [0232.0.0.27] to
[0282.0.0.27]
[13479] [0283.0.30.30] Moreover, native polypeptide conferring the
increase of the fine chemical in an organism or part thereof can be
isolated from cells (e.g., endothelial cells), for example using
the antibody of the present invention as described below, in
particular, an anti-YKR057W, YNL135C, b1343 and/or b3983 protein
antibody or an antibody against polypeptides as shown in Table XII,
application no. 30, columns 5 or 7, which can be produced by
standard techniques utilizing the polypeptid of the present
invention or fragment thereof, i.e., the polypeptide of this
invention. Preferred are monoclonal antibodies. [0284.0.0.30]: see
[0284.0.0.27]
[13480] [0285.0.30.30] In one embodiment, the present invention
relates to a polypeptide having the sequence shown in Table XII,
application no. 30, columns 5 or 7, or as coded by the nucleic acid
molecule shown in Table XI, application no. 30, columns 5 or 7, or
functional homologues thereof.
[13481] [0286.0.30.30] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased comprising or consisting of the consensus sequence shown
in Table XIV, application no. 30, and in one another embodiment,
the present invention relates to a polypeptide comprising or
consisting of the consensus sequence shown in Table XIV,
application no. 30, whereby 20 or less, preferably 15 or 10,
preferably 9, 8, 7, or 6, more preferred 5 or 4, even more
preferred 3, even more preferred 2, even more preferred 1, most
preferred 0 of the amino acids positions indicated can be replaced
by any amino acid or, in an further embodiment, can be replaced
and/or absent.
[13482] [0287.0.0.30] to [0290.0.0.30]: see [0287.0.0.27] to
[0290.0.0.27]
[13483] [0291.0.30.30] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. In one embodiment, said polypeptide of the
invention distinguishes over a sequence as indicated in Table XII,
application no. 30, columns 5 or 7, by one or more amino acids. In
one embodiment, polypeptide distinguishes form a sequence as
indicated in Table XII, application no. 30, columns 5 or 7, by more
than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, even more preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table XII, application no. 30, columns 5 or 7, by not
more than 80% or 70% of the amino acids, preferably not more than
60% or 50%, more preferred not more than 40% or 30%, even more
preferred not more than 20% or 10%. In an other embodiment, said
polypeptide of the invention does not consist of a sequence as
indicated in Table XII, application no. 30, columns 5 or 7.
[13484] [0292.0.0.30]: see [0292.0.0.27]
[13485] [0293.0.30.30] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, the polypeptide of
the invention has a sequence which distinguishes from a sequence as
indicated in Table XII, application no. 30, columns 5 or 7, by one
or more amino acids. In an other embodiment, said polypeptide of
the invention does not consist of the sequence as indicated in
Table XII, application no. 30, columns 5 or 7.
[13486] In a further embodiment, said polypeptide of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by a nucleic acid molecules as indicated in
Table XI, application no. 30, columns 5 or 7.
[13487] [0294.0.30.30] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 30, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 30, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[13488] [0295.0.0.30] to [0297.0.0.30]: see [0295.0.0.27] to
[0297.0.0.27]
[13489] [0297.0.30.30] The language "substantially free of cellular
material" includes preparations of the polypeptide of the invention
in which the protein is separated from cellular components of the
cells in which it is naturally or recombinantly produced. In one
embodiment, the language "substantially free of cellular material"
includes preparations having less than about 30% (by dry weight) of
"contaminating protein", more preferably less than about 20% of
"contaminating protein", still more preferably less than about 10%
of "contaminating protein", and most preferably less than about 5%
"contaminating protein". The term "Contaminating protein" relates
to polypeptides, which are not polypeptides of the present
invention. When the polypeptide of the present invention or
biologically active portion thereof is recombinantly produced, it
is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less
than about 10%, and most preferably less than about 5% of the
volume of the protein preparation. The language "substantially free
of chemical precursors or other chemicals" includes preparations in
which the polypeptide of the present invention is separated from
chemical precursors or other chemicals which are involved in the
synthesis of the protein. The language "substantially free of
chemical precursors or other chemicals" includes preparations
having less than about 30% (by dry weight) of chemical precursors
or non-YKR057W, YNL135C, b1343 and/or b3983 chemicals, more
preferably less than about 20% chemical precursors or non-YKR057W,
YNL135C, b1343 and/or b3983 chemicals, still more preferably less
than about 10% chemical precursors or non-YKR057W, YNL135C, b1343
and/or b3983 chemicals, and most preferably less than about 5%
chemical precursors or non-YKR057W, YNL135C, b1343 and/or b3983
chemicals. In preferred embodiments, isolated proteins or
biologically active portions thereof lack contaminating proteins
from the same organism from which the polypeptide of the present
invention is derived. Typically, such proteins are produced by
recombinant techniques
[13490] [00297.1.30.30] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity and/or the amino acid
sequence of a polypeptide indicated in Table XII, application no.
30, columns 3, 5 or 7.
[13491] [0298.0.30.30] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
shown in Table XII, application no. 30, columns 5 or 7, such that
the protein or portion thereof maintains the ability to confer the
activity of the present invention. The portion of the protein is
preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical as shown in Table XII,
application no. 30, columns 5 or 7.
[13492] [0299.0.30.30] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences of Table XII, application no. 30, columns 5 or
7. The preferred polypeptide of the present invention preferably
possesses at least one of the activities according to the invention
and described herein. A preferred polypeptide of the present
invention includes an amino acid sequence encoded by a nucleotide
sequence which hybridizes, preferably hybridizes under stringent
conditions, to a nucleotide sequence of Table XI, application no.
30, columns 5 or 7, or which is homologous thereto, as defined
above.
[13493] [0300.0.30.30] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 30, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 30, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[13494] [0301.0.0.30]: see [0301.0.0.27]
[13495] [0302.0.30.30] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., the amino acid sequence shown in Table
XII, application no. 30, columns 5 or 7, or the amino acid sequence
of a protein homologous thereto, which include fewer amino acids
than a full length polypeptide of the present invention or used in
the process of the present invention or the full length protein
which is homologous to an polypeptide of the present invention or
used in the process of the present invention depicted herein, and
exhibit at least one activity of polypeptide of the present
invention or used in the process of the present invention.
[13496] [0303.0.0.30]: see [0303.0.0.27]
[13497] [0304.0.30.30] Manipulation of the nucleic acid molecule of
the invention may result in the production of YKR057W, YNL135C,
b1343 and/or b3983 protein having differences from the wild-type
YKR057W, YNL135C, b1343 and/or b3983 protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[13498] [0305.0.0.30]: see [0305.0.0.27]
[13499] [0305.1.30.30] Any mutagenesis strategies for the
polypeptide of the present invention or the polypeptide used in the
process of the present invention to result in increasing said
activity are not meant to be limiting; variations on these
strategies will be readily apparent to one skilled in the art.
Using such strategies, and incorporating the mechanisms disclosed
herein, the nucleic acid molecule and polypeptide of the invention
may be utilized to generate plants or parts thereof, expressing
wildtype YKR057W, YNL135C, b1343 and/or b3983 proteins or mutated
YKR057W, YNL135C, b1343 and/or b3983 protein encoding nucleic acid
molecules and polypeptide molecules of the invention such that the
yield, production, and/or efficiency of production of a desired
compound is improved.
[13500] [0306.0.0.30] to [0308.0.0.30]: see [0306.0.0.27] to
[0308.0.0.27]
[13501] [0309.0.30.30] In one embodiment, an "YKR057W, YNL135C,
b1343 and/or b3983 protein (=polypeptide)" refers to a polypeptide
having an amino acid sequence corresponding to the polypeptide of
the invention or used in the process of the invention, whereas a
"non-YKR057W, YNL135C, b1343 and/or b3983 polypeptide" or "other
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a protein which is not substantially homologous a
polypeptide of the invention, preferably which is not substantially
homologous to a polypeptide having an YKR057W, YNL135C, b1343
and/or b3983 protein activity, e.g., a protein which does not
confer the activity described herein and which is derived from the
same or a different organism.
[13502] [0310.0.0.30] to [0334.0.0.30]: see [0310.0.0.27] to
[0334.0.0.27]
[13503] [0335.0.30.30] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of the nucleic acid sequences of the Table XI, application no. 30,
columns 5 or 7, and/or homologs thereof. As described inter alia in
WO 99/32619, dsRNAi approaches are clearly superior to traditional
antisense approaches. The invention therefore furthermore relates
to double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived therefrom), bring about altered
metabolic activity by the reduction in the expression of the
nucleic acid sequences of the Table XI, application no. 30, columns
5 or 7, and/or homologs thereof. In a double-stranded RNA molecule
for reducing the expression of an protein encoded by a nucleic acid
sequence of one of the Table XI, application no. 30, columns 5 or
7, and/or homologs thereof, one of the two RNA strands is
essentially identical to at least part of a nucleic acid sequence,
and the respective other RNA strand is essentially identical to at
least part of the complementary strand of a nucleic acid
sequence.
[13504] [0336.0.0.30] to [0342.0.0.30]: see [0336.0.0.27] to
[0342.0.0.27]
[13505] [0343.0.30.30] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence of one of the Table XI, application no. 30, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence of one
of Table XI, application no. 30, columns 5 or 7, or homologs
thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[13506] [0344.0.0.30] to [0361.0.0.30]: see [0344.0.0.27] to
[0361.0.0.27]
[13507] [0362.0.30.30] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the fine chemical in
a cell or an organism or a part thereof, e.g. the nucleic acid
molecule of the invention, the nucleic acid construct of the
invention, the antisense molecule of the invention, the vector of
the invention or a nucleic acid molecule encoding the polypeptide
of the invention, e.g. encoding a polypeptide having an YKR057W,
YNL135C, b1343 and/or b3983 protein activity. Due to the above
mentioned activity the fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an YKR057W, YNL135C, b1343
and/or b3983 protein or activity means herein that due to
modulation or manipulation of the genome, the activity of YKR057W,
YNL135C, b1343 and/or b3983 or a YKR057W, YNL135C, b1343 and/or
b3983-like activity is increased in the cell or organism or part
thereof. Examples are described above in context with the process
of the invention.
[13508] [0363.0.0.30]: see [0363.0.0.27]
[13509] [0364.0.30.30] A naturally occurring expression
cassette--for example the naturally occurring combination of the
YKR057W, YNL135C, b1343 and/or b3983 protein promoter with the
corresponding YKR057W, YNL135C, b1343 and/or b3983 protein
gene--becomes a transgenic expression cassette when it is modified
by non-natural, synthetic "artificial" methods such as, for
example, mutagenization. Such methods have been described (U.S.
Pat. No. 5,565,350; WO 00/15815; also see above).
[13510] [0365.0.0.30] to [0382.0.0.30]: see [0365.0.0.27] to
[0382.0.0.27]
[13511] [0383.0.30.30] For preparing branched-chain amino acid
compound-containing fine chemicals, in particular the fine
chemical, it is possible to use as branched-chain amino acid source
organic branched-chain amino acid-containing compounds such as, for
example, isovalerate, isopropylmalate, oxoisocaproate,
isovaleryl-compounds, methyl-valerate, isobutyrate,
methyl-butyryl-compounds, isopropyrate, isopropyl-compounds or else
organic branched-chain amino acid precursor compounds.
[13512] [0384.0.0.30] to [0392.0.0.30]: see [0384.0.0.27] to
[0392.0.0.27]
[13513] [0393.0.30.30] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [13514] a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [13515] b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table XI, application no. 30, columns 5 or 7, and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[13516] c) introducing the candidate nucleic acid molecules in host
cells, preferably in a plant cell or a microorganism, appropriate
for producing the fine chemical; [13517] d) expressing the
identified nucleic acid molecules in the host cells; [13518] e)
assaying the the fine chemical level in the host cells; and [13519]
f) identifying the nucleic acid molecule and its gene product which
expression confers an increase in the the fine chemical level in
the host cell after expression compared to the wild type.
[13520] [0394.0.28.30] to [0552.0.28.30]: see [0394.0.0.28] to
[0552.0.0.28]
[13521] [00553.0.30.30]
1. A process for the production of valine and/or leucine resp.,
which comprises (a) increasing or generating the activity of a
protein as indicated in Table XII, application no. 30, columns 5 or
7, or a functional equivalent thereof in a non-human organism, or
in one or more parts thereof; and (b) growing the organism under
conditions which permit the production of valine and/or leucine
resp. in said organism. 2. A process for the production of valine
and/or leucine resp., comprising the increasing or generating in an
organism or a part thereof the expression of at least one nucleic
acid molecule comprising a nucleic acid molecule selected from the
group consisting of: a) nucleic acid molecule encoding of a
polypeptide as indicated in Table XII, application no. 30, columns
5 or 7, or a fragment thereof, which confers an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of valine and/or leucine resp., in an organism or a part
thereof; b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table XI, application no. 30, columns 5 or
7, c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of valine and/or leucine
resp., in an organism or a part thereof; d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of valine and/or leucine resp., in an organism or a part
thereof; e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of valine and/or
leucine resp., in an organism or a part thereof; f) nucleic acid
molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table XIII, application no. 30, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of valine and/or leucine resp., in an organism
or a part thereof; g) nucleic acid molecule encoding a polypeptide
which is isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of valine and/or leucine
resp., in an organism or a part thereof; h) nucleic acid molecule
encoding a polypeptide comprising a consensus sequence as indicated
in Table XIV, application no. 30, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of valine and/or leucine resp., in an organism
or a part thereof; and i) nucleic acid molecule which is obtainable
by screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of valine and/or leucine resp., in an organism or a part
thereof. or comprising a sequence which is complementary thereto.
3. The process of claim 1 or 2, comprising recovering of the free
or bound valine and/or leucine resp. 4. The process of any one of
claims 1 to 3, comprising the following steps: (a) selecting an
organism or a part thereof expressing a polypeptide encoded by the
nucleic acid molecule characterized in claim 2; (b) mutagenizing
the selected organism or the part thereof; (c) comparing the
activity or the expression level of said polypeptide in the
mutagenized organism or the part thereof with the activity or the
expression of said polypeptide of the selected organisms or the
part thereof; (d) selecting the mutated organisms or parts thereof,
which comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
(e) optionally, growing and cultivating the organisms or the parts
thereof; and (f) recovering, and optionally isolating, the free or
bound valine and/or leucine resp., produced by the selected mutated
organisms or parts thereof. 5. The process of any one of claims 1
to 4, wherein the activity of said protein or the expression of
said nucleic acid molecule is increased or generated transiently or
stably. 6. An isolated nucleic acid molecule comprising a nucleic
acid molecule selected from the group consisting of: a) nucleic
acid molecule encoding of a polypeptide as indicated in Table XII,
application no. 30, columns 5 or 7, or a fragment thereof, which
confers an increase in the amount of the fine chemical as indicated
in table XII, column 6, e.g of valine and/or leucine resp., in an
organism or a part thereof; b) nucleic acid molecule comprising of
a nucleic acid molecule as indicated in Table XI, application no.
30, columns 5 or 7, c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of valine and/or leucine
resp., in an organism or a part thereof; d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of valine and/or leucine resp., in an organism or a part
thereof; e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of valine and/or
leucine resp., in an organism or a part thereof; f) nucleic acid
molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table XIII, application no. 30, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of valine and/or leucine resp., in an organism
or a part thereof; g) nucleic acid molecule encoding a polypeptide
which is isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of valine and/or leucine
resp., in an organism or a part thereof; h) nucleic acid molecule
encoding a polypeptide comprising a consensus sequence as indicated
in Table XIV, application no. 30, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of valine and/or leucine resp., in an organism
or a part thereof; and i) nucleic acid molecule which is obtainable
by screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of valine and/or leucine resp., in an organism or a part
thereof. whereby the nucleic acid molecule distinguishes over the
sequence as indicated in Table XI, application no. 30, columns 5 or
7, by one or more nucleotides. 7. A nucleic acid construct which
confers the expression of the nucleic acid molecule of claim 6,
comprising one or more regulatory elements. 8. A vector comprising
the nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7. 9. The vector as claimed in claim 8, wherein
the nucleic acid molecule is in operable linkage with regulatory
sequences for the expression in a prokaryotic or eukaryotic, or in
a prokaryotic and eukaryotic, host. 10. A host cell, which has been
transformed stably or transiently with the vector as claimed in
claim 8 or 9 or the nucleic acid molecule as claimed in claim 6 or
the nucleic acid construct of claim 7 or produced as described in
claim any one of claims 2 to 5. 11. The host cell of claim 10,
which is a transgenic host cell. 12. The host cell of claim 10 or
11, which is a plant cell, an animal cell, a microorganism, or a
yeast cell, a fungus cell, a prokaryotic cell, an eukaryotic cell
or an archaebacterium. 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. 14. A polypeptide produced by the
process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table XII,
application no. 30, columns 5 or 7, by one or more amino acids
[13522] 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. 16. A plant tissue, propagation
material, harvested material or a plant comprising the host cell as
claimed in claim 12 which is plant cell or an Agrobacterium. 17. A
method for screening for agonists and antagonists of the activity
of a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of valine and/or leucine
resp., in an organism or a part thereof comprising: (a) contacting
cells, tissues, plants or microorganisms which express the a
polypeptide encoded by the nucleic acid molecule of claim 5
conferring an increase in the amount of valine and/or leucine
resp., in an organism or a part thereof with a candidate compound
or a sample comprising a plurality of compounds under conditions
which permit the expression the polypeptide; (b) assaying the
valine and/or leucine resp., level or the polypeptide expression
level in the cell, tissue, plant or microorganism or the media the
cell, tissue, plant or microorganisms is cultured or maintained in;
and (c) identifying a agonist or antagonist by comparing the
measured valine and/or leucine resp., level or polypeptide
expression level with a standard valine and/or leucine resp., or
polypeptide expression level measured in the absence of said
candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. 18. A process for the identification
of a compound conferring increased valine and/or leucine resp.,
production in a plant or microorganism, comprising the steps: (a)
culturing a plant cell or tissue or microorganism or maintaining a
plant expressing the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of valine
and/or leucine resp., in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with dais readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of valine
and/or leucine resp., in an organism or a part thereof; (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. 19. A method for the identification of a gene
product conferring an increase in valine and/or leucine resp.,
production in a cell, comprising the following steps: (a)
contacting the nucleic acid molecules of a sample, which can
contain a candidate gene encoding a gene product conferring an
increase in valine and/or leucine resp., after expression with the
nucleic acid molecule of claim 6; (b) identifying the nucleic acid
molecules, which hybridise under relaxed stringent conditions with
the nucleic acid molecule of claim 6; (c) introducing the candidate
nucleic acid molecules in host cells appropriate for producing
valine and/or leucine resp.; (d) expressing the identified nucleic
acid molecules in the host cells; (e) assaying the valine and/or
leucine resp., level in the host cells; and (f) identifying nucleic
acid molecule and its gene product which expression confers an
increase in the valine and/or leucine resp., level in the host cell
in the host cell after expression compared to the wild type. 20. A
method for the identification of a gene product conferring an
increase in valine and/or leucine resp., production in a cell,
comprising the following steps: (a) identifying in a data bank
nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the valine and/or leucine resp., amount or level in an organism or
a part thereof after expression, and which are at least 20% homolog
to the nucleic acid molecule of claim 6; (b) introducing the
candidate nucleic acid molecules in host cells appropriate for
producing valine and/or leucine resp.; (c) expressing the
identified nucleic acid molecules in the host cells; (d) assaying
the valine and/or leucine resp., level in the host cells; and (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the valine and/or leucine resp.,
level in the host cell after expression compared to the wild type.
21. A method for the production of an agricultural composition
comprising the steps of the method of any one of claims 17 to 20
and formulating the compound identified in any one of claims 17 to
20 in a form acceptable for an application in agriculture. 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. 23. Use of the
nucleic acid molecule as claimed in claim 6 for the identification
of a nucleic acid molecule conferring an increase of valine and/or
leucine resp., after expression. 24. Use of the polypeptide of
claim 14 or the nucleic acid construct claim 7 or the gene product
identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of valine
and/or leucine resp., levels in an organism. 25. Food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. 26. Use of the nucleic acid molecule
of claim 6, the polypeptide of claim 14, the nucleic acid construct
of claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the harvested material of claim
16, the host cell of claim 10 to 12 or the gene product identified
according to the method of claim 19 or 20 for the protection of a
plant against a valine and/or leucine synthesis inhibiting
herbicide.
[13523] [00554.0.0.30] Abstract: see [00554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[13524] [0000.0.31.31] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and corresponding embodiments as
described herein as follows.
[13525] [0001.0.0.31] to [0008.0.0.31]: see [0001.0.0.27] to
[0008.0.0.27]
[13526] [0009.0.31.31] As described above, the essential amino
acids are necessary for humans and many mammals, for example for
livestock. Arginine is a semi-essential amino acid involved in
multiple areas of human physiology and metabolism. It is not
considered essential because humans can synthesize it de novo from
glutamine, glutamate, and proline. However, dietary intake remains
the primary determinant of plasma arginine levels, since the rate
of arginine biosynthesis does not increase to compensate for
depletion or inadequate supply. Dietary arginine intake regulates
whole body arginine synthesis from proline in the neonatal piglet.
The maximal rate of arginine synthesis (0.68 g/kg/d) is not enough
to supply the whole body metabolic requirement for arginine in the
young pig. In animals, glutamate functions as a neurotransmitter
and activates glutamate receptor cation channels (iGluRs), which
trigger electrical or Ca.sup.2+ signal cascades. In plants, amino
acids are involved in signalling of both plant nitrogen status and
plant nitrogen:carbon ratios. Endogenous glutamine has been
implicated in feedback inhibition of root N uptake, via the
suppression of transcription of genes encoding inorganic nitrogen
transporters (Rawat et al., Plant Journal 19: 143-152, 1999; Zhuo
et al., Plant Journal 17: 563-568, 1999). The nonessential amino
acid, proline, is synthesized from L-ornithine or L-glutamate. The
proline from L-ornithine is linked to protein metabolism in the
urea cycle and the proline from L-glutamate is linked to
carbohydrate metabolism. Collagen is the major reservoir for
proline in the body. Vitamin C should be used with proline for
collagen problems.
[13527] [0010.0.0.31] to [0011.0.0.31]: see [0010.0.0.27] to
[0011.0.0.27]
[13528] [0012.0.31.31] It is an object of the present invention to
develop an inexpensive process for the synthesis of arginine and/or
glutamate and/or glutamine and/or proline, preferably L-arginine
and/or L-glutamate and/or L-glutamine and/or L-proline.
[13529] [0013.0.0.31]: see [0013.0.0.27]
[13530] [0014.0.31.31] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is arginine and/or glutamate
and/or glutamine and/or proline, preferably L-arginine and/or
L-glutamate and/or L-glutamine and/or L-proline. Accordingly, in
the present invention, the term "the fine chemical" as used herein
relates to "arginine and/or glutamate and/or glutamine and/or
proline". Further, the term "the fine chemicals" as used herein
also relates to fine chemicals comprising arginine and/or glutamate
and/or glutamine and/or proline.
[13531] [0015.0.31.31] In one embodiment, the term "the fine
chemical" means arginine and/or glutamate and/or glutamine and/or
proline, preferably L-arginine and/or L-glutamate and/or
L-glutamine and/or L-proline. Throughout the specification the term
"the fine chemical" means arginine and/or glutamate and/or
glutamine and/or proline, preferably L-arginine and/or L-glutamate
and/or L-glutamine and/or L-proline, its salts, ester or amids in
free form or bound to proteins. In a preferred embodiment, the term
"the fine chemical" means arginine and/or glutamate and/or
glutamine and/or proline, preferably L-arginine and/or L-glutamate
and/or L-glutamine and/or L-proline, in free form or its salts or
bound to proteins.
[13532] [0016.0.31.31]
[13533] Accordingly, the present invention relates to a process for
the production of the respective fine chemical comprising [13534]
(a) increasing or generating the activity of one or more [13535] of
a protein as shown in table XII, application no. 31, column 3
encoded by the nucleic acid sequences as shown in table XI,
application no. 31, column 5, in a non-human organism or in one one
or more parts thereof or [13536] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
amino acids of the invention or fine chemicals comprising amino
acids of the invention, in said organism or in the culture medium
surrounding the organism.
[13537] [0016.1.31.31] Accordingly, the term "the fine chemical"
means in one embodiment "arginine" in relation to all sequences
listed in Tables XI to XIV, line 10 or homologs thereof and
means in one embodiment "proline" in relation to all sequences
listed in Table XI to XIV, line 11 or homologs thereof and means in
one embodiment "glutamine" in relation to all sequences listed in
Table XI to XIV, lines 12 and/or 14 to 15 or homologs thereof and
means in one embodiment "glutamate" in relation to all sequences
listed in Table XI to XIV, line 13 or homologs thereof.
[13538] Accordingly, in one embodiment the term "the fine chemical"
means "arginine" and "glutamine" in relation to all sequences
listed in Table XI to XIV, lines 10 and/or 12;
in one embodiment the term "the fine chemical" means "glutamate"
and "glutamine" in relation to all sequences listed in Table XI to
XIV, lines 13 and/or 14;
[13539] [0017.0.0.31] to [0019.0.0.31]: see [0017.0.0.27] to
[0019.0.0.27]
[13540] [0020.0.31.31] Surprisingly it was found, that the
transgenic expression of the Zea mays protein as indicated in Table
XII, column 5, lines 10 and 12 in a plant conferred an increase in
arginine and/or glutamine (or the respective fine chemical) content
of the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of arginine; in one
embodiment, said protein or its homologs are used for the
production of glutamine, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of arginine and/or
glutamine.
[13541] Surprisingly it was found, that the transgenic expression
of the Brassica napus protein as indicated in Table XII, column 5,
line 11 in a plant conferred an increase in proline content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of proline.
[13542] Surprisingly it was found, that the transgenic expression
of the Linum usitatissimum protein as indicated in Table XII,
column 5, lines 13 and 14 in a plant conferred an increase in
glutamate and/or glutamine (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of glutamate;
in one embodiment, said protein or its homologs are used for the
production of glutamine, in one embodiment, said protein or its
homologs are used for the production of one or more fine chemical
selected from the group consisting of glutamate and/or
glutamine.
[13543] Surprisingly it was found, that the transgenic expression
of the Brassica napus protein as indicated in Table XII, column 5,
line 15 in a plant conferred an increase in glutamine content of
the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of glutamine.
[13544] [0021.0.0.31]: see [0021.0.0.27]
[13545] [0022.0.31.31] The sequence of YGR135W from Saccharomyces
cerevisiae has been published in Goffeau st al., Science 274
(5287), 546-547, 1996 and Tettelin et al., Nature 387 (6632 Suppl),
81-84 (1997) and its activity is being defined as a "proteasome
component Y13". Accordingly, in one embodiment, the process of the
present invention comprises the use of a a gene product with an
activity of multicatalytic endopeptidase complex chain C9
superfamily, preferably a protein with proteasome component Y13
activity, from Saccharomyces cerevisiae or a plant or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of proline, in particular for increasing the amount of
proline, preferably proline in free or bound form in an organism or
a part thereof, as mentioned.
[13546] The sequence of YKR057W from Saccharomyces cerevisiae has
been published in Dujon et al., Nature 369 (6479), 371-378, 1994
and Goffeau et al., Science 274 (5287), 546-547, 1996 and its
activity is being defined as a ribosomal protein, similar to S21
ribosomal proteins, involved in ribosome biogenesis and
translation. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of rat ribosomal protein S21 superfamily, preferably a
protein with a ribosomal protein, similar to S21 ribosomal
proteins, involved in ribosome biogenesis and translation activity
from Saccharomyces cerevisiae or a plant or its homolog, e.g. as
shown herein, for the production of the fine chemical, meaning of
arginine and/or glutamine, in particular for increasing the amount
of arginine and/or glutamine, in particular for increasing the
amount of arginine, in particular for increasing the amount of
glutamine, in particular for increasing the amount of arginine and
glutamine, preferably arginine and/or glutamine in free or bound
form in an organism or a part thereof, as mentioned.
[13547] The sequence of b1343 (Accession number NP.sub.--415490)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a ATP-dependent RNA helicase, stimulated by 23S rRNA.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
Escherichia coli b1343 protein, preferably a protein with the
activity of a ATP-dependent RNA helicase, stimulated by 23S rRNA
from E. coli or a plant or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of glutamine and/or
glutamate, in particular for increasing the amount of glutamine, in
particular for increasing the amount of glutamate, in particular
for increasing the amount of glutamine and glutamate, preferably
glutamine and/or glutamate in free or bound form in an organism or
a part thereof, as mentioned. The sequence of b2426 (Accession
number NP.sub.--416921) from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a putative oxidoreductase with
NAD(P)-binding domain. Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of ribitol dehydrogenase, short-chain alcohol
dehydrogenase homology superfamily, preferably a protein with the
activity of a putative oxidoreductase with NAD(P)-binding domain
from E. coli or a plant or its homolog, e.g. as shown herein, for
the production of the fine chemical, meaning of glutamine, in
particular for increasing the amount of glutamine, preferably
glutamine in free or bound form in an organism or a part thereof,
as mentioned.
[13548] [0022.1.0.31] to [0023.0.0.31] see [0022.1.0.27] to
[0023.0.0.27]
[13549] [0023.1.31.31] Homologs of the polypeptide disclosed in
table XII, application no. 31, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 31, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 31, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 31,
column 7, resp.
[13550] [0024.0.0.31]: see [0024.0.0.27]
[13551] [0025.0.31.31] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 31, column 3" if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 31, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[13552] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 31,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 31,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[13553] [0025.1.0.31] to [0033.0.0.31]: see [0025.1.0.27] to
[0033.0.0.27]
[13554] [0034.0.31.31] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 31, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[13555] [0035.0.0.31] to [0044.0.0.31]: see [0035.0.0.27] to
[0044.0.0.27]
[13556] [0045.0.31.31] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
31, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line,
between 10% and 50%, 10% and 100%, 10% and 200%, 10% and 300%, 10%
and 400%, 10% and 500%, 20% and 50%, 20% and 250%, 20% and 500%,
50% and 100%, 50% and 250%, 50% and 500%, 500% and 1000% or
more.
[13557] [0046.0.31.31] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
31, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line
confers an increase of the respective fine chemical and of further
amino acids or their precursors.
[13558] [0047.0.0.31] and [0048.0.0.31]: see [0047.0.0.27] and
[0048.0.0.27]
[13559] [0049.0.31.31] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 31, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 31, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 31, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[13560] [0050.0.31.31] For the purposes of the present invention,
the term "arginine" and/or "glutamate" and/or "glutamine" and/or
"proline" and "L-arginine" and/or" L-glutamate" and/or
"L-glutamine" and/or "L-proline" also encompass the corresponding
salts, such as, for example, arginine- and/or glutamate- and/or
glutamine- and/or proline-hydrochloride or arginine and/or
glutamate and/or glutamine and/or proline sulfate.
[13561] Preferably the term arginine and/or glutamate and/or
glutamine and/or proline is intended to encompass the term
L-arginine and/or L-glutamate and/or L-glutamine and/or
L-proline.
[13562] [0051.0.0.31] and [0052.0.0.31]: see [0051.0.0.27] and
[0052.0.0.27]
[13563] [0053.0.31.31] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[13564] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 31, columns 5 and 7 or its homologs activity
having herein-mentioned amino acids of the invention increasing
activity; and/or [13565] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 31,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 31, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned amino acids of the invention increasing
activity; and/or [13566] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned amino acid increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 31, columns 5 and 7 or its
homologs activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [13567] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned amino acids of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 31, columns 5
and 7 or its homologs activity; and/or [13568] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned amino acids of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 31, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [13569] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned amino acids of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 31, columns 5 and 7 or its
homologs activity, and/or [13570] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned amino acids of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 31, columns 5 and 7 or its
homologs activity; and/or [13571] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 31, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[13572] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [13573] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[13574] [0054.0.31.31] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 31 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 31, column 3 or its homologs.
[13575] [0055.0.0.31] to [0071.0.0.31]: see [0055.0.0.27] to
[0071.0.0.27]
[13576] [0072.0.31.31] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to arginine and/or glutamate and/or glutamine and/or
proline Argininosuccinate, Citrulline, Ornithine, Urea,
Pyrroline-5-carboxylate, Hydroxy-proline,
Hydroxypyrroline-carboxylate, 3-Hydroxypyrroline-5-carboxylate,
.gamma.-Glutamylcysteine, Glutathione, Hydroxyglutamate,
4-Hydroxyglutamate, Oxoglutarate, 4-Hydroxy-2-oxoglutarate,
Glutamine.
[13577] [0073.0.31.31] Accordingly, in one embodiment, the process
according to the invention relates to a process which comprises:
[13578] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [13579] b) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table XII, application no. 31, columns 5 or 7, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the respective fine chemical in an organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [13580] c) growing the organism,
preferably the microorganism, the non-human animal, the plant or
animal cell, the plant or animal tissue or the plant under
conditions which permit the production of the fine chemical in the
organism, preferably the microorganism, the plant cell, the plant
tissue or the plant; and [13581] d) if desired, revovering,
optionally isolating, the free and/or bound the fine chemical and,
optionally further free and/or bound amino acids synthetized by the
organism, the microorganism, the non-human animal, the plant or
animal cell, the plant or animal tissue or the plant.
[13582] [0074.0.0.31] to [0084.0.0.31]: see [0074.0.0.27] to
[0084.0.0.27]
[13583] [0085.0.31.31] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [13584] a) the nucleic acid sequence as
indicated in Table XI, application no. 31, columns 5 or 7, or a
derivative thereof, or [13585] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 31, columns
5 or 7, or a derivative thereof, or [13586] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[13587] [0086.0.0.31] and [0088.1.0.31]: see [0086.0.0.27] and
[0088.1.0.27]
[13588] [0089.0.0.31] to [0097.0.0.31]: see [0089.0.0.27] to
[0097.0.0.27]
[13589] [0098.0.31.31] In a preferred embodiment, the fine chemical
(arginine and/or glutamate and/or glutamine and/or proline) is
produced in accordance with the invention and, if desired, is
isolated. The production of further amino acids such as methionine,
lysine and/or threonine mixtures of amino acid by the process
according to the invention is advantageous.
[13590] [0099.0.0.31] to [0102.0.0.31]: see [0099.0.0.27] to
[0102.0.0.27]
[13591] [0103.0.31.31] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[13592] a) nucleic acid molecule encoding, preferably at least the
mature form, of the polypeptide having a sequence as indicated in
Table XII, application no. 31, columns 5 or 7, or a fragment
thereof, which confers an increase in the amount of the respective
fine chemical in an organism or a part thereof; [13593] b) nucleic
acid molecule comprising, preferably at least the mature form, of a
nucleic acid molecule having a sequence as indicated in Table XI,
application no. 31. [13594] c) nucleic acid molecule whose sequence
can be deduced from a polypeptide sequence encoded by a nucleic
acid molecule of (a) or (b) as result of the degeneracy of the
genetic code and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [13595] d) nucleic acid
molecule encoding a polypeptide which has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[13596] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [13597] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c) and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [13598] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to [13599]
(c) and and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [13600] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers pairs having a sequence as indicated in
Table XIII, application no. 31; column 7, and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [13601] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from an expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[13602] j) nucleic acid molecule which encodes a polypeptide
comprising the consensus sequence having a sequences as indicated
in Table XIV, application no. 31, column 7, and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [13603] k) nucleic acid molecule
comprising one or more of the nucleic acid molecule encoding the
amino acid sequence of a polypeptide encoding a domain of the
polypeptide indicated in Table XII, application no. 31, columns 5
or 7, and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; and [13604] l)
nucleic acid molecule which is obtainable by screening a suitable
library under stringent conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; or which comprises a
sequence which is complementary thereto.
[13605] [00103.1.31.31.] In one embodiment, the nucleic acid
molecule used in the process of the invention distinguishes over
the sequence indicated in Table XI, application no. 31, columns 5
or 7 by one or more nucleotides. In one embodiment, the nucleic
acid molecule used in the process of the invention does not consist
of the sequence shown in indicated in Table XI, application no. 31,
columns 5 or 7. In one embodiment, the nucleic acid molecule used
in the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI,
application no. 31, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table XII, application no. 31, columns 5 or 7.
[13606] [0104.0.31.31] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence indicated in
Table XI, application no. 31, columns 5 or 7, by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of the sequence
indicated in Table XI, application no. 31, columns 5 or 7, In one
embodiment, the nucleic acid molecule of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to the
sequence indicated in Table XI, application no. 31, columns 5 or 7,
In another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table XII, application no.
31, columns 5 or 7
[13607] [0105.0.0.31] to [0107.0.0.31]: see [0105.0.0.27] to
[0107.0.0.27]
[13608] [0108.0.31.31] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 31, columns 5 or 7, nucleic
acid molecules which are derived from a amino acid sequences as
indicated in Table XII, application no. 31, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 31, column 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of a polypeptide as indicated in Table XI, application no.
31, column 3, 5 or 7, or e.g. conferring a increase of the fine
chemical after increasing its expression or activity are
advantageously increased in the process according to the
invention.
[13609] [0109.0.0.31]: see [0109.0.0.27]
[13610] [0110.0.31.31] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
used in the process of the invention, e.g. of a protein as
indicated in Table XII, application no. 31, column 5, or being
encoded by a nucleic acid molecule indicated in Table XI,
application no. 31, column 5, or of its homologs, e.g. as indicated
in Table XII, application no. 31, column 7, can be determined from
generally accessible databases.
[13611] [0111.0.0.31]: see [0111.0.0.27]
[13612] [0112.0.31.31] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with protein
activity of proteins as indicated in Table XII, application no. 31,
columns 5 or 7, and conferring a arginine and/or glutamate and/or
proline and/or glutamine increase.
[13613] [0113.0.0.31] to [0120.0.0.31]: see [0113.0.0.27] to
[0120.0.0.27]
[13614] [0121.0.31.31] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 31, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a increase of the respective fine
chemical after increasing its activity.
[13615] [0122.0.0.31] to [0127.0.0.31]: see [0122.0.0.27] to
[0127.0.0.27]
[13616] [0128.0.31.31] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 31, column 7, by means of polymerase chain reaction
can be generated on the basis of a sequence shown herein, for
example the sequence as indicated in Table XI, application no. 31,
columns 5 or 7, or the sequences derived from a sequence as
indicated in Table XII, application no. 31, columns 5 or 7.
[13617] [0129.0.31.31] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence indicated in Table XIV,
application no. 31, column 7, is derived from said alignments.
[13618] [0130.0.0.31] to [0138.0.0.31]: see [0130.0.0.27] to
[0138.0.0.27]
[13619] [0139.0.31.31] Polypeptides having above-mentioned
activity, i.e. conferring the increase of the respective fine
chemical level, derived from other organisms, can be encoded by
other DNA sequences which hybridize to a sequences indicated in
Table XI, application no. 31, columns 5 or 7, under relaxed
hybridization conditions and which code on expression for peptides
having the respective fine chemical, in particular, of arginine
and/or glutamate and/or proline and/or glutamine, resp., increasing
activity.
[13620] [0140.0.0.31] to [0146.0.0.31]: see [0140.0.0.27] to
[0146.0.0.27]
[13621] [0147.0.31.31] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 31, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences s
such that it can hybridize to one of said nucleotide sequences
thereby forming a stable duplex. Preferably, the hybridisation is
performed under stringent hybridization conditions. However, a
complement of one of the herein disclosed sequences is preferably a
sequence complement thereto according to the base pairing of
nucleic acid molecules well known to the skilled person. For
example, the bases A and G undergo base pairing with the bases T
and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[13622] [0148.0.31.31] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 31,
columns 5 or 7 or a portion thereof and preferably has above
mentioned activity, in particular, of arginine and/or glutamate
and/or proline and/or glutamine increasing activity after
increasing the activity or an activity of a product of a gene
encoding said sequences or their homologs.
[13623] [0149.0.31.31] The nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 31,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity and as indicated in indicated in Table
XII.
[13624] [00149.1.31.31] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table
XI, application no. 31, columns 5 or 7, has further one or more of
the activities annotated or known for the a protein as indicated in
Table XII, application no. 31, column 3.
[13625] [0150.0.31.31] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table XI, application no. 31, columns
5 or 7, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of arginine and/or glutamate
and/or proline and/or glutamine, resp., if its activity is
increased. The nucleotide sequences determined from the cloning of
the present protein-according-to-the-invention-encoding gene allows
for the generation of probes and primers designed for use in
identifying and/or cloning its homologues in other cell types and
organisms. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, 15 preferably about 20 or 25, more
preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one of the sequences set forth, e.g., as indicated in
Table XI, application no. 31, columns 5 or 7, anti-sense sequence
of one of the sequences, e.g., as indicated in Table XI,
application no. 31, columns 5 or 7, or naturally occurring mutants
thereof. Primers based on a nucleotide of invention can be used in
PCR reactions to clone homologues of the polypeptide of the
invention or of the polypeptide used in the process of the
invention, e.g. as the primers described in the examples of the
present invention, e.g. as shown in the examples. A PCR with the
primer pairs indicated in Table XIII, application no. 31, column 7,
will result in a fragment of a polynucleotide sequence as indicated
in Table XI, application no. 31, columns 5 or 7 or its gene
product.
[13626] [0151.0.0.31]: see [0151.0.0.27]
[13627] [0152.0.31.31] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 31, columns 5
or 7, such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular an activity increasing the level of arginine and/or
glutamate and/or proline and/or glutamine, resp., as mentioned
above or as described in the examples in plants or microorganisms
is comprised.
[13628] [0153.0.31.31] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 31,
columns 5 or 7, such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 31, columns 5 or 7, has for
example an activity of a polypeptide as indicated in Table XII,
application no. 31, column 3.
[13629] [0154.0.31.31] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 31, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[13630] [0155.0.0.31] and [0156.0.0.31]: see [0155.0.0.27] and
[0156.0.0.27]
[13631] [0157.0.31.31] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences
indicated in Table XI, application no. 31, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides comprising the consensus sequences as indicated
in Table XIV, application no. 31, column 7 or of the polypeptide as
indicated in Table XII, application no. 31, columns 5 or 7, or
their functional homologues. Advantageously, the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, a consensus sequences as indicated in Table XIV,
application no. 31, column 7, or of the polypeptide as indicated in
Table XII, application no. 31, columns 5 or 7, or the functional
homologues. In a still further embodiment, the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention encodes a full length protein which is
substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table XIV, application no. 31,
column 7, or of a polypeptide as indicated in Table XII,
application no. 31, columns 5 or 7, or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 31, columns 5 or 7.
Preferably the nucleic acid molecule of the invention is a
functional homologue or identical to a nucleic acid molecule
indicated in Table XI, application no. 31, columns 5 or 7.
[13632] [0158.0.0.31] to [0160.0.0.31]: see [0158.0.0.27] to
[0160.0.0.27]
[13633] [0161.0.31.31] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 31,
columns 5 or 7. The nucleic acid molecule is preferably at least
20, 30, 50, 100, 250 or more nucleotides in length.
[13634] [0162.0.0.31]: see [0162.0.0.27]
[13635] [0163.0.31.31] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 31, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[13636] [0164.0.0.31]: see [0164.0.0.27]
[13637] [0165.0.31.31] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 31, columns 5 or 7.
[13638] [0166.0.0.31] and [0167.0.0.31]: see [0166.0.0.27] and
[0167.0.0.27]
[13639] [0168.0.31.31] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 31, columns 5 or 7, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 31,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table XII, application no. 31, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 31, columns 5
or 7, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table XII, application no.
31, columns 5 or 7, and most preferably at least about 96%, 97%,
98%, or 99% identical to the sequence as indicated in Table XII,
application no. 31, columns 5 or 7.
[13640] [0169.0.0.31] to [0172.0.0.31]: see [0169.0.0.27] to
[0172.0.0.27]
[13641] [0173.0.31.31] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108442 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108442 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[13642] [0174.0.0.31]: see [0174.0.0.27]
[13643] [0175.0.31.31] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108443 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108443 by the above program algorithm with the
above parameter set, has a 80% homology.
[13644] [0176.0.31.31] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 31,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 31, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 31, columns
5 or 7.,
[13645] [0177.0.31.31] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 31,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptide as indicated in Table XII,
application no. 31, columns 5 or according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 31, columns
5 or 7.
[13646] [0178.0.0.31]: see [0178.0.0.27]
[13647] [0179.0.31.31] A nucleic acid molecule encoding an
homologous to a protein sequence as indicated in Table XII,
application no. 31, columns 5 or 7, can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table XI, application no.
31, columns 5 or 7, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences of a
sequences as indicated in Table XI, application no. 31, columns 5
or 7 by standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[13648] [0180.0.0.31] to [0183.0.0.31]: see [0180.0.0.27] to
[0183.0.0.27]
[13649] [0184.0.31.31] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 31,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 31, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 31,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[13650] [0185.0.31.31] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 31, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table XI, application no. 31, columns 5 or 7, In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequences as indicated in Table XI, application no. 31, columns 5
or 7.
[13651] [0186.0.31.31] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table XII,
application no. 31, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 31, columns 5 or 7.
[13652] [0187.0.31.31] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table XII, application no.
31, columns 5 or 7, comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence as
indicated in Table XII, application no. 31, columns 5 or 7.
[13653] [0188.0.31.31] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 31, columns 5 or 7 and is expressed under
identical conditions.
[13654] [0189.0.31.31] Homologues of a sequences as indicated in
Table XI, application no. 31, columns 5 or 7, or of a derived
sequences as indicated in Table XII, application no. 31, columns 5
or 7 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[13655] [0190.0.0.31] to [0203.0.0.31]: see [0190.0.0.27] to
[0203.0.0.27]
[13656] [0204.0.31.31] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule which comprises a
nucleic acid molecule selected from the group consisting of:
[13657] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 31, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII, application no. 31,
column 6 in an organism or a part thereof [13658] b) nucleic acid
molecule comprising, preferably at least the mature form, of a
nucleic acid molecule as indicated in Table XI, application no. 31,
columns 5 or 7, or a fragment thereof conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [13659] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [13660] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[13661] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [13662] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [13663] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13664] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table XIII, application no. 31, column 7, and
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII, application no. 31,
column 6 in an organism or a part thereof; [13665] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[13666] j) nucleic acid molecule which encodes a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 31, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical, in particular according
to table XII, application no. 31, column 6 in an organism or a part
thereof; [13667] k) nucleic acid molecule encoding the amino acid
sequence of a polypeptide encoding a domaine of a polypeptide as
indicated in Table XII, application no. 31, columns 5 or 7, and
conferring an increase in the amount of the respective fine
chemical, in particular accccording to table XII, application no.
31, column 6 in an organism or a part thereof; and [13668] l)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (h) or of a nucleic
acid molecule as indicated in Table XI, application no. 31, columns
5 or 7, or a nucleic acid molecule encoding, preferably at least
the mature form of, a polypeptide as indicated in Table XII,
application no. 31, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical according to table XII,
application no. 31, column 6 in an organism or a part thereof;
[13669] or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over the sequence indicated in Table
XI, application no. 31, columns 5 or 7, by one or more nucleotides.
In one embodiment, the nucleic acid molecule does not consist of
the sequence shown and indicated in Table XI, application no. 31,
columns 5 or 7, In one embodiment, the nucleic acid molecule is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 31, columns 5 or
7.
[13670] In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table XII,
application no. 31, columns 5 or 7.
[13671] In an other embodiment, the nucleic acid molecule of the
present invention is at least 30%, 40%, 50%, or 60% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 31, columns 5 or 7.
In a further embodiment the nucleic acid molecule does not encode a
polypeptide sequence as indicated in Table XII, application no. 31,
columns 5 or 7.
[13672] Accordingly, in one embodiment, the nucleic acid molecule
of the differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 31, columns 5 or
7.
[13673] Accordingly, in one embodiment, the nucleic acid molecule
of the present invention encodes a polypeptide, which differs at
least in one or more amino acids from a polypeptide indicated in
Table XII, application no. 31, columns 5 or 7.
[13674] In another embodiment, a nucleic acid molecule indicated in
Table XI, application no. 31, columns 5 or 7.
[13675] Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table XII, application no.
31, columns 5 or 7.
[13676] In a further embodiment, the protein of the present
invention is at least 30%, 40%, 50%, or 60% identical to a protein
sequence indicated in Table XII, application no. 31, columns 5 or
7, and less than 100%, preferably less than 99.999%, 99.99% or
99.9%, more preferably less than 99%, 985, 97%, 96% or 95%
identical to a sequence as indicated in Table XI, columns 5 or
7.
[13677] [0205.0.0.31] to [0226.0.0.31]: see [0205.0.0.27] to
[0226.0.0.27]
[13678] [0227.0.31.31] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[13679] In addition to a sequence indicated in Table XI,
application no. 31, columns 5 or 7, or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the amino acid biosynthetic pathway such as for
L-lysine, L-threonine and/or L-methionine and/or L-leucine and/or
isoleucine and/or valine is expressed in the organisms such as
plants or microorganisms. It is also possible that the regulation
of the natural genes has been modified advantageously so that the
gene and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the amino acids desired since, for example, feedback
regulations no longer exist to the same extent or not at all. In
addition it might be advantageously to combine one or more of the
sequences indicated in Table XI, application no. 31, columns 5 or
7, with genes which generally support or enhances to growth or
yield of the target organisms, for example genes which lead to
faster growth rate of microorganisms or genes which produces
stress-, pathogen, or herbicide resistant plants.
[13680] [0228.0.0.31] to [0230.0.0.31]: see[0228.0.0.27] to
[0230.0.0.27]
[13681] [0231.0.31.31] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously an arginine and/or glutamate and/or
glutamine and/or proline degrading protein is attenuated, in
particular by reducing the rate of expression of the corresponding
gene.
[13682] [0232.0.0.31] to [0282.0.0.31]: see [0232.0.0.27] to
[0282.0.0.27]
[13683] [0283.0.31.31] Moreover, a native polypeptide conferring
the increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table
XII, application no. 31, column 3, or an antibody against a
polypeptide as indicated in Table XII, application no. 31, columns
5 or 7, which can be produced by standard techniques utilizing the
polypeptid of the present invention or fragment thereof, i.e., the
polypeptide of this invention. Preferred are monoclonal
antibodies.
[13684] [0284.0.0.31]: see [0284.0.0.27].
[13685] [0285.0.31.31] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 31, columns 5 or 7, or as encoded by a nucleic
acid molecule as indicated in Table XI, application no. 31, columns
5 or 7, or functional homologues thereof.
[13686] [0286.0.31.31] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 31, column 7. In another
embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 31, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table XIV, application no. 31, column 7.
[13687] [0287.0.0.31] to [0290.0.0.31]: see [0287.0.0.27] to
[0290.0.0.27]
[13688] [0291.0.31.31] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[13689] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table XII,
application no. 31, columns 5 or 7, by one or more amino acids. In
one embodiment, polypeptide distinguishes form a sequence as
indicated in Table XII, application no. 31, columns 5 or 7, by more
than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, even more preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table XII, application no. 31, columns 5 or 7, by not
more than 80% or 70% of the amino acids, preferably not more than
60% or 50%, more preferred not more than 40% or 30%, even more
preferred not more than 20% or 10%. In an other embodiment, said
polypeptide of the invention does not consist of a sequence as
indicated in Table XII, application no. 31, columns 5 or 7.
[13690] [0292.0.0.31]: see [0292.0.0.27]
[13691] [0293.0.31.31] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, the polypeptide of
the invention has a sequence which distinguishes from a sequence as
indicated in Table XII, application no. 31, columns 5 or 7, by one
or more amino acids. In an other embodiment, said polypeptide of
the invention does not consist of the sequence as indicated in
Table XII, application no. 31, columns 5 or 7.
[13692] In a further embodiment, said polypeptide of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by a nucleic acid molecules as indicated in
Table XI, application no. 31, columns 5 or 7.
[13693] [0294.0.31.31] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 31, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 31, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[13694] [0295.0.0.31] and [0297.0.0.31]: see [0295.0.0.27] and
[0297.0.0.27]
[13695] [0297.1.31.31] Non polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table XII, application no. 31, columns 3, 5 or 7.
[13696] [0298.0.31.31] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 31, columns 5 or 7. The
portion of the protein is preferably a biologically active portion
as described herein. Preferably, the polypeptide used in the
process of the invention has an amino acid sequence identical to a
sequence as indicated in Table XII, application no. 31, columns 5
or 7.
[13697] [0299.0.31.31] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 31,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 31, columns 5 or 7, or which is
homologous thereto, as defined above.
[13698] [0300.0.31.31] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 31, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 31, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[13699] [0301.0.0.31]: see [0301.0.0.27]
[13700] [0302.0.31.31] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 31, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[13701] [0303.0.0.31]: see [0303.0.0.27]
[13702] [0304.0.31.31] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 31, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[13703] [0305.0.0.31] to [0308.0.0.31]: see [0306.0.0.27] to
[0308.0.0.27]
[13704] [0309.0.31.31] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table XII,
application no. 31, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table XII, application no. 31, columns 5 or 7, refers
to a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous a polypeptide of the
invention, preferably which is not substantially homologous to a
polypeptide as indicated in Table XII, application no. 31,
[13705] columns 5 or 7, e.g., a protein which does not confer the
activity described herein or annotated or known for as indicated in
Table XII, application no. 31, column 3, and which is derived from
the same or a different organism. In one embodiment, a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table XII, application no. 31, columns 5 or 7, does
not confer an increase of the respective fine chemical in an
organism or part therof.
[13706] [0310.0.0.31] to [0334.0.0.31]: see [0310.0.0.27] to
[0334.0.0.27]
[13707] [0335.0.31.31] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XI, application
no. 31, columns 5 or 7, and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
application no. 31, columns 5 or 7, and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of an
protein encoded by a nucleic acid sequence sequences as indicated
in Table XI, application no. 31, columns 5 or 7, and/or homologs
thereof, one of the two RNA strands is essentially identical to at
least part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[13708] [0336.0.0.31] to [0342.0.0.31]: see [0336.0.0.27] to
[0342.0.0.27]
[13709] [0343.0.31.31] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 31, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table XI, application no. 31, columns 5 or 7, or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[13710] [0344.0.0.31] to [0361.0.0.31]: see [0344.0.0.27] to
[0361.0.0.27]
[13711] [0362.0.31.31] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 31, columns 5 or 7, e.g. encoding a
polypeptide having protein activity, as indicated in Table XII,
application no. 31, columns 3. Due to the above mentioned activity
the respective fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity of the polypeptide of the invention or nucleic
acid molecule of the invention is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a polypeptide as
indicated in Table XII, application no. 31, columns 5 or 7, means
herein that due to modulation or manipulation of the genome, an
activity as annotated for a polypeptide as indicated in Table XII,
application no. 31, column 3, e.g. having a sequence as indicated
in Table XII, application no. 31, columns 5 or 7, is increased in a
cell or an organism or a part thereof. Examples are described above
in context with the process of the invention [0363.0.0.31]: to
[0382.0.0.31]: see [0363.0.0.27] to [0382.0.0.27]
[13712] [0383.0.31.31] For preparing arginine and/or glutamate
and/or glutamine and/or proline compound-containing fine chemicals,
in particular the fine chemical, it is possible to use as arginine
and/or glutamate and/or glutamine and/or proline amino acid source
organic compounds such as, for example, citrulline,
argininosuccinate, ornithine, aspartate, 2-Oxoglutarate, glutamyl,
glutamic-semialdehyde, Pyrroline-5-carboxylate, Glutamine or else
organic arginine and/or glutamate and/or glutamine and/or proline
acid precursor compounds.
[13713] [0384.0.0.31]: see [0384.0.0.27]
[13714] [0385.0.31.31] The fermentation broths obtained in this
way, containing in particular L-arginine and/or L-glutamate and/or
L-proline and/or L-tryptophane, L-methionine, L-threonine and/or
L-lysine, normally have a dry matter content of from 7.5 to 25% by
weight. Sugar-limited fermentation is additionally advantageous, at
least at the end, but especially over at least 30% of the
fermentation time. This means that the concentration of utilizable
sugar in the fermentation medium is kept at, or reduced to, 0 to 3
g/l during this time. The fermentation broth is then processed
further. Depending on requirements, the biomass can be removed
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation or a combination of these
methods, from the fermentation broth or left completely in it. The
fermentation broth can then be thickened or concentrated by known
methods, such as, for example, with the aid of a rotary evaporator,
thin-film evaporator, falling film evaporator, by reverse osmosis
or by nanofiltration. This concentrated fermentation broth can then
be worked up by freeze-drying, spray drying, spray granulation or
by other processes.
[13715] [0386.0.0.31] to [0392.0.0.31]: see [0386.0.0.27] to
[0392.0.0.27]
[13716] [0393.0.31.31] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [13717] a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [13718] b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table XI, application no. 31, columns 5 or 7, and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[13719] c) introducing the candidate nucleic acid molecules in host
cells, preferably in a plant cell or a microorganism, appropriate
for producing the fine chemical; [13720] d) expressing the
identified nucleic acid molecules in the host cells; [13721] e)
assaying the the fine chemical level in the host cells; and [13722]
f) identifying the nucleic acid molecule and its gene product which
expression confers an increase in the the fine chemical level in
the host cell after expression compared to the wild type.
[13723] [0394.0.28.31] to [0552.0.28.31]: see [0394.0.0.28] to
[0552.0.0.28]
[13724] [0553.0.31.31]
1. A process for the production of arginine, proline, glutamine
and/or glutamate resp., which comprises (a) increasing or
generating the activity of a protein as indicated in Table XII,
application no. 31, columns 5 or 7, or a functional equivalent
thereof in a non-human organism, or in one or more parts thereof;
and (b) growing the organism under conditions which permit the
production of arginine, proline, glutamine and/or glutamate resp.
in said organism. 2. A process for the production of arginine,
proline, glutamine and/or glutamate resp., comprising the
increasing or generating in an organism or a part thereof the
expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: a)
nucleic acid molecule encoding of a polypeptide as indicated in
Table XII, application no. 31, columns 5 or 7, or a fragment
thereof, which confers an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of arginine,
proline, glutamine and/or glutamate resp., in an organism or a part
thereof; b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table XI, application no. 31, columns 5 or
7, nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of arginine, proline,
glutamine and/or glutamate resp., in an organism or a part thereof;
d) nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical as indicated in
table XII, column 6, e.g of arginine, proline, glutamine and/or
glutamate resp., in an organism or a part thereof; e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of the fine chemical as indicated in
table XII, column 6, e.g of arginine, proline, glutamine and/or
glutamate resp., in an organism or a part thereof; f) nucleic acid
molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table XIII, application no. 31, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of arginine, proline, glutamine and/or glutamate
resp., in an organism or a part thereof; g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
the fine chemical as indicated in table XII, column 6, e.g of
arginine, proline, glutamine and/or glutamate resp., in an organism
or a part thereof; h) nucleic acid molecule encoding a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 31, column 7, and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of arginine, proline, glutamine and/or glutamate resp., in an
organism or a part thereof; and i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of arginine, proline, glutamine and/or glutamate resp., in an
organism or a part thereof. or comprising a sequence which is
complementary thereto. 3. The process of claim 1 or 2, comprising
recovering of the free or bound arginine, proline, glutamine and/or
glutamate resp. 4. The process of any one of claims 1 to 3,
comprising the following steps: (a) selecting an organism or a part
thereof expressing a polypeptide encoded by the nucleic acid
molecule characterized in claim 2; (b) mutagenizing the selected
organism or the part thereof; (c) comparing the activity or the
expression level of said polypeptide in the mutagenized organism or
the part thereof with the activity or the expression of said
polypeptide of the selected organisms or the part thereof; (d)
selecting the mutated organisms or parts thereof, which comprise an
increased activity or expression level of said polypeptide compared
to the selected organism or the part thereof; (e) optionally,
growing and cultivating the organisms or the parts thereof; and (f)
recovering, and optionally isolating, the free or bound arginine,
proline, glutamine and/or glutamate resp., produced by the selected
mutated organisms or parts thereof. 5. The process of any one of
claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: a) nucleic acid molecule encoding of a polypeptide
as indicated in Table XII, application no. 31, columns 5 or 7, or a
fragment thereof, which confers an increase in the amount of the
fine chemical as indicated in table XII, column 6, e.g of arginine,
proline, glutamine and/or glutamate resp., in an organism or a part
thereof; b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table XI, application no. 31, columns 5 or
7, c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of arginine, proline,
glutamine and/or glutamate resp., in an organism or a part thereof;
d) nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical as indicated in
table XII, column 6, e.g of arginine, proline, glutamine and/or
glutamate resp., in an organism or a part thereof; e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of the fine chemical as indicated in
table XII, column 6, e.g of arginine, proline, glutamine and/or
glutamate resp., in an organism or a part thereof; f) nucleic acid
molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table XIII, application no. 31, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of arginine, proline, glutamine and/or glutamate
resp., in an organism or a part thereof; g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
the fine chemical as indicated in table XII, column 6, e.g of
arginine, proline, glutamine and/or glutamate resp., in an organism
or a part thereof; h) nucleic acid molecule encoding a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 31, column 7, and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of arginine, proline, glutamine and/or glutamate resp., in an
organism or a part thereof; and i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of arginine, proline, glutamine and/or glutamate resp., in an
organism or a part thereof. whereby the nucleic acid molecule
distinguishes over the sequence as indicated in Table XI,
application no. 31, columns 5 or 7, by one or more nucleotides. 7.
A nucleic acid construct which confers the expression of the
nucleic acid molecule of claim 6, comprising one or more regulatory
elements. 8. A vector comprising the nucleic acid molecule as
claimed in claim 6 or the nucleic acid construct of claim 7. 9. The
vector as claimed in claim 8, wherein the nucleic acid molecule is
in operable linkage with regulatory sequences for the expression in
a prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. 11. The host cell of claim 10, which is a transgenic
host cell. 12. The host cell of claim 10 or 11, which is a plant
cell, an animal cell, a microorganism, or a yeast cell, a fungus
cell, a prokaryotic cell, an eukaryotic cell or an archaebacterium.
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. 14. A polypeptide produced by the process as claimed in claim
13 or encoded by the nucleic acid molecule as claimed in claim 6
whereby the polypeptide distinguishes over a sequence as indicated
in Table XII, application no. 31, columns 5 or 7, by one or more
amino acids 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. 16. A plant tissue, propagation
material, harvested material or a plant comprising the host cell as
claimed in claim 12 which is plant cell or an Agrobacterium. 17. A
method for screening for agonists and antagonists of the activity
of a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of arginine, proline,
glutamine and/or glutamate resp., in an organism or a part thereof
comprising: (a) contacting cells, tissues, plants or microorganisms
which express the a polypeptide encoded by the nucleic acid
molecule of claim 5 conferring an increase in the amount of
arginine, proline, glutamine and/or glutamate resp., in an organism
or a part thereof with a candidate compound or a sample comprising
a plurality of compounds under conditions which permit the
expression the polypeptide; (b) assaying the arginine, proline,
glutamine and/or glutamate resp., level or the polypeptide
expression level in the cell, tissue, plant or microorganism or the
media the cell, tissue, plant or microorganisms is cultured or
maintained in; and (c) identifying a agonist or antagonist by
comparing the measured arginine, proline, glutamine and/or
glutamate resp., level or polypeptide expression level with a
standard arginine, proline, glutamine and/or glutamate resp., or
polypeptide expression level measured in the absence of said
candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. 18. A process for the identification
of a compound conferring increased arginine, proline, glutamine
and/or glutamate resp., production in a plant or microorganism,
comprising the steps: (a) culturing a plant cell or tissue or
microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of arginine, proline, glutamine and/or
glutamate resp., in an organism or a part thereof and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
dais readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
arginine, proline, glutamine and/or glutamate resp., in an organism
or a part thereof; (b) identifying if the compound is an effective
agonist by detecting the presence or absence or increase of a
signal produced by said readout system. 19. A method for the
identification of a gene product conferring an increase in
arginine, proline, glutamine and/or glutamate resp., production in
a cell, comprising the following steps: (a) contacting the nucleic
acid molecules of a sample, which can contain a candidate gene
encoding a gene product conferring an increase in arginine,
proline, glutamine and/or glutamate resp., after expression with
the nucleic acid molecule of claim 6; (b) identifying the nucleic
acid molecules, which hybridise under relaxed stringent conditions
with the nucleic acid molecule of claim 6; (c) introducing the
candidate nucleic acid molecules in host cells appropriate for
producing arginine, proline, glutamine and/or glutamate resp.; (d)
expressing the identified nucleic acid molecules in the host cells;
(e) assaying the arginine, proline, glutamine and/or glutamate
resp., level in the host cells; and (f) identifying nucleic acid
molecule and its gene product which expression confers an increase
in the arginine, proline, glutamine and/or glutamate resp., level
in the host cell in the host cell after expression compared to the
wild type. 20. A method for the identification of a gene product
conferring an increase in arginine, proline, glutamine and/or
glutamate resp., production in a cell, comprising the following
steps: (a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the arginine, proline, glutamine
and/or glutamate resp., amount or level in an organism or a part
thereof after expression, and which are at least 20% homolog to the
nucleic acid molecule of claim 6; (b) introducing the candidate
nucleic acid molecules in host cells appropriate for producing
arginine, proline, glutamine and/or glutamate resp.; (c) expressing
the identified nucleic acid molecules in the host cells; (d)
assaying the arginine, proline, glutamine and/or glutamate resp.,
level in the host cells; and (e) identifying nucleic acid molecule
and its gene product which expression confers an increase in the
arginine, proline, glutamine and/or glutamate resp., level in the
host cell after expression compared to the wild type. 21. A method
for the production of an agricultural composition comprising the
steps of the method of any one of claims 17 to 20 and formulating
the compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. 22. A composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of any
one of claim 8 or 9, an antagonist or agonist identified according
to claim 17, the compound of claim 18, the gene product of claim 19
or 20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. 23. Use of the nucleic acid molecule as claimed
in claim 6 for the identification of a nucleic acid molecule
conferring an increase of arginine, proline, glutamine and/or
glutamate resp., after expression. 24. Use of the polypeptide of
claim 14 or the nucleic acid construct claim 7 or the gene product
identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of
arginine, proline, glutamine and/or glutamate resp., levels in an
organism. 25. Food or feed composition comprising the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the harvested material of
claim 16, the host cell of claim 10 to 12 or the gene product
identified according to the method of claim 19 or 20. 26. Use of
the nucleic acid molecule of claim 6, the polypeptide of claim 14,
the nucleic acid construct of claim 7, the vector of claim 8 or 9,
the antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20 for the protection of a plant against a arginine and/or proline
and/or glutamine and/or glutamate synthesis inhibiting
herbicide.
[13725] [0554.0.0.31] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[13726] [0000.0.32.32] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[13727] [0001.0.0.32] for the disclosure of this paragraph see
[0001.0.0.27].
[13728] [0002.0.32.32] Fatty acids and triglycerides have numerous
applications in the food and feed industry, in cosmetics and in the
drug sector. Depending on whether they are free saturated or
unsaturated fatty acids or triglycerides with an increased content
of saturated or unsaturated fatty acids, they are suitable for the
most varied applications; thus, for example, polyunsaturated fatty
acids (=PUFAs) are added to infant formula to increase its
nutritional value. The various fatty acids and triglycerides are
mainly obtained from microorganisms such as fungi or from
oil-producing plants including phytoplankton and algae, such as
soybean, oilseed rape, sunflower and others, where they are usually
obtained in the form of their triacylglycerides.
[13729] [0003.0.32.32] Stearic acid (=octadecanoic acid) is one of
the many useful types of saturated fatty acids that comes from many
animal and vegetable fats and oils. It is a waxy solid that melts
at around 70.degree. C. Commenly stearic acid is either prepared by
treating animal fat with water at a high pressure and temperature
or starting with vegetable oils by hydrogenation of said oils. It
is useful as an ingredient in making candles, soaps, and cosmetics
and for softening rubber.
[13730] [0004.0.32.32] Principally microorganisms such as
Mortierella or oil producing plants such as soybean, rapeseed or
sunflower or algae such as Crytocodinium or Phaeodactylum are a
common source for oils containing fatty acids, where they are
usually obtained in the form of their triacyl glycerides.
Alternatively, they are obtained advantageously from animals, such
as fish. The free fatty acids are prepared advantageously by
hydrolysis with a strong base such as potassium or sodium
hydroxide.
[13731] [0005.0.32.32] Whether oils with unsaturated or with
saturated fatty acids are preferred depends on the intended
purpose; thus, for example, lipids with unsaturated fatty acids,
specifically polyunsaturated fatty acids, are preferred in human
nutrition since they have a positive effect on the cholesterol
level in the blood and thus on the possibility of heart disease.
They are used in a variety of dietetic foodstuffs or medicaments.
In addition PUFAs are commonly used in food, feed and in the
cosmetic industry. Poly unsaturated .omega.-3- and/or
.omega.-6-fatty acids are an important part of animal feed and
human food. Because of the common composition of human food poly
unsaturated w-3-fatty acids, which are an essential component of
fish oil, should be added to the food to increase the nutritional
value of the food; thus, for example, polyunsaturated fatty acids
such as DHA or EPA are added as mentioned above to infant formula
to increase its nutritional value. The true essential fatty acids
linoleic and linolenic fatty acid have a lot of positive effects in
the human body such as a positive effect on healthy heart, arteries
and skin. They bring for example relieve from eczema, diabetic
neuropathy or PMS and cyclical breast pain.
[13732] [0006.0.32.32] Unlike most saturated fats, stearic acid
does not seem to increase cholesterol levels in the blood, because
liver enzymes convert it to an unsaturated fat during
digestion.
[13733] [0007.0.32.32] Stearic acid is the most common one of the
long-chain fatty acids. It is found in many foods, such as beef
fat, and cocoa butter. It is widely used as mentioned above as a
lubricant, in soaps, cosmetics, food packaging, deodorant sticks,
toothpastes, and as a softener in rubber.
[13734] [0008.0.32.32] Encouraging research shows that stearic
acid, one of the components of the fat found in the cocoa butter of
chocolate, may have some positive effects on platelets. The
mechanism believed to be responsible for the potential platelet
activation by stearic acid involves Arachidonic metabolism, which
includes thromboxane A2, a potent aggregating compound, and
prostaglandin 12, a potent anti-aggregating compound.
[13735] [0009.0.32.32] As described above, fatty acids are used in
a lot of different applications, for example in cosmetics,
pharmaceuticals and in feed and food.
[13736] [0010.0.32.32] Therefore improving the productivity of such
fatty acids and improving the quality of foodstuffs and animal
feeds is an important task of the different industries.
[13737] [0011.0.32.32] To ensure a high productivity of certain
fatty acids in plants or microorganism, it is necessary to
manipulate the natural biosynthesis of fatty acids in said
organism.
[13738] [0012.0.32.32] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes which
participate in the biosynthesis of fatty acids and make it possible
to produce certain fatty acids specifically on an industrial scale
without unwanted byproducts forming. In the selection of genes for
biosynthesis two characteristics above all are particularly
important. On the one hand, there is as ever a need for improved
processes for obtaining the highest possible contents of fatty
acids on the other hand as less as possible byproducts should be
produced in the production process.
[13739] [0013.0.0.32] for the disclosure of this paragraph see
[0013.0.0.27] above.
[13740] [0014.0.32.32] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is stearic acid or
tryglycerides, lipids, oils or fats containing stearic acid.
Accordingly, in the present invention, the term "the fine chemical"
as used herein relates to "stearic acid and/or tryglycerides,
lipids, oils and/or fats containing stearic acid". Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising stearic acid and/or triglycerides, lipids,
oils and/or fats containing stearic acid.
[13741] [0015.0.32.32] In one embodiment, the term "the fine
chemical" means stearic acid and/or tryglycerides, lipids, oils
and/or fats containing stearic acid. Throughout the specification
the term "the fine chemical" means stearic acid and/or
tryglycerides, lipids, oils and/or fats containing stearic acid,
stearic acid and its salts, ester, thioester or stearic acid in
free form or bound to other compounds such as triglycerides,
glycolipids, phospholipids etc. In a preferred embodiment, the term
"the fine chemical" means stearic acid, in free form or its salts
or bound to triglycerides. Triglycerides, lipids, oils, fats or
lipid mixture thereof shall mean any triglyceride, lipid, oil
and/or fat containing any bound or free stearic acid for example
sphingolipids, phosphoglycerides, lipids, glycolipids such as
glycosphingolipids, phospholipids such as phosphatidylethanolamine,
phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol or diphosphatidylglycerol, or as
monoacylglyceride, diacylglyceride or triacylglyceride or other
fatty acid esters such as acetyl-Coenzym A thioester, which contain
further saturated or unsaturated fatty acids in the fatty acid
molecule.
[13742] In one embodiment, the term "the fine chemical" and the
term "the respective fine chemical" mean at least one chemical
compound with an activity of the above mentioned fine chemical.
[13743] [0016.0.32.32] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [13744] (a) increasing or generating the activity of one
or more of a protein as shown in table XII, application no. 32,
column 3 encoded by the nucleic acid sequences as shown in table
XI, application no. 32, column 5, in a non-human organism or in one
or more parts thereof or [13745] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
fatty acid of the invention or fine chemicals comprising the fatty
acid of the invention, in said organism or in the culture medium
surrounding the organism.
[13746] [0017.0.0.32] and [0018.0.0.32] for the disclosure of the
paragraphs [0017.0.0.32] and [0018.0.0.32] see paragraphs
[0017.0.0.27] and [0018.0.0.27] above.
[13747] [0019.0.0.32] for the disclosure of the paragraph
[0019.0.0.32] see paragraph [0019.0.0.27] above.
[13748] [0020.0.32.32] Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, application no. 32, column 5, line 16 in a plant
conferred an increase in Stearic Acid C18:0 content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of Stearic Acid C18:0.
[13749] Surprisingly it was found, that the transgenic expression
of the Glycine max protein as indicated in Table XII, application
no. 32, column 5, line 17 in thaliana plant conferred an increase
in Stearic Acid C18:0 content of the transformed plants. Thus, in
one embodiment, said protein or its homologs are used for the
production of Stearic Acid C18:0.
[13750] [0021.0.0.32] for the disclosure of this paragraph see
[0021.0.0.27] above.
[13751] [0022.0.32.32] The sequence of b1093 from Escherichia coli
K12 has been published in Blattner et al., Science 277(5331),
1453-1474, 1997, and its activity is being defined as
3-oxoacyl-[acyl-carrier-protein] reductase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a 3-oxoacyl-[acyl-carrier-protein] reductase from E. coli or a
plant or its homolog, e.g. as shown herein, for the production of
the fine chemical, meaning of stearic acid and/or tryglycerides,
lipids, oils and/or fats containing stearic acid, in particular for
increasing the amount of stearic acid and/or tryglycerides, lipids,
oils and/or fats containing stearic acid, preferably stearic acid
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a 3-oxoacyl-[acyl-carrier-protein]
reductase is increased or generated, e.g. from E. coli or a plant
or a homolog thereof.
[13752] The sequence of YDR513w from Saccharomyces cerevisiae has
been published in Jacq et al., Nature 387 (6632 Suppl), 75-78
(1997) and in Goffeau et al., Science 274 (5287), 546-547, 1996 and
its cellular activity has characterized as glutaredoxin
(thioltransferase, glutathione reductase. Accordingly, in one
embodiment, the process of the present invention comprises the use
of YDR513w, from Saccharomyces cerevisiae or a plant or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of stearic acid and/or tryglycerides, lipids,
oils and/or fats containing stearic acid, in particular for
increasing the amount of stearic acid and/or tryglycerides, lipids,
oils and/or fats containing stearic acid, preferably stearic acid
in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a glutaredoxin is increased or generated,
e.g. from Saccharomyces cerevisiae or a plant or a homolog
thereof.
[13753] [0022.1.0.32] see [0022.1.0.27]
[13754] [0023.0.0.32] for the disclosure of the paragraph
[0023.0.0.32] see paragraph [0023.0.0.27] above.
[13755] [0023.1.32.32] Homologs of the polypeptide disclosed in
table XII, application no. 32, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 32, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 32, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 32,
column 7, resp.
[13756] [0024.0.0.32] for the disclosure of the paragraph
[0024.0.0.32] see [0024.0.0.27] above.
[13757] [0025.0.32.32] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 32, column 3 "if its de novo activity, or its
increased expression directly or indirectly leads to an increase in
the level of the fine chemical indicated in the respective line of
table XII, application no. 32, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[13758] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 32,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 32,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[13759] [0025.1.0.32] and [0025.2.0.32] for the disclosure of the
paragraphs [0025.1.0.32] and [0025.2.0.32] see paragraphs
[0025.1.0.27] and [0025.2.0.27] above.
[13760] [0026.0.0.32] to [0033.0.0.32] for the disclosure of the
paragraphs [0026.0.0.32] to [0033.0.0.32] see paragraphs
[0026.0.0.27] to [0033.0.0.27] above.
[13761] [0034.0.32.32] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 32, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[13762] [0035.0.0.32] to [0038.0.0.32] and [0039.0.0.32] for the
disclosure of the paragraphs [0035.0.0.32] to [0038.0.0.32] and
[0039.0.0.32] see paragraphs [0035.0.0.27] to [0039.0.0.27]
above.
[13763] [0040.0.0.32] to [0044.0.0.32] for the disclosure of the
paragraphs [0040.0.0.32] to [0044.0.0.32] see paragraphs
[0040.0.0.27] to [0044.0.0.27] above.
[13764] [0045.0.32.32] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
32, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 32, column 6 of
the respective line, between 10% and 50%, 10% and 100%, 10% and
200%, 10% and 300%, 10% and 400%, 10% and 500%, 20% and 50%, 20%
and 250%, 20% and 500%, 50% and 100%, 50% and 250%, 50% and 500%,
500% and 1000% or more
[13765] [0046.0.32.32] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
32, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 32, column 6 of
the respective line confers an increase of the respective fine
chemical and of further fatty acid or their precursors.
[13766] [0047.0.0.32] to [0048.0.0.32] for the disclosure of the
paragraphs [0047.0.0.32] and [0048.0.0.32] see paragraphs
[0047.0.0.27] and [0048.0.0.27] above.
[13767] [0049.0.32.32] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 32, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 32, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 32, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[13768] [0050.0.32.32] For the purposes of the present invention,
the term "stearic acid" also encompasses the corresponding salts,
such as, for example, the potassium or sodium salts of stearic acid
or the salts of stearic acid with amines such as diethylamine.
[13769] [0051.0.0.32] and [0052.0.0.32] for the disclosure of the
paragraphs [0051.0.0.32] and [0052.0.0.32] see paragraphs
[0051.0.0.27] and [0052.0.0.27] above.
[13770] [0053.0.32.32] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[13771] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 32, columns 5 and 7 or its homologs activity
having herein-mentioned fatty acid of the invention increasing
activity; and/or [13772] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 32,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 32, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned fatty acid of the invention increasing
activity; and/or [13773] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned fatty acid increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 32, columns 5 and 7 or its
homologs activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [13774] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned fatty acid of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 32, columns 5
and 7 or its homologs activity; and/or [13775] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned fatty acid of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 32, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [13776] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned fatty acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 32, columns 5 and 7 or its
homologs activity, and/or [13777] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned fatty acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 32, columns 5 and 7 or its
homologs activity; and/or [13778] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 32, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[13779] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [13780] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[13781] [0054.0.32.32] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 32 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 32, column 3 or its homologs.
[13782] [0055.0.0.32] to [0067.0.0.32] for the disclosure of the
paragraphs [0055.0.0.32] to [0067.0.0.32] see paragraphs
[0055.0.0.27] to [0067.0.0.27] above.
[13783] [0068.0.32.32] The mutation is introduced in such a way
that the production of the fatty acids is not adversely
affected.
[13784] [0069.0.0.32] for the disclosure of this paragraph see
[0069.0.0.27]
[13785] [0070.0.32.32] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention or the polypeptide of the invention or the
polypeptide used in the method of the invention as described below,
for example the nucleic acid construct mentioned below into an
organism alone or in combination with other genes, it is possible
not only to increase the biosynthetic flux towards the end product,
but also to increase, modify or create de novo an advantageous,
preferably novel metabolites composition in the organism, e.g. an
advantageous fatty acid composition comprising a higher content of
(from a viewpoint of nutritional physiology limited) fatty acids,
like palmitate, palmitoleate, stearate and/or oleate.
[13786] [0071.0.32.32] Preferably the composition further comprises
higher amounts of metabolites positively affecting or lower amounts
of metabolites negatively affecting the nutrition or health of
animals or humans provided with said compositions or organisms of
the invention or parts thereof. Likewise, the number or activity of
further genes which are required for the import or export of
nutrients or metabolites, including fatty acids or its precursors,
required for the cell's biosynthesis of fatty acids may be
increased so that the concentration of necessary or relevant
precursors, cofactors or intermediates within the cell(s) or within
the corresponding storage compartments is increased. Owing to the
increased or novel generated activity of the polypeptide of the
invention or the polypeptide used in the method of the invention or
owing to the increased number of nucleic acid sequences of the
invention and/or to the modulation of further genes which are
involved in the biosynthesis of the fatty acids, e.g. by increasing
the activity of enzymes synthesizing precursors or by destroying
the activity of one or more genes which are involved in the
breakdown of the fatty acids, it is possible to increase the yield,
production and/or production efficiency of fatty acids in the host
organism, such as the plants or the microorganisms.
[13787] [0072.0.32.32] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to stearic acid, triglycerides, lipids, oils and/or fats
containing stearic acid compounds such as palmitate, palmitoleate,
stearate, oleate, .alpha.-linolenic acid and/or linoleic acid.
[13788] [0073.0.32.32] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[13789] (a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [13790] (b) increasing the activity of
a protein having the activity of a polypeptide of the invention or
the polypeptide used in the method of the invention or a homolog
thereof, e.g. as shown in Table XII, application no. 32, columns 5
or 7, or of a polypeptide being encoded by the nucleic acid
molecule of the present invention and described below, i.e.
conferring an increase of the respective fine chemical in the
organism, preferably a microorganism, the a non-human animal, a
plant or animal cell, a plant or animal tissue or the plant,
[13791] (c) growing the organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or the plant under conditions which permit the production of the
fine chemical in the organism, preferably a microorganism, a plant
cell, a plant tissue or the plant; and [13792] (d) if desired,
revovering, optionally isolating, the free and/or bound the
respective fine chemical and, optionally further free and/or bound
fatty acids synthetized by the organism, the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[13793] [0074.0.32.32] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the fine chemical or the free and bound the fine
chemical but as option it is also possible to produce, recover and,
if desired isolate, other free or/and bound fatty acids, in
particular oleic acid.
[13794] [0075.0.0.32] to [0084.0.0.32] for the disclosure of the
paragraphs [0075.0.0.32] to [0084.0.0.32] see paragraphs
[0075.0.0.27] to [0084.0.0.27] above.
[13795] [0085.0.32.32] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [13796] a) the nucleic acid sequence as
shown in table XI, application no. 32, columns 5 and 7 or a
derivative thereof, or [13797] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as shown table XI, application no. 32, columns 5 and
7 or a derivative thereof, or [13798] c) (a) and (b) is/are not
present in its/their natural genetic environment or has/have been
modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[13799] [0086.0.0.32] and [0087.0.0.32] for the disclosure of the
paragraphs [0086.0.0.32] and [0087.0.0.32] see paragraphs
[0086.0.0.27] and [0087.0.0.27] above.
[13800] [0088.0.32.32] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose fatty acid
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for poultry is dependent on the abovementioned essential fatty
acids and the general amount of fatty acids as energy source in
feed. After the activity of a protein as shown in Table XII,
application no. 32, columns 5 or 7, has been increased or
generated, or after the expression of nucleic acid molecule or
polypeptide according to the invention has been generated or
increased, the transgenic plant generated thus is grown on or in a
nutrient medium or else in the soil and subsequently harvested.
[13801] [0088.1.0.32], [0089.0.0.32], [0090.0.0.32] and
[0091.0.0.32] for the disclosure of the paragraphs [0088.1.0.32],
[0089.0.0.32], [0090.0.0.32] and [0091.0.0.32] see paragraphs
[0088.1.0.27], [0089.0.0.27], [0090.0.0.27] and [0091.0.0.27]
above.
[13802] [0092.0.0.32] to [0094.0.0.32] for the disclosure of the
paragraphs [0092.0.0.32] to [0094.0.0.32] see paragraphs
[0092.0.0.27] to [0094.0.0.27] above.
[13803] [0095.0.32.32] It may be advantageous to increase the pool
of free fatty acids in the transgenic organisms by the process
according to the invention in order to isolate high amounts of the
pure fine chemical [0096.0.32.32] In another preferred embodiment
of the invention a combination of the increased expression of the
nucleic acid sequence or the protein of the invention together with
the transformation of a protein or polypeptid for example a fatty
acid transporter protein or a compound, which functions as a sink
for the desired fatty acid for example for linoleic or linolenic
acid in the organism is useful to increase the production of the
respective fine chemical (see Bao and Ohlrogge, Plant Physiol. 1999
August; 120 (4): 1057-1062). Such fatty acid transporter protein
may serve as a link between the location of fatty acid synthesis
and the socalled sink tissue, in which fatty acids, triglycerides,
oils and fats are stored.
[13804] [0097.0.0.32] for the disclosure of the paragraph
[0097.0.0.32] see paragraph
[13805] [0097.0.0.27] above.
[13806] [0098.0.32.32] In a preferred embodiment, the fine chemical
(stearic acid) is produced in accordance with the invention and, if
desired, is isolated. The production of further fatty acids such as
palmitic acid, stearic acid, palmitoleic acid, oleic acid and/or
linoleic acid mixtures thereof or mixtures of other fatty acids by
the process according to the invention is advantageous.
[13807] [0099.0.32.32] In the case of the fermentation of
microorganisms, the abovementioned fatty acids may accumulate in
the medium and/or the cells. If microorganisms are used in the
process according to the invention, the fermentation broth can be
processed after the cultivation. Depending on the requirement, all
or some of the biomass can be removed from the fermentation broth
by separation methods such as, for example, centrifugation,
filtration, decanting or a combination of these methods, or else
the biomass can be left in the fermentation broth. The fermentation
broth can subsequently be reduced, or concentrated, with the aid of
known methods such as, for example, rotary evaporator, thin-layer
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. Afterwards advantageously further compounds for
formulation can be added such as corn starch or silicates. This
concentrated fermentation broth advantageously together with
compounds for the formulation can subsequently be processed by
lyophilization, spray drying, spray granulation or by other
methods. Preferably the fatty acids or the fatty acid compositions
are isolated from the organisms, such as the microorganisms or
plants or the culture medium in or on which the organisms have been
grown, or from the organism and the culture medium, in the known
manner, for example via extraction, distillation, crystallization,
chromatography or a combination of these methods. These
purification methods can be used alone or in combination with the
aforementioned methods such as the separation and/or concentration
methods.
[13808] [0100.0.32.32] Transgenic plants which comprise the fatty
acids such as saturated or polyunsaturated fatty acids synthesized
in the process according to the invention can advantageously be
marketed directly without there being any need for the oils, lipids
or fatty acids synthesized to be isolated. Plants for the process
according to the invention are listed as meaning intact plants and
all plant parts, plant organs or plant parts such as leaf, stem,
seeds, root, tubers, anthers, fibers, root hairs, stalks, embryos,
calli, cotelydons, petioles, harvested material, plant tissue,
reproductive tissue and cell cultures which are derived from the
actual transgenic plant and/or can be used for bringing about the
transgenic plant. In this context, the seed comprises all parts of
the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue. However, the fine chemical produced
in the process according to the invention can also be isolated from
the organisms, advantageously plants, in the form of their oils,
fats, lipids and/or free fatty acids. Fatty acids produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts, preferably the plant
seeds. To increase the efficiency of oil extraction it is
beneficial to clean, to temper and if necessary to hull and to
flake the plant material especially the seeds. In this context, the
oils, fats, lipids and/or free fatty acids can be obtained by what
is known as cold beating or cold pressing without applying heat. To
allow for greater ease of disruption of the plant parts,
specifically the seeds, they are previously comminuted, steamed or
roasted. The seeds, which have been pretreated in this manner can
subsequently be pressed or extracted with solvents such as warm
hexane. The solvent is subsequently removed. In the case of
microorganisms, the latter are, after harvesting, for example
extracted directly without further processing steps or else, after
disruption, extracted via various methods with which the skilled
worker is familiar. In this manner, more than 96% of the compounds
produced in the process can be isolated. Thereafter, the resulting
products are processed further, i.e. degummed and/or refined. In
this process, substances such as the plant mucilages and suspended
matter are first removed. What is known as desliming can be
affected enzymatically or, for example, chemico-physically by
addition of acid such as phosphoric acid. Thereafter otionally, the
free fatty acids are removed by treatment with a base like alkali,
for example aqueous KOH or NaOH, or acid hydrolysis, advantageously
in the presence of an alcohol such as methanol or ethanol, or via
enzymatic cleavage, and isolated via, for example, phase separation
and subsequent acidification via, for example, H.sub.2SO.sub.4. The
fatty acids can also be liberated directly without the
above-described processing step. If desired the resulting product
can be washed thoroughly with water to remove traces of soap and
the alkali remaining in the product and then dried. To remove the
pigment remaining in the product, the products can be subjected to
bleaching, for example using filler's earth or active charcoal. At
the end, the product can be deodorized, for example using steam
distillation under vacuum. These chemically pure fatty acids or
fatty acid compositions are advantageous for applications in the
food industry sector, the cosmetic sector and especially the
pharmacological industry sector.
[13809] [00101.0.0.32] for the disclosure of the paragraph
[0101.0.0.32] see paragraph [0101.0.0.27] above.
[13810] [0102.0.32.32] Fatty acids can for example be detected
advantageously via GC separation methods. The unambiguous detection
for the presence of fatty acid products can be obtained by
analyzing recombinant organisms using analytical standard methods:
GC, GC-MS or TLC, as described on several occasions by Christie and
the references therein (1997, in: Advances on Lipid Methodology,
Fourth Edition:
[13811] Christie, Oily Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfah ren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). One
example is the analysis of fatty acids via FAME and GC-MS or TLC
(abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer chromatography.
The material to be analyzed can be disrupted by sonication,
grinding in a glass mill, liquid nitrogen and grinding or via other
applicable methods. After disruption, the material must be
centrifuged. The sediment is resuspended in distilled water, heated
for 10 minutes at 100.degree. C., cooled on ice and recentrifuged,
followed by extraction for one hour at 90.degree. C. in 0.5 M
sulfuric acid in methanol with 2% dimethoxypropane, which leads to
hydrolyzed oil and lipid compounds, which give transmethylated
lipids. These fatty acid methyl esters are extracted in petroleum
ether and finally subjected to a GC analysis using a capillary
column (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 .mu.m, 0.32
mm) at a temperature gradient of between 170.degree. C. and
240.degree. C. for 20 minutes and 5 minutes at 240.degree. C. The
identity of the resulting fatty acid methyl esters must be defined
using standards which are available from commercial sources (i.e.
Sigma).
[13812] [0103.0.32.32] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[13813] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide having a sequence as shown in Table
XII, application no. 32, columns 5 or 7, in or a fragment thereof,
which confers an increase in the amount of the respective fine
chemical in an organism or a part thereof; [13814] b) nucleic acid
molecule comprising, preferably at least the mature form, of the
nucleic acid molecule having a sequence as shown in Table XI,
application no. 32, columns 5 or 7, [13815] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[13816] d) nucleic acid molecule encoding a polypeptide which has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13817] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[13818] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[13819] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [13820] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primer pairs having a sequence as shown in Table
XIII, application no. 32, column 7, and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [13821] i) nucleic acid molecule encoding a polypeptide
which is isolated, e.g. from an expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (h), preferably to (a) to (c), and
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [13822] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequence as shown in Table XIV, application no.
32, column 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [13823]
k) nucleic acid molecule comprising one or more of the nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domain of a polypeptide as shown in Table XII, application no.
32, columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[13824] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; or which comprises a
sequence which is complementary thereto.
[13825] [0103.1.0.32] and [0103.2.0.32] for the disclosure of the
paragraphs [0103.1.0.32] and [0103.2.0.32] see paragraphs
[0103.1.0.27] and [0103.2.0.27] above.
[13826] [0104.0.32.32] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence shown in Table
XI, application no. 32, columns 5 or 7, by one or more nucleotides
or does not consist of the sequence shown in Table XI, application
no. 32, columns 5 or 7. In one embodiment, the nucleic acid
molecule of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to the sequence shown in Table XI,
application no. 32, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of the sequence
shown in Table XII, application no. 32, columns 5 or 7.
[13827] [0105.0.0.32] to [0107.0.0.32] for the disclosure of the
paragraphs [0105.0.0.32] to [0107.0.0.32] see paragraphs
[0105.0.0.27] and [0107.0.0.27] above.
[13828] [0108.0.32.32] Nucleic acid molecules with the sequence
shown in Table XI, application no. 32, columns 5 or 7, nucleic acid
molecules which are derived from the amino acid sequences shown in
Table XII, application no. 32, columns 5 or 7, or from polypeptides
comprising the consensus sequence shown in Table XIV, application
no. 32, column 7, or their derivatives or homologues encoding
polypeptides with the enzymatic or biological activity of a protein
as shown in Table XII, application no. 32, columns 5 or 7, or e.g.
conferring a linoleic acid increase after increasing its expression
or activity are advantageously increased in the process according
to the invention.
[13829] [0109.0.0.32] for the disclosure of this paragraph see
[0109.0.0.27] above.
[13830] [0110.0.32.32] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide used in the
method of the invention or used in the process of the invention,
e.g. of a protein as shown in Table XII, application no. 32,
columns 5 or 7, or being encoded by a nucleic acid molecule
indicated in Table XI, application no. 32, columns 5 or 7, or of
its homologs, e.g. as indicated in Table XII, application no. 32,
columns 5 or 7, can be determined from generally accessible
databases.
[13831] [0111.0.0.32] for the disclosure of this paragraph see
[0111.0.0.27] above.
[13832] [0112.0.32.32] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XII, application no. 32,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 32, columns 5 and 7, and conferring an
increase of the respective fine chemical.
[13833] [0113.0.0.32] to [0120.0.0.32] for the disclosure of the
paragraphs [0113.0.0.32] to [0120.0.0.32] see paragraphs
[0113.0.0.27] and [0120.0.0.27] above.
[13834] [0121.0.32.32] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences shown in Table XII,
application no. 32, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring an increase of the respective fine
chemical after increasing its activity.
[13835] [0122.0.0.32] to [0127.0.0.32] for the disclosure of the
paragraphs [0122.0.0.32] to [0127.0.0.32] see paragraphs
[0122.0.0.27] and [0127.0.0.27] above.
[13836] [0128.0.32.32] Synthetic oligonucleotide primers for the
amplification, e.g. as shown in Table XIII, application no. 32,
column 7, by means of polymerase chain reaction can be generated on
the basis of a sequence shown herein, for example the sequence
shown in Table XI, application no. 32, columns 5 or 7, or the
sequences as shown in Table XII, application no. 32, columns 5 or
7.
[13837] [0129.0.32.32] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence shown in Table XIV,
application no. 32, column 7, is derived from said alignments.
[13838] [0130.0.0.32] for the disclosure of this paragraph see
[0130.0.0.27].
[13839] [0131.0.0.32] to [0138.0.0.32] for the disclosure of the
paragraphs [0131.0.0.32] to [0138.0.0.32] see paragraphs
[0131.0.0.27] to [0138.0.0.27] above.
[13840] [0139.0.32.32] Polypeptides having above-mentioned
activity, i.e. conferring the respective fine chemical increase,
derived from other organisms, can be encoded by other DNA sequences
which hybridize to the sequences shown in Table XI, application no.
32, columns 5 or 7, under relaxed hybridization conditions and
which code on expression for peptides having the stearic acid
increasing activity.
[13841] [0140.0.0.32] to [0146.0.0.32] for the disclosure of the
paragraphs [0140.0.0.32] to [0146.0.0.32] see paragraphs
[0140.0.0.27] and [0146.0.0.27] above.
[13842] [0147.0.32.32] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 32, columns 5 or 7 is one which is sufficiently
complementary to one of said nucleotide sequences such that it can
hybridize to one of said nucleotide sequences thereby forming a
stable duplex. Preferably, the hybridisation is performed under
stringent hybridization conditions. However, a complement of one of
the herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[13843] [0148.0.32.32] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 32,
columns 5 or 7, or a functional portion thereof and preferably has
above mentioned activity, in particular has
the--fine-chemical-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homologs.
[13844] [0149.0.32.32] The nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 32,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity as indicated in Table XII, application no.
32, columns 5 or 7, e.g. conferring an increase of the respective
fine chemical.
[13845] [00149.1.32.32] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table
XI, application no. 32, columns 5 or 7, has further one or more of
the activities annotated or known for the a protein as indicated in
Table XII, application no. 32, column 3.
[13846] [0150.0.32.32] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table XI, application no. 32, columns 5 or 7, for example a
fragment which can be used as a probe or primer or a fragment
encoding a biologically active portion of the polypeptide of the
present invention or of a polypeptide used in the process of the
present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of methionine if its activity is increased.
The nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences indicated in Table XI, application no. 32, columns
5 or 7, an anti-sense sequence of one of the sequences indicated in
Table XI, application no. 32, columns 5 or 7, or naturally
occurring mutants thereof. Primers based on a nucleotide sequence
of the invention can be used in PCR reactions to clone homologues
of the polypeptide of the invention or of the polypeptide used in
the process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primer pairs indicated in Table XIII, application no.
32, column 7, will result in a fragment of a polynucleotide
sequence as indicated in Table XI, application no. 32, columns 5 or
7.
[13847] [0151.0.0.32] for the disclosure of this paragraph see
[0151.0.0.27] above.
[13848] [0152.0.32.32] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to the amino acid
sequence shown in Table XII, application no. 32, columns 5 or 7,
such that the protein or portion thereof maintains the ability to
participate in the fine chemical production, in particular a
stearic acid increasing the activity as mentioned above or as
described in the examples in plants or microorganisms is
comprised.
[13849] [0153.0.32.32] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence shown in Table XII, application no. 32, columns 5 or
7, such that the protein or portion thereof is able to participate
in the increase of the respective fine chemical production. In one
embodiment, a protein or protion thereof as shown in Table XII,
application no. 32, columns 5 or 7, has for example an activity of
a polypeptide as indicated in Table XII, application no. 32,
columns 5 or 7 are described herein.
[13850] [0154.0.32.32] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention.
[13851] The protein is at least about 30%, 35%, 40%, 45% or 50%,
preferably at least about 55%, 60%, 65% or 70%, and more preferably
at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94% and most
preferably at least about 95%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence as shown in Table XII, application
no. 32, columns 5 or 7, and having above-mentioned activity, e.g.
conferring preferably the increase of the respective fine
chemical.
[13852] [0155.0.0.32] and [0156.0.0.32] for the disclosure of the
paragraphs [0155.0.0.32] and [0156.0.0.32] see paragraphs
[0155.0.0.27] and [0156.0.0.27] above.
[13853] [0157.0.32.32] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences
indicated in Table XI, application no. 32, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides comprising the consensus sequences as indicated
in Table XIV, application no. 32, column 7, or of the polypeptide
as indicated in Table XII, application no. 32, columns 5 or 7, or
their functional homologues. Advantageously, the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, a consensus sequences as indicated in Table XIV,
application no. 32, column 7, or of the polypeptide as indicated in
Table XII, application no. 32, columns 5 or 7, or the functional
homologues. In a still further embodiment, the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention encodes a full length protein which is
substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table XIV, application no. 32,
column 7, or of a polypeptide as indicated in Table XII,
application no. 32, columns 5 or 7, or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 32, columns 5 or 7,
[13854] [0158.0.0.32] to [0160.0.0.32] for the disclosure of the
paragraphs [0158.0.0.32] to [0160.0.0.32] see paragraphs
[0158.0.0.27] to [0160.0.0.27] above.
[13855] [0161.0.32.32] Accordingly, in another embodiment, a
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising the
sequence shown in Table XI, application no. 32, columns 5 or 7. The
nucleic acid molecule is preferably at least 20, 30, 50, 100, 250
or more nucleotides in length.
[13856] [0162.0.0.32] for the disclosure of this paragraph see
paragraph [0162.0.0.27] above.
[13857] [0163.0.32.32] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 32, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[13858] [0164.0.0.32] for the disclosure of this paragraph see
paragraph [0164.0.0.27] above.
[13859] [0165.0.32.32] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
shown in Table XI, application no. 32, columns 5 or 7.
[13860] [0166.0.0.32] and [0167.0.0.32] for the disclosure of the
paragraphs [0166.0.0.32] and [0167.0.0.32] see paragraphs
[0166.0.0.27] and [0167.0.0.27] above.
[13861] [0168.0.32.32] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the
respective fine chemical in an organism or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 32, columns 5 or 7, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 32,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to the
sequence as indicated in Table XII, application no. 32, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 32, columns 5
or 7, even more preferably at least about 80%, 90%, 95% homologous
to the sequence as indicated in Table XII, application no. 32,
columns 5 or 7, and most preferably at least about 96%, 97%, 98%,
or 99% identical to the sequence as indicated in Table XII,
application no. 32, columns 5 or 7.
[13862] Accordingly, the invention relates to nucleic acid
molecules encoding a polypeptide having above-mentioned activity,
e.g. conferring an increase in the the respective fine chemical in
an organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table XII, application no.
32, columns 5 or 7 yet retain said activity described herein. The
nucleic acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table XII, application no. 32, columns 5 or 7, and is
capable of participation in the increase of production of the
respective fine chemical after increasing its activity, e.g. its
expression. Preferably, the protein encoded by the nucleic acid
molecule is at least about 60% identical to a sequence as indicated
in Table XII, application no. 32, columns 5 or 7, more preferably
at least about 70% identical to one of the sequences as indicated
in Table XII, application no. 32, columns 5 or 7, even more
preferably at least about 80%, 90%, or 95% homologous to a sequence
as indicated in Table XII, application no. 32, columns 5 or 7, and
most preferably at least about 96%, 97%, 98%, or 99% identical to
the sequence as indicated in Table XII, application no. 32, columns
5 or 7.
[13863] [0169.0.0.32] to [0172.0.0.32] for the disclosure of the
paragraphs [0169.0.0.32] to [0172.0.0.32] see paragraphs
[0169.0.0.27] to [0172.0.0.27] above.
[13864] [0173.0.32.32] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108401 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108401 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[13865] [0174.0.0.32] for the disclosure of this paragraph see
[0174.0.0.27]
[13866] [0175.0.32.32] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108402 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108402 by the above program algorithm with the
above parameter set, has a 80% homology.
[13867] [0176.0.32.32] Functional equivalents derived from one of
the polypeptides as shown in Table XII, application no. 32, columns
5 or 7, according to the invention by substitution, insertion or
deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at
least 55%, 60%, 65% or 70% by preference at least 80%, especially
preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very
especially preferably at least 95%, 97%, 98% or 99% homology with
one of the polypeptides as shown in Table XII, application no. 32,
columns 5 or 7, according to the invention and are distinguished by
essentially the same properties as the polypeptide as shown in
Table XII, application no. 32, columns 5 or 7.
[13868] [0177.0.32.32] Functional equivalents derived from the
nucleic acid sequence as shown in Table XI, application no. 32,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as shown in Table XII,
application no. 32, columns 5 or 7, according to the invention and
encode polypeptides having essentially the same properties as the
polypeptide as shown in Table XII, application no. 32, columns 5 or
7.
[13869] [0178.0.0.32] for the disclosure of this paragraph see
[0178.0.0.27] above.
[13870] [0179.0.32.32] A nucleic acid molecule encoding a
homologous to a protein sequence of as indicated in Table XII,
application no. 32, columns 5 or 7, can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table XI, application no.
32, columns 5 or 7, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences for example
into a sequences as indicated in Table XI, application no. 32,
columns 5 or 7, by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis.
[13871] [0180.0.0.32] to [0183.0.0.32] for the disclosure of the
paragraphs [0180.0.0.32] to [0183.0.0.32] see paragraphs
[0180.0.0.27] to [0183.0.0.27] above.
[13872] [0184.0.32.32] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 32,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 32, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 32,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[13873] [0185.0.32.32] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 32, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotide sequences not shown in any one of
sequences as indicated in Table XI, application no. 32, columns 5
or 7. In one embodiment, the nucleic acid molecule comprises less
than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or 40 further
nucleotides. In a further embodiment, the nucleic acid molecule
comprises less than 30, 20 or 10 further nucleotides. In one
embodiment, a nucleic acid molecule used in the process of the
invention is identical to a sequences as indicated in Table XI,
application no. 32, columns 5 or 7.
[13874] [0186.0.32.32] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encode a
polypeptide comprising a sequence as indicated in Table XII,
application no. 32, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 32, columns 5 or 7.
[13875] [0187.0.32.32] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table XII, application no.
32, columns 5 or 7, comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence
encoding a sequences as indicated in Table XII, application no. 32,
columns 5 or 7.
[13876] [0188.0.32.32] Polypeptides (=proteins), which still have
the essential biological activity of the polypeptide of the present
invention conferring an increase of the fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide shown in Table XII, application no. 32, columns 5
or 7, expressed under identical conditions.
[13877] [0189.0.32.32] Homologues of sequences as indicated in
Table XI, application no. 32, columns 5 or 7, or of the derived
sequences shown in Table XII, application no. 32, columns 5 or 7
also mean truncated sequences, cDNA, single-stranded DNA or RNA of
the coding and noncoding DNA sequence. Homologues of said sequences
are also understood as meaning derivatives, which comprise
noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[13878] [0190.0.0.32], [0191.0.0.32], [00191.1.0.32] and
[0192.0.0.32] to [0203.0.0.32] for the disclosure of the paragraphs
[0190.0.0.32], [0191.0.0.32], [0191.1.0.32] and [0192.0.0.32] to
[0203.0.0.32] see paragraphs [0190.0.0.27], [0191.0.0.27],
[13879] [0191.1.0.27] and [0192.0.0.27] to [0203.0.0.27] above.
[13880] [0204.0.32.32] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule, which comprises a
nucleic acid molecule selected from the group consisting of:
[13881] a) nucleic acid molecule encoding, preferably at least the
mature form, of the polypeptide shown in Table XII, application no.
32, columns 5 or 7; or a fragment thereof conferring an increase in
the amount of the fine chemical in an organism or a part thereof
[13882] b) nucleic acid molecule comprising, preferably at least
the mature form, of the nucleic acid molecule shown in Table XI,
application no. 32, columns 5 or 7; or a fragment thereof
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [13883] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [13884]
d) nucleic acid molecule encoding a polypeptide whose sequence has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [13885] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [13886] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [13887] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [13888]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
the primers or primer pairs as indicated in Table XIII, application
no. 32, column 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [13889]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from a expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (g), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [13890] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence shown in
Table XIV, application no. 32, column 7, and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [13891] k) nucleic acid molecule encoding the amino
acid sequence of a polypeptide encoding a domaine of the
polypeptide shown in Table XII, application no. 32, columns 5 or 7;
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; and [13892] l) nucleic
acid molecule which is obtainable by screening a suitable nucleic
acid library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment of at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (h) or of the nucleic acid molecule shown
in Table XI, application no. 32, columns 5 or 7, or a nucleic acid
molecule encoding, preferably at least the mature form of, the
polypeptide shown in Table XII, application no. 32, columns 5 or 7,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which encompasses a
sequence which is complementary thereto; whereby, preferably, the
nucleic acid molecule according to (a) to (l) distinguishes over
the sequence as depicted in Table XI, application no. 32, columns 5
or 7, by one or more nucleotides. In one embodiment, the nucleic
acid molecule of the invention does not consist of the sequence
shown in Table XI, application no. 32, columns 5 or 7. In an other
embodiment, the nucleic acid molecule of the present invention is
at least 30 identical and less than 100%, 99.999%, 99.99%, 99.9% or
99% identical to the sequence shown in Table XI, application no.
32, columns 5 or 7. In a further embodiment the nucleic acid
molecule does not encode the polypeptide sequence shown in Table
XII, application no. 32, columns 5 or 7. Accordingly, in one
embodiment, the nucleic acid molecule of the present invention
encodes in one embodiment a polypeptide which differs at least in
one or more amino acids from the polypeptide as depicted in Table
XII, application no. 32, columns 5 or 7, and therefore does not
encode a protein of the sequence shown in Table XII, application
no. 32, columns 5 or 7. Accordingly, in one embodiment, the protein
encoded by a sequence of a nucleic acid according to (a) to (l)
does not consist of the sequence shown in Table XII, application
no. 32, columns 5 or 7. In a further embodiment, the protein of the
present invention is at least 30% identical to protein sequence
depicted in Table XII, application no. 32, columns 5 or 7, and less
than 100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 985, 97%, 96% or 95% identical to the
sequence shown in Table XII, application no. 32, columns 5 or
7.
[13893] [0205.0.0.32] to [0226.0.0.32] for the disclosure of the
paragraphs [0205.0.0.32] to [0226.0.0.32] see paragraphs
[0205.0.0.27] to [0226.0.0.27] above.
[13894] [0227.0.32.32] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[13895] In addition to the sequence mentioned in Table XI,
application no. 32, columns 5 or 7, or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the fatty acid biosynthetic pathway such as for
palmitate, palmitoleate, stearate and/or oleate is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine the sequences shown in Table XI,
application no. 32, columns 5 or 7, with genes which generally
support or enhances to growth or yield of the target organisms, for
example genes which lead to faster growth rate of microorganisms or
genes which produces stress-, pathogen, or herbicide resistant
plants.
[13896] [0228.0.32.32] In a further embodiment of the process of
the invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the fatty acid
metabolism, in particular in fatty acid synthesis.
[13897] [0229.0.32.32] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the saturated, poly unsaturated
fatty acid biosynthesis such as desaturases like
.DELTA.-4-desaturases, .DELTA.-5-desaturases,
.DELTA.-6-desaturases, .DELTA.-8-desaturases,
.DELTA.-9-desaturases, .DELTA.-12-desaturases,
.DELTA.-17-desaturases, .omega.-3-desaturases, elongases like
.DELTA.-5-elongases, .DELTA.-6-elongases, A-9-elongases,
acyl-CoA-dehydrogenases, acyl-ACP-desaturases,
acyl-ACP-thioesterases, fatty acid acyl-transferases, acyl-CoA
lysophospholipid-acyltransferases, acyl-CoA carboxylases, fatty
acid synthases, fatty acid hydroxylases, acyl-CoA oxydases,
acetylenases, lipoxygenases, triacyl-lipases etc. as described in
WO 98/46765, WO 98/46763, WO 98/46764, WO 99/64616, WO 00/20603, WO
00/20602, WO 00/40705, US 20040172682, US 20020156254, U.S. Pat.
No. 6,677,145 US 20040053379 or US 20030101486. These genes lead to
an increased synthesis of the essential fatty acids.
[13898] [0230.0.0.32] for the disclosure of this paragraph see
[0230.0.0.27].
[13899] [0231.0.32.32] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a stearic acid degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[13900] [0232.0.0.32] to [0276.0.0.32] for the disclosure of the
paragraphs [0232.0.0.32] to [0276.0.0.32] see paragraphs
[0232.0.0.27] to [0276.0.0.27] above.
[13901] [0277.0.32.32] The fatty acids produced can be isolated
from the organism by methods with which the skilled worker is
familiar. For example via extraction, salt precipitation and/or
different chromatography methods. The process according to the
invention can be conducted batchwise, semibatchwise or
continuously. The fine chemical produced in the process according
to the invention can be isolated as mentioned above from the
organisms, advantageously plants, in the form of their oils, fats,
lipids and/or free fatty acids. Fatty acids produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts, preferably the plant
seeds. Hexane is preferably used as solvent in the process, in
which more than 96% of the compounds produced in the process can be
isolated. Thereafter, the resulting products are processed further,
i.e. degummed, refined, bleached and/or deodorized.
[13902] [0278.0.0.32] to [0282.0.0.32] for the disclosure of the
paragraphs [0278.0.0.32] to [0282.0.0.32] see paragraphs
[0278.0.0.27] to [0282.0.0.27] above.
[13903] [0283.0.32.32] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an antibody against polypeptides as shown in
Table XII, application no. 32, columns 5 or 7, or an antigenic part
thereof, which can be produced by standard techniques utilizing
polypeptides comprising or consisting of abovementioned sequences,
e.g. the polypeptid of the present invention or fragment thereof.
Preferred are monoclonal antibodies specifically binding to
polypeptides as indicated in Table XII, application no. 32, columns
5 or 7.
[13904] [0284.0.0.32] for the disclosure of this paragraph see
[0284.0.0.27] above.
[13905] [0285.0.32.32] In one embodiment, the present invention
relates to a polypeptide having the sequence shown in Table XII,
application no. 32, columns 5 or 7, or as coded by the nucleic acid
molecule shown in Table XI, application no. 32, columns 5 or 7, or
functional homologues thereof.
[13906] [0286.0.32.32] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 32, column 7, and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 32, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a poylpeptide or to a polypeptide comprising more than
one consensus sequences as indicated in Table XIV, application no.
32, column 7.
[13907] [0287.0.0.32] to [0290.0.0.32] for the disclosure of the
paragraphs [0287.0.0.32] to [0290.0.0.32] see paragraphs
[0287.0.0.27] to [0290.0.0.27] above.
[13908] [0291.0.32.32] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of a
plant or microorganism specific consensus sequences.
[13909] In one embodiment, said polypeptide of the invention
distinguishes over the sequence as indicated in Table, application
no. 32, columns 5 or 7, by one or more amino acids. In one
embodiment, polypeptide distinguishes form the sequence shown in
Table XII, application no. 32, columns 5 or 7, by more than 5, 6,
7, 8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or 30
amino acids, evenmore preferred are more than 40, 50, or 60 amino
acids and, preferably, the sequence of the polypeptide of the
invention distinguishes from the sequence shown in Table XII,
application no. 32, columns 5 or 7, by not more than 80% or 70% of
the amino acids, preferably not more than 60% or 50%, more
preferred not more than 40% or 30%, even more preferred not more
than 20% or 10%. In another embodiment, said polypeptide of the
invention does not consist of the sequence shown in Table XII,
application no. 32, columns 5 or 7.
[13910] [0292.0.0.32] for the disclosure of this paragraph see
[0292.0.0.27] above.
[13911] [0293.0.32.32] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the
process.
[13912] In one embodiment, the polypeptide of the invention has a
sequence, which distinguishes from the sequence as shown in Table
XII, application no. 32, columns 5 or 7, by one or more amino
acids. In another embodiment, said polypeptide of the invention
does not consist of the sequence shown in Table XII, application
no. 32, columns 5 or 7. In a further embodiment, said polypeptide
of the present invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical. In one embodiment, said polypeptide does not
consist of the sequence encoded by the nucleic acid molecules shown
in Table XI, application no. 32, columns 5 or 7.
[13913] [0294.0.32.32] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 32, column 3, which
distinguishes over the sequence as indicated in Table XII,
application no. 32, columns 5 or 7 by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[13914] [0295.0.0.32], [0296.0.0.32] and [0297.0.0.32] for the
disclosure of the paragraphs [0295.0.0.32], [0296.0.0.32] and
[0297.0.0.32] see paragraphs [0295.0.0.27] to [0297.0.0.27]
above.
[13915] [00297.1.32.32] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity and/or the amino acid
sequence of a polypeptide indicated in Table XII, application no.
32, columns 3, 5 or 7.
[13916] [0298.0.32.32] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 32, columns 5 or 7, such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table XII, application no. 32, columns 5 or 7.
[13917] [0299.0.32.32] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences of Table XII, application no. 32, columns 5 or
7. The preferred polypeptide of the present invention preferably
possesses at least one of the activities according to the invention
and described herein. A preferred polypeptide of the present
invention includes an amino acid sequence encoded by a nucleotide
sequence which hybridizes, preferably hybridizes under stringent
conditions, to a nucleotide sequence of Table XI, application no.
32, columns 5 or 7, or which is homologous thereto, as defined
above.
[13918] [0300.0.32.32] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table XII,
columns 5 or 7, lines 72 to 81 and 460 to 462 in amino acid
sequence due to natural variation or mutagenesis, as described in
detail herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of a sequence as indicated in Table
XII, application no. 32, columns 5 or 7.
[13919] [0301.0.0.32] for the disclosure of this paragraph see
[0301.0.0.27] above.
[13920] [0302.0.32.32] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence shown in Table XII,
application no. 32, columns 5 or 7, or the amino acid sequence of a
protein homologous thereto, which include fewer amino acids than a
full length polypeptide of the present invention or used in the
process of the present invention or the full length protein which
is homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[13921] [0303.0.0.32] for the disclosure of this paragraph see
[0303.0.0.27] above.
[13922] [0304.0.32.32] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 32, columns 5 or 7, but having differences in
the sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[13923] [0305.0.0.32] and [0306.0.0.32] for the disclosure of the
paragraphs [0305.0.0.32] and [0306.0.0.32] see paragraphs
[0305.0.0.27] and [0306.0.0.27] above.
[13924] [0306.1.32.32] Preferably, the compound is a composition
comprising the stearic acid or a recovered stearic acid, in
particular, the fine chemical, free or in protein-bound form.
[13925] [0307.0.0.32] and [0308.0.0.32] for the disclosure of the
paragraphs [0307.0.0.32] and [0308.0.0.32] see paragraphs
[0307.0.0.27] and [0308.0.0.27] above.
[13926] [0309.0.32.32] In one embodiment, a reference to protein
(=polypeptide)" of the invention e.g. as indicated in Table XII,
application no. 32, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas a
"non-polypeptide" or "other polypeptide" e.g. not indicated in
Table XII, application no. 32, columns 5 or 7 refers to a
polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous to a polypeptide of
the invention, preferably which is not substantially homologous to
a polypeptide having a protein activity, e.g., a protein which does
not confer the activity described herein and which is derived from
the same or a different organism.
[13927] [0310.0.0.32] to [0334.0.0.32] for the disclosure of the
paragraphs [0310.0.0.32] to [0334.0.0.32] see paragraphs
[0310.0.0.27] to [0334.0.0.27] above.
[13928] [0335.0.32.32] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequence as indicated in Table XI, application
no. 32, columns 5 or 7, and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
application no. 32, columns 5 or 7, and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of an
protein encoded by a nucleic acid sequence of one of the sequences
as indicated in Table XI, application no. 32, columns 5 or 7 and/or
homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[13929] [0336.0.0.32] to [0342.0.0.32] for the disclosure of the
paragraphs [0336.0.0.32] to [0342.0.0.32] see paragraphs
[0336.0.0.27] to [0342.0.0.27] above.
[13930] [0343.0.32.32] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 32, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence of one
of the sequences as indicated in Table XI, application no. 32,
columns 5 or 7, or homologs thereof of the one organism, may be
used to suppress the corresponding expression in another
organism.
[13931] [0344.0.0.32] to [0361.0.0.32] for the disclosure of the
paragraphs [0344.0.0.32] to [0361.0.0.32] see paragraphs
[0344.0.0.27] to [0361.0.0.27] above.
[13932] [0362.0.32.32] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, the nucleic acid construct of
the invention, the antisense molecule of the invention, the vector
of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. encoding a polypeptide having an
activity of a protein such as the polypeptides as indicated in
Table XII, application no. 32, column 3. Due to the above mentioned
activity the fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity is increased, e.g. due to an increased expression
or specific activity of the subject matters of the invention in a
cell or an organism or a part thereof. Transgenic for a polypeptide
having an activity of a protein such as the polypeptides as
indicated in Table XII, application no. 32, column 3. Activity
means herein that due to modulation or manipulation of the genome,
the activity of polypeptide having an activity of a protein such as
the polypeptides as indicated in Table XII, application no. 32,
column 3, or a similar activity, which is increased in the cell or
organism or part thereof. Examples are described above in context
with the process of the invention.
[13933] [0363.0.0.32] to [0373.0.0.32] for the disclosure of the
paragraphs [0363.0.0.32] to [0373.0.0.32] see paragraphs
[0363.0.0.27] to [0373.0.0.27] above.
[13934] [0374.0.32.32] Transgenic plants comprising the fatty acids
synthesized in the process according to the invention can be
marketed directly without isolation of the compounds synthesized.
In the process according to the invention, plants are understood as
meaning all plant parts, plant organs such as leaf, stalk, root,
tubers or seeds or propagation material or harvested material or
the intact plant. In this context, the seed encompasses all parts
of the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue. The fatty acids produced in the
process according to the invention may, however, also be isolated
from the plant in the form of their free fatty acids or bound in
proteins. Fatty acids produced by this process can be harvested by
harvesting the organisms either from the culture in which they grow
or from the field. This can be done via expressing, grinding and/or
extraction, salt precipitation and/or ion-exchange chromatography
of the plant parts, preferably the plant seeds, plant fruits, plant
tubers and the like.
[13935] [0375.0.0.32] to [0376.0.0.32] for the disclosure of the
paragraphs [0375.0.0.32] to [0376.0.0.32] see paragraphs
[0375.0.0.27] to [0376.0.0.27] above.
[13936] [0377.0.32.32] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced fatty acid composition or the produced respective fine
chemical is isolated.
[13937] [0378.0.0.32] to [0379.0.0.32] for the disclosure of the
paragraphs [0378.0.0.32] to [0379.0.0.32] see paragraphs
[0378.0.0.27] to [0379.0.0.27] above.
[13938] [0380.0.32.32] The fatty acids obtained in the process are
suitable as starting material for the synthesis of further products
of value. For example, they can be used in combination with each
other or alone for the production of pharmaceuticals, foodstuffs,
animal feeds or cosmetics. Accordingly, the present invention
relates a method for the production of a pharmaceuticals, food
stuff, animal feeds, nutrients or cosmetics comprising the steps of
the process according to the invention, including the isolation of
the fatty acid composition produced or the fine chemical produced
if desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the fatty acids produced in the
process or of the transgenic organisms in animal feeds, foodstuffs,
medicines, food supplements, cosmetics or pharmaceuticals.
[13939] [0381.0.0.32] and [0382.0.0.32] for the disclosure of the
paragraphs [0381.0.0.32] and [0382.0.0.32] see paragraphs
[0381.0.0.27] and [0382.0.0.27] above.
[13940] [0383.0.32.32] For preparing fatty acid compound-containing
fine chemicals, in particular the fine chemical, it is possible to
use as fatty acid source organic compounds such as, for example,
oils, fats and/or lipids comprising fatty acids such as fatty acids
having a carbon back bone between C.sub.10- to C.sub.16-carbon
atoms and/or small organic acids such acetic acid, propionic acid
or butanoic acid as precursor compounds.
[13941] [0384.0.0.32] for the disclosure of the paragraph
[0384.0.0.32] see paragraph [0384.0.0.27] above.
[13942] [0385.0.0.32] for the disclosure of the paragraph
[0385.0.0.32] see paragraph [0385.0.0.27] above.
[13943] [0386.0.32.32] However, it is also possible to purify the
fatty acid produced further. For this purpose, the
product-containing composition is subjected for example to a thin
layer chromatography on silica gel plates or to a chromatography
such as a Florisil column (Bouhours J. F., J. Chromatrogr. 1979,
169, 462), in which case the desired product or the impurities are
retained wholly or partly on the chromatography resin. These
chromatography steps can be repeated if necessary, using the same
or different chromatography resins. The skilled worker is familiar
with the choice of suitable chromatography resins and their most
effective use. An alternative method to purify the fatty acids is
for example a crystallization in the presence of urea. These
methods can be combined with each other.
[13944] [0387.0.0.32] to [0392.0.0.32] for the disclosure of the
paragraphs [0387.0.0.32] to [0392.0.0.32] see paragraphs
[0387.0.0.27] to [0392.0.0.27] above.
[13945] [0393.0.32.32] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [13946] (a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the respective fine chemical after expression, with the
nucleic acid molecule of the present invention; [13947] (b)
identifying the nucleic acid molecules, which hybridize under
relaxed stringent conditions with the nucleic acid molecule of the
present invention in particular to the nucleic acid molecule
sequence shown in Table XI, application no. 32, columns 5 or 7,
and, optionally, isolating the full length cDNA clone or complete
genomic clone; [13948] (c) introducing the candidate nucleic acid
molecules in host cells, preferably in a plant cell or a
microorganism, appropriate for producing the respective fine
chemical; [13949] (d) expressing the identified nucleic acid
molecules in the host cells; [13950] (e) assaying the respective
fine chemical level in the host cells; and [13951] (f) identifying
the nucleic acid molecule and its gene product which expression
confers an increase in the respective fine chemical level in the
host cell after expression compared to the wild type.
[13952] [0394.0.0.32] to [0415.0.0.32] and [0416.0.0.32] for the
disclosure of the paragraphs [0394.0.0.32] to [0415.0.0.32] and
[0416.0.0.32] see paragraphs [0394.0.0.27] to [0416.0.0.27]
above.
[13953] [0417.0.32.32] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the fatty acid production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect an organism such as a
microorganism or a plant against inhibitors, which block the fatty
acid, in particular the fine chemical, synthesis in said organism.
Examples of inhibitors or herbicides blocking the fatty acid
synthesis in organism such as microorganism or plants are for
example cerulenin, Thiolactomycin, Diazoborines or Triclosan, which
inhibit the fatty acids (beta-ketoacyl thioester synthetase
inhibitors) and sterol biosynthesis inhibitors,
aryloxyphenoxypropionates such as diclofop, fenoxaprop, haloxyfop,
fluazifop or quizalofop or cyclohexanediones such as clethodim or
sethoxydim
[(2-[1-{ethoxyimino}butyl]-542-{ethylthio}propyl]-3-hydroxy-2-cyclohexen--
1-one], which inhibit the plant acetyl-coenzyme A carboxylase or
thiocarbamates such as butylate, EPTC [=S-ethyl
dipropylcarbamothioat] or vernolate.
[13954] [0418.0.0.32] to [0430.0.0.32] for the disclosure of the
paragraphs [0418.0.0.32] to [0430.0.0.32] see paragraphs
[0418.0.0.27] to [0430.0.0.27] above.
[13955] [0431.0.0.32], [0432.0.0.32], [0433.0.0.32] and
[0434.0.0.32] for the disclosure of the paragraphs [0431.0.0.32],
[0432.0.0.32], [0433.0.0.32] and [0434.0.0.32] see paragraphs
[0431.0.0.27] to [0434.0.0.27] above.
[0435.0.32.32] Example 3
In-Vivo and In-Vitro Mutagenesis
[13956] [0436.0.32.32] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of fatty acids can be
carried out by passing a plasmid DNA (or another vector DNA)
containing the desired nucleic acid sequence or nucleic acid
sequences through E. coli and other microorganisms (for example
Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are
not capable of maintaining the integrity of its genetic
information. Usual mutator strains have mutations in the genes for
the DNA repair system [for example mutHLS, mutD, mutT and the like;
for comparison, see Rupp, W. D. (1996) DNA repair mechanisms in
Escherichia coli and Salmonella, pp. 2277-2294, ASM: Washington].
The skilled worker knows these strains. The use of these strains is
illustrated for example in Greener, A. and Callahan, M. (1994)
Strategies 7; 32-34.
[13957] In-vitro mutation methods such as increasing the
spontaneous mutation rates by chemical or physical treatment are
well known to the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[13958] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[13959] Positive mutation events can be selected by screening the
organisms for the production of the desired fine chemical.
[0437.0.32.32] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpina
[13960] [0438.0.32.32] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology, VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[13961] [0439.0.32.32] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255269) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[13962] [0440.0.32.32] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002) teaches a homolog recombination system dependent on
rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this system
for homologous recombination are vectors derived from the Ylp
series. Plasmid vectors derived for example from the 2.mu.-Vector
are known by the skilled worker and used for the expression in
yeasts. Other preferred vectors are for example pART1, pCHY21 or
pEVP11 as they have been described by McLeod et al. (EMBO J. 1987,
6:729-736) and Hoffman et al. (Genes Dev. 5, 1991: :561-571.) or
Russell et al. (J. Biol. Chem. 258, 1983: 143-149.). Other
beneficial yeast vectors are plasmids of the REP, REP-X, pYZ or RIP
series.
[13963] [0441.0.0.32] for the disclosure of the paragraph
[0441.0.0.32] see [0441.0.0.27] above.
[13964] [0442.0.32.32] The observations of the acivity of a
mutated, or transgenic, protein in a transformed host cell are
based on the fact that the protein is expressed in a similar manner
and in a similar quantity as the wild-type protein. A suitable
method for determining the transcription quantity of the mutant, or
transgenic, gene (a sign for the amount of mRNA which is available
for the translation of the gene product) is to carry out a Northern
blot (see, for example, Ausubel et al., (1988) Current Protocols in
Molecular Biology, Wiley: New York), where a primer which is
designed in such a way that it binds to the gene of interest is
provided with a detectable marker (usually a radioactive or
chemiluminescent marker) so that, when the total RNA of a culture
of the organism is extracted, separated on a gel, applied to a
stable matrix and incubated with this probe, the binding and
quantity of the binding of the probe indicates the presence and
also the amount of mRNA for this gene. Another method is a
quantitative PCR. This information detects the extent to which the
gene has been transcribed. Total cell RNA can be isolated for
example from yeasts or E. coli by a variety of methods, which are
known in the art, for example with the Ambion kit according to the
instructions of the manufacturer or as described in Edgington et
al., Promega Notes Magazine Number 41, 1993, p. 14.
[13965] [0443.0.0.32] for the disclosure of this paragraph see
[0443.0.0.27] above.
[0444.0.32.32] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[13966] [0445.0.32.32] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[13967] [0446.0.0.32] to [0453.0.0.32] for the disclosure of the
paragraphs [0446.0.0.32] to [0453.0.0.32] see paragraphs
[0446.0.0.27] to [0453.0.0.27] above.
[0454.0.32.32] Example 8
Analysis of the Effect of the Nucleic Acid Molecule on the
Production of the Fatty Acid
[13968] [0455.0.32.32] The effect of the genetic modification in
plants, fungi, algae or ciliates on the production of a desired
compound (such as a fatty acid) can be determined by growing the
modified microorganisms or the modified plant under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the elevated production of
desired product (i.e. of the lipids or a fatty acid). These
analytical techniques are known to the skilled worker and comprise
spectroscopy, thin-layer chromatography, various types of staining
methods, enzymatic and microbiological methods and analytical
chromatography such as high-performance liquid chromatography (see,
for example, Ullman, Encyclopedia of Industrial Chemistry, Vol. A2,
p. 89-90 and p. 443-613, VCH: Weinheim (1985); Fallon, A., et al.,
(1987) "Applications of HPLC in Biochemistry" in: Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 17; Rehm et
al. (1993) Biotechnology, Vol. 3, Chapter III: "Product recovery
and purification", p. 469-714, VCH: Weinheim; Better, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[13969] In addition to the abovementioned processes, plant lipids
are extracted from plant material as described by Cahoon et al.
(1999) Proc. Natl. Acad. Sci. USA 96 (22): 12935-12940 and Browse
et al. (1986) Analytic Biochemistry 152:141-145. The qualitative
and quantitative analysis of lipids or fatty acids is described by
Christie, William W., Advances in Lipid Methodology, Ayr/Scotland:
Oily Press (Oily Press Lipid Library; 2); Christie, William W., Gas
Chromatography and Lipids. A Practical Guide--Ayr, Scotland: Oily
Press, 1989, Repr. 1992, IX, 307 pp. (Oily Press Lipid Library; 1);
"Progress in Lipid Research, Oxford: Pergamon Press, 1 (1952)--16
(1977) under the title: Progress in the Chemistry of Fats and Other
Lipids CODEN.
[13970] [0456.0.0.32] for the disclosure of this paragraph see
[0456.0.0.27] above.
[0457.0.32.32] Example 9
Purification of the Fatty Acid
[13971] [0458.0.32.32] One example is the analysis of fatty acids
(abbreviations: FAME, fatty acid methyl ester; GC-MS, gas liquid
chromatography/mass spectrometry; TAG, triacylglycerol; TLC,
thin-layer chromatography).
[13972] The unambiguous detection for the presence of fatty acid
products can be obtained by analyzing recombinant organisms using
analytical standard methods: GC, GC-MS or TLC, as described on
several occasions by Christie and the references therein (1997, in:
Advances on Lipid Methodology, Fourth Edition: Christie, Oily
Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353).
[13973] The total fatty acids produced in the organism for example
in yeasts used in the inventive process can be analysed for example
according to the following procedure: The material such as yeasts,
E. coli or plants to be analyzed can be disrupted by sonication,
grinding in a glass mill, liquid nitrogen and grinding or via other
applicable methods. After disruption, the material must be
centrifuged (1000.times.g, 10 min., 4.degree. C.) and washed once
with 100 mM NaHCO.sub.3, pH 8.0 to remove residual medium and fatty
acids. For preparation of the fatty acid methyl esters (FAMES) the
sediment is resuspended in distilled water, heated for 10 minutes
at 100.degree. C., cooled on ice and recentrifuged, followed by
extraction for one hour at 90.degree. C. in 0.5 M sulfuric acid in
methanol with 2% dimethoxypropane, which leads to hydrolyzed oil
and lipid compounds, which give transmethylated lipids.
[13974] The FAMES are then extracted twice with 2 ml petrolether,
washed once with 100 mM NaHCO.sub.3, pH 8.0 and once with distilled
water and dried with Na.sub.2SO.sub.4. The organic solvent can be
evaporated under a stream of Argon and the FAMES were dissolved in
50 .mu.l of petrolether. The samples can be separated on a ZEBRON
ZB-Wax capillary column (30 m, 0.32 mm, 0.25 .mu.m; Phenomenex) in
a Hewlett Packard 6850 gas chromatograph with a flame ionisation
detector. The oven temperature is programmed from 70.degree. C. (1
min. hold) to 200.degree. C. at a rate of 20.degree. C./min., then
to 250.degree. C. (5 min. hold) at a rate of 5.degree. C./min and
finally to 260.degree. C. at a rate of 5.degree. C./min. Nitrogen
is used as carrier gas (4.5 ml/min. at 70.degree. C.). The identity
of the resulting fatty acid methyl esters can be identified by
comparison with retention times of FAME standards, which are
available from commercial sources (i.e. Sigma).
[13975] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[13976] This is followed by heating at 100.degree. C. for 10
minutes and, after cooling on ice, by resedimentation. The cell
sediment is hydrolyzed for one hour at 90.degree. C. with 1 M
methanolic sulfuric acid and 2% dimethoxypropane, and the lipids
are transmethylated. The resulting fatty acid methyl esters (FAMEs)
are extracted in petroleum ether. The extracted FAMEs are analyzed
by gas liquid chromatography using a capillary column
[13977] (Chrompack, WCOT Fused Silica, CP-Wax-52 CB, 25 m, 0.32 mm)
and a temperature gradient of from 170.degree. C. to 240.degree. C.
in 20 minutes and 5 minutes at 240.degree. C. The identity of the
fatty acid methyl esters is confirmed by comparison with
corresponding FAME standards (Sigma). The identity and position of
the double bond can be analyzed further by suitable chemical
derivatization of the FAME mixtures, for example to give
4,4-dimethoxyoxazoline derivatives (Christie, 1998) by means of
GC-MS.
[13978] The methodology is described for example in Napier and
Michaelson, 2001, Lipids. 36(8):761-766; Sayanova et al., 2001,
Journal of Experimental Botany. 52(360):1581-1585, Sperling et al.,
2001, Arch. Biochem. Biophys. 388(2):293-298 and Michaelson et al.,
1998, FEBS Letters. 439(3):215-218.
[13979] [0459.0.32.32] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously the fatty acids can be further purified with a
so-called RTHPLC. As eluent different an acetonitrile/water or
chloroform/acetonitrile mixtures are advantageously is used. For
example canola oil can be separated said HPLC method using an
RP-18-column (ET 250/3 Nucleosil 120-5 C.sub.18 Macherey and Nagel,
Duren, Germany). A chloroform/acetonitrile mixture (v/v 30:70) is
used as eluent. The flow rate is beneficial 0.8 ml/min. For the
analysis of the fatty acids an ELSD detector (evaporative
light-scattering detector) is used. MPLC, dry-flash chromatography
or thin layer chromatography are other beneficial chromatography
methods for the purification of fatty acids. If necessary, these
chromatography steps may be repeated, using identical or other
chromatography resins. The skilled worker is familiar with the
selection of suitable chromatography resin and the most effective
use for a particular molecule to be purified.
[13980] [0460.0.32.32] In addition depending on the produced fine
chemical purification is also possible with cristalisation or
destilation. Both methods are well known to a person skilled in the
art.
[0461.0.32.32] Example 10
Cloning SEQ ID NO: 108401 for the Expression in Plants
[13981] [0462.0.0.32] for the disclosure of this paragraph see
[0462.0.0.27] above.
[13982] [0463.0.32.32] SEQ ID NO: 108401 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[13983] [0464.0.32.32] The composition for the protocol of the Pfu
Turbo DNA polymerase was as follows: 1.times.PCR buffer
(Stratagene), 0.2 mM of each dNTP, 100 ng genomic DNA of
Saccharomyces cerevisiae (strain S288C; Research Genetics, Inc.,
now Invitrogen) or Escherichia coli (strain MG1655; E. coli Genetic
Stock Center), 50 .mu.mol forward primer, 50 .mu.mol reverse
primer, 2.5 u Pfu Turbo DNA polymerase. The amplification cycles
were as follows:
[13984] [0465.0.32.32] 1 cycle of 3 minutes at 94-95.degree. C.,
followed by 25-36 cycles of in each case 1 minute at 95.degree. C.
or 30 seconds at 94.degree. C., 45 seconds at 50.degree. C., 30
seconds at 50.degree. C. or 30 seconds at 55.degree. C. and 210-480
seconds at 72.degree. C., followed by 1 cycle of 8 minutes at
72.degree. C., then 4.degree. C.
[13985] [0466.0.0.32] for the disclosure of the paragraphs
[0466.0.0.32] see paragraphs [0466.0.0.27] above.
[13986] [0467.0.32.32] The following primer sequences were selected
for the gene SEQ ID NO: 108401: [13987] i) forward primer SEQ ID
NO: 108403 [13988] ii) reverse primer SEQ ID NO: 108404
[13989] [0468.0.0.32] to [0479.0.0.32] for the disclosure of the
paragraphs [0468.0.0.32] to [0479.0.0.32] see paragraphs
[0468.0.0.27] to [0479.0.0.27] above.
[0480.0.32.32] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 108401
[13990] [0481.0.0.32] for the disclosure of this paragraph see
[0481.0.0.27] above.
[13991] [0482.0.0.32] to [0513.0.0.32] for the disclosure of the
paragraphs [0482.0.0.32] to [0513.0.0.32] see paragraphs
[0482.0.0.27] to [0513.0.0.27] above.
[13992] [0514.0.32.32] As an alternative, the fatty acids can be
detected and purfied advantageously as described in I. Brondz,
"Development of fatty acid analysis by high-performance liquid
chromatography, gas chromatography, and related techniques
[Review]", Analytica Chimica Acta 2002, 465, 1-37
[13993] [0515.0.32.32] %
[13994] [0516.0.0.32] and [0517.0.0.32] for the disclosure of the
paragraphs [0516.0.0.32] and [0517.0.0.32] see paragraphs
[0516.0.0.27] and [0517.0.0.27] above.
[13995] [0518.0.0.32] to [0529.0.0.32] and [0530.0.0.32] for the
disclosure of the paragraphs [0518.0.0.32] to [0529.0.0.32] and
[0530.0.0.32] see paragraphs [0518.0.0.27] to [0530.0.0.27]
above.
[13996] [0530.1.0.32] to [0530.6.0.32] for the disclosure of the
paragraphs [0530.1.0.32] to [0530.6.0.32] see paragraphs
[0530.1.0.27] to [0530.6.0.27] above.
[13997] [0531.0.0.32] to [0533.0.0.32] and [0534.0.0.32] for the
disclosure of the paragraphs [0531.0.0.32] to [0533.0.0.32] and
[0534.0.0.32] see paragraphs [0531.0.0.27] to [0534.0.0.27]
above.
[13998] [0535.0.0.32] to [0537.0.0.32] and [0538.0.0.32] for the
disclosure of the paragraphs [0535.0.0.32] to [0537.0.0.32] and
[0538.0.0.32] see paragraphs [0535.0.0.27] to [0538.0.0.27]
above.
[13999] [0539.0.0.32] to [0542.0.0.32] and [0543.0.0.32] for the
disclosure of the paragraphs [0539.0.0.32] to [0542.0.0.32] and
[0543.0.0.32] see paragraphs [0539.0.0.27] to [0543.0.0.27]
above.
[14000] [0544.0.0.32] to [0547.0.0.32] and [0548.0.0.32] to
[0552.0.0.32] for the disclosure of the paragraphs [0544.0.0.32] to
[0547.0.0.32] and [0548.0.0.32] to [0552.0.0.32] see paragraphs
[0544.0.0.27] to [0552.0.0.27] above.
[14001] [0553.0.32.32] [14002] 1. A process for the production of
stearic acid, which comprises [14003] a) increasing or generating
the activity of a protein as indicated in Table XII, application
no. 32, columns 5 or 7, or a functional equivalent thereof in a
non-human organism, or in one or more parts thereof; and [14004] b)
growing the organism under conditions which permit the production
of stearic acid in said organism. [14005] 2. A process for the
production of stearic acid, comprising the increasing or generating
in an organism or a part thereof the expression of at least one
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: [14006] a) nucleic acid molecule
encoding of a polypeptide as indicated in Table XII, application
no. 32, columns 5 or 7, or a fragment thereof, which confers an
increase in the amount of stearic acid in an organism or a part
thereof; [14007] b) nucleic acid molecule comprising of a nucleic
acid molecule as indicated in Table XI, application no. 32, columns
5 or 7; [14008] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of stearic acid in an
organism or a part thereof; [14009] d) nucleic acid molecule which
encodes a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
stearic acid in an organism or a part thereof; [14010] e) nucleic
acid molecule which hybridizes with a nucleic acid molecule of (a)
to (c) under stringent hybridisation conditions and conferring an
increase in the amount of stearic acid in an organism or a part
thereof; [14011] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table XIII, application no.
32, column 7, and conferring an increase in the amount of stearic
acid in an organism or a part thereof; [14012] g) nucleic acid
molecule encoding a polypeptide which is isolated with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (f) and conferring an increase in
the amount of stearic acid in an organism or a part thereof;
[14013] h) nucleic acid molecule encoding a polypeptide comprising
a consensus sequence as indicated in Table XIV, application no. 32,
column 7, and conferring an increase in the amount of stearic acid
in an organism or a part thereof; and [14014] i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of stearic acid in an organism or a part thereof.
[14015] or comprising a sequence which is complementary thereto.
[14016] 3. The process of claim 1 or 2, comprising recovering of
the free or bound stearic acid. [14017] 4. The process of any one
of claims 1 to 3, comprising the following steps: [14018] (a)
selecting an organism or a part thereof expressing a polypeptide
encoded by the nucleic acid molecule characterized in claim 2;
[14019] (b) mutagenizing the selected organism or the part thereof;
[14020] (c) comparing the activity or the expression level of said
polypeptide in the mutagenized organism or the part thereof with
the activity or the expression of said polypeptide of the selected
organisms or the part thereof; [14021] (d) selecting the mutated
organisms or parts thereof, which comprise an increased activity or
expression level of said polypeptide compared to the selected
organism or the part thereof; [14022] (e) optionally, growing and
cultivating the organisms or the parts thereof; and [14023] (f)
recovering, and optionally isolating, the free or bound stearic
acid produced by the selected mutated organisms or parts thereof.
[14024] 5. The process of any one of claims 1 to 4, wherein the
activity of said protein or the expression of said nucleic acid
molecule is increased or generated transiently or stably. [14025]
6. An isolated nucleic acid molecule comprising a nucleic acid
molecule selected from the group consisting of: [14026] a) nucleic
acid molecule encoding of a polypeptide as indicated in Table XII,
application no. 32, columns 5 or 7, or a fragment thereof, which
confers an increase in the amount of stearic acid in an organism or
a part thereof; [14027] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table XI, application no. 32,
columns 5 or 7; [14028] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of stearic acid in an
organism or a part thereof; [14029] d) nucleic acid molecule which
encodes a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
stearic acid in an organism or a part thereof; [14030] e) nucleic
acid molecule which hybridizes with a nucleic acid molecule of (a)
to (c) under stringent hybridisation conditions and conferring an
increase in the amount of stearic acid in an organism or a part
thereof; [14031] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table XIII, application no.
32, column 7, and conferring an increase in the amount of stearic
acid in an organism or a part thereof; [14032] g) nucleic acid
molecule encoding a polypeptide which is isolated with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (f) and conferring an increase in
the amount of stearic acid in an organism or a part thereof;
[14033] h) nucleic acid molecule encoding a polypeptide comprising
a consensus sequence as indicated in Table XIV, application no. 32,
column 7, and conferring an increase in the amount of stearic acid
in an organism or a part thereof; and [14034] i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of stearic acid in an organism or a part thereof.
[14035] whereby the nucleic acid molecule distinguishes over the
sequence as indicated in Table XI, application no. 32, columns 5 or
7, by one or more nucleotides. [14036] 7. A nucleic acid construct
which confers the expression of the nucleic acid molecule of claim
6, comprising one or more regulatory elements. [14037] 8. A vector
comprising the nucleic acid molecule as claimed in claim 6 or the
nucleic acid construct of claim 7. [14038] 9. The vector as claimed
in claim 8, wherein the nucleic acid molecule is in operable
linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [14039] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [14040] 11. The host cell of claim 10, which is a
transgenic host cell. [14041] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [14042] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [14043] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table XII,
application no. 32, columns 5 or 7 by one or more amino acids
[14044] 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. [14045] 16. A plant tissue,
propagation material, harvested material or a plant comprising the
host cell as claimed in claim 12 which is plant cell or an
Agrobacterium. [14046] 17. A method for screening for agonists and
antagonists of the activity of a polypeptide encoded by the nucleic
acid molecule of claim 6 conferring an increase in the amount of
stearic acid in an organism or a part thereof comprising: [14047]
(a) contacting cells, tissues, plants or microorganisms which
express the a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of stearic acid in an
organism or a part thereof with a candidate compound or a sample
comprising a plurality of compounds under conditions which permit
the expression the polypeptide; [14048] (b) assaying the stearic
acid level or the polypeptide expression level in the cell, tissue,
plant or microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [14049] (c)
identifying a agonist or antagonist by comparing the measured
stearic acid level or polypeptide expression level with a standard
of stearic acid or polypeptide expression level measured in the
absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. [14050] 18. A process for
the identification of a compound conferring increased stearic acid
production in a plant or microorganism, comprising the steps:
[14051] (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of stearic acid in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with dais readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of stearic
acid in an organism or a part thereof; [14052] (b) identifying if
the compound is an effective agonist by detecting the presence or
absence or increase of a signal produced by said readout system.
[14053] 19. A method for the identification of a gene product
conferring an increase in stearic acid production in a cell,
comprising the following steps: [14054] a) contacting the nucleic
acid molecules of a sample, which can contain a candidate gene
encoding a gene product conferring an increase in stearic acid
after expression with the nucleic acid molecule of claim 6; [14055]
b) identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; [14056] c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing stearic acid;
[14057] d) expressing the identified nucleic acid molecules in the
host cells; [14058] e) assaying the stearic acid level in the host
cells; and [14059] f) identifying nucleic acid molecule and its
gene product which expression confers an increase in the stearic
acid level in the host cell in the host cell after expression
compared to the wild type. [14060] 20. A method for the
identification of a gene product conferring an increase in stearic
acid production in a cell, comprising the following steps: [14061]
(a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the stearic acid amount or level
in an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; [14062]
(b) introducing the candidate nucleic acid molecules in host cells
appropriate for producing stearic acid; [14063] (c) expressing the
identified nucleic acid molecules in the host cells; [14064] (d)
assaying the stearic acid level in the host cells; and [14065] (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the stearic acid level in the
host cell after expression compared to the wild type. [14066] 21. A
method for the production of an agricultural composition comprising
the steps of the method of any one of claims 17 to 20 and
formulating the compound identified in any one of claims 17 to 20
in a form acceptable for an application in agriculture. [14067] 22.
A composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [14068] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
stearic acid after expression. [14069] 24. Use of the polypeptide
of claim 14 or the nucleic acid construct claim 7 or the gene
product identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of stearic
acid levels in an organism. [14070] 25. Food or feed composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20.
[14071] 26. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the host cell of claim 10 to 12 or the gene
product identified according to the method of claim 19 or 20 for
the protection of a plant against a stearic acid synthesis
inhibiting herbicide.
[14072] [0554.0.0.32] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[14073] [0000.0.33.33] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[14074] [0001.0.0.33] for the disclosure of this paragraph see
[0001.0.0.27] above.
[14075] [0002.0.32.33] for the disclosure of this paragraph see
[0002.0.32.32] above.
[14076] [0003.0.33.33] Palmitic acid is a major component for
manufacturing of soaps, lubricating oils and waterproofing
materials. Furhermore it is used for the synthesis of metallic
palmitates. Additional applications are as food additive and in the
synthesis of food-grade additives; as a constituent of cosmetic
formulations. Palmitic acid is a major component of many natural
fats and oils in the form of a glyceryl ester, e.g. palm oil, and
in most commercial-grade stearic acid products.
[14077] [0004.0.32.33] and [0005.0.32.33] for the disclosure of the
paragraphs [0004.0.32.33] and [0005.0.32.33] see paragraphs
[0004.0.32.32] and [0005.0.32.32] above.
[14078] [0006.0.33.33] Palmitic acid is as mentioned above the
major fat in meat and dairy products.
[14079] [0007.0.33.33] Further uses or palmitic acid are as food
ingredients raw material for emulsifiers or personal care
emulsifier for facial creams and lotions. Palmitic acid is also
used in shaving cream formulations, waxes or fruit wax
formulations.
[14080] [0008.0.33.33] Palmitic acid is also used in shaving cream
formulations, waxes or fruit wax formulations.
[14081] [0009.0.32.33] to [0012.0.32.33] for the disclosure of the
paragraphs [0009.0.32.33] to [0012.0.32.33] see paragraphs
[0009.0.32.32] and [0012.0.32.32] above.
[14082] [0013.0.0.33] for the disclosure of this paragraph see
[0013.0.0.27] above.
[14083] [0014.0.33.33] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is palmitic acid or
tryglycerides, lipids, oils or fats containing palmitic acid.
Accordingly, in the present invention, the term "the fine chemical"
as used herein relates to "palmitic acid and/or tryglycerides,
lipids, oils and/or fats containing palmitic acid". Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising palmitic acid and/or triglycerides, lipids,
oils and/or fats containing palmitic acid.
[14084] [0015.0.33.33] In one embodiment, the term "the fine
chemical" means palmitic acid and/or tryglycerides, lipids, oils
and/or fats containing palmitic acid. Throughout the specification
the term "the fine chemical" means palmitic acid and/or
tryglycerides, lipids, oils and/or fats containing palmitic acid,
palmitic acid and its salts, ester, thioester or palmitic acid in
free form or bound to other compounds such as triglycerides,
glycolipids, phospholipids etc. In a preferred embodiment, the term
"the fine chemical" means palmitic acid, in free form or its salts
or bound to triglycerides. Triglycerides, lipids, oils, fats or
lipid mixture thereof shall mean any triglyceride, lipid, oil
and/or fat containing any bound or free palmitic acid for example
sphingolipids, phosphoglycerides, lipids, glycolipids such as
glycosphingolipids, phospholipids such as phosphatidylethanolamine,
phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol or diphosphatidylglycerol, or as
monoacylglyceride, diacylglyceride or triacylglyceride or other
fatty acid esters such as acetyl-Coenzym A thioester, which contain
further saturated or unsaturated fatty acids in the fatty acid
molecule.
[14085] In one embodiment, the term "the fine chemical" and the
term "the respective fine chemical" mean at least one chemical
compound with an activity of the above mentioned fine chemical.
[14086] [0016.0.33.33] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [14087] (a) increasing or generating the activity of one
or more of a protein as shown in table XII, application no. 33,
column 3 encoded by the nucleic acid sequences as shown in table
XI, application no. 33, column 5, in a non-human organism or in one
or more parts thereof or [14088] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
fatty acid of the invention or fine chemicals comprising the fatty
acid of the invention, in said organism or in the culture medium
surrounding the organism.
[14089] [0017.0.0.33] to [0019.0.0.33] for the disclosure of the
paragraphs [0017.0.0.33] to [0019.0.0.33] see paragraphs
[0017.0.0.27] and [0019.0.0.27] above.
[14090] [0020.0.33.33] Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, application no. 33, column 5, line 18 in a plant
conferred an increase in Palmitic Acid C16:0 content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of Palmitic Acid C16:0.
[14091] [0021.0.0.33] for the disclosure of this paragraph see
[0021.0.0.27] above.
[14092] [0022.0.33.33] ./. The sequence of YDR513W from
Saccharomyces cerevisiae has been published in Jacq et al., Nature
387 (6632 Suppl), 75-78 (1997) and in Goffeau et al., Science 274
(5287), 546-547, 1996 and its cellular activity has characterized
as glutaredoxin (thioltransferase, glutathione reductase).
Accordingly, in one embodiment, the process of the present
invention comprises the use of a glutaredoxin, from Saccharomyces
cerevisiae or a plant or its homolog, e.g. as shown herein, for the
production of the fine chemical, meaning of palmitic acid and/or
tryglycerides, lipids, oils and/or fats containing palmitic acid,
in particular for increasing the amount of palmitic acid and/or
tryglycerides, lipids, oils and/or fats containing palmitic acid,
preferably palmitic acid in free or bound form in an organism or a
part thereof, as mentioned. In one embodiment, in the process of
the present invention the activity of a glutaredoxin is increased
or generated, e.g. from Saccharomyces cerevisiae or a plant or a
homolog thereof.
[14093] [0022.1.0.33] to [0023.0.0.33] for the disclosure of this
paragraphs see [0022.1.0.27] to [0023.0.0.27] above.
[14094] [0023.1.33.33] Homologs of the polypeptide disclosed in
table XII, application no. 33, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 33, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 33, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 33,
column 7, resp.
[14095] [0024.0.0.33] for the disclosure of the paragraphs
[0024.0.0.33] see [0024.0.0.27] above.
[14096] [0025.0.33.33] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 33, column 3 "if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 33, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[14097] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 33,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 33,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[14098] [0025.1.0.33] and [0025.2.0.33] for the disclosure of the
paragraphs [0025.1.0.33] and [0025.2.0.33] see paragraphs
[0025.1.0.27] and [0025.2.0.27] above.
[14099] [0026.0.0.33] to [0033.0.0.33] for the disclosure of the
paragraphs [0026.0.0.33] to [0033.0.0.33] see paragraphs
[0026.0.0.27] to [0033.0.0.27] above.
[14100] [0034.0.33.33] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 33, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[14101] [0035.0.0.33] to [0038.0.0.33] and [0039.0.0.33] for the
disclosure of the paragraphs [0035.0.0.33] to [0038.0.0.33] and
[0039.0.0.33] see paragraphs [0035.0.0.27] to [0039.0.0.27]
above.
[14102] [0040.0.0.33] to [0044.0.0.33] for the disclosure of the
paragraphs [0040.0.0.33] to [0044.0.0.33] see paragraphs
[0040.0.0.27] and [0044.0.0.27] above.
[14103] [0045.0.33.33] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
33, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 33, column 6 of
the respective line, between 10% and 50%, 10% and 100%, 10% and
200%, 10% and 300%, 10% and 400%, 10% and 500%, 20% and 50%, 20%
and 250%, 20% and 500%, 50% and 100%, 50% and 250%, 50% and 500%,
500% and 1000% or more
[14104] [0046.0.33.33] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
33, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 33, column 6 of
the respective line confers an increase of the respective fine
chemical and of further fatty acid or their precursors.
[14105] [0047.0.0.33] to [0048.0.0.33] for the disclosure of the
paragraphs [0047.0.0.33] and [0048.0.0.33] see paragraphs
[0047.0.0.27] and [0048.0.0.27] above.
[14106] [0049.0.33.33] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 33, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 33, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 33, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[14107] [0050.0.33.33] For the purposes of the present invention,
the term "palmitic acid" also encompasses the corresponding salts,
such as, for example, the potassium or sodium salts of palmitic
acid or the salts of palmitic acid with amines such as
diethylamine.
[14108] [0051.0.0.33] and [0052.0.0.33] for the disclosure of the
paragraphs [0051.0.0.33] and [0052.0.0.33] see paragraphs
[0051.0.0.27] and [0052.0.0.27] above.
[14109] [0053.0.33.33] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[14110] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 33, columns 5 and 7 or its homologs activity
having herein-mentioned fatty acid of the invention increasing
activity; and/or [14111] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 33,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 33, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned fatty acid of the invention increasing
activity; and/or [14112] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned fatty acid increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 33, columns 5 and 7 or its
homologs activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [14113] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned fatty acid of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 33, columns 5
and 7 or its homologs activity; and/or [14114] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned fatty acid of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 33, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [14115] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned fatty acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 33, columns 5 and 7 or its
homologs activity, and/or [14116] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned fatty acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 33, columns 5 and 7 or its
homologs activity; and/or [14117] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 33, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[14118] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [14119] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[14120] [0054.0.33.33] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of palmitic acid after
increasing the expression or activity of the encoded polypeptide or
having the activity of a polypeptide having an activity of a
protein as indicated in Table XII, application no. 33, column 3, or
its homolog's activity, e.g. as indicated in Table XII, application
no. 33, columns 5 or 7. In general, the amount of mRNA or
polypeptide in a cell or a compartment of a organism correlates
with the amount of encoded protein and thus with the overall
activity of the encoded protein in said volume. Said correlation is
not always linear, the activity in the volume is dependent on the
stability of the molecules or the presence of activating or
inhibiting co-factors. Further, product and educt inhibitions of
enzymes are well known and described in Textbooks, e.g. Stryer,
Biochemistry.
[14121] [0055.0.0.33] to [0067.0.0.33] for the disclosure of the
paragraphs [0055.0.0.33] to [0067.0.0.33] see paragraphs
[0055.0.0.27] to [0067.0.0.27] above.
[14122] [0068.0.32.33] for the disclosure of this paragraph see
[0068.0.32.32] above.
[14123] [0069.0.0.33] for the disclosure of this paragraph see
[0069.0.0.27] above.
[14124] [0070.0.32.33] and [0071.0.32.33] for the disclosure of the
paragraphs [0070.0.32.33] and [0071.0.32.33] see paragraphs
[0070.0.32.32] and [0071.0.32.32] above.
[14125] [0072.0.33.33] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to palmitic acid, triglycerides, lipids, oils and/or fats
containing palmitic acid compounds such as palmitate, palmitoleate,
stearate, oleate, .alpha.-linolenic acid and/or linoleic acid.
[14126] [0073.0.33.33] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
a) providing a non-human organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant; b) increasing the activity of a protein having the
activity of a polypeptide of the invention or the polypeptide used
in the method of the invention or a homolog thereof, e.g. as shown
in Table XII, application no. 33, columns 5 or 7, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, i.e. conferring an increase
of the respective fine chemical in the organism, preferably a
microorganism, the a non-human animal, a plant or animal cell, a
plant or animal tissue or the plant, c) growing the organism,
preferably a microorganism, a non-human animal, a plant or animal
cell, a plant or animal tissue or the plant under conditions which
permit the production of the fine chemical in the organism,
preferably a microorganism, a plant cell, a plant tissue or the
plant; and d) if desired, recovering, optionally isolating, the
free and/or bound the respective fine chemical and, optionally
further free and/or bound fatty acids synthetized by the organism,
the microorganism, the non-human animal, the plant or animal cell,
the plant or animal tissue or the plant.
[14127] [0074.0.32.33] for the disclosure of this paragraph see
[0074.0.32.32] above.
[14128] [0075.0.0.33] to [0084.0.0.33] for the disclosure of the
paragraphs [0075.0.0.33] to [0084.0.0.33] see paragraphs
[0075.0.0.27] to [0084.0.0.27] above.
[14129] [0085.0.33.33] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [14130] a) the nucleic acid sequence as
shown in table XI, application no. 33, columns 5 and 7 or a
derivative thereof, or [14131] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as shown table XI, application no. 33, columns 5 and
7 or a derivative thereof, or [14132] c) (a) and (b) is/are not
present in its/their natural genetic environment or has/have been
modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[14133] [0086.0.0.33] and [0087.0.0.33] for the disclosure of the
paragraphs [0086.0.0.33] and [0087.0.0.33] see paragraphs
[0086.0.0.27] and [0087.0.0.27] above.
[14134] [0088.0.33.33] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose fatty acid
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for poultry is dependent on the abovementioned essential fatty
acids and the general amount of fatty acids as energy source in
feed. After the activity of a protein as shown in Table XII,
application no. 33, columns 5 or 7, has been increased or
generated, or after the expression of nucleic acid molecule or
polypeptide according to the invention has been generated or
increased, the transgenic plant generated thus is grown on or in a
nutrient medium or else in the soil and subsequently harvested.
[14135] [0088.1.0.33], [0089.0.0.33], [0090.0.0.33] and
[0091.0.0.33] for the disclosure of the paragraphs [0088.1.0.33],
[0089.0.0.33], [0090.0.0.33] and [0091.0.0.33] see paragraphs
[0088.1.0.27], [0089.0.0.27], [0090.0.0.27] and [0091.0.0.27]
above.
[14136] [0092.0.0.33] to [0094.0.0.33] for the disclosure of the
paragraphs [0092.0.0.33] to [0094.0.0.33] see paragraphs
[0092.0.0.27] to [0094.0.0.27] above.
[14137] [0095.0.32.33] and [0096.0.32.33] for the disclosure of the
paragraphs [0095.0.32.33] and [0096.0.32.33] see paragraphs
[0095.0.32.32] and [0096.0.32.32] above.
[14138] [0097.0.0.33] for the disclosure of this paragraph see
[0097.0.0.27] above.
[14139] [0098.0.33.33] In a preferred embodiment, the fine chemical
(palmitic acid) is produced in accordance with the invention and,
if desired, is isolated. The production of further fatty acids such
as stearic acid, palmitoleic acid, oleic acid, .alpha.-linolenic
acid and/or linoleic acid mixtures thereof or mixtures of other
fatty acids by the process according to the invention is
advantageous.
[14140] [0099.0.32.33] and [0100.0.32.33] for the disclosure of the
paragraphs [0099.0.32.33] and [0100.0.32.33] see paragraphs
[0099.0.32.32] and [0100.0.32.32] above.
[14141] [0101.0.0.33] for the disclosure of this paragraph see
[0101.0.0.27] above.
[14142] [0102.0.32.33] for the disclosure of this paragraph see
[0102.0.32.32] above.
[14143] [0103.0.33.33] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[14144] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide having a sequence as shown in Table
XII, application no. 33, columns 5 or 7, in or a fragment thereof,
which confers an increase in the amount of the respective fine
chemical in an organism or a part thereof; [14145] b) nucleic acid
molecule comprising, preferably at least the mature form, of the
nucleic acid molecule having a sequence as shown in Table XI,
application no. 33, columns 5 or 7, [14146] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[14147] d) nucleic acid molecule encoding a polypeptide which has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [14148] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[14149] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[14150] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [14151] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primer pairs having a sequence as shown in Table
XIII, application no. 33, column 7, and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [14152] i) nucleic acid molecule encoding a polypeptide
which is isolated, e.g. from an expression library, with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (h), preferably to (a) to (c), and
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [14153] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequence as shown in Table XIV, application no.
33, column 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [14154]
k) nucleic acid molecule comprising one or more of the nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domain of a polypeptide as shown in Table XII, application no.
33, columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[14155] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; or which comprises a
sequence which is complementary thereto.
[14156] [0103.1.0.33] and [0103.2.0.33] for the disclosure of the
paragraphs [0103.1.0.33] and [0103.2.0.33] see paragraphs
[0103.1.0.27] and [0103.2.0.27] above.
[14157] [0104.0.33.33] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence shown in Table
XI, application no. 33, columns 5 or 7, by one or more nucleotides
or does not consist of the sequence shown in Table XI, application
no. 33, columns 5 or 7. In one embodiment, the nucleic acid
molecule of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to the sequence shown in Table XI,
application no. 33, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of the sequence
shown in Table XII, application no. 33, columns 5 or 7.
[14158] [0105.0.0.33] to [0107.0.0.33] for the disclosure of the
paragraphs [0105.0.0.33] to [0107.0.0.33] see paragraphs
[0105.0.0.27] and [0107.0.0.27] above.
[14159] [0108.0.33.33] Nucleic acid molecules with the sequence
shown in Table XI, application no. 33, columns 5 or 7, nucleic acid
molecules which are derived from the amino acid sequences shown in
Table XII, application no. 33, columns 5 or 7, or from polypeptides
comprising the consensus sequence shown in Table XIV, application
no. 33, column 7, or their derivatives or homologues encoding
polypeptides with the enzymatic or biological activity of a protein
as shown in Table XII, application no. 33, columns 5 or 7, or e.g.
conferring a linoleic acid increase after increasing its expression
or activity are advantageously increased in the process according
to the invention.
[14160] [0109.0.0.33] for the disclosure of this paragraph see
[0109.0.0.27] above.
[14161] [0110.0.33.33] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide used in the
method of the invention or used in the process of the invention,
e.g. of a protein as shown in Table XII, application no. 33,
columns 5 or 7 or being encoded by a nucleic acid molecule
indicated in Table XI, application no. 33, columns 5 or 7, or of
its homologs, e.g. as indicated in Table XII, application no. 33,
columns 5 or 7, can be determined from generally accessible
databases.
[14162] [0111.0.0.33] for the disclosure of this paragraph see
[0111.0.0.27] above.
[14163] [0112.0.33.33] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XII, application no. 33,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 33, columns 5 and 7, and conferring an
increase of the respective fine chemical.
[14164] [0113.0.0.33] to [0120.0.0.33] for the disclosure of the
paragraphs [0113.0.0.33] to [0120.0.0.33] see paragraphs
[0113.0.0.27] and [0120.0.0.27] above.
[14165] [0121.0.33.33] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences shown in Table XII,
application no. 33, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring an increase of the respective fine
chemical after increasing its activity.
[14166] [0122.0.0.33] to [0127.0.0.33] for the disclosure of the
paragraphs [0122.0.0.33] to [0127.0.0.33] see paragraphs
[0122.0.0.27] and [0127.0.0.27] above.
[14167] [0128.0.33.33] Synthetic oligonucleotide primers for the
amplification, e.g. as shown in Table XIII, application no. 33,
column 7, by means of polymerase chain reaction can be generated on
the basis of a sequence shown herein, for example the sequence
shown in Table XI, application no. 33, columns 5 or 7, or the
sequences as shown in Table XII, application no. 33, columns 5 or
7.
[14168] [0129.0.33.33] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequences shown in Table XIV,
application no. 33, column 7, are derived from said alignments.
[14169] [0130.0.0.33] for the disclosure of this paragraph see
[0130.0.0.27].
[14170] [0131.0.0.33] to [0138.0.0.33] for the disclosure of the
paragraphs [0131.0.0.33] to [0138.0.0.33] see paragraphs
[0131.0.0.27] to [0138.0.0.27] above.
[14171] [0139.0.33.33] Polypeptides having above-mentioned
activity, i.e. conferring the respective fine chemical increase,
derived from other organisms, can be encoded by other DNA sequences
which hybridize to the sequences shown in Table XI, application no.
33, columns 5 or 7, under relaxed hybridization conditions and
which code on expression for peptides having the palmitic acid
increasing activity.
[14172] [0140.0.0.33] to [0146.0.0.33] for the disclosure of the
paragraphs [0140.0.0.33] to [0146.0.0.33] see paragraphs
[0140.0.0.27] and [0146.0.0.27] above.
[14173] [0147.0.33.33] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 33, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences such
that it can hybridize to one of said nucleotide sequences thereby
forming a stable duplex. Preferably, the hybridisation is performed
under stringent hybridization conditions. However, a complement of
one of the herein disclosed sequences is preferably a sequence
complement thereto according to the base pairing of nucleic acid
molecules well known to the skilled person. For example, the bases
A and G undergo base pairing with the bases T and U or C, resp. and
visa versa. Modifications of the bases can influence the
base-pairing partner.
[14174] [0148.0.33.33] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 33,
columns 5 or 7, or a functional portion thereof and preferably has
above mentioned activity, in particular has
the--fine-chemical-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homologs.
[14175] [0149.0.33.33] The nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 33,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity as indicated in Table XII, application no.
33, columns 5 or 7, e.g. conferring an increase of the respective
fine chemical.
[14176] [00149.1.33.33] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table
XI, application no. 33, columns 5 or 7, has further one or more of
the activities annotated or known for the a protein as indicated in
Table XII, application no. 33, column 3.
[14177] [0150.0.33.33] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table XI, application no. 33, columns 5 or 7, for example a
fragment which can be used as a probe or primer or a fragment
encoding a biologically active portion of the polypeptide of the
present invention or of a polypeptide used in the process of the
present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of methionine if its activity is increased.
The nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences indicated in Table XI, application no. 33, columns
5 or 7, an anti-sense sequence of one of the sequences indicated in
Table XI, application no. 33, columns 5 or 7, or naturally
occurring mutants thereof. Primers based on a nucleotide sequence
of the invention can be used in PCR reactions to clone homologues
of the polypeptide of the invention or of the polypeptide used in
the process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primer pairs indicated in Table XIII, application no.
33, column 7, will result in a fragment of a polynucleotide
sequence as indicated in Table XI, application no. 33, columns 5 or
7. Preferred is Table XI, application no. 33, column 7.
[14178] [0151.0.0.33] for the disclosure of this paragraph see
[0151.0.0.27] above.
[14179] [0152.0.33.33] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to the amino acid
sequence shown in Table XII, application no. 33, columns 5 or 7,
such that the protein or portion thereof maintains the ability to
participate in the fine chemical production, in particular a
palmitic acid increasing the activity as mentioned above or as
described in the examples in plants or microorganisms is
comprised.
[14180] [0153.0.33.33] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence shown in Table XII, application no. 33, columns 5 or
7, such that the protein or portion thereof is able to participate
in the increase of the respective fine chemical production. In one
embodiment, a protein or portion thereof as shown in Table XII,
application no. 33, columns 5 or 7, has for example an activity of
a polypeptide as indicated in Table XII, application no. 33,
columns 5 or 7.
[14181] [0154.0.33.33] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as shown in Table XII, application no. 33, columns 5 or 7,
and having above-mentioned activity, e.g. conferring preferably the
increase of the respective fine chemical.
[14182] [0155.0.0.33] and [0156.0.0.33] for the disclosure of the
paragraphs [0155.0.0.33] and [0156.0.0.33] see paragraphs
[0155.0.0.27] and [0156.0.0.27] above.
[14183] [0157.0.33.33] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences
indicated in Table XI, application no. 33, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides comprising the consensus sequences as indicated
in Table XIV, application no. 33, column 7, or of the polypeptide
as indicated in Table XII, application no. 33, columns 5 or 7, or
their functional homologues. Advantageously, the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, a consensus sequences as indicated in Table XIV,
application no. 33, column 7, or of the polypeptide as indicated in
Table XII, application no. 33, columns 5 or 7, or the functional
homologues. In a still further embodiment, the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention encodes a full length protein which is
substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table XIV, application no. 33,
column 7, or of a polypeptide as indicated in Table XII,
application no. 33, columns 5 or 7, or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 33, columns 5 or 7,
[14184] [0158.0.0.33] to [0160.0.0.33] for the disclosure of the
paragraphs [0158.0.0.33] to [0160.0.0.33] see paragraphs
[0158.0.0.27] to [0160.0.0.27] above.
[14185] [0161.0.33.33] Accordingly, in another embodiment, a
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising the
sequence shown in Table XI, application no. 33, columns 5 or 7. The
nucleic acid molecule is preferably at least 20, 30, 50, 100, 250
or more nucleotides in length.
[14186] [0162.0.0.33] for the disclosure of this paragraph see
paragraph [0162.0.0.27] above.
[14187] [0163.0.33.33] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 33, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[14188] [0164.0.0.33] for the disclosure of this paragraph see
paragraph [0164.0.0.27] above.
[14189] [0165.0.33.33] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
shown in Table XI, application no. 33, columns 5 or 7.
[14190] [0166.0.0.33] and [0167.0.0.33] for the disclosure of the
paragraphs [0166.0.0.33] and [0167.0.0.33] see paragraphs
[0166.0.0.27] and [0167.0.0.27] above.
[14191] [0168.0.33.33] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the
respective fine chemical in an organism or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 33, columns 5 or 7, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 33,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to the
sequence as indicated in Table XII, application no. 33, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 33, columns 5
or 7, even more preferably at least about 80%, 90%, 95% homologous
to the sequence as indicated in Table XII, application no. 33,
columns 5 or 7 and most preferably at least about 96%, 97%, 98%, or
99% identical to the sequence as indicated in Table XII,
application no. 33, columns 5 or 7.
[14192] Accordingly, the invention relates to nucleic acid
molecules encoding a polypeptide having above-mentioned activity,
e.g. conferring an increase in the the respective fine chemical in
an organisms or parts thereof that contain changes in amino acid
residues that are not essential for said activity. Such
polypeptides differ in amino acid sequence from a sequence
contained in a sequence as indicated in Table XII, application no.
33, columns 5 or 7, yet retain said activity described herein. The
nucleic acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table XII, application no. 33, columns 5 or 7, and is
capable of participation in the increase of production of the
respective fine chemical after increasing its activity, e.g. its
expression. Preferably, the protein encoded by the nucleic acid
molecule is at least about 60% identical to a sequence as indicated
in Table XII, application no. 33, columns 5 or 7, more preferably
at least about 70% identical to one of the sequences as indicated
in Table XII, application no. 33, columns 5 or 7, even more
preferably at least about 80%, 90%, or 95% homologous to a sequence
as indicated in Table XII, application no. 33, columns 5 or 7, and
most preferably at least about 96%, 97%, 98%, or 99% identical to
the sequence as indicated in Table XII, application no. 33, columns
5 or 7.
[14193] [0169.0.0.33] to [0172.0.0.33] for the disclosure of the
paragraphs [0169.0.0.33] to [0172.0.0.33] see paragraphs
[0169.0.0.27] to [0172.0.0.27] above.
[14194] [0173.0.33.33] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108401 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108401 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[14195] [0174.0.0.33] for the disclosure of this paragraph see
[0174.0.0.27] above.
[14196] [0175.0.33.33] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108402 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108402 by the above program algorithm with the
above parameter set, has a 80% homology.
[14197] [0176.0.33.33] Functional equivalents derived from one of
the polypeptides as shown in Table XII, application no. 33, columns
5 or 7 according to the invention by substitution, insertion or
deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at
least 55%, 60%, 65% or 70% by preference at least 80%, especially
preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very
especially preferably at least 95%, 97%, 98% or 99% homology with
one of the polypeptides as shown in Table XII, application no. 33,
columns 5 or 7, according to the invention and are distinguished by
essentially the same properties as the polypeptide as shown in
Table XII, application no. 33, columns 5 or 7.
[14198] [0177.0.33.33] Functional equivalents derived from the
nucleic acid sequence as shown in Table XI, application no. 33,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as shown in Table XII,
application no. 33, columns 5 or 7, according to the invention and
encode polypeptides having essentially the same properties as the
polypeptide as shown in Table XII, application no. 33, columns 5 or
7.
[14199] [0178.0.0.33] for the disclosure of this paragraph see
[0178.0.0.27] above.
[14200] [0179.0.33.33] A nucleic acid molecule encoding a
homologous protein to a protein sequence of as indicated in Table
XII, application no. 33, columns 5 or 7, can be created by
introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of the nucleic acid molecule
of the present invention, in particular as indicated in Table XI,
application no. 33, columns 5 or 7, such that one or more amino
acid substitutions, additions or deletions are introduced into the
encoded protein. Mutations can be introduced into the encoding
sequences for example into a sequences as indicated in Table XI,
application no. 33, columns 5 or 7, by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
[14201] [0180.0.0.33] to [0183.0.0.33] for the disclosure of the
paragraphs [0180.0.0.33] to [0183.0.0.33] see paragraphs
[0180.0.0.27] to [0183.0.0.27] above.
[14202] [0184.0.33.33] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 33,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 33, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 33,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[14203] [0185.0.33.33] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 33, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotide sequences not shown in any one of
sequences as indicated in Table XI, application no. 33, columns 5
or 7. In one embodiment, the nucleic acid molecule comprises less
than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or 40 further
nucleotides. In a further embodiment, the nucleic acid molecule
comprises less than 30, 20 or 10 further nucleotides. In one
embodiment, a nucleic acid molecule used in the process of the
invention is identical to a sequences as indicated in Table XI,
application no. 33, columns 5 or 7.
[14204] [0186.0.33.33] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encode a
polypeptide comprising a sequence as indicated in
[14205] Table XII, application no. 33, columns 5 or 7. In one
embodiment, the nucleic acid molecule encodes less than 150, 130,
100, 80, 60, 50, 40 or 30 further amino acids. In a further
embodiment, the encoded polypeptide comprises less than 20, 15, 10,
9, 8, 7, 6 or 5 further amino acids. In one embodiment, the encoded
polypeptide used in the process of the invention is identical to
the sequences as indicated in Table XII, application no. 33,
columns 5 or 7.
[14206] [0187.0.33.33] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table XII, application no.
33, columns 5 or 7, and comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a sequences as indicated in Table XII,
application no. 33, columns 5 or 7.
[14207] [0188.0.33.33] Polypeptides (=proteins), which still have
the essential biological activity of the polypeptide of the present
invention conferring an increase of the fine chemical i.e. whose
activity is essentially not reduced, are polypeptides with at least
10% or 20%, by preference 30% or 40%, especially preferably 50% or
60%, very especially preferably 80% or 90 or more of the wild type
biological activity or enzyme activity, advantageously, the
activity is essentially not reduced in comparison with the activity
of a polypeptide shown in Table XII, application no. 33, columns 5
or 7, expressed under identical conditions.
[14208] [0189.0.33.33] Homologues of sequences as indicated in
Table XI, application no. 33, columns 5 or 7, or of the derived
sequences shown in Table XII, application no. 33, columns 5 or 7,
also mean truncated sequences, cDNA, single-stranded DNA or RNA of
the coding and noncoding DNA sequence. Homologues of said sequences
are also understood as meaning derivatives, which comprise
noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[14209] [0190.0.0.33], [0191.0.0.33], [00191.1.0.33] and
[0192.0.0.33] to [0203.0.0.33] for the disclosure of the paragraphs
[0190.0.0.33], [0191.0.0.33], [0191.1.0.33] and [0192.0.0.33] to
[0203.0.0.33] see paragraphs [0190.0.0.27], [0191.0.0.27],
[14210] [0191.1.0.27] and [0192.0.0.27] to [0203.0.0.27] above.
[14211] [0204.0.33.33] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule, which comprises a
nucleic acid molecule selected from the group consisting of:
[14212] a) nucleic acid molecule encoding, preferably at least the
mature form, of the polypeptide shown in Table XII, application no.
33, columns 5 or 7; or a fragment thereof conferring an increase in
the amount of the fine chemical in an organism or a part thereof
[14213] b) nucleic acid molecule comprising, preferably at least
the mature form, of the nucleic acid molecule shown in Table XI,
application no. 33, columns 5 or 7; or a fragment thereof
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [14214] c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as result of the degeneracy of
the genetic code and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [14215]
d) nucleic acid molecule encoding a polypeptide whose sequence has
at least 50% identity with the amino acid sequence of the
polypeptide encoded by the nucleic acid molecule of (a) to (c) and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [14216] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [14217] f) nucleic acid molecule
encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [14218] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [14219]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
the primers or primer pairs as indicated in Table XIII, application
no. 33, column 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [14220]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from a expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (g), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [14221] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence shown in
Table XIV, application no. 33, column 7, and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [14222] k) nucleic acid molecule encoding the amino
acid sequence of a polypeptide encoding a domaine of the
polypeptide shown in Table XII, application no. 33, columns 5 or 7;
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; and [14223] l) nucleic
acid molecule which is obtainable by screening a suitable nucleic
acid library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment of at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (h) or of the nucleic acid molecule shown
in Table XI, application no. 33, columns 5 or 7, or a nucleic acid
molecule encoding, preferably at least the mature form of, the
polypeptide shown in Table XII, application no. 33, columns 5 or 7,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which encompasses a
sequence which is complementary thereto; whereby, preferably, the
nucleic acid molecule according to (a) to (l) distinguishes over
the sequence as depicted in Table XI, application no. 33, columns 5
or 7, by one or more nucleotides. In one embodiment, the nucleic
acid molecule of the invention does not consist of the sequence
shown in Table XI, application no. 33, columns 5 or 7. In an other
embodiment, the nucleic acid molecule of the present invention is
at least 30 identical and less than 100%, 99.999%, 99.99%, 99.9% or
99% identical to the sequence shown in Table XI, application no.
33, columns 5 or 7. In a further embodiment the nucleic acid
molecule does not encode the polypeptide sequence shown in Table
XII, application no. 33, columns 5 or 7. Accordingly, in one
embodiment, the nucleic acid molecule of the present invention
encodes in one embodiment a polypeptide which differs at least in
one or more amino acids from the polypeptide as depicted in Table
XII, application no. 33, columns 5 or 7, and therefore does not
encode a protein of the sequence shown in Table XII, application
no. 33, columns 5 or 7. Accordingly, in one embodiment, the protein
encoded by a sequence of a nucleic acid according to (a) to (l)
does not consist of the sequence shown in Table XII, application
no. 33, columns 5 or 7. In a further embodiment, the protein of the
present invention is at least 30% identical to protein sequence
depicted in Table XII, application no. 33, columns 5 or 7, and less
than 100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 985, 97%, 96% or 95% identical to the
sequence shown in Table XII, application no. 33, columns 5 or
7.
[14224] [0205.0.0.33] to [0226.0.0.33] for the disclosure of the
paragraphs [0205.0.0.33] to [0226.0.0.33] see paragraphs
[0205.0.0.27] to [0226.0.0.27] above.
[14225] [0227.0.33.33] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[14226] In addition to the sequence mentioned in Table XI,
application no. 33, columns 5 or 7, or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the fatty acid biosynthetic pathway such as for
palmitate, palmitoleate, stearate and/or oleate is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine the sequences shown in Table XI,
application no. 33, columns 5 or 7, with genes which generally
support or enhances to growth or yield of the target organisms, for
example genes which lead to faster growth rate of microorganisms or
genes which produces stress-, pathogen, or herbicide resistant
plants.
[14227] [0228.0.32.33] to [0229.0.32.33] for the disclosure of the
paragraphs [0228.0.32.33] to [0229.0.32.33] see paragraphs
[0228.0.32.32] to [0229.0.32.32] above.
[14228] [0230.0.0.33] for the disclosure of this paragraph see
[0230.0.0.27] above.
[14229] [0231.0.33.33] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a palmitic acid degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[14230] [0232.0.0.33] to [0276.0.0.33] for the disclosure of the
paragraphs [0232.0.0.33] to [0276.0.0.33] see paragraphs
[0232.0.0.27] to [0276.0.0.27] above.
[14231] [0277.0.32.33] for the disclosure of this paragraph see
paragraph [0277.0.32.32] above.
[14232] [0278.0.0.33] to [0282.0.0.33] for the disclosure of the
paragraphs [0278.0.0.33] to [0282.0.0.33] see paragraphs
[0278.0.0.27] to [0282.0.0.27] above.
[14233] [0283.0.33.33] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an antibody against polypeptides as shown in
Table XII, application no. 33, columns 5 or 7, or an antigenic part
thereof, which can be produced by standard techniques utilizing
polypeptides comprising or consisting of abovementioned sequences,
e.g. the polypeptid of the present invention or fragment thereof.
Preferred are monoclonal antibodies specifically binding to
polypeptides as indicated in Table XII, application no. 33, columns
5 or 7.
[14234] [0284.0.0.33] for the disclosure of this paragraph see
[0284.0.0.27] above.
[14235] [0285.0.33.33] In one embodiment, the present invention
relates to a polypeptide having the sequence shown in Table XII,
application no. 33, columns 5 or 7, or as coded by the nucleic acid
molecule shown in Table XI, application no. 33, columns 5 or 7, or
functional homologues thereof.
[14236] [0286.0.33.33] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 33, column 7, and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 33, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a poylpeptide or to a polypeptide comprising more than
one consensus sequences as indicated in Table XIV, application no.
33, column 7.
[14237] [0287.0.0.33] to [0290.0.0.33] for the disclosure of the
paragraphs [0287.0.0.33] to [0290.0.0.33] see paragraphs
[0287.0.0.27] to [0290.0.0.27] above.
[14238] [0291.0.33.33] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of a
plant or microorganism specific consensus sequences.
[14239] In one embodiment, said polypeptide of the invention
distinguishes over the sequence as indicated in Table XII,
application no. 33, columns 5 or 7, by one or more amino acids. In
one embodiment, polypeptide distinguishes form the sequence shown
in Table XII, application no. 33, columns 5 or 7, by more than 5,
6, 7, 8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or
30 amino acids, evenmore preferred are more than 40, 50, or 60
amino acids and, preferably, the sequence of the polypeptide of the
invention distinguishes from the sequence shown in Table XII,
application no. 33, columns 5 or 7, by not more than 80% or 70% of
the amino acids, preferably not more than 60% or 50%, more
preferred not more than 40% or 30%, even more preferred not more
than 20% or 10%. In another embodiment, said polypeptide of the
invention does not consist of the sequence shown in Table XII,
application no. 33, columns 5 or 7.
[14240] [0292.0.0.33] for the disclosure of this paragraph see
[0292.0.0.27] above.
[14241] [0293.0.33.33] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the
process.
[14242] In one embodiment, the polypeptide of the invention has a
sequence, which distinguishes from the sequence as shown in Table
XII, application no. 33, columns 5 or 7, by one or more amino
acids. In another embodiment, said polypeptide of the invention
does not consist of the sequence shown in Table XII, application
no. 33, columns 5 or 7. In a further embodiment, said polypeptide
of the present invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical. In one embodiment, said polypeptide does not
consist of the sequence encoded by the nucleic acid molecules shown
in Table XI, application no. 33, columns 5 or 7.
[14243] [0294.0.33.33] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 33, column 3, which
distinguishes over the sequence as indicated in Table XII, columns
5 or 7, by one or more amino acids, preferably by more than 5, 6,
7, 8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or 30
amino acids, evenmore preferred are more than 40, 50, or 60 amino
acids but even more preferred by less than 70% of the amino acids,
more preferred by less than 50%, even more preferred my less than
30% or 25%, more preferred are 20% or 15%, even more preferred are
less than 10%.
[14244] [0295.0.0.33], [0296.0.0.33] and [0297.0.0.33] for the
disclosure of the paragraphs [0295.0.0.33], [0296.0.0.33] and
[0297.0.0.33] see paragraphs [0295.0.0.27] to [0297.0.0.27]
above.
[14245] [00297.1.33.33] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity and/or the amino acid
sequence of a polypeptide indicated in Table XII, application no.
33, columns 3, 5 or 7.
[14246] [0298.0.33.33] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 33, columns 5 or 7, such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table XII, application no. 33, columns 5 or 7.
[14247] [0299.0.33.33] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences of Table XII, application no. 33, columns 5 or
7. The preferred polypeptide of the present invention preferably
possesses at least one of the activities according to the invention
and described herein. A preferred polypeptide of the present
invention includes an amino acid sequence encoded by a nucleotide
sequence which hybridizes, preferably hybridizes under stringent
conditions, to a nucleotide sequence of Table XI, application no.
33, columns 5 or 7, or which is homologous thereto, as defined
above.
[14248] [0300.0.33.33] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table XII,
application no. 33, columns 5 or 7 in amino acid sequence due to
natural variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprise an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence of a sequence as indicated in Table XII,
application no. 33, columns 5 or 7.
[14249] [0301.0.0.33] for the disclosure of this paragraph see
[0301.0.0.27] above.
[14250] [0302.0.33.33] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence shown in Table XII,
application no. 33, columns 5 or 7, or the amino acid sequence of a
protein homologous thereto, which include fewer amino acids than a
full length polypeptide of the present invention or used in the
process of the present invention or the full length protein which
is homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[14251] [0303.0.0.33] for the disclosure of this paragraph see
[0303.0.0.27] above.
[14252] [0304.0.33.33] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 33, columns 5 or 7, but having differences in
the sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[14253] [0305.0.0.33] and [0306.0.0.33] for the disclosure of the
paragraphs [0305.0.0.33] and [0306.0.0.33] see paragraphs
[0305.0.0.27] and [0306.0.0.27] above.
[14254] [0306.1.32.33] for the disclosure of this paragraph see
[0306.1.32.32] above.
[14255] [0307.0.0.33] and [0308.0.0.33] for the disclosure of the
paragraphs [0307.0.0.33] and [0308.0.0.33] see paragraphs
[0307.0.0.27] and [0308.0.0.27] above.
[14256] [0309.0.33.33] In one embodiment, a reference to protein
(=polypeptide)" of the invention e.g. as indicated in Table XII,
application no. 33, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas a
"non-polypeptide" or "other polypeptide" e.g. not indicated in
Table XII, application no. 33, columns 5 or 7, refers to a
polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous to a polypeptide of
the invention, preferably which is not substantially homologous to
a polypeptide having a protein activity, e.g., a protein which does
not confer the activity described herein and which is derived from
the same or a different organism.
[14257] [0310.0.0.33] to [0334.0.0.33] for the disclosure of the
paragraphs [0310.0.0.33] to [0334.0.0.33] see paragraphs
[0310.0.0.27] to [0334.0.0.27] above.
[14258] [0335.0.33.33] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequence as indicated in Table XI, application
no. 33, columns 5 or 7 and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
application no. 33, columns 5 or 7, and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of an
protein encoded by a nucleic acid sequence of one of the sequences
as indicated in Table XI, application no. 33, columns 5 or 7,
and/or homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[14259] [0336.0.0.33] to [0342.0.0.33] for the disclosure of the
paragraphs [0336.0.0.33] to [0342.0.0.33] see paragraphs
[0336.0.0.27] to [0342.0.0.27] above.
[14260] [0343.0.33.33] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 33, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence of one
of the sequences as indicated in Table XI, application no. 33,
columns 5 or 7, or homologs thereof of the one organism, may be
used to suppress the corresponding expression in another
organism.
[14261] [0344.0.0.33] to [0361.0.0.33] for the disclosure of the
paragraphs [0344.0.0.33] to [0361.0.0.33] see paragraphs
[0344.0.0.27] to [0361.0.0.27] above.
[14262] [0362.0.33.33] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or the nucleic acid molecule
used in the method of the invention, the nucleic acid construct of
the invention, the antisense molecule of the invention, the vector
of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. encoding a polypeptide having an
activity of a protein such as the polypeptides as indicated in
Table XII, application no. 33, column 3. Due to the above mentioned
activity the fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity is increased, e.g. due to an increased expression
or specific activity of the subject matters of the invention in a
cell or an organism or a part thereof. Transgenic for a polypeptide
having an activity of a protein such as the polypeptides as
indicated in Table XII, application no. 33, column 3. Activity
means herein that due to modulation or manipulation of the genome,
the activity of polypeptide having an activity of a protein such as
the polypeptides as indicated in Table XII, application no. 33,
column 3, or a similar activity, which is increased in the cell or
organism or part thereof. Examples are described above in context
with the process of the invention.
[14263] [0363.0.0.33] to [0373.0.0.33] for the disclosure of the
paragraphs [0363.0.0.33] to [0373.0.0.33] see paragraphs
[0363.0.0.27] to [0373.0.0.27] above.
[14264] [0374.0.32.33] for the disclosure of this paragraph see
[0374.0.32.32] above.
[14265] [0375.0.0.33] to [0376.0.0.33] for the disclosure of the
paragraphs [0375.0.0.33] to [0376.0.0.33] see paragraphs
[0375.0.0.27] to [0376.0.0.27] above.
[14266] [0377.0.32.33] for the disclosure of this paragraph see
[0377.0.32.32] above.
[14267] [0378.0.0.33] to [0379.0.0.33] for the disclosure of the
paragraphs [0378.0.0.33] to [0379.0.0.33] see paragraphs
[0378.0.0.27] to [0379.0.0.27] above.
[14268] [0380.0.32.33] for the disclosure of this paragraph see
[0380.0.32.32] above.
[14269] [0381.0.0.33] to [0382.0.0.33] for the disclosure of the
paragraphs [0381.0.0.33] to [0382.0.0.33] see paragraphs
[0381.0.0.27] to [0382.0.0.27] above.
[14270] [0383.0.32.33] for the disclosure of this paragraph see
[0383.0.32.32] above.
[14271] [0384.0.0.33] to [0385.0.0.33] for the disclosure of the
paragraphs [0384.0.0.33] to [0385.0.0.33] see paragraphs
[0384.0.0.27] to [0385.0.0.27] above.
[14272] [0386.0.32.33] for the disclosure of this paragraph see
[0386.0.32.32] above.
[14273] [0387.0.0.33] to [0392.0.0.33] for the disclosure of the
paragraphs [0387.0.0.33] to [0392.0.0.33] see paragraphs
[0387.0.0.27] to [0392.0.0.27] above.
[14274] [0393.0.33.33] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps:
(a) contacting e.g. hybridising, the nucleic acid molecules of a
sample, e.g. cells, tissues, plants or microorganisms or a nucleic
acid library, which can contain a candidate gene encoding a gene
product conferring an increase in the respective fine chemical
after expression, with the nucleic acid molecule of the present
invention; (b) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence shown in Table XI, application no. 33, columns 5
or 7 and, optionally, isolating the full length cDNA clone or
complete genomic clone; (c) introducing the candidate nucleic acid
molecules in host cells, preferably in a plant cell or a
microorganism, appropriate for producing the respective fine
chemical; (d) expressing the identified nucleic acid molecules in
the host cells; (e) assaying the respective fine chemical level in
the host cells; and (f) identifying the nucleic acid molecule and
its gene product which expression confers an increase in the
respective fine chemical level in the host cell after expression
compared to the wild type.
[14275] [0394.0.32.33] to [0552.0.32.33] for the disclosure of the
paragraphs [0394.0.32.33] to [0552.0.32.33] see paragraphs
[0394.0.0.32] to [0552.0.0.32] above.
[14276] [0553.0.33.33] [14277] 1. A process for the production of
palmitic acid, which comprises [14278] (a) increasing or generating
the activity of a protein as indicated in Table XII, application
no. 33, columns 5 or 7, or a functional equivalent thereof in a
non-human organism, or in one or more parts thereof; and [14279]
(b) growing the organism under conditions which permit the
production of palmitic acid in said organism. [14280] 2. A process
for the production of palmitic acid, comprising the increasing or
generating in an organism or a part thereof the expression of at
least one nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [14281] a) nucleic acid
molecule encoding of a polypeptide as indicated in Table XII,
application no. 33, columns 5 or 7, or a fragment thereof, which
confers an increase in the amount of palmitic acid in an organism
or a part thereof; [14282] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table XI, application no. 33,
columns 5 or 7; [14283] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of palmitic acid in
an organism or a part thereof; [14284] d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of palmitic acid in an organism or a part thereof; [14285]
e) nucleic acid molecule which hybridizes with a nucleic acid
molecule of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of palmitic acid in an
organism or a part thereof; [14286] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table XIII,
application no. 33, column 7, and conferring an increase in the
amount of palmitic acid in an organism or a part thereof; [14287]
g) nucleic acid molecule encoding a polypeptide which is isolated
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (f) and conferring
an increase in the amount of palmitic acid in an organism or a part
thereof; [14288] h) nucleic acid molecule encoding a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 33, column 7, and conferring an increase in the
amount of palmitic acid in an organism or a part thereof; and
[14289] i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of palmitic acid in an
organism or a part thereof. [14290] or comprising a sequence which
is complementary thereto. [14291] 3. The process of claim 1 or 2,
comprising recovering of the free or bound palmitic acid. [14292]
4. The process of any one of claims 1 to 3, comprising the
following steps: [14293] (a) selecting an organism or a part
thereof expressing a polypeptide encoded by the nucleic acid
molecule characterized in claim 2; [14294] (b) mutagenizing the
selected organism or the part thereof; [14295] (c) comparing the
activity or the expression level of said polypeptide in the
mutagenized organism or the part thereof with the activity or the
expression of said polypeptide of the selected organisms or the
part thereof; [14296] (d) selecting the mutated organisms or parts
thereof, which comprise an increased activity or expression level
of said polypeptide compared to the selected organism or the part
thereof; [14297] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [14298] (f) recovering, and
optionally isolating, the free or bound palmitic acid produced by
the selected mutated organisms or parts thereof. [14299] 5. The
process of any one of claims 1 to 4, wherein the activity of said
protein or the expression of said nucleic acid molecule is
increased or generated transiently or stably. [14300] 6. An
isolated nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [14301] a) nucleic acid
molecule encoding of a polypeptide as indicated in Table XII,
application no. 33, columns 5 or 7, or a fragment thereof, which
confers an increase in the amount of palmitic acid in an organism
or a part thereof; [14302] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table XI, application no. 33,
columns 5 or 7; [14303] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of palmitic acid in
an organism or a part thereof; [14304] d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of palmitic acid in an organism or a part thereof; [14305]
e) nucleic acid molecule which hybridizes with a nucleic acid
molecule of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of palmitic acid in an
organism or a part thereof; [14306] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table XIII,
application no. 33, column 7, and conferring an increase in the
amount of palmitic acid in an organism or a part thereof; [14307]
g) nucleic acid molecule encoding a polypeptide which is isolated
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (f) and conferring
an increase in the amount of palmitic acid in an organism or a part
thereof; [14308] h) nucleic acid molecule encoding a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 33, column 7, and conferring an increase in the
amount of palmitic acid in an organism or a part thereof; and
[14309] i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of palmitic acid in an
organism or a part thereof. [14310] whereby the nucleic acid
molecule distinguishes over the sequence as indicated in Table XI,
application no. 33, columns 5 or 7, by one or more nucleotides.
[14311] 7. A nucleic acid construct which confers the expression of
the nucleic acid molecule of claim 6, comprising one or more
regulatory elements. [14312] 8. A vector comprising the nucleic
acid molecule as claimed in claim 6 or the nucleic acid construct
of claim 7. [14313] 9. The vector as claimed in claim 8, wherein
the nucleic acid molecule is in operable linkage with regulatory
sequences for the expression in a prokaryotic or eukaryotic, or in
a prokaryotic and eukaryotic, host. [14314] 10. A host cell, which
has been transformed stably or transiently with the vector as
claimed in claim 8 or 9 or the nucleic acid molecule as claimed in
claim 6 or the nucleic acid construct of claim 7 or produced as
described in claim any one of claims 2 to 5. [14315] 11. The host
cell of claim 10, which is a transgenic host cell. [14316] 12. The
host cell of claim 10 or 11, which is a plant cell, an animal cell,
a microorganism, or a yeast cell, a fungus cell, a prokaryotic
cell, an eukaryotic cell or an archaebacterium. [14317] 13. A
process for producing a polypeptide, wherein the polypeptide is
expressed in a host cell as claimed in any one of claims 10 to 12.
[14318] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table XII, application no. 33, columns 5 or 7, by one
or more amino acids [14319] 15. An antibody, which binds
specifically to the polypeptide as claimed in claim 14. [14320] 16.
A plant tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [14321] 17. A method for screening for
agonists and antagonists of the activity of a polypeptide encoded
by the nucleic acid molecule of claim 6 conferring an increase in
the amount of palmitic acid in an organism or a part thereof
comprising: [14322] (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of palmitic acid in an organism or a part thereof with a
candidate compound or a sample comprising a plurality of compounds
under conditions which permit the expression the polypeptide;
[14323] (b) assaying the palmitic acid level or the polypeptide
expression level in the cell, tissue, plant or microorganism or the
media the cell, tissue, plant or microorganisms is cultured or
maintained in; and [14324] (c) identifying a agonist or antagonist
by comparing the measured palmitic acid level or polypeptide
expression level with a standard of palmitic acid or polypeptide
expression level measured in the absence of said candidate compound
or a sample comprising said plurality of compounds, whereby an
increased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an agonist and
a decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[14325] 18. A process for the identification of a compound
conferring increased palmitic acid production in a plant or
microorganism, comprising the steps: [14326] (a) culturing a plant
cell or tissue or microorganism or maintaining a plant expressing
the polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of palmitic acid in an
organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with dais readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of palmitic acid in an
organism or a part thereof; [14327] (b) identifying if the compound
is an effective agonist by detecting the presence or absence or
increase of a signal produced by said readout system. [14328] 19. A
method for the identification of a gene product conferring an
increase in palmitic acid production in a cell, comprising the
following steps: [14329] (a) contacting the nucleic acid molecules
of a sample, which can contain a candidate gene encoding a gene
product conferring an increase in palmitic acid after expression
with the nucleic acid molecule of claim 6; [14330] (b) identifying
the nucleic acid molecules, which hybridise under relaxed stringent
conditions with the nucleic acid molecule of claim 6; [14331] (c)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing palmitic acid; [14332] (d) expressing the
identified nucleic acid molecules in the host cells; [14333] (e)
assaying the palmitic acid level in the host cells; and [14334] (f)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the palmitic acid level in the
host cell in the host cell after expression compared to the wild
type. [14335] 20. A method for the identification of a gene product
conferring an increase in palmitic acid production in a cell,
comprising the following steps: [14336] (a) identifying in a data
bank nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the palmitic acid amount or level in an organism or a part thereof
after expression, and which are at least 20% homolog to the nucleic
acid molecule of claim 6; [14337] (b) introducing the candidate
nucleic acid molecules in host cells appropriate for producing
palmitic acid; [14338] (c) expressing the identified nucleic acid
molecules in the host cells; [14339] (d) assaying the palmitic acid
level in the host cells; and [14340] (e) identifying nucleic acid
molecule and its gene product which expression confers an increase
in the palmitic acid level in the host cell after expression
compared to the wild type. [14341] 21. A method for the production
of an agricultural composition comprising the steps of the method
of any one of claims 17 to 20 and formulating the compound
identified in any one of claims 17 to 20 in a form acceptable for
an application in agriculture. [14342] 22. A composition comprising
the nucleic acid molecule of claim 6, the polypeptide of claim 14,
the nucleic acid construct of claim 7, the vector of any one of
claim 8 or 9, an antagonist or agonist identified according to
claim 17, the compound of claim 18, the gene product of claim 19 or
20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. [14343] 23. Use of the nucleic acid molecule as
claimed in claim 6 for the identification of a nucleic acid
molecule conferring an increase of palmitic acid after expression.
[14344] 24. Use of the polypeptide of claim 14 or the nucleic acid
construct claim 7 or the gene product identified according to the
method of claim 19 or 20 for identifying compounds capable of
conferring a modulation of palmitic acid levels in an organism.
[14345] 25. Food or feed composition comprising the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the harvested material of
claim 16, the host cell of claim 10 to 12 or the gene product
identified according to the method of claim 19 or 20.
[14346] 26. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the host cell of claim 10 to 12 or the gene
product identified according to the method of claim 19 or 20 for
the protection of a plant against a palmitic acid synthesis
inhibiting herbicide.
[14347] [0554.0.0.33] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[14348] [0000.0.34.34] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and corresponding embodiments as
described herein as follows.
[14349] [0001.0.0.34] for the disclosure of this paragraph see
[0001.0.0.27].
[14350] [0002.0.34.34] Sterols are a class of essential, natural
compounds required by all eukaryotes to complete their life cycle.
In animals, cholesterol is typically the major sterol while in
fungi it is ergosterol. Plants produce a class of sterols called
phytosterols. Phytosterols are natural components of many
vegetables and grains. The term "phytosterols" covers plant sterols
and plant stanols. Plant sterols are naturally occurring substances
present in the diet as minor components of vegetable oils. The
structures of these plant sterols are similar to that of
cholesterol with an extra methyl or ethyl group and a double bond
in the side chain. Saturated plant sterols, referred to as stanols,
have no double bond in the ring structure.
[14351] Phytosterols (including plant sterols and stanols) are
natural components of plant foods, especially plant oils, seeds and
nuts, cereals and legumes specially of edible vegetable oils such
as sunflower seed oil and, as such are natural constituents of the
human diet. The most common phytosterols are beta-sitosterol,
campesterol, and stigmasterol. Beta-sitosterol is found in high
amounts in nuts.
[14352] [0003.0.34.34] A high concentration of cholesterol in
serum, i.e., hypercholesterolemia, is a wellknown risk factor for
coronary heart disease (CHD). Blood cholesterol levels can be
decreased by following diets, which are low in saturated fat, high
in polyunsaturated fat and low in cholesterol. Although
considerable achievements have been made in terms of knowledge and
education, consumers still find it difficult to follow healthy
eating advice.
[14353] Both plant sterols and plant stanols are effective in
lowering plasma total and low density lipoprotein (LDL) cholesterol
and this occurs by inhibiting the absorption of cholesterol from
the small intestine. The plasma cholesterol-lowering properties of
plant sterols have been known since the 1950s (Pollak, Circulation,
7, 702-706.1953). They have been used as cholesterol-lowering
agents, first in a free form (Pollak and Kritchevsky, Sitosterol.
In: Monographs on Aherosclerosis. Clarkson T B, Kritchevsky D,
Pollak O J, eds. New York, Basel, Karger 1981; 1-219) and recently
mainly as esterified phytosterols (Katan et al., Mayo Clin Proc
2003; 78: 965-978). The consumption of plant sterols and plant
stanols lowers blood cholesterol levels by inhibiting the
absorption of dietary and endogenously-produced cholesterol from
the small intestine and the plant sterols/stanols are only very
poorly absorbed themselves. This inhibition is related to the
similarity in physico-chemical properties of plant sterols and
stanols and cholesterol. Plant sterols and plant stanols appear to
be without hazard to health, having been shown without adverse
effects in a large number of human studies. They show no evidence
of toxicity even at high dose levels and gastro-intestinal
absorption is low.
[14354] [0004.0.34.34] The most abundant sterols of vascular plants
are campesterol, sitosterol and stigmasterol, all of which contain
a double bond between the carbon atoms at positions 5 and 6 and are
classified as delta-5 sterols.
[14355] Exemplary naturally occurring delta-5 plant sterols
isofucosterol, sitosterol, stigmasterol, campesterol, cholesterol,
and di hydrobrassicasterol. Exemplary naturally occurring
non-delta-5 plant sterols are cycloartenol, 24-methylene
cycloartenol, cycloeucalenol, and obtusifoliol.
[14356] The ratio of delta-5 to non-delta-5 sterols in plants can
be an important factor relating to insect pest resistance. Insect
pests are unable to synthesize de novo the steroid nucleus and
depend upon external sources of sterols in their food source for
production of necessary steroid compounds. In particular, insect
pests require an external source of delta-5 sterols. By way of
example, externally provided delta-5 sterols are necessary for the
production of ecdysteroids, hormones that control reproduction and
development. See, e.g., Costet et al., Proc. Natl. Acad. Sci. USA,
84:643 (1987) and Corio-Costet et al., Archives of Insect Biochem.
Physiol., 11:47 (1989).
[14357] [0005.0.34.34] US 20020148006 and WO 98/45457 describes the
modulation of phytosterol compositions to confer resistance to
insects, nematodes, fungi and/or environmental stresses, and/or to
improve the nutritional value of plants by using a DNA sequence
encoding a first enzyme; which binds a first sterol and is
preferably selected from the group consisting of
S-adenosyl-L-methionine-.sub.24(25)-sterol methyl transferase, a
C-4 demethylase, a cycloeucalenol to obtusifoliol-isomerase, a
14-demethylase, a .sub.8 to .sub.7-isomerase, a
.sub.7-C-S-desaturase and a 24,25-reductase, and produces a second
sterol and a 3' non-translated region which causes polyadenylation
at the 3' end of the RNA.
[14358] WO 93/16187 discloses new plants containing in its genome
one or more genes involved in the early stages of phytosterol
biosynthesis, preferably the genes encode mevalonate kinase.
[14359] U.S. Pat. No. 5,306,862, U.S. Pat. No. 5,589,619, U.S. Pat.
No. 5,365,017, U.S. Pat. No. 5,349,126 and US 20030150008 describe
a method of increasing sterol (and squalene) accumulation in a
plant based on an increased HMG-CoA reductase activity to increase
the pest resistance of transgenic plants.
[14360] WO 97/48793 discloses a C-14 sterol reductase polypeptide
for the genetic manipulation of a plant sterol biosynthetic
pathway.
[14361] US 20040172680 disclose the use of a gene expressing a SMT1
(sterol methyltransferase) to increase the level of sterols in the
seeds of plants. A DNA sequence encoding sterol methyltransferase
isolated from Zea mays is disclosed in WO 00/08190. Bouvier-Nav et
al in Eur. J. Biochem. 256, 88-96 (1988) describes two families of
sterol methyl transf erases (SMTs), The first (SMT1) applying to
cycloartenol and the second (SMT2) to 24-methylene lophenol.
Schaller et al (Plant Physiology (1998) 118: 461-169) describes the
over-expression of SMT2 from Arabidopsis in tobacco resulting in a
change in the ratio of 24-methyl cholesterol to sitosterol in the
tobacco leaf.
[14362] U.S. Pat. No. 6,723,837 and US 20040199940 disclose nucleic
acid molecules encoding proteins and fragments of proteins
associated with sterol and phytosterol metabolism as well as cells,
that have been manipulated to contain increased levels or
overexpress at least one sterol or phytosterol compound. The
protein or fragment is selected from the group consisting of a
HES1, HMGCoA reductase, squalene synthase, cycloartenol synthase,
SMTI, SMTII and UPC, preferably from member of the KES1/HES1/OSH1
family of oxysterol-binding (OSBP) proteins comprising an
oxysterol-binding protein consensus sequence--E(K, Q).times.SH (H,
R) PPx (S, T, A, C, F)A. One class of proteins, oxysterol-binding
proteins, have been reported in humans and yeast (Jiang et al.,
Yeast 10: 341-353 (1994), the entirety of which is herein
incorporated by reference). These proteins have been reported to
modulate ergosterol levels in yeast (Jiang et al., Yeast 10:
341-353 (1994)). In particular, Jiang et al., reported three genes
KES1, HES1 and OSH1, which encode proteins containing an
oxysterol-binding region.
[14363] [0006.0.34.34] Transgenic plants having altered sterol
profiles could be instrumental in establishing a dietary approach
to cholesterol management and cardiovascular disease prevention.
The altered phytosterol profile further leads to pest
resistance.
[14364] [0007.0.34.34] Although people consume plant sterols every
day in their normal diet, the amount is not great enough to have a
significant blood cholesterol lowering effect. The intake of
phytosterols varies among different populations according to the
food products being consumed, but the average daily Western diet is
reported to contain 150-300 mg of these sterols (de Vries et al., J
Food Comp Anal 1997; 19: 115-141; Bjorkhem et al. Inborn errors in
bile acid biosynthesis and storage of sterols other than
cholesterol. In: The Metabolic and Molecular Bases of Inherited
Disease. Scriver C S, Beaudet A L, Sly W S, Valle D, eds. New York,
McGraw-Hill 2001; 2961-2988). In order to achieve a
cholesterol-lowering benefit, approximately 1 g/day of plant
sterols need to be consumed (Hendriks et al., European Journal of
Clinical Nutrition, 53, 319-327.1999).
[14365] [0008.0.34.34] Phytosterols are found naturally in plant
foods at low levels. The enrichment of foods such as margarines
with plant sterols and stanols is one of the recent developments in
functional foods to enhance the cholesterol-lowering ability of
traditional food products. Incorporation of additional phytosterols
into the diet may be an effective way of lowering total and LDL
cholesterol levels. The non-esterified phytosterols can be used as
novel food ingredients in: [14366] (f) bakery products and cereals
(eg, breakfast cereals, breakfast bars); [14367] (g) dairy products
such as low and reduced fat liquid milk, low and reduced fat
yoghurt and yoghurt products, and dairy based desserts; [14368] (h)
non-carbonated soft drinks like low and reduced fat soy beverages
and low and reduced fat soy-based yoghurts; [14369] (i) meat
products or edible fats and oils (eg, mayonnaise, spice sauces,
salad dressings); [14370] (j) margarine; and [14371] table spreads
or dietary fats.
[14372] [0009.0.34.34] When edible oils undergo normal refining,
plant sterols are partially extracted. It is estimated that 2500
tonnes of vegetable oil needs to be refined to produce 1 tonne of
plant sterols. Plant stanols are obtained by hydrogenation of the
plant sterols.
[14373] [0010.0.34.34] Another source of plant sterols is tall oil,
derived from the process of paper production from wood and
approximately 2500 tonnes of pine is required to produce 1 tonne of
plant sterols. Tall oil also contains a higher proportion of plant
stanols (primarily b-sitostanol) than do vegetable oils.
[14374] [0011.0.34.34] As described above, phytosterols are used in
a lot of different applications, for example in cosmetics,
pharmaceuticals and in feed and food. Therefore improving the
quality of foodstuffs and animal feeds is an important task of the
food-and-feed industry. Especially advantageous for the quality of
foodstuffs and animal feeds is as balanced as possible a sterol
profile in the diet.
[14375] [0012.0.34.34] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes which
participate in the biosynthesis of phytosterols and make it
possible to produce them specifically on an industrial scale
without unwanted byproducts forming. In the selection of genes for
or regulators of biosynthesis two characteristics above all are
particularly important. On the one hand, there is as ever a need
for improved processes for obtaining the highest possible contents
of phytosterols; on the other hand as less as possible by products
should be produced in the production process.
[14376] [0013.0.0.34] for the disclosure of this paragraph see
[0013.0.0.27] above.
[14377] [0014.0.34.34] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is/are phytosterols. The term
"phytosterols" covers plant sterols and plant stanols. Accordingly,
in the present invention, the term "the fine chemical" as used
herein relates to "phytosterols". Further, the term "the fine
chemicals" as used herein also relates to fine chemicals comprising
plant sterols and plant stanols, preferably beta-sitosterol,
campesterol, and/or stigmasterol.
[14378] [0015.0.34.34] In one embodiment, the term "the fine
chemical" means phytosterols, plant sterols and plant stanols.
Throughout the specification the term "the fine chemical" means
phytosterols and ester, thioester or sterols in free form or bound
to other compounds. For the purpose of this description, the term
sterol/stanol refers both to free sterols/stanols and conjugated
sterols/stanols, for example, where the 3-hydroxy group is
esterified by a fatty acid chain or phenolic acid to give a
steryl/stanyl ester. As used herein, the term "phytosterol"
includes all phytosterols without limitation, for example:
sitosterol, campesterol, stigmasterol, brassicasterol, desmosterol,
chalinosterol, poriferasterol, clionasterol, the corresponding
stanols and all natural or synthesized forms and derivatives
thereof, including isomers. It is to be understood that
modifications to the phytosterols i.e. to include side chains also
falls within the purview of this invention. All those derivates
forms are summarized as "conjugates". In an preferred embodiment,
the term "the fine chemical" or the term "phytosterol" or the term
"the respective fine chemical" means at least one chemical compound
plant sterols and plant stanols selected from the group
"beta-sitosterol, sitostanol, stigmasterol, brassicasterol,
campestanol, isofucosterol and campesterol", preferred
"beta-sitosterol, campesterol, and/or stigmasterol", most preferred
"beta-sitosterol and/or campesterol". Also preferably, are esters
of sterols/stanols with C10-24 fatty acids.
[14379] Increased phytosterol content normally means an increased
total phytosterol content. However, an increased phytosterol
content also means, in particular, a modified content of the
above-described compounds ("beta-sitosterol, sitostanol,
stigmasterol, brassicasterol, campestanol, isofucosterol and
campesterol") with phytosterol activity, without the need for an
inevitable Xlncrease in the total phytosterol content.
[14380] [0016.0.34.34] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [14381] (a) increasing or generating the activity of one
or more [14382] of a protein as shown in table XII, application no.
34, column 3 encoded by the nucleic acid sequences as shown in
table XI, application no. 34, column 5, in a non-human organism or
in one one or more parts thereof or [14383] (b) growing the
organism under conditions which permit the production of the fine
chemical, thus beta-sitosterol and/or campesterol of the invention
or fine chemicals comprising beta-sitosterol and/or campesterol of
the invention, in said organism or in the culture medium
surrounding the organism.
[14384] [0016.1.34.34] Accordingly, the term "the fine chemical"
means in one embodiment "beta-Sitosterol" in relation to all
sequences listed in Tables XI to XIV, line 19 or homologs thereof
and
[14385] means in one embodiment "Campesterol" in relation to all
sequences listed in Table XI to XIV, line 20, or homologs
thereof.
[14386] Accordingly, in one embodiment the term "the fine chemical"
means "beta-Sitosterol" and "Campesterol" in relation to all
sequences listed in Table XI to XIV, lines 19 and/or 20.
[14387] [0017.0.0.34] and [0019.0.0.34] for the disclosure of the
paragraphs [0017.0.0.34] and [0019.0.0.34] see paragraphs
[0017.0.0.27] and [0019.0.0.27] above.
[14388] [0020.0.34.34] Surprisingly it was found, that the
transgenic expression of the Zea mays protein as indicated in Table
XII, column 5, lines 19 and 20 in a plant conferred an increase in
beta-Sitosterol and/or Campesterol (or the respective fine
chemical) content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of beta-Sitosterol; in one embodiment, said protein or
its homologs are used for the production of Campesterol, in one
embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of beta-Sitosterol and/or Campesterol.
[14389] [0021.0.0.34] for the disclosure of this paragraph see
[0021.0.0.27] above.
[14390] [0022.0.34.34] The sequence of YKR057W from Saccharomyces
cerevisiae has been published in Dujon et al., Nature 369 (6479),
371-378, 1994 and Goffeau et al., Science 274 (5287), 546-547, 1996
and its activity is being defined as a ribosomal protein, similar
to S21 ribosomal proteins, involved in ribosome biogenesis and
translation. Accordingly, in one embodiment, the process of the
present invention comprises the use of a protein involved in the
ribosome biogenesis and translation, in particular of the
superfamiliy of the ribosomal protein, preferably having a S21
ribosomal protein activity or its homolog, e.g. as shown herein,
for the production of the respective fine chemical, meaning of
phytosterol, e.g. beta-sitosterol and/or campesterol and/or
conjugates, preferably in free or bound form in an organism or a
part thereof, as mentioned. In one further embodiment the YKR057W
protein expression is increased together with the increase of
another gene of the phytosterol biosynthesis pathway, preferably
with a gene encoding a protein being involved in the production of
phytosterol from the intermediates like squalene and squalene
epoxide or cycloartenol. In one embodiment, in the process of the
present invention said activity, e.g. of a protein of the yeast
ribosomal protein superfamily or preferably having a activity
involved in ribosome biogenesis and translation is increased or
generated, e.g. from Saccharomyces cerevisiae or a plant or a
homolog thereof.
[14391] [0022.1.0.34] to [0023.0.34.34] for the disclosure of this
paragraphs see
[14392] [0022.1.0.27] to [0023.0.0.27] above.
[14393] [0023.1.34.34] Homologs of the polypeptide disclosed in
table XII, application no. 34, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 34, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 34, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 34,
column 7, resp.
[14394] [0024.0.0.34] for the disclosure of this paragraph see
[0024.0.0.27] above.
[14395] [0025.0.34.34] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 34, column 3" if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 34, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said organism.
The protein has the above mentioned activities of a protein as
shown in table XII, application no. 34, column 3, preferably in the
event the nucleic acid sequences encoding said proteins is
functionally joined to the nucleic acid sequence of a transit
peptide. Throughout the specification the activity or preferably
the biological activity of such a protein or polypeptide or an
nucleic acid molecule or sequence encoding such protein or
polypeptide is identical or similar if it still has the biological
or enzymatic activity of a protein as shown in table XII,
application no. 34, column 3, or which has at least 10% of the
original enzymatic or biological activity, preferably 20%,
particularly preferably 30%, most particularly preferably 40% in
comparison to a protein as shown in the respective line of table
XII, application no. 34, column 3 of a E. coli or Saccharomyces
cerevisae, respectively, protein as mentioned in table XI to XIV,
column 3 respectively and as disclosed in paragraph [0022] of the
respective invention.
[14396] [0025.1.0.34] and [0025.2.0.34] for the disclosure of the
paragraphs [0025.1.0.34] and [0025.2.0.34] see [0025.1.0.27] and
[0025.2.0.27] above.
[14397] [0026.0.0.34] to [0033.0.0.34] for the disclosure of the
paragraphs [0026.0.0.34] to [0033.0.0.34] see paragraphs
[0026.0.0.27] to [0033.0.0.27] above.
[14398] [0034.0.34.34] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 34, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[14399] [0035.0.0.34] to [0038.0.0.34] and [0039.0.5.12] for the
disclosure of the paragraphs [0035.0.0.34] to [0038.0.0.34] and
[0039.0.5.12] see paragraphs [0035.0.0.27] to [0039.0.0.27]
above.
[14400] [0040.0.0.34] to [0044.0.0.34] for the disclosure of the
paragraphs [0040.0.0.34] to [0044.0.0.34] see paragraphs
[0035.0.0.27] and [0044.0.0.27] above.
[14401] [0045.0.34.34.] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
34, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line,
between 10% and 50%, 10% and 100%, 10% and 200%, 10% and 300%, 10%
and 400%, 10% and 500%, 20% and 50%, 20% and 250%, 20% and 500%,
50% and 100%, 50% and 250%, 50% and 500%, 500% and 1000% or
more.
[14402] [0046.0.34.34] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
34, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line
confers an increase of the respective fine chemical or their
precursors.
[14403] [0047.0.0.34] to [0048.0.0.34] for the disclosure of the
paragraphs [0047.0.0.34] and [0048.0.0.34] see paragraphs
[0047.0.0.27] and [0048.0.0.27] above.
[14404] [0049.0.34.34] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 34, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 34, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 34, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[14405] [0050.0.34.34] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of phytosterols or their ester.
[14406] [0051.0.0.34] and [0052.0.0.34] for the disclosure of the
paragraphs [0051.0.5.12] and [0052.0.0.34] see paragraphs
[0051.0.0.27] and [0052.0.0.27] above.
[14407] [0053.0.34.34] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[14408] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 34, columns 5 and 7 or its homologs activity
having herein-mentioned beta-sitosterol and/or campesterol of the
invention increasing activity; and/or [14409] b) stabilizing a mRNA
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention as shown in table XI,
application no. [14410] 34, columns 5 and 7, e.g. a nucleic acid
sequence encoding a polypeptide having the activity of a protein as
indicated in table XII, application no. 34, columns 5 and 7 or its
homologs activity or of a mRNA encoding the polypeptide of the
present invention having herein-mentioned beta-sitosterol and/or
campesterol of the invention increasing activity; and/or [14411] c)
increasing the specific activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention or of the polypeptide of the present
invention having herein-mentioned beta-sitosterol and/or
campesterol increasing activity, e.g. of a polypeptide having the
activity of a protein as indicated in table XII, application no.
34, columns 5 and 7 or its homologs activity, or decreasing the
inhibiitory regulation of the polypeptide of the invention; and/or
[14412] d) generating or increasing the expression of an endogenous
or artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned beta-sitosterol and/or
campesterol of the invention increasing activity, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 34, columns 5 and 7 or its homologs activity;
and/or [14413] e) stimulating activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention having herein-mentioned beta-sitosterol and/or
campesterol of the invention increasing activity, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 34, columns 5 and 7 or its homologs activity,
by adding one or more exogenous inducing factors to the organisms
or parts thereof; and/or [14414] f) expressing a transgenic gene
encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned beta-sitosterol and/or campesterol of the
invention increasing activity, e.g. of a polypeptide having the
activity of a protein as indicated in table XII, application no.
34, columns 5 and 7 or its homologs activity, and/or [14415] g)
increasing the copy number of a gene conferring the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or the
polypeptide of the invention having herein-mentioned
beta-sitosterol and/or campesterol of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 34, columns 5 and 7 or its
homologs activity; and/or [14416] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 34, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[14417] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [14418] k) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[14419] [0054.0.34.34] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 34 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 34, column 5 or 7. In general, the amount of
mRNA or polypeptide in a cell or a compartment of a organism
correlates with the amount of encoded protein and thus with the
overall activity of the encoded protein in said volume. Said
correlation is not always linear, the activity in the volume is
dependent on the stability of the molecules or the presence of
activating or inhibiting co-factors. Further, product and educt
inhibitions of enzymes are well known and described in Textbooks,
e.g. Stryer, Biochemistry.
[14420] [0055.0.0.34] to [0067.0.0.34] for the disclosure of the
paragraphs [0055.0.0.34] to [0067.0.0.34] see paragraphs
[0055.0.0.27] to [0067.0.0.27] above.
[14421] [0068.0.34.34] The mutation is introduced in such a way
that the production of the phytosterol(s) is not adversely
affected.
[14422] [0069.0.0.34] for the disclosure of this paragraph see
paragraphs [0069.0.0.27] above.
[14423] [0070.0.34.34] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding the
YKR057W protein into an organism alone or in combination with other
genes, it is possible not only to increase the biosynthetic flux
towards the end product, but also to increase, modify or create de
novo an advantageous, preferably novel metabolites composition in
the organism, e.g. an advantageous fatty acid composition
comprising a higher content of (from a viewpoint of nutritional
physiology limited) phytosterol(s) etc.
[14424] [0071.0.0.34] for the disclosure of this paragraph see
[0071.0.0.27] above.
[14425] [0072.0.34.34] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to phytosterols further sterols, stanols or squalene,
squalene epoxide or cycloartenol.
[14426] [0073.0.34.34] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[14427] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [14428] b) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table XII, application no. 34, columns 5 or 7, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the respective fine chemical in an organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [14429] c) growing an organism,
preferably a microorganism, a non-human animal, a plant or animal
cell, a plant or animal tissue or a plant under conditions which
permit the production of the respective fine chemical in the
organism, preferably the microorganism, the plant cell, the plant
tissue or the plant; and [14430] d) if desired, revovering,
optionally isolating, the free and/or bound the respective fine
chemical and, optionally further free and/or bound phytosterol or
its conjugate synthesised by the organism, the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[14431] [0074.0.34.34] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the respective fine chemical or the free and
bound respective fine chemical but as option it is also possible to
produce, recover and, if desired isolate, other free or/and bound
of phytosterol(s) or its/their conjugates.
[14432] [0075.0.0.34] to [0077.0.0.34] for the disclosure of the
paragraphs [0075.0.0.34] to [0077.0.0.34] see paragraphs
[0075.0.0.27] to [0077.0.0.27] above.
[14433] [0078.0.34.34] The organism such as microorganisms or
plants or the recovered, and if desired isolated, respective fine
chemical can then be processed further directly into foodstuffs or
animal feeds or for other applications, for example according to
the disclosures made in:
US 20040101829, which disclose a methods for treating
hyperlipidemia and to reduce Low Density Lipoprotein ("LDL") levels
in a subject, US 20040047971, which disclose the preparation of a
fat composition containing sterol esters characterised by direct
interesterification of sterol with triglyceride, U.S. Pat. No.
5,965,449, which describes phytosterol-based compositions useful in
preventing and treating cardiovascular disease and other disorders,
U.S. Pat. No. 5,523,087, which is for a pharmaceutical composition
containing beta-sitosterol for the treatment of diabetic male
sexual dysfunction; U.S. Pat. No. 5,747,464, which discloses a
composition for inhibiting absorption of fat and cholesterol from
the gut comprising beta.-sitosterol bound irreversibly to pectin,
U.S. Pat. No. 4,588,717, which describes a vitamin supplement which
comprises a fatty acid ester of a phytosterol, U.S. Pat. No.
5,270,041, which teaches the use of small amounts of sterols, their
fatty acid esters and glucosides for the treatment of tumours, U.S.
Pat. No. 6,087,353, which comprises methods of making a composition
suitable for incorporation into foods, beverages, pharmaceuticals,
nutraceuticals and the like which comprises condensing a suitable
aliphatic acid with a phytosterol to form a phytosterol ester and
subsequently hydrogenating the phytosterol ester to form a
hydrogenated phytosterol ester, which are expressly incorporated
herein by reference.
[14434] The fermentation broth, fermentation products, plants or
plant products can be treated with water and a mixture of organic
solvents (hexane and acetone) in order to extract the phytosterols.
Crude phytosterols are obtained from the organic phase by removal
of the solvents, complexation of the sterols in the extract with
calcium chloride in methanol, separation of the sterol-complexes by
centrifugation, dissociation of the complexes by heating in water
and removal of the water. The crude phytosterols can be further
purified by crystallisation from isopropanol. According to an other
production process the tall oil soap is first subjected to
fractional distillation which removes volatile compounds. The
resulting residue (tall oil pitch) containing sterols in esterified
form is treated with alkali to liberate these sterols. After
neutralisation, the material is subjected to a two-stage
distillation process. The distillate is then dissolved in
methanol/methylethylketone solvent and the sterols crystallising
from this solution are obtained by filtration, washed with solvent
and dried. U.S. Pat. No. 4,420,427 teaches the preparation of
sterols from vegetable oil sludge using solvents such as methanol.
Alternatively, phytosterols may be obtained from tall oil pitch or
soap, by-products of the forestry practise as described in
PCT/CA95/00555, incorporated herein by reference. The extraction
and crystallization may be performed by other methods known to the
person skilled in the art and described herein below. To form a
phytosterol ester in accordance with the U.S. Pat. No. 6,087,353,
the selected phytosterol and aliphatic acid or its ester with
volatile alcohol are mixed together under reaction conditions to
permit condensation of the phytosterol with the aliphatic acid to
produce an ester. A most preferred method of preparing these esters
which is widely used in the edible fat and oil industry is
described in U.S. Pat. No. 5,502,045 (which is incorporated herein
by reference). The stanol and/or sterol esters with the desired
fatty acid composition can also be produced by direct, preferably
catalytic esterification methods, e.g. U.S. Pat. No. 5,892,068,
between free fatty acids or fatty acid blends of the composition
and the stanol and/or sterol. In addition, stanol and/or sterol
esters can also be produced by enzymatic esterification e.g. as
outlined in EP 195 311 (which are incorporated herein by
reference).
[14435] Products of these different work-up procedures are
phytosterols and/or esters and/or conjugates or compositions which
still comprise fermentation broth, plant particles and cell
components in different amounts, advantageously in the range of
from 0 to 99% by weight, preferably below 80% by weight, especially
preferably between below 50% by weight.
[14436] [0079.0.0.34] to [0084.0.0.34] for the disclosure of the
paragraphs [0079.0.0.34] to [0084.0.0.34] see paragraphs
[0079.0.0.27] to [0084.0.0.27] above.
[14437] [0085.0.34.34] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [14438] a) a nucleic acid sequence as
indicated in Table XI, application no. 34, columns 5 or 7, or a
derivative thereof, or [14439] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 34, columns
5 or 7, or a derivative thereof, or [14440] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[14441] [0086.0.0.34] and [0087.0.0.34] for the disclosure of the
paragraphs [0086.0.0.34] and [0087.0.0.34] see paragraphs
[0086.0.0.27] and [0087.0.0.27] above.
[14442] [0088.0.34.34] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose phytosterol
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for poultry is dependent on the abovementioned phytosterol and the
general amount of phytosterol as source in feed and/or food.
Further, this is also important since, for example a balanced
content of different phytosterols induces stress resistance to
plants. After the YKR057W, protein activity has been increased or
generated, or after the expression of nucleic acid molecule or
polypeptide according to the invention has been generated or
increased, the transgenic plant generated thus is grown on or in a
nutrient medium or else in the soil and subsequently harvested.
[14443] [0088.1.0.34], [0089.0.0.34] and [0090.0.0.34] for the
disclosure of the paragraphs [0088.1.0.34], [0089.0.0.34] and
[0090.0.0.34] see paragraphs [0088.1.0.27],
[14444] [0089.0.0.27] and [0090.0.0.27] above.
[14445] [0091.0.34.34] Thus, the content of plant components and
preferably also further impurities is as low as possible, and the
abovementioned phytosterols are obtained in as pure form as
possible. In these applications, the content of plant components
advantageously amounts to less than 10%, preferably 1%, more
preferably 0.1%, very especially preferably 0.01% or less.
[14446] [0092.0.0.34] to [0094.0.0.34] for the disclosure of the
paragraphs [0092.0.0.34] to [0094.0.0.34] see paragraphs
[0092.0.0.27] to [0094.0.0.27] above.
[14447] [0095.0.34.34] It may be advantageous to increase the pool
of said phytosterols in the transgenic organisms by the process
according to the invention in order to isolate high amounts of the
pure respective fine chemical.
[14448] [0096.0.34.34] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example
beta-sitosterol or campesterol in the organism, is useful to
increase the production of the respective fine chemical.
[14449] [0097.0.0.34] for the disclosure of this paragraph see
paragraph [0097.0.0.27] above.
[14450] [0098.0.34.34] In a preferred embodiment, the respective
fine chemical (beta-sitosterol or campesterol) is produced in
accordance with the invention and, if desired, is isolated. The
production of further phytosterols or conjugates or mixtures
thereof or mixtures with other compounds by the process according
to the invention is advantageous.
[14451] [0099.0.34.34] In the case of the fermentation of
microorganisms, the abovementioned phytosterol (preferably
beta-sitosterol and/or campesterol) accumulate in the medium and/or
the cells. If microorganisms are used in the process according to
the invention, the fermentation broth can be processed after the
cultivation. Depending on the requirement, all or some of the
biomass can be removed from the fermentation broth by separation
methods such as, for example, centrifugation, filtration, decanting
or a combination of these methods, or else the biomass can be left
in the fermentation broth. The fermentation broth can subsequently
be reduced, or concentrated, with the aid of known methods such as,
for example, rotary evaporator, thin-layer evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. Afterwards
advantageously further compounds for formulation can be added such
as corn starch or silicates. This concentrated fermentation broth
advantageously together with compounds for the formulation can
subsequently be processed by lyophilization, spray drying, and
spray granulation or by other methods. Preferably the respective
fine chemical or the phytosterol (preferably beta-sitosterol and/or
campesterol) comprising compositions are isolated from the
organisms, such as the microorganisms or plants or the culture
medium in or on which the organisms have been grown, or from the
organism and the culture medium, in the known manner, for example
via extraction, distillation, crystallization, chromatography or a
combination of these methods. These purification methods can be
used alone or in combination with the aforementioned methods such
as the separation and/or concentration methods.
[14452] [0100.0.34.34] Transgenic plants which comprise the
phytosterol (preferably beta-sitosterol and/or campesterol)
synthesized in the process according to the invention can
advantageously be marketed directly without there being any need
for the phytosterols (preferably beta-sitosterol and/or
campesterol) (oils, lipids or fatty acids synthesized to be
isolated). Plants for the process according to the invention are
listed as meaning intact plants and all plant parts, plant organs
or plant parts such as leaf, stem, seeds, root, tubers, anthers,
fibers, root hairs, stalks, embryos, calli, cotelydons, petioles,
harvested material, plant tissue, reproductive tissue and cell
cultures which are derived from the actual transgenic plant and/or
can be used for bringing about the transgenic plant. In this
context, the seed comprises all parts of the seed such as the seed
coats, epidermal cells, seed cells, endosperm or embryonic tissue.
However, the respective fine chemical produced in the process
according to the invention can also be isolated from the organisms,
advantageously plants, in the form of their oils, fats, lipids,
esters and/or as extracts, e.g. ether, alcohol, or other organic
solvents or water containing extract and/or free phytosterol(s).
The respective fine chemical produced by this process can be
obtained by harvesting the organisms, either from the crop in which
they grow, or from the field. This can be done via pressing or
extraction of the plant parts, preferably the plant seeds. To
increase the efficiency of extraction it is beneficial to clean, to
temper and if necessary to hull and to flake the plant material
especially the seeds. In this context, the oils, fats, lipids,
esters and/or free phytosterols can be obtained by what is known as
cold beating or cold pressing without applying heat. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they are previously comminuted, steamed or roasted. The
seeds, which have been pretreated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. In this manner, more than 96% of the compounds produced
in the process can be isolated. Thereafter, the resulting products
are processed further, i.e. degummed and/or refined. In this
process, substances such as the plant mucilages and suspended
matter are first removed. What is known as desliming can be
affected enzymatically or, for example, chemico-physically by
addition of acid such as phosphoric acid.
[14453] Plant sterols (phytosterols) are by-products of traditional
vegetable oil refining. The source may be commonly a blend of crude
edible oils, consisting of soy bean oil or of other edible oils,
e.g. corn, rapeseed, olive and palm oil in varying proportions.
Hemp may also be a source of new oilseed, oil and food ingredients
as well as Sea buckthorn (hippophae rhamnoides). The crude oil,
which is obtained by pressing or solvent extraction, may undergoes
a series of refining processes to remove solvents, lecithins, free
fatty acids, color bodies, off-odors and off-flavors. In one of
these steps, the oil may be subjected to steam distillation at
reduced pressure (deodorisation) and the resulting distillate
contains the phytosterol fraction. From this fraction, fatty acids,
lecithins and other compounds are removed by fractional
distillation, ethanolysis/transesterification, distillation and
crystallisation from a heptane solution, and the phytosterols are
further purified by recrystallisation using food grade materials
and good manufacturing practices. The extraction and purification
steps are standard methods and similar to the procedures used
traditionally by the food industry for the production of plant
sterols. Phytosterol esters may be produced from the sterols using
food grade vegetable oil-derived fatty acids or triglycerides and
applying standard methods for esterification or transesterification
commonly used in the fats and oils industry.
[14454] Phytosterol in microorganisms may be localized
intracellularly, therefor their recovery essentials comes down to
the isolation of the biomass. Well-establisthed approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Determination of
tocopherols in cells has been described by Tan and Tsumura 1989,
see also Biotechnology of Vitamins, Pigments and Growth Factors,
Edited by Erik J. Vandamme, London, 1989, p. 96 to 103. Many
further methods to determine the tocopherol content are known to
the person skilled in the art.
[14455] [0101.0.34.34] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[14456] [0102.0.34.34] Phytosterols can for example be analyzed
advantageously via HPLC or GC separation methods and detected by MS
oder MSMS methods. The unambiguous detection for the presence of
beta-sitosterol and/or campesterol containing products can be
obtained by analyzing recombinant organisms using analytical
standard methods: GC, GC-MS or TLC, as described on several
occasions by Christie and the references therein (1997, in:
Advances on Lipid Methodology, Fourth Edition: Christie, Oily
Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfah ren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). The
material to be analyzed can be disrupted by sonication, grinding in
a glass mill, liquid nitrogen and grinding, cooking, or via other
applicable methods; see also Biotechnology of Vitamins, Pigments
and Growth Factors, Edited by Erik J. Vandamme, London, 1989, p. 96
to 103.
[14457] For the sterol isolation and analysis the German standard
method F III (1) may be used. The method comprises: saponification
of fat, isolation of unsaponifiable matter using an aliminium oxide
column, separation of sterol fraction by preparative TLC and
determination of the composition of sterols as trimetysilyl ethers
by GLC.
[14458] [0103.0.34.34] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [14459] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide having a
sequence as indicated in Table XII, application no. 34, columns 5
or 7, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [14460] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table XI, application no. 34, columns 5 or 7.
[14461] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [14462] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[14463] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [14464]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [14465] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [14466] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers pairs
having a sequence as indicated in Table XIII, application no. 34,
column 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [14467]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [14468] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequences as indicated in Table XIV, application
no. 34, column 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [14469]
k) nucleic acid molecule comprising one or more of the nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domain of the polypeptide indicated in Table XII, application no.
34, columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[14470] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [14471]
or which comprises a sequence which is complementary thereto.
[14472] [00103.1.34.34.] In one embodiment, the nucleic acid
molecule used in the process of the invention distinguishes over
the sequence indicated in Table XI, application no. 34, columns 5
or 7 by one or more nucleotides. In one embodiment, the nucleic
acid molecule used in the process of the invention does not consist
of the sequence shown in indicated in Table XI, application no. 34,
columns 5 or 7. In one embodiment, the nucleic acid molecule used
in the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI,
application no. 34, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table XII, application no. 34, columns 5 or 7.
[14473] [0104.0.34.34] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence indicated in
Table XI, application no. 34, columns 5 or 7, y one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of the sequence
indicated in Table XI, application no. 34, columns 5 or 7, In one
embodiment, the nucleic acid molecule of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to the
sequence indicated in Table XI, application no. 34, columns 5 or 7.
In another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table XII, application no.
34, columns 5 or 7.
[14474] [0105.0.0.34] to [0107.0.0.34] for the disclosure of the
paragraphs [0105.0.0.34] to [0107.0.0.34] see paragraphs
[0105.0.0.27] and [0107.0.0.27] above.
[14475] [0108.0.34.34] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 34, columns 5 or 7, nucleic
acid molecules which are derived from an amino acid sequences as
indicated in Table XII, application no. 34, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 34, column 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of an activity of a polypeptide as indicated in Table XII,
application no. 34, column 3, 5 or 7, or conferring an increase of
the respective fine chemical, meaning phytosterol, in particular,
beta-sitosterol and/or campesterol after increasing its expression
or activity are advantageously increased in the process according
to the invention.
[14476] [0109.0.0.34] for the disclosure of this paragraph see
[0109.0.0.27] above.
[14477] [0110.0.34.34] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide used in the
method of the invention or used in the process of the invention,
e.g. of a protein as shown in Table XII, application no. 34,
columns 5 or 7, or being encoded by a nucleic acid molecule
indicated in Table XI, application no. 34, columns 5 or 7, or of
its homologs, e.g. as indicated in Table XII, application no. 34,
columns 5 or 7, can be determined from generally accessible
databases.
[14478] [0111.0.0.34] for the disclosure of this paragraph see
[0111.0.0.27] above.
[14479] [0112.0.34.34] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XII, application no. 34,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 34, columns 5 and 7, and conferring an
increase in the level of phytosterols, preferably beta-sitosterol
and/or campesterol.
[14480] [0113.0.0.34] to [0120.0.0.34] for the disclosure of the
paragraphs [0113.0.0.34] to [0120.0.0.34] see paragraphs
[0113.0.0.27] and [0120.0.0.27] above.
[14481] [0121.0.34.34] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 34, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring an increase in the level of
beta-sitosterol after increasing the activity of the polypeptide
sequences indicated in Table XII, application no. 34, columns 5 or
7, or conferring increase in the level of campesterol after
increasing the activity of the polypeptide sequences indicated in
Table XII, application no. 34, columns 5 or 7.
[14482] [0122.0.0.34] to [0127.0.0.34] for the disclosure of the
paragraphs [0122.0.0.34] to [0127.0.0.34] see paragraphs
[0122.0.0.27] and [0127.0.0.27] above.
[14483] [0128.0.34.34] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 34, column 7, by means of polymerase chain reaction
can be generated on the basis of a sequence shown herein, for
example the sequence as indicated in Table XI, application no. 34,
columns 5 or 7, or the sequences derived from a sequence as
indicated in Table XII, application no. 34, columns 5 or 7.
[14484] [0129.0.34.34] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved regions are those, which show a
very little variation in the amino acid in one particular position
of several homologs from different origin. The consensus sequences
indicated in Table XIV, application no. 34, column 7, are derived
from said alignments.
[14485] [0130.0.34.34] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of
phytosterol, preferably beta-sitosterol and/or campesterol resp
after increasing the expression or activity of the protein
comprising said fragment.
[14486] [0131.0.0.34] to [0138.0.0.34] for the disclosure of the
paragraphs [0131.0.0.34] to [0138.0.0.34] see paragraphs
[0131.0.0.27] to [0138.0.0.27] above.
[14487] [0139.0.34.34] Polypeptides having above-mentioned
activity, i.e. conferring the respective fine chemical increase,
derived from other organisms, can be encoded by other DNA sequences
which hybridize to a sequences indicated in Table XI, application
no. 34, columns 5 or 7, under relaxed hybridization conditions and
which code on expression for peptides having the phytosterol,
preferably beta-sitosterol and/or campesterol increasing
activity.
[14488] [0140.0.0.34] to [0146.0.0.34] for the disclosure of the
paragraphs [0140.0.0.34] to [0146.0.0.34] see paragraphs
[0140.0.0.27] to [0146.0.0.27] above.
[14489] [0147.0.34.34] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 34, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences s
such that it can hybridize to one of said nucleotide sequences
thereby forming a stable duplex. Preferably, the hybridisation is
performed under stringent hybridization conditions. However, a
complement of one of the herein disclosed sequences is preferably a
sequence complement thereto according to the base pairing of
nucleic acid molecules well known to the skilled person. For
example, the bases A and G undergo base pairing with the bases T
and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[14490] [0148.0.34.34] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 34,
columns 5 or 7 or a portion thereof and preferably has above
mentioned activity, in particular, of beta-Sitosterol and/or
Campesterol increasing activity after increasing the activity or an
activity of a product of a gene encoding said sequences or their
homologs.
[14491] [0149.0.34.34] The nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 34,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity and as indicated in indicated in Table
XII.
[14492] [00149.1.34.34] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table XI, application no. 34, columns 5 or
7, has further one or more of the activities annotated or known for
the a protein as indicated in Table XII, application no. 34, column
3.
[14493] [0150.0.34.34] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table XI, application no. 34, columns
5 or 7, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of beta-Sitosterol and/or
Campesterol, resp., if its activity is increased. The nucleotide
sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table XI,
application no. 34, columns 5 or 7, anti-sense sequence of one of
the sequences, e.g., as indicated in Table XI, application no. 34,
columns 5 or 7, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table XIII, application no. 34, column 7, will result in a fragment
of a polynucleotide sequence as indicated in Table XI, application
no. 34, columns 5 or 7 or its gene product.
[14494] [0151.0.0.34] for the disclosure of this paragraph see
paragraph [0151.0.0.27] above.
[14495] [0152.0.34.34] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 34, columns 5
or 7, such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular an activity increasing the level of phytosterol, in
particular, of beta-sitosterol and/or campesterol, resp., as
mentioned above or as described in the examples in plants or
microorganisms is comprised.
[14496] [0153.0.34.34] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 34,
columns 5 or 7, such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 34, columns 5 or 7, has for
example an activity of a polypeptide as indicated in Table XII,
application no. 34, column 3.
[14497] [0154.0.34.34] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 34, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[14498] [0155.0.0.34] and [0156.0.0.34] for the disclosure of the
paragraphs [0155.0.0.34] and [0156.0.0.34] see paragraphs
[0155.0.0.27] and [0156.0.0.27] above.
[14499] [0157.0.34.34] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences as
indicated in Table XI, application no. 34, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides encoded by the sequence as indicated in Table XI,
application no. 34, columns 5 or 7, or of the polypeptide as
indicated in Table XII, application no. 34, columns 5 or 7, or the
functional homologues. Advantageously, the nucleic acid molecule of
the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, an amino acid sequence of a consensus sequences
as indicated in Table XIV, application no. 34, column 7, or of the
polypeptide as indicated in Table XII, application no. 34, columns
5 or 7, or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
XIV, application no. 34, column 7, or of a polypeptide as indicated
in Table XII, application no. 34, columns 5 or 7, or the functional
homologues. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 34, columns 5 or 7,
Preferably the nucleic acid molecule of the invention is a
functional homologue or identical to a nucleic acid molecule
indicated in Table XI, application no. 34, column 7.
[14500] [0158.0.0.34] to [0160.0.0.34] for the disclosure of the
paragraphs [0158.0.0.34] to [0160.0.0.34] see paragraphs
[0158.0.0.27] to [0160.0.0.27] above.
[14501] [0161.0.34.34] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 34, columns 5 or
7. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[14502] [0162.0.0.34] for the disclosure of this paragraph see
paragraph [0162.0.0.27] above.
[14503] [0163.0.34.34] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 34, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[14504] [0164.0.0.34] for the disclosure of this paragraph see
paragraph [0164.0.0.27] above.
[14505] [0165.0.34.34] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 34, columns 5 or 7.
[14506] [0166.0.0.34] and [0167.0.0.34] for the disclosure of the
paragraphs [0166.0.0.34] and [0167.0.0.34] see paragraphs
[0166.0.0.27] and [0167.0.0.27] above.
[14507] [0168.0.34.34] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 34, columns 5 or 7, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 34,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table XII, application no. 34, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 34, columns 5
or 7, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table XII, application no.
34, columns 5 or 7, and most preferably at least about 96%, 97%,
98%, or 99% identical to the sequence as indicated in Table XII,
application no. 34, columns 5 or 7.
[14508] [0169.0.0.34] to [0172.0.0.34] for the disclosure of the
paragraphs [0169.0.0.34] to [0172.0.0.34] see paragraphs
[0169.0.0.27] to [0172.0.0.27] above.
[14509] [0173.0.34.34] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108442 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108442 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[14510] [0174.0.0.34] for the disclosure of the paragraph
[0174.0.0.34] see paragraph [0174.0.0.27] above.
[14511] [0175.0.34.34] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108443 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108443 by the above program algorithm with the
above parameter set, has a 80% homology.
[14512] [0176.0.34.34] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 34,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 34, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 34, columns
5 or 7.
[14513] [0177.0.34.34] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 34,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptide as indicated in Table XII,
application no. 34, columns 5 or according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 34, columns
5 or 7.
[14514] [0178.0.0.34] for the disclosure of this paragraph see
[0178.0.0.27] above.
[14515] [0179.0.34.34] A nucleic acid molecule encoding an
homologous to a protein sequence as indicated in Table XII,
application no. 34, columns 5 or 7, can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table XI, application no.
34, columns 5 or 7, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences of a
sequence as indicated in Table XI, application no. 34, columns 5 or
7, by standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[14516] [0180.0.0.34] to [0183.0.0.34] for the disclosure of the
paragraphs [0180.0.0.34] to [0183.0.0.34] see paragraphs
[0180.0.0.27] to [0183.0.0.27] above.
[14517] [0184.0.34.34] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 34,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 34, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 34,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[14518] [0185.0.34.34] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 34, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table XI, application no. 34, columns 5 or 7. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequences as indicated in Table XI, application no. 34, columns 5
or 7,
[14519] [0186.0.34.34] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table XII,
application no. 34, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 34, columns 5 or 7.
[14520] [0187.0.34.34] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table XII, application no.
34, columns 5 or 7, comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence of
the amino sequences indicated in Table XII, application no. 34,
columns 5 or 7.
[14521] [0188.0.34.34] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 34, columns 5 or 7 and is expressed under
identical conditions.
[14522] [0189.0.34.34] Homologues of a sequences as indicated in
Table XI, application no. 34, columns 5 or 7, or of a derived
sequences as indicated in Table XII, application no. 34, columns 5
or 7 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[14523] [0190.0.0.34] to [0203.0.0.34] for the disclosure of the
paragraphs [0190.0.0.34] to [0203.0.0.34] see paragraphs
[0190.0.0.27] to [0203.0.0.27] above.
[14524] [0204.0.34.34] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule which comprises a
nucleic acid molecule selected from the group consisting of:
[14525] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 34, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII, application no. 34,
column 6 in an organism or a part thereof [14526] b) nucleic acid
molecule comprising, preferably at least the mature form, of a
nucleic acid molecule as indicated in Table XI, application no. 34,
columns 5 or 7, or a fragment thereof conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [14527] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [14528] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[14529] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [14530] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [14531] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to [14532]
(c) and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [14533] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table XIII, application no. 34, column 7, and
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII, application no. 34,
column 6 in an organism or a part thereof; [14534] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[14535] j) nucleic acid molecule which encodes a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 34, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical, in particular according
to table XII, application no. 34, column 6 in an organism or a part
thereof; [14536] k) nucleic acid molecule encoding the amino acid
sequence of a polypeptide encoding a domaine of a polypeptide as
indicated in Table XII, application no. 34, columns 5 or 7, and
conferring an increase in the amount of the respective fine
chemical, in particular accccording to table XII, application no.
34, column 6 in an organism or a part thereof; and [14537] l)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (h) or of a nucleic
acid molecule as indicated in Table XI, application no. 34, columns
5 or 7, or a nucleic acid molecule encoding, preferably at least
the mature form of, a polypeptide as indicated in Table XII,
application no. 34, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical according to table XII,
application no. 34, column 6 in an organism or a part thereof;
[14538] or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over the sequence indicated in Table
XI, application no. 34, columns 5 or 7, by one or more nucleotides.
In one embodiment, the nucleic acid molecule does not consist of
the sequence shown and indicated in Table XI, application no. 34,
columns 5 or 7,
[14539] In one embodiment, the nucleic acid molecule is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table XI, application no. 34, columns 5 or 7.
[14540] In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table XII,
application no. 34, columns 5 or 7.
[14541] In an other embodiment, the nucleic acid molecule of the
present invention is at least 30%, 40%, 50%, or 60% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 34, columns 5 or
7.
[14542] In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table XII,
application no. 34, columns 5 or 7.
[14543] Accordingly, in one embodiment, the nucleic acid molecule
of the differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 34, columns 5 or
7.
[14544] Accordingly, in one embodiment, the nucleic acid molecule
of the present invention encodes a polypeptide, which differs at
least in one or more amino acids from a polypeptide indicated in
Table XII, application no. 34, columns 5 or 7.
[14545] In another embodiment, a nucleic acid molecule indicated in
Table XI, application no. 34, columns 5 or 7.
[14546] Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table XII, application no.
34, columns 5 or 7.
[14547] In a further embodiment, the protein of the present
invention is at least 30%, 40%, 50%, or 60% identical to a protein
sequence indicated in Table XII, application no. 34, columns 5 or
7, and less than 100%, preferably less than 99.999%, 99.99% or
99.9%, more preferably less than 99%, 985, 97%, 96% or 95%
identical to a sequence as indicated in Table XI, columns 5 or
7.
[14548] [0205.0.0.34] and [0206.0.0.34] for the disclosure of the
paragraphs [0205.0.0.34] and [0206.0.0.34] see paragraphs
[0205.0.0.27] and [0206.0.0.27] above.
[14549] [0207.0.34.34] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the sterol metabolism, the squalen
metabolism, the amino acid metabolism, of glycolysis, of the
tricarboxylic acid metabolism or their combinations. As described
herein, regulator sequences or factors can have a positive effect
on preferably the gene expression of the genes introduced, thus
increasing it. Thus, an enhancement of the regulator elements may
advantageously take place at the transcriptional level by using
strong transcription signals such as promoters and/or enhancers. In
addition, however, an enhancement of translation is also possible,
for example by increasing mRNA stability or by inserting a
translation enhancer sequence.
[14550] [0208.0.0.34] to [0226.0.0.34] for the disclosure of the
paragraphs [0208.0.0.34] to [0226.0.0.34] see paragraphs
[0208.0.0.27] to [0226.0.0.27] above.
[14551] [0227.0.34.34] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[14552] In addition to a sequence indicated in Table XI,
application no. 34, columns 5 or 7, or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the sterol biosynthetic pathway such as for a
phytosterol precursor, for example squalene epoxide is expressed in
the organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine one or more of the sequences indicated in
Table XI, application no. 34, columns 5 or 7, with genes which
generally support or enhances to growth or yield of the target
organisms, for example genes which lead to faster growth rate of
microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[14553] [0228.0.34.34] In a further embodiment of the process of
the invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the
sterol/phytosterol metabolism, in particular in synthesis of of
beta-sitosterol and/or campesterol.
[14554] [0229.0.34.34] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the phytosterol biosynthetic
pathway, such as the enzymes catalyzing the production of acetyl
CoA HMGCoA, mevalonate, mevalonate 5 phosphate, mevalonate
5-pyrophosphate, isopentyl diphosphate, 5-pyrophosphatemevalonate,
isopentyl pyrophosphate (PIP), dimethylallyl pyrophosphate (DMAPP),
PIP+DMAPP, geranyl pyrophosphate+IPP, farnesyl pyrophosphate, 2
farnesyl pyrophosphate, squalene (squalene synthase) and squalene
epoxide, or cycloartenol synthase controling the cyclization of
squalene epoxide, S-adenosyl-L-methionine:sterol C-24 methyl
transferase (EC 2.1.1.41) (SMT1) catalyzing the transfer of a
methyl group from a cofactor, SMT2 catalyzing the second methyl
transfer reaction, sterol C-14 demethylase catalyzing the
demethylation at C-14, removing the methyl group and creating a
double bond. These genes can lead to an increased synthesis of the
essential phytosterol, in particular, of beta-sitosterol and/or
campesterol resp.
[14555] [0230.0.0.34] for the disclosure of this paragraph see
paragraph [0230.0.0.27] above.
[14556] [0231.0.34.34] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a protein degrading phytosterol, in
particular, of beta-sitosterol and/or campesterol, resp., is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[14557] [0232.0.0.5] to [0276.0.0.5] for the disclosure of the
paragraphs [0232.0.0.5] to [0276.0.0.5] see paragraphs
[0232.0.0.27] to [0276.0.0.27] above.
[14558] [0277.0.34.34] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
is familiar. For example, via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously. The respective fine chemical produced by this process
can be obtained by harvesting the organisms, either from the crop
in which they grow, or from the field. This can be done via
pressing or extraction of the plant parts.
[14559] [0278.0.0.34] to [0282.0.0.34] for the disclosure of the
paragraphs [0278.0.0.34] to [0282.0.0.34] see paragraphs
[0278.0.0.27] to [0282.0.0.27] above.
[14560] [0283.0.34.34] Moreover, a native polypeptide conferring
the increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table
XII, application no. 34, column 3, or an antibody against a
polypeptide as indicated in Table XII, application no. 34, columns
5 or 7, which can be produced by standard techniques utilizing the
polypeptid of the present invention or fragment thereof, i.e., the
polypeptide of this invention. Preferred are monoclonal
antibodies.
[14561] [0284.0.0.34] for the disclosure of this paragraph see
[0284.0.0.27] above.
[14562] [0285.0.34.34] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 34, columns 5 or 7, or as encoded by a nucleic
acid molecule as indicated in Table XI, application no. 34, columns
5 or 7, or functional homologues thereof.
[14563] [0286.0.34.34] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 34, column 7. In another
embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 34, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table XIV, application no. 34, column 7.
[14564] [0287.0.0.34] to [0290.0.0.34] for the disclosure of the
paragraphs [0287.0.0.34] to [0290.0.0.34] see paragraphs
[0287.0.0.27] to [0290.0.0.27] above.
[14565] [0291.0.34.34] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. In one embodiment, said polypeptide of the
invention distinguishes over a sequence as indicated in Table XII,
application no. 34, columns 5 or 7, by one or more amino acids.
[14566] In one embodiment, polypeptide distinguishes form a
sequence as indicated in Table XII, application no. 34, columns 5
or 7, by more than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, even more
preferred are more than 40, 50, or 60 amino acids and, preferably,
the sequence of the polypeptide of the invention distinguishes from
a sequence as indicated in Table XII, application no. 34, columns 5
or 7, by not more than 80% or 70% of the amino acids, preferably
not more than 60% or 50%, more preferred not more than 40% or 30%,
even more preferred not more than 20% or 10%. In an other
embodiment, said polypeptide of the invention does not consist of a
sequence as indicated in Table XII, application no. 34, columns 5
or 7.
[14567] [0292.0.0.34] for the disclosure of this paragraph see
[0292.0.0.27] above.
[14568] [0293.0.34.34] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, the polypeptide of
the invention has a sequence which distinguishes from a sequence as
indicated in Table XII, application no. 34, columns 5 or 7, by one
or more amino acids. In an other embodiment, said polypeptide of
the invention does not consist of the sequence as indicated in
Table XII, application no. 34, columns 5 or 7.
[14569] In a further embodiment, said polypeptide of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by a nucleic acid molecules as indicated in
Table XI, application no. 34, columns 5 or 7.
[14570] [0294.0.34.34] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 34, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 34, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[14571] [0295.0.0.34] to [0297.0.0.34] for the disclosure of the
paragraphs [0295.0.0.34] to [0297.0.0.34] see paragraphs
[0295.0.0.27] to [0297.0.0.27] above.
[14572] [0297.1.34.34] Non polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table XII, application no. 34, columns 3, 5 or 7.
[14573] [0298.0.34.34] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence, which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 34, columns 5
or 7. The portion of the protein is preferably a biologically
active portion as described herein. Preferably, the polypeptide
used in the process of the invention has an amino acid sequence
identical to a sequence as indicated in Table XII, application no.
34, columns 5 or 7.
[14574] [0299.0.34.34] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 34,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 34, or which is homologous thereto, as
defined above.
[14575] [0300.0.34.34] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 34, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 34, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[14576] [0301.0.0.34] for the disclosure of this paragraph see
[0301.0.0.27] above.
[14577] [0302.0.34.34] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., the amino acid sequence as indicated in
Table XII, application no. 34, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[14578] [0303.0.0.34] for the disclosure of this paragraph see
[0303.0.0.27] above.
[14579] [0304.0.34.34] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 34, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[14580] [0305.0.0.34], [0306.0.0.34] and [0306.1.0.34] for the
disclosure of the paragraphs [0305.0.0.34], [0306.0.0.34] and
[0306.1.0.34] see paragraphs [0305.0.0.27], [0306.0.0.27] and
[0306.1.0.27] above.
[14581] [0307.0.0.34] and [0308.0.0.34] for the disclosure of the
paragraphs [0307.0.0.34] and [0308.0.0.34] see paragraphs
[0307.0.0.27 and [0308.0.0.27] above.
[14582] [0309.0.34.34] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table XII,
application no. 34, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table XII, application no. 34, columns 5 or 7, refers
to a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous a polypeptide of the
invention, preferably which is not substantially homologous to a
polypeptide as indicated in Table XII, application no. 34, columns
5 or 7, e.g., a protein which does not confer the activity
described herein or annotated or known for as indicated in Table
XII, application no. 34, column 3, and which is derived from the
same or a different organism. In one embodiment, a "non-polypeptide
of the invention" or "other polypeptide" not being indicated in
Table XII, application no. 34, columns 5 or 7, does not confer an
increase of the respective fine chemical in an organism or part
therof.
[14583] [0310.0.0.34] to [0334.0.0.34] for the disclosure of the
paragraphs [0310.0.0.34] to [0334.0.0.34] see paragraphs
[0310.0.0.27] to [0334.0.0.27] above.
[14584] [0335.0.34.34] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XI, application
no. 34, columns 5 or 7, and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
application no. 34, columns 5 or 7, and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of an
protein encoded by a nucleic acid sequence sequences as indicated
in Table XI, application no. 34, columns 5 or 7, and/or homologs
thereof, one of the two RNA strands is essentially identical to at
least part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[14585] [0336.0.0.34] to [0342.0.0.34] for the disclosure of the
paragraphs [0336.0.0.34] to [0342.0.0.34] see paragraphs
[0336.0.0.27] to [0342.0.0.27] above.
[14586] [0343.0.34.34] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 34, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table XI, application no. 34, columns 5 or 7, or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[14587] [0344.0.0.34] to [0361.0.0.34] for the disclosure of the
paragraphs [0344.0.0.34] to [0361.0.0.34] see paragraphs
[0344.0.0.27] to [0361.0.0.27] above.
[14588] [0362.0.34.34] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 34, columns 5 or 7, e.g. encoding a
polypeptide having protein activity, as indicated in Table XII,
application no. 34, column 3. Due to the above mentioned activity
the respective fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity of the polypeptide of the invention or nucleic
acid molecule of the invention is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a polypeptide as
indicated in Table XII, application no. 34, columns 5 or 7, means
herein that due to modulation or manipulation of the genome, an
activity as annotated for a polypeptide as indicated in Table XII,
application no. 34, column 3, e.g. having a sequence as indicated
in Table XII, application no. 34, columns 5 or 7, is increased in a
cell or an organism or a part thereof. Examples are described above
in context with the process of the invention
[14589] [0363.0.0.34] for the disclosure of this paragraph see
paragraph [0363.0.0.27] above.
[14590] [0364.0.34.34] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a polypeptide of the invention as indicated in Table
XII, application no. 34, column 3, with the corresponding
protein-encoding sequence as indicated in Table XI, application no.
34, column 3, --becomes a transgenic expression cassette when it is
modified by non-natural, synthetic "artificial" methods such as,
for example, mutagenization. Such methods have been described (U.S.
Pat. No. 5,565,350; WO 00/15815; also see above).
[14591] [0365.0.0.34] to [0373.0.0.34] for the disclosure of the
paragraphs [0365.0.0.34], to [0373.0.0.34] see paragraphs
[0365.0.0.27] to [0373.0.0.27] above.
[14592] [0374.0.34.34] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. Phytosterol, in particular,
of beta-sitosterol and/or campesterol resp., produced in the
process according to the invention may, however, also be isolated
from the plant in the form of their free form or bound in or to
compounds or moieties. Phytosterol, in particular, of
beta-sitosterol and/or campesterol resp., produced by this process
can be harvested by harvesting the organisms either from the
culture in which they grow or from the field. This can be done via
expressing, grinding and/or extraction, salt precipitation and/or
ion-exchange chromatography or other chromatographic methods of the
plant parts, preferably the plant seeds, plant fruits, plant tubers
and the like.
[14593] [0375.0.0.34] and [0376.0.0.34] for the disclosure of the
paragraphs [0375.0.0.34] and [0376.0.0.34] see paragraphs
[0375.0.0.27] and [0376.0.0.27] above.
[14594] [0377.0.34.34] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced phytosterol, in particular, of beta-sitosterol and/or
campesterol comprising composition or the produced the respective
fine chemical is isolated.
[14595] [0378.0.34.34] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the phytosterol,
in particular, of beta-sitosterol and/or campesterol, resp.,
produced in the process can be isolated. The resulting phytosterol,
in particular, of beta-sitosterol and/or campesterol resp., can, if
appropriate, subsequently be further purified, if desired mixed
with other active ingredients such as vitamins, amino acids,
carbohydrates, antibiotics and the like, and, if appropriate,
formulated.
[14596] [0379.0.34.34] In one embodiment, the phytosterol, in
particular, of beta-sitosterol and/or campesterol resp., is a
mixture comprising of one or more the respective fine chemicals. In
one embodiment, the respective fine chemical means here
phytosterol, in particular, of beta-sitosterol and/or campesterol.
In one embodiment, phytosterol means here a mixture of the
respective fine chemicals.
[14597] [0380.0.34.34] The phytosterol, in particular, the
beta-sitosterol and/or campesterol resp., obtained in the process
are suitable as starting material for the synthesis of further
products of value. For example, they can be used in combination
with each other or alone for the production of pharmaceuticals,
foodstuffs, animal feeds or cosmetics. Accordingly, the present
invention relates a method for the production of pharmaceuticals,
food stuff, animal feeds, nutrients or cosmetics comprising the
steps of the process according to the invention, including the
isolation of the phytosterol comprising composition produced or the
respective fine chemical produced if desired and formulating the
product with a pharmaceutical acceptable carrier or formulating the
product in a form acceptable for an application in agriculture. A
further embodiment according to the invention is the use of the
phytosterol resp., produced in the process or of the transgenic
organisms in animal feeds, foodstuffs, medicines, food supplements,
cosmetics or pharmaceuticals.
[14598] [0381.0.0.34] and [0382.0.0.34] for the disclosure of the
paragraphs [0381.0.0.34] and [0382.0.0.34] see paragraphs
[0381.0.0.27] and [0382.0.0.27] above.
[14599] [0383.0.34.34] For preparing sterol compound-containing
fine chemicals, in particular the respective fine chemical of the
invention, it is possible to use as source organic compounds such
as, for example, acetyl CoA HMGCoA, mevalonate, mevalonate 5
phosphate, mevalonate 5-pyrophosphate, isopentyl diphosphate,
5-pyrophosphatemevalonate, isopentyl pyrophosphate (PIP),
dimethylallyl pyrophosphate (DMAPP), PIP+DMAPP, geranyl
pyrophosphate+IPP, farnesyl pyrophosphate, 2 farnesyl
pyrophosphate, squalene and squalene epoxide, cycloartenol and for
preparing esters comprising the phytosterols of the invention oils,
fats and/or lipids comprising fatty acids such as fatty acids
having a carbon back bone between C.sub.10- to C.sub.16-carbon
atoms and/or small organic acids such acetic acid, propionic acid
or butanoic acid as precursor compounds.
[14600] [0384.0.0.34] for the disclosure of this paragraph see
paragraph [0384.0.0.27] above.
[14601] [0385.0.34.34] The fermentation broths obtained in this
way, containing in particular phytosterol, in particular, of
beta-sitosterol and/or campesterol resp., in mixtures with other
compounds, in particular with other sterols or vitamins, e.g. with
carotenoids, e.g. with astaxanthin, or fatty acids or containing
microorganisms or parts of microorganisms, like plastids,
containing phytosterol, in particular, of beta-sitosterol and/or
campesterol resp., in mixtures with other compounds, e.g. with
vitamins, normally have a dry matter content of from 7.5 to 25% by
weight. Sugar-limited fermentation is additionally advantageous,
e.g. at the end, for example over at least 30% of the fermentation
time. This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 3 g/l during
this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed or isolated
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation, coagulation/flocculation
or a combination of these methods, from the fermentation broth or
left completely in it. The fermentation broth can then be thickened
or concentrated by known methods, such as, for example, with the
aid of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[14602] As phytosterol is often localized in membranes or plastids,
in one embodiment it is advantageous to avoid a leaching of the
cells when the biomass is isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth. The dry biomass can directly
be added to animal feed; provided the vitamin E concentration is
sufficiently high and no toxic compounds are present.
[14603] [0386.0.34.34] Accordingly, it is possible to further
purify the produced phytosterol, in particular, of beta-sitosterol
and/or campesterol resp. For this purpose, the product-containing
composition, e.g. a total or partial lipid extraction fraction
using organic solvents, e.g. as described above, is subjected for
example to a saponification to remove triglycerides, partition
between e.g. hexane/methanol (separation of non-polar epiphase from
more polar hypophasic derivates) and separation via e.g. an open
column chromatography, preparative thin layer chromatography or
HPLC in which case the desired product or the impurities are
retained wholly or partly on the chromatography resin (see e.g.
Kaluzny et al., J Lipid Res 1985; 26: 135-140). These
chromatography steps can be repeated if necessary, using the same
or different chromatography resins. The skilled worker is familiar
with the choice of suitable chromatography resins and their most
effective use.
[14604] [0387.0.0.34] to [0392.0.0.34] for the disclosure of the
paragraphs [0387.0.0.34] to [0392.0.0.34] see paragraphs
[0387.0.0.27] to [0392.0.0.27] above.
[14605] [0393.0.34.34] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [14606] a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [14607] b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table XI, application no. 34, columns 5 or 7, and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[14608] c) introducing the candidate nucleic acid molecules in host
cells, preferably in a plant cell or a microorganism, appropriate
for producing the fine chemical; [14609] d) expressing the
identified nucleic acid molecules in the host cells; [14610] e)
assaying the the fine chemical level in the host cells; and [14611]
f) identifying the nucleic acid molecule and its gene product which
expression confers an increase in the the fine chemical level in
the host cell after expression compared to the wild type.
[14612] [0394.0.0.34] to [0399.0.0.34] for the disclosure of the
paragraphs [0394.0.0.34] to [0399.0.0.34] see paragraphs
[0394.0.0.27] to [0399.0.0.27] above.
[14613] [0399.1.34.34] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table XII,
application no. 34, columns 5 or 7, or a homolog thereof, e.g.
comparing the phenotyp of nearly identical organisms with low and
high activity of a protein as indicated in Table XII, application
no. 34, columns 5 or 7, after incubation with the drug.
[14614] [0400.0.0.34] to [0416.0.0.34] for the disclosure of the
paragraphs [0400.0.0.34] to [0416.0.0.34] see paragraphs
[0400.0.0.27] to [0416.0.0.27] above.
[14615] [0417.0.34.34] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the phytosterol production biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect an organism such as a
microorganism or a plant against inhibitors, which block the
phytosterol, in particular the respective fine chemical, synthesis
in said organism.
[14616] Examples of inhibitors or herbicides blocking the
phytosterol synthesis in organism such as microorganism or plants
are for example compounds which inhibit the cytochrom P450 such as
Tetcyclasis, triazoles like Paclobutrazol or Epoxiconazol,
pyridines like Obtusifoliol, demethylases inhibitors, or compounds
like Mevilonin, which inhibits the HMG-CoA reductase.
[14617] [0418.0.0.34] to [0423.0.0.34] for the disclosure of the
paragraphs [0418.0.0.34] to [0423.0.0.34] see paragraphs
[0418.0.0.27] to [0423.0.0.27] above.
[14618] [0424.0.34.34] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other sterols,
phytosterols, carotenoids, vitamins or fatty acids. Accordingly,
the nucleic acid of the invention, or the nucleic acid molecule
identified with the method of the present invention or the
complement sequences thereof, the polypeptide of the invention, the
nucleic acid construct of the invention, the organisms, the host
cell, the microorganisms, the plant, plant tissue, plant cell, or
the part thereof of the invention, the vector of the invention, the
antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the respective
fine chemical in a organism or part thereof, e.g. in a cell.
[14619] [0425.0.0.34] to [0430.0.0.34] for the disclosure of the
paragraphs [0425.0.0.34] to [0430.0.0.34] see paragraphs
[0425.0.0.27] to [0430.0.0.27] above.
[14620] [0431.0.0.34] to [0434.0.0.34] for the disclosure of the
paragraphs [0431.0.0.34] to [0434.0.0.34] see paragraphs
[0431.0.0.27] to [0434.0.0.27] above.
[0435.0.34.34] Example 3
In-Vivo and In-Vitro Mutagenesis
[14621] [0436.0.34.34] An in vivo mutagenesis of organisms such as
green algae (e.g. Spongiococcum sp, e.g. Spongiococcum exentricum,
Chlorella sp., Haematococcus,
[14622] Phaedactylum tricornatum, Volvox or Dunaliella),
Synchocytic spec. PLL 6803, Physocmetrella patens, Saccharomyces,
Mortierella, Escherichia and others mentioned above, which are
beneficial for the production of phytosterol can be carried out by
passing a plasmid DNA (or another vector DNA) containing the
desired nucleic acid sequences, e.g. the nucleic acid molecule of
the invention or the vector of the invention, or nucleic acid
sequences through E. coli and other microorganisms (for example
Bacillus spp. or yeasts such as Saccharomyces cerevisiae) which are
not capable of maintaining the integrity of its genetic
information. Usual mutator strains have mutations in the genes for
the DNA repair system [for example mutHLS, mutD, mutT and the like;
for comparison, see Rupp, W. D. (1996) DNA repair mechanisms in
Escherichia coli and Salmonella, pp. 2277-2294, ASM: Washington].
The skilled worker knows these strains. The use of these strains is
illustrated for example in Greener, A. and Callahan, M. (1994)
Strategies 7; 32-34.
[14623] In-vitro mutation methods such as increasing the
spontaneous mutation rates by chemical or physical treatment are
well known to the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below. Site-directed mutagensis method such as the
introduction of desired mutations with an M13 or phagemid vector
and short oligonucleotides primers is a well-known approach for
site-directed mutagensis. The clou of this method involves cloning
of the nucleic acid sequence of the invention into an M13 or
phagemid vector, which permits recovery of single-stranded
recombinant nucleic acid sequence. A mutagenic oligonucleotide
primer is then designed whose sequence is perfectly complementary
to nucleic acid sequence in the region to be mutated, but with a
single difference: at the intended mutation site it bears a base
that is complementary to the desired mutant nucleotide rather than
the original. The mutagenic oligonucleotide is then allowed to
prime new DNA synthesis to create a complementary full-length
sequence containing the desired mutation. Another site-directed
mutagensis method is the PCR mismatch primer mutagensis method also
known to the skilled person. Dpnl site-directed mutagensis is a
further known method as described for example in the Stratagene
Quickchange.TM. site-directed mutagenesis kit protocol. A huge
number of other methods are also known and used in common
practice.
[14624] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[14625] [0437.0.5.12] to [0440.0.5.12] and [0441.0.0.34] for the
disclosure of the paragraphs [0437.0.5.12] to [0440.0.5.12] and
[0441.0.0.34] see paragraphs [0437.0.5.5] to [0444.0.5.5] and
[0441.0.0.27] above.
[14626] [0442.0.5.12], [0443.0.0.34], [0444.0.5.12] and
[0445.0.5.12] for the disclosure of the paragraphs [0442.0.5.12],
[0443.0.0.34], [0444.0.5.12] and [0445.0.5.12] see paragraphs
[0442.0.5.5], [0443.0.0.27], [0444.0.5.5] and [0445.0.5.5]
above.
[14627] [0446.0.0.34] to [0450.0.0.34] and [0451.0.5.12] for the
disclosure of the paragraphs [0446.0.0.34] to [0450.0.0.34] and
[0451.0.5.12] see paragraphs [0446.0.0.27] to [0450.0.0.27] and
[0451.0.5.5] above.
[14628] [0452.0.0.34] to [0454.0.0.34], [0455.0.5.12] and
[0456.0.0.34] for the disclosure of the paragraphs [0452.0.0.34] to
[0454.0.0.34], [0455.0.5.12] and [0456.0.0.34] see
[14629] [0452.0.0.27] to [0454.0.0.27], [0455.0.5.5] and
[0456.0.0.27] above.
[0457.0.34.34] Example 9
Purification of the Phytosterol
[14630] [0458.0.34.34] One example is the analysis of phytosterol:
the content of the phytosterols of the invention can be
determinated by gas chromatography with flame ionisation detection
(GC-FID; column SAC-5, 30 m.times.0.25 mm, 0.25 .mu.m, samples not
silylated) using standards for these phytosterols. Another method
is the detection by gas chromatography-mass spectrometry (GC-MS)
using the same type of column as indicated above.
[14631] For the analysis of the concentrations of sterols by gas
chromatography mass spectrometry a Hewlett-Packard (HP) 5890 gas
chromatograph equipped with an NB-54 fused-silica capillary column
(15 mx0.20 mm I.D.; Nordion, Helsinki, Finland) and interfaced with
an HP 5970A mass spectrometry detector operating in electron impact
mode (70 eV) can be used. The column oven is programmed from
230.degree. C. to 285.degree. C. at 10.degree. C./min and injector
and detector should be at 285.degree. C. The lipids from the
samples (200 .mu.l) are extracted with chloroform/methanol (2:1)
and transesterified with sodium methoxide. The released free
sterols are trimethylsilylated as described previously (Gylling et
al. J. Lipid Res 40: 593-600, 1999) and quantified by single ion
monitoring technique using m/z 129 (cholesterol, campesterol and
.beta.-sitosterol), m/z 215 (.beta.-sitostanol), m/z 343
(desmosterol), m/z 255 (lathosterol) and m/z 217
(5-.alpha.-cholestane, internal standard) as selected ions
(Vaskonen, Dissertation, Biomedicum Helsinki, Jun. 19, 2002).
[14632] [0459.0.34.34] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the phytosterol, in particular, of beta-sitosterol
and/or campesteroll, can be further purified with a so-called
RTHPLC. As eluent acetonitrile/water or chloroform/acetonitrile
mixtures can be used. If necessary, these chromatography steps may
be repeated, using identical or other chromatography resins. The
skilled worker is familiar with the selection of suitable
chromatography resin and the most effective use for a particular
molecule to be purified.
[14633] [0460.0.0.34] for the disclosure of this paragraph see
[0460.0.0.27] above.
[0461.0.34.34] Example 10
Cloning SEQ ID NO: 108442 for the Expression in Plants
[14634] [0462.0.0.34] for the disclosure of this paragraph see
[0462.0.0.27] above.
[14635] [0463.0.34.34] SEQ ID NO: 108442 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[14636] [0464.0.0.34] to [0466.0.0.34] for the disclosure of the
paragraphs [0464.0.0.34] to [0466.0.0.34] see paragraphs
[0464.0.0.27] to [0466.0.0.27] above.
[14637] [0467.0.34.34] The following primer sequences were selected
for the gene SEQ ID NO: 108442:
i) forward primer: SEQ ID NO: 108448 ii) reverse primer: SEQ ID NO:
108449
[14638] [0468.0.0.34] to [0479.0.0.34] for the disclosure of the
paragraphs [0468.0.0.34] to [0479.0.0.34] see paragraphs
[0468.0.0.27] to [0479.0.0.27] above.
[0480.0.34.34] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 108442
[14639] [0481.0.0.34] to [0513.0.0.34] for the disclosure of the
paragraphs [0481.0.0.34] to [0513.0.0.34] see paragraphs
[0482.0.0.27] to [0513.0.0.27] above.
[14640] [0514.0.34.34] As an alternative, phytosterols can be
detected via HPLC, e.g. reversed-phase HPLC, as described by
Heftmann, E. and Hunter, I. R. (J Chromatogr 1979; 165: 283-299).
As separating principles of HPLC and GC are complementary,
preparative reversed-phase HPLC followed by GC-MS analysis of the
obtained sterol fractions is a preferred method to analyze sterols
from natural products (Bianchini, J.-P. et al.; J Chromatogr 1985;
329: 231-246).
[14641] [0515.0.0.34] to [0552.0.0.34] for the disclosure of the
paragraphs [0515.0.0.34] to [0552.0.0.34] see paragraphs
[0515.0.0.27] to [0552.0.0.27] above.
[14642] [0553.0.34.34]
1. A process for the production of beta-Sitosterol and/or
Campesterol resp., which comprises (a) increasing or generating the
activity of a protein as indicated in Table XII, application no.
34, columns 5 or 7, or a functional equivalent thereof in a
non-human organism, or in one or more parts thereof; and (b)
growing the organism under conditions which permit the production
of beta-Sitosterol and/or Campesterol resp. in said organism. 2. A
process for the production of beta-Sitosterol and/or Campesterol
resp., comprising the increasing or generating in an organism or a
part thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: a) nucleic acid molecule encoding of a polypeptide
as indicated in Table XII, application no. 34, columns 5 or 7, or a
fragment thereof, which confers an increase in the amount of the
fine chemical as indicated in table XII, column 6, e.g of
beta-Sitosterol and/or Campesterol resp., in an organism or a part
thereof; b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table XI, application no. 34, columns 5 or
7, c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of beta-Sitosterol and/or
Campesterol resp., in an organism or a part thereof; d) nucleic
acid molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the fine chemical as indicated in table XII,
column 6, e.g of beta-Sitosterol and/or Campesterol resp., in an
organism or a part thereof; e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of beta-Sitosterol and/or Campesterol resp., in an organism
or a part thereof; f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table XIII, application no.
34, column 7, and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of
beta-Sitosterol and/or Campesterol resp., in an organism or a part
thereof; g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of beta-Sitosterol and/or
Campesterol resp., in an organism or a part thereof; h) nucleic
acid molecule encoding a polypeptide comprising a consensus
sequence as indicated in Table XIV, application no. 34, column 7,
and conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of beta-Sitosterol and/or
Campesterol resp., in an organism or a part thereof; and i) nucleic
acid molecule which is obtainable by screening a suitable nucleic
acid library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of beta-Sitosterol and/or Campesterol resp., in an organism
or a part thereof. or comprising a sequence which is complementary
thereto. 3. The process of claim 1 or 2, comprising recovering of
the free or bound beta-Sitosterol and/or Campesterol resp. 4. The
process of any one of claims 1 to 3, comprising the following
steps: (a) selecting an organism or a part thereof expressing a
polypeptide encoded by the nucleic acid molecule characterized in
claim 2; (b) mutagenizing the selected organism or the part
thereof; (c) comparing the activity or the expression level of said
polypeptide in the mutagenized organism or the part thereof with
the activity or the expression of said polypeptide of the selected
organisms or the part thereof; (d) selecting the mutated organisms
or parts thereof, which comprise an increased activity or
expression level of said polypeptide compared to the selected
organism or the part thereof; (e) optionally, growing and
cultivating the organisms or the parts thereof; and (f) recovering,
and optionally isolating, the free or bound beta-Sitosterol and/or
Campesterol resp., produced by the selected mutated organisms or
parts thereof. 5. The process of any one of claims 1 to 4, wherein
the activity of said protein or the expression of said nucleic acid
molecule is increased or generated transiently or stably. 6. An
isolated nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: a) nucleic acid molecule
encoding of a polypeptide as indicated in Table XII, application
no. 34, columns 5 or 7, or a fragment thereof, which confers an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of beta-Sitosterol and/or Campesterol resp., in
an organism or a part thereof; b) nucleic acid molecule comprising
of a nucleic acid molecule as indicated in Table XI, application
no. 34, columns 5 or 7, c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of beta-Sitosterol and/or
Campesterol resp., in an organism or a part thereof; d) nucleic
acid molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the fine chemical as indicated in table XII,
column 6, e.g of beta-Sitosterol and/or Campesterol resp., in an
organism or a part thereof; e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of beta-Sitosterol and/or Campesterol resp., in an organism
or a part thereof; f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table XIII, application no.
34, column 7, and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g beta-Sitosterol
and/or Campesterol resp., in an organism or a part thereof; g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of beta-Sitosterol and/or Campesterol resp., in
an organism or a part thereof; h) nucleic acid molecule encoding a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 34, column 7, and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of beta-Sitosterol and/or Campesterol resp., in an organism
or a part thereof; and i) nucleic acid molecule which is obtainable
by screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of beta-Sitosterol and/or Campesterol resp., in an organism or
a part thereof. whereby the nucleic acid molecule distinguishes
over the sequence as indicated in Table XI, application no. 34,
columns 5 or 7, by one or more nucleotides. 7. A nucleic acid
construct which confers the expression of the nucleic acid molecule
of claim 6, comprising one or more regulatory elements. 8. A vector
comprising the nucleic acid molecule as claimed in claim 6 or the
nucleic acid construct of claim 7. 9. The vector as claimed in
claim 8, wherein the nucleic acid molecule is in operable linkage
with regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic, host. 10. A host
cell, which has been transformed stably or transiently with the
vector as claimed in claim 8 or 9 or the nucleic acid molecule as
claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 2 to 5. 11. The
host cell of claim 10, which is a transgenic host cell. 12. The
host cell of claim 10 or 11, which is a plant cell, an animal cell,
a microorganism, or a yeast cell, a fungus cell, a prokaryotic
cell, an eukaryotic cell or an archaebacterium. 13. A process for
producing a polypeptide, wherein the polypeptide is expressed in a
host cell as claimed in any one of claims 10 to 12. 14. A
polypeptide produced by the process as claimed in claim 13 or
encoded by the nucleic acid molecule as claimed in claim 6 whereby
the polypeptide distinguishes over a sequence as indicated in Table
XII, application no. 34, columns 5 or 7, by one or more amino acids
15. An antibody, which binds specifically to the polypeptide as
claimed in claim 14. 16. A plant tissue, propagation material,
harvested material or a plant comprising the host cell as claimed
in claim 12 which is plant cell or an Agrobacterium. 17. A method
for screening for agonists and antagonists of the activity of a
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of beta-Sitosterol and/or
Campesterol resp., in an organism or a part thereof comprising: (a)
contacting cells, tissues, plants or microorganisms which express
the a polypeptide encoded by the nucleic acid molecule of claim 5
conferring an increase in the amount of beta-Sitosterol and/or
Campesterol resp., in an organism or a part thereof with a
candidate compound or a sample comprising a plurality of compounds
under conditions which permit the expression the polypeptide; (b)
assaying the beta-Sitosterol and/or Campesterol resp., level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and (c) identifying a
agonist or antagonist by comparing the measured beta-Sitosterol
and/or Campesterol resp., level or polypeptide expression level
with a standard beta-Sitosterol and/or Campesterol resp., or
polypeptide expression level measured in the absence of said
candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. 18. A process for the identification
of a compound conferring increased beta-Sitosterol and/or
Campesterol resp., production in a plant or microorganism,
comprising the steps: (a) culturing a plant cell or tissue or
microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of beta-Sitosterol and/or Campesterol resp.,
in an organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with dais readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of beta-Sitosterol and/or
Campesterol resp., in an organism or a part thereof; (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. 19. A method for the identification of a gene
product conferring an increase in beta-Sitosterol and/or
Campesterol resp., production in a cell, comprising the following
steps: (a) contacting the nucleic acid molecules of a sample, which
can contain a candidate gene encoding a gene product conferring an
increase in beta-Sitosterol and/or Campesterol resp., after
expression with the nucleic acid molecule of claim 6; (b)
identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; (c) introducing the candidate nucleic acid molecules in
host cells appropriate for producing beta-Sitosterol and/or
Campesterol resp.; (d) expressing the identified nucleic acid
molecules in the host cells; (e) assaying the beta-Sitosterol
and/or Campesterol resp., level in the host cells; and (f)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the beta-Sitosterol and/or
Campesterol resp., level in the host cell in the host cell after
expression compared to the wild type. 20. A method for the
identification of a gene product conferring an increase in
beta-Sitosterol and/or Campesterol resp., production in a cell,
comprising the following steps: (a) identifying in a data bank
nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the beta-Sitosterol and/or Campesterol resp., amount or level in an
organism or a part thereof after expression, and which are at least
20% homolog to the nucleic acid molecule of claim 6; (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing beta-Sitosterol and/or Campesterol resp.;
(c) expressing the identified nucleic acid molecules in the host
cells; (d) assaying the beta-Sitosterol and/or Campesterol resp.,
level in the host cells; and (e) identifying nucleic acid molecule
and its gene product which expression confers an increase in the
beta-Sitosterol and/or Campesterol resp., level in the host cell
after expression compared to the wild type. 21. A method for the
production of an agricultural composition comprising the steps of
the method of any one of claims 17 to 20 and formulating the
compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. 22. A composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of any
one of claim 8 or 9, an antagonist or agonist identified according
to claim 17, the compound of claim 18, the gene product of claim 19
or 20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. 23. Use of the nucleic acid molecule as claimed
in claim 6 for the identification of a nucleic acid molecule
conferring an increase of beta-Sitosterol and/or Campesterol resp.,
after expression. 24. Use of the polypeptide of claim 14 or the
nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of beta-Sitosterol and/or
Campesterol resp., levels in an organism. 25. Food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. 26. Use of the nucleic acid molecule
of claim 6, the polypeptide of claim 14, the nucleic acid construct
of claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the harvested material of claim
16, the host cell of claim 10 to 12 or the gene product identified
according to the method of claim 19 or 20 for the protection of a
plant against a beta-Sitosterol and/or Campesterol synthesis
inhibiting herbicide.
[14643] [0554.0.0.34] Abstract: see [0554.0.0.27]:
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[14644] [0000.0.35.35] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and corresponding embodiments as
described herein as follows.
[14645] [0001.0.0.35]: see [0001.0.0.27]
[14646] [0002.0.32.35]: see [0002.0.32.32]
[14647] [0003.0.35.35] Straight- or normal-chain (even-numbered),
monoenoic components, i.e. with one double bond, make up a high
proportion of the total fatty acids in most natural lipids.
Normally the double bond is of the cis- or Z-configuration,
although some fatty acids with trans- or E-double bonds are known.
The most abundant monoenoic fatty acids in animal and plant tissues
are straight-chain compounds with 16 or 18 carbon atoms, but
analogous fatty acids with 10 to 36 carbon atoms have been found in
nature in esterified form. Very long-chain (20:1 upwards)
cis-monoenoic fatty acids have relatively high melting points, but
the more common C.sub.18 monoenes tend to be liquid at room
temperature. Triacylglycerols (or oils and fats) containing high
proportions of monoenoic fatty acids are usually liquid at ambient
temperature. Analogous fatty acids with trans double bonds are
normally higher melting. Very-long-chain monoenoic fatty acids of
the (n-9) family occur in a variety of natural sources, often
accompanied by analogous fatty acids of the (n-7) family),
especially in animal tissues. For example, monoenes from 20:1 to
26:1 are normal constituents of animal sphingolipids. Odd-numbered
very-long chain monoenes (23:1 upwards) from brain belong to (n-8)
and (n-10) families, presumably because they are formed by
chain-elongation of 9-17:1 (17:1(n-8)) and 9-19:1 (19:1(n-10)),
respectively (see below). An even wider range of chain-lengths is
found in monoenes from plant waxes and sponge lipids.
[14648] In animals and yeasts, stearoyl-CoA is converted directly
to oleoyl-CoA by a concerted removal of hydrogen atoms from carbons
9 and 10 (D-stereochemistry in each instance). Subsequently, oleate
can be chain elongated by two carbon atoms to give longer-chain
fatty acids of the (n-9) family, while palmitoleate is the
precursor of the (n-7) family of fatty acids. Certain bacteria
produce mono-unsaturated fatty acids by an anaerobic mechanism that
involves the fatty acid synthetase. During the fourth cycle of
chain elongation, a branch point occurs in fatty acid synthesis
following the dehydrase step. Chain elongation can proceed as
normal, or an isomerase can convert the trans-2-decanoyl-ACP too
cis-3-decanoyl-ACP. The latter is not a substrate for the enoyl-ACP
reductase, but it can be further elongated with eventual formation
of a cis-11-18:1 fatty acid. For many years, this was thought to be
the major pathway for biosynthesis of unsaturated fatty acids in
bacteria, but it is now recognised that it is restricted to a few
proteobacteria, such as E. coli. Aerobic mechanisms certainly
exist, but other mechanisms and enzymes have yet to be adequately
characterized for most bacterial species.
[14649] [0004.0.35.35] Principally microorganisms such as
Mortierella or oil producing plants such as soybean, rapeseed or
sunflower or algae such as Crytocodinium or Phaeodactylum are a
common source for oils containing fatty acids, where they are
usually obtained in the form of their triacyl glycerides.
Alternatively, they are obtained advantageously from animals, such
as fish. The free fatty acids are prepared advantageously by
hydrolysis with a strong base such as potassium or sodium
hydroxide.
[14650] [0005.0.35.35] ./.
[14651] [0006.0.35.35] 11-cis-Eicosenoic acid (gadoleic acid) is a
common if minor constituent of animal tissues and fish oils, often
accompanied by the 13-isomer. It is also found in rapeseed oil and
seed oils of related species. cis-5-20:1 can amount to 67% of the
total fatty acids in meadowfoam oil.
[14652] [0007.0.35.35] cis-Monoenoic acids obviously have desirable
physical properties for membranes lipids, and they are now
recognised by nutritionists as being beneficial in the human
diet.
[14653] [0008.0.35.35] The exception is erucic acid as there is
evidence from studies with laboratory rats that it may adversely
affect the metabolism of the heart. Erucic acid is used in the
manufacture of industrial oils, e.g. for production of polyethylene
film.
[14654] [0009.0.35.35] As described above, cis-Monoenoic acids are
used in a lot of different applications, for example in cosmetics,
pharmaceuticals and in feed and food.
[14655] [0010.0.35.35] Therefore improving the productivity of such
cis-Monoenoic acids and improving the quality of foodstuffs and
animal feeds is an important task of the different industries.
[14656] [0011.0.35.35] To ensure a high productivity of certain
cis-Monoenoic acids in plants or microorganism, it is necessary to
manipulate the natural biosynthesis of fatty acids in said
organism.
[14657] [0012.0.35.35] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
regulators which participate in the biosynthesis of cis-Monoenoic
acids and make it possible to produce certain cis-Monoenoic acids
specifically on an industrial scale without unwanted byproducts
forming. In the selection of genes for biosynthesis two
characteristics above all are particularly important. On the one
hand, there is as ever a need for improved processes for obtaining
the highest possible contents of cis-Monoenoic acids on the other
hand as less as possible byproducts should be produced in the
production process.
[14658] [0013.0.0.35] see [0013.0.0.27]
[14659] [0014.0.35.35] It was found that the overexpression of the
nucleic acid molecule characterized herein confers an increase in
the content of Eicosenic acid (20:1) in plants. Accordingly, in a
first embodiment, the invention relates to a process for the
production of Eicosenic acid. In Arabidopsis thaliana, Eicosenic
acid is predominately found in the stereoisomeric form 20:1 delta
9c (gadoleic acid). Accordingly, in a further embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is gadoleic acid or
tryglycerides, lipids, oils or fats containing gadoleic acid.
Accordingly, in the present invention, the term "the fine chemical"
as used herein relates to "gadoleic acid and/or tryglycerides,
lipids, oils and/or fats containing gadoleic acid". Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising gadoleic acid and/or triglycerides, lipids,
oils and/or fats containing gadoleic acid.
[14660] Advantageously, the increase of the Eicosenic acid (20:1
fatty acid) content, e.g. the gadoleic acid content, in a
composition produced according to the process of the invention
results in an incrase in the melting point of the composition, in
particular if the composition is a fatty composition as a oil or a
wax. As other monoenoic fatty acids, eicosenic acid, in particular
gadoleic acid, may be used as anti-foaming agent in detergents or
as an anti-blocking agent in the production of platics or used
preservation agent, flouering agent, plastic softener, formulation
agent, flotation agent, wetting agent emulsifer agend and/or
lubricating agents, as e.g. erucic acid, arachinic acid, pelagonic
acid, brassylic acid or erucic acid amidses. Furthermore,
monounsaturated fatty acids as eicosenic acid, in particular
gadolenic acid, are cholesterol lowering when they replace
significant levels of saturated fatty acids in the diet. Some
studies have found that diets high in monounsaturated fatty acids
compared with polyunsaturated fatty acids decrease LDL cholsterol
while maintaining HDL cholesterol levels. However, other studies
suggested that the effect of consuming polyunsaturated fat and
monounsaturated fat is similar and results in a decrease in both
LDL and HDL cholesterol (see e.g. homepage of the Instiute of
Shorting and Edible Oils for references).
[14661] [0015.0.35.35] In one embodiment, the term "the respective
fine chemical" means gadoleic acid, eicosenic acid, C20:1 fatty
acidand/or tryglycerides, lipids, oils and/or fats containing
gadoleic acid. Throughout the specification the term "the
respective fine chemical" means gadoleic acid and/or tryglycerides,
lipids, oils and/or fats containing gadoleic acid, gadoleic acid
and its salts, ester, thioester or gadoleic acid in free form or
bound to other compounds such as triglycerides, glycolipids,
phospholipids etc. In a preferred embodiment, the term "the
respective fine chemical" means gadoleic acid, in free form or its
salts or bound to triglycerides. Triglycerides, lipids, oils, fats
or lipid mixture thereof shall mean any triglyceride, lipid, oil
and/or fat containing any bound or free gadoleic acid for example
sphingolipids, phosphoglycerides, lipids, glycolipids such as
glycosphingolipids, phospholipids such as phosphatidylethanolamine,
phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol or diphosphatidylglycerol, or as
monoacylglyceride, diacylglyceride or triacylglyceride or other
fatty acid esters such as acetyl-Coenzym A thioester, which contain
further saturated or unsaturated fatty acids in the fatty acid
molecule.
[14662] [0016.0.35.35] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [14663] (a) increasing or generating the activity of one
or more of a protein as shown in table XII, application no. 35,
column 3 encoded by the nucleic acid sequences as shown in table
XI, application no. 35, column 5, in a non-human organism or in one
or more parts thereof or [14664] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
the acid of the invention or fine chemicals comprising the acid of
the invention, in said organism or in the culture medium
surrounding the organism.
[14665] [0017.0.0.35] to [0019.0.0.35]: see [0017.0.0.27] to
[0019.0.0.27]
[14666] [0020.0.35.35] Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, application no. 35, column 5, line 21 in a plant
conferred an increase in C20:1 fatty acid, putative content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of C20:1 fatty acid,
putative.
[14667] [0021.0.0.35] see [0021.0.0.27]
[14668] [0022.0.35.35] The sequence of YDR513W from Saccharomyces
cerevisiae has been published in Jacq et al., Nature 387 (6632
Suppl), 75-78 (1997) and in Goffeau et al., Science 274 (5287),
546-547, 1996 and its cellular activity has characterized as
glutathione reductase. Accordingly, in one embodiment, the process
of the present invention comprises the use of glutathione reductase
or a protein of the glutaredoxin superfamily for the production of
gadoleic acid. Accordingly, in one embodiment, the process of the
present invention comprises the use of YDR513W, from Saccharomyces
cerevisiae, e.g. as indicated herein in Table XII, line 125,
columns 3 or 5, or its homologue, e.g. as shown herein in Table
XII, line 125, column 7, for the production of the respective fine
chemical, meaning of gadoleic acid and/or tryglycerides, lipids,
oils and/or fats containing gadoleic acid, in particular for
increasing the amount of gadoleic acid and/or tryglycerides,
lipids, oils and/or fats containing gadoleic acid, preferably
gadoleic acid in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a glutathione reductase is
increased or generated, e.g. from Saccharomyces cerevisiae or a
plant or a homolog thereof.
[14669] [0022.1.0.35] to [0023.0.0.35]: see [0022.1.0.27] to
[0023.0.0.27]
[14670] [0023.1.35.35] Homologs of the polypeptide disclosed in
table XII, application no. 35, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 35, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 35, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 35,
column 7, resp.
[14671] [0024.0.0.35] see [0024.0.0.27]
[14672] [0025.0.35.35] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 35, column 3 "if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 35, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[14673] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 35,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 35,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[14674] [0025.1.0.35] see [0025.1.0.27]
[14675] [0026.0.0.35] to [0033.0.0.35]: see [0026.0.0.27] to
[0033.0.0.27]
[14676] [0034.0.35.35] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 35, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[14677] [0035.0.0.35] to [0044.0.0.35]: see [0035.0.0.27] to
[0044.0.0.27]
[14678] [0045.0.35.35] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
35, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 35, column 6 of
the respective line, between 10% and 50%, 10% and 100%, 10% and
200%, 10% and 300%, 10% and 400%, 10% and 500%, 20% and 50%, 20%
and 250%, 20% and 500%, 50% and 100%, 50% and 250%, 50% and 500%,
500% and 1000% or more [0046.0.35.35] In one embodiment, the
activity of the protein as indicated in Table XII, columns 5 or 7,
application no. 35, is increased conferring an increase of the
respective fine chemical, indicated in Table XII, application no.
35, column 6 of the respective line confers an increase of the
respective fine chemical and of further acids, like erucic acid or
their precursors.
[14679] [0047.0.0.35] to [0048.0.0.35]: see [0047.0.0.27] to
[0048.0.0.27]
[14680] [0049.0.35.35] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 35, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 35, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 35, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[14681] [0050.0.35.35] For the purposes of the present invention,
the term "gadoleic acid" also encompasses the corresponding salts,
such as, for example, the potassium or sodium salts of gadoleic
acid or the salts of gadoleic acid with amines such as
diethylamine.
[14682] [0051.0.35.35] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g monoenoic fatty acid compositions.
Depending on the choice of the organism used for the process
according to the present invention, for example a microorganism or
a plant, compositions or mixtures of various monoenoic fatty acid
can be produced.
[14683] [0052.0.0.35]: see [0052.0.0.27]
[14684] [0053.0.35.35] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[14685] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 35, columns 5 and 7 or its homologs activity
having herein-mentioned 229,230 of the invention increasing
activity; and/or [14686] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 35,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 35, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned 229,230 of the invention increasing
activity; and/or [14687] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned 229,230 increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 35, columns 5 and 7 or its
homologs activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [14688] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned 229,230 of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 35, columns 5
and 7 or its homologs activity; and/or [14689] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned 229,230 of the invention increasing activity, e.g.
of a polypeptide having the activity of a protein as indicated in
table XII, application no. 35, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [14690] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned 229,230 of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 35, columns 5 and 7 or its
homologs activity, and/or [14691] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned 229,230 of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 35, columns 5 and 7 or its
homologs activity; and/or [14692] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 35, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[14693] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [14694] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[14695] [0054.0.35.35] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 35 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 35, column 3 or its homologs.
[14696] [0055.0.0.35] to [0067.0.0.35]: see [0055.0.0.27] to
[0067.0.0.27]
[14697] [0068.0.35.35] The mutation is introduced in such a way
that the production of the fatty acids is not adversely
affected.
[14698] [0069.0.0.35] see [0069.0.0.27]
[14699] [0070.0.32.35] to [0071.0.32.35]: see [0070.0.32.32] to
[0071.0.32.32]
[14700] [0072.0.35.35] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to gadoleic acid, triglycerides, lipids, oils and/or fats
containing gadoleic acid compounds such as palmitate, palmitoleate,
oleate and/or linoleic acid.
[14701] [0073.0.32.35] to [0084.0.32.35] see [0073.0.32.32] to
[0084.0.0.32]
[14702] [0084.1.35.35] It was found that the level of eicosenic
acid is high in the following plants seeds with decreasing level:
Selenia grandis, Teesdalia nudicaulis (L.)R. Br., Teesdalia
nudicaulis (L.) R. Br., Cardiospermum canescens, Lesquerella
fendleri, Leavenworthia torulosa, Leavenworthia torulosa,
Cardiospermum grandiflorum, Cardiospermum halicacaburn, Paullinia
elegans, Cupania anacardloides, Koelreuteria eleaans, Koelreuteria
apiculata, Lobularia maritima, Iberis odorata, Alyssum maritirnum,
Cardiospermum corindum, Biscutella laevigata, Biscutella
auriculata, Lobularia maritima, Dithyrea californica, Dithyrea
wislizenii, Thysanocarpus radians, Cheirantus maritimus, Cordia
verbenacea DC., Conringia orientalis (L.) Dumort., Tropaeolum
majus, Conringia orientalis (L.) Dumort., Tropaeolum majus, Arachis
hypogaea, Neslia paniculata, Malcolmia maritima, Malcolmia
flexuosa, Arachisperuviana peruviana, Sapindus mukorossi, Conringia
orientalis, Arachis fastigiata, Arabidopsis thaliana, Arabidopsis
thaliana Schur, Sapindus mukorossi Azirna tetracantha, Tropaeolum
majus, Tropaeolum maius, Brassica repanda (Willd.) DC., Calepina
irregularis, Arabidopsis thaliana (L.) Heynh. Columbia, Crambe
orientalis, Nerisyrenia camporum, Crambe maritima L., Delphinium
ajacis, Tropaeolum minus L., Cardamine imatiens, Brassica oleracea
L. ssp. robertiana (Gay) Rouy et Foug., Delphinium spp., Camelina
rumelica, Juniperus chinensis, Capsella rubella, Capsella rubella,
Cordia myxa, Sapindus emarginatus, Lepidium cuneiforme, Caulanthus
inflatus, Malcolmia chia, Camelina sativa, Cardamine bellidifolia
L., Atalaya herniglauca, Aesculus assamica, Leonurus sibiricus,
Capsella grandiflora, Capsella grandiflora, Sinapis arvensis L.,
Erysimum perovskianum, Camelina microcarpa, Schleichera trijuga,
Schleichera trijuaa, Brassica juncea, Tropaeolum majis, Schleichera
trijuga, Cardamine hirsuta, Cardamine amare, Lepidium lasiocarpum,
Stanleyella texana, Sapindus mukorossi, Goldbachia laevigata D. C.,
Descurainia bourgaeana Webb., Crambe maritima, Consolida
orientalis, Tropaeolum majus, Tropaeolum majus, Lunaria rediviva,
Tristellateia australasica, Lepidium perfoliatum, Camelina safiva,
Camelina sativa, Lepidium apetalum, Sapindus saponaria, Neslia
paniculata, Sapindus saponaria, Brassica sp., Brassica juncea,
Coronopus didymus, Fezia pterocara pitard, Arabis glabra, Eruca
sativa, Descurainia innata var. innata, Hornungia petraea,
Descurainia sophia, Phoenix dactylifera L., Descurainia sophia,
Thlaspi alpinum, Capsella bursa-pastoris, Capsella bursa-pastoris,
Capsella spp., Capsella spp, Descurainia sophia, Capsella spp.,
Capsella spp, Tropaeolum minus L., Capsella bursa-pastoris,
Brassica campestris, Lunaria rediviva, Capsella spp., Descurainia
sophia, Capsella spp., Capsella bursa-pastoris, Clitoria
rubiginosa, Arabis laevigata, Thlaspi alpestre L., Camelina saliva,
Crambe cordifolia, Capsella bursa-pastoris, Savigna parviflora,
Brassica campestris, Lepidium sativum, Arabis virginica, Sophia
ochroleuca, Sapindus emarginatus, Brassica cossoneana (Boiss. et
Reuter) Maire, Parrya menziesii, Diplotaxis tenuifolia. For
example, it was found that the following content of eicosenic acid
is comprised in the oil of seeds: 58.50, GLC-Area-%, Selenia
grandis, 56.10, GLC-Area-%, Teesdalia nudicaulis (L.) R. Br.,
56.00, GLC-Area-%, Teesdalia nudicalis (L.) R. Br., 55.70, GLC area
%, Cardiospermum canescens, 54.50, GLC-Area-%, Lesquerella
fendleri, 53.00, GLC-Area-%, Leavenworthia torulosa, 53.00,
GLC-Area-%, Leavenworthia torulosa, 52.60, GLC-Area-%,
Cardiospermum grandiflorum, 49.10, GLC-Area-%, Cardiospermum
halicacabum, 48.70, GLC-Area-%, Paullinia elegans, 46.00,
GLC-Area-%, Cupania anacardioides, 45.30, GLC-Area-%, Koelreuteria
elegans, 44.60, GLC area %, Koelreuteria apiculata, 42.00,
GLC-Area-%, Lobularia maritima, 41.90, GLC-Area-%, Iberis odorata,
41.80, GLC-Area-%, Alyssum maritimum, 41.60, GLC-Area-%,
Cardiospermum corindum, 40.00, GLC-Area-%, Biscutella laevigata,
36.10, GLC-area-%, Biscutella auriculata, 35.60, GLC-area-%,
Lobularia maritima. A further list of plants with high gadoleic
acid can be found on webpage of the Agricultural Research service
(www.ars-grin.gov/cqi bin/duke/chemical.pl?gadoleicacid) showing
plant species with the highest amount of gadoleic acid. The content
of fatty acids in the oil of plants and plants which have a high
level of distinct fatty acids can also be identified on the webpage
of the Institute of Chemistry and Physics of Lipids,
http://www.bagkf.de/sofa/.
[14703] Thus, in on embodiment, the process of the present
invention is performed in a plant of the above lists of identified
in above webpages, preferably in a plant with a high amount of
eicosenic acid, in particular with high gadoleic level and/or
together with further genes isolated from a plant with a high
eicosenic acid level, in particular a high gadoleic level. In a
preferred embodiment, the process of the present invention is
performed in a plant identified in above webpages with a low amount
of erucic acid, in particular with high gadoleic level and/or
together with further genes isolated from a plant with a high
eicosenic acid level, in particular a high gadoleic level. In
another embodiment, the process of the present invention is
performed in a plant identified in above webpages, preferably in a
plant with a high amount of erucic acid, in particular with high
gadoleic level and/or together with further genes isolated from a
plant with a high eicosenic acid level, in particular a high
gadoleic level.
[14704] [0085.0.35.35] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [14705] a) a nucleic acid sequence as
shown in table XI, application no. 35, columns 5 or 7 or a
derivative thereof, or [14706] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as shown table XI, application no. 35, columns 5 or 7
or a derivative thereof, or [14707] c) (a) and (b) is/are not
present in its/their natural genetic environment or has/have been
modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[14708] [0086.0.0.35] to [0087.0.0.35]: see [0086.0.0.27] to
[0087.0.0.27]
[14709] [0088.0.35.35] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose fatty acid
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for poultry is dependent on the abovementioned essential fatty
acids and the general amount of fatty acids as energy source in
feed. After the YDR513W protein activity, e.g. of a protein as
indicated in Table XII, column 5 or 7, application no. 35 or being
encoded by a nucleic acid molecule indicated in Table XI, column 5,
application no. 35 or of its homologs, e.g. as indicated in Table
XII, column 7, line 125, has been increased or generated, or after
the expression of nucleic acid molecule or polypeptide according to
the invention has been generated or increased, the transgenic plant
generated thus is grown on or in a nutrient medium or else in the
soil and subsequently harvested.
[14710] [0088.1.0.35]: see [0088.1.0.27]
[14711] [0089.0.32.35] to [0102.0.32.35]: see [0089.0.0.32] to
[0102.0.32.32]
[14712] [0103.0.35.35] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [14713] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide shown in
table XII, application no. 35, columns 5 or 7 or a fragment
thereof, which confers an increase in the amount of the respective
fine chemical in an organism or a part thereof; [14714] b) nucleic
acid molecule comprising, preferably at least the mature form, of
the nucleic acid molecule shown in table XI, application no. 35,
columns 5 or 7; [14715] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [14716] d) nucleic
acid molecule encoding a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [14717] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[14718] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[14719] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [14720] h) nucleic acid
molecule comprising a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers shown in Table XIII, application no. 35,
column 7 and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [14721] i) nucleic
acid molecule encoding a polypeptide which is isolated, e.g. from
an expression library, with the aid of monoclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (h), preferably to (a) to (c), and and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [14722] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence shown in
table XIV, application no. 35, column 7 and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [14723] k) nucleic acid molecule comprising one or
more of the nucleic acid molecule encoding the amino acid sequence
of a polypeptide encoding a domain of the polypeptide shown in
table XII, application no. 35, columns 5 or 7 and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; and [14724] l) nucleic acid molecule
which is obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably to (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; or which comprises a sequence which is complementary
thereto.
[14725] [0103.1.35.35.] In one embodiment, the nucleic acid
molecule used in the process of the invention distinguishes over
the sequence indicated in Table XI, columns 5 or 7, application no.
35, by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence shown in indicated in Table XI, columns 5 or 7,
application no. 35. In one embodiment, the nucleic acid molecule
used in the process of the invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to a sequence indicated in Table XI,
columns 5 or 7, application no. 35. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table XII, columns 5 or 7, application no. 35.
[14726] [0104.0.35.35] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence shown in table
XI, application no. 35, columns 5 or 7 by one or more nucleotides
or does not consist of the sequence shown in table XI, application
no. 35, columns 5 or 7. In one embodiment, the nucleic acid
molecule of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to the sequence shown in table XI,
application no. 35, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of the sequence
shown in table XII, application no. 35, columns 5 or 7.
[14727] [0105.0.0.35] to [0107.0.0.35]: see [0105.0.0.27] to
[0107.0.0.27]
[14728] [0108.0.35.35] Nucleic acid molecules with the sequence
shown in table XI, application no. 35, columns 5 or 7, nucleic acid
molecules which are derived from the amino acid sequences shown in
table XII, application no. 35, columns 5 or 7 or from polypeptides
comprising the consensus sequence shown in table XIV, application
no. 35, column 7, or their derivatives or homologues encoding
polypeptides with the enzymatic or biological activity of an
YDR513W protein or conferring of eicosenic acid, in particular of
gadoleic acid increase after increasing its expression or activity
are advantageously increased in the process according to the
invention.
[14729] [0109.0.0.35]: see [0109.0.0.27]
[14730] [0110.0.35.35] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with YDR513W protein activity, e.g. of a
protein as indicated in Table XII, column 5, application no. 35 or
being encoded by a nucleic acid molecule indicated in Table XI,
column 5, application no. 35 or of its homologs, e.g. as indicated
in Table XII, column 7, application no. 35, can be determined from
generally accessible databases.
[14731] [0111.0.0.35] see [0111.0.0.27]
[14732] [0112.0.35.35] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with YDR513W
protein activity and conferring gadoleic acid increase, e.g. a
protein as indicated in Table XII, column 5, application no. 35 or
being encoded by a nucleic acid molecule indicated in Table XI,
column 5, application no. 35, or of their homologs, e.g. as
indicated in Table XI or XII, column 7, application no. 35.
[14733] [0113.0.32.35] to [0147.0.32.35]: see [0113.0.0.32] to
[0147.0.32.32]
[14734] [0148.0.35.35] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence shown in table XI, application no. 35,
columns 5 or 7, or a functional portion thereof and preferably has
above mentioned activity, in particular having a the fine chemical,
in particular gadoleic acid-increasing activity after increasing
the activity or an activity of an YDR513W gene product, e.g. a gene
encoding a protein as indicated in Table XII, column 5, application
no. 35 or comprising or expressing a nucleic acid molecule
indicated in Table XI, column 5, application no.
[14735] 35, or of their homologs, e.g. as indicated in Table XI or
XII, column 7, application no.
[14736] 35.
[14737] [0149.0.35.35] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences shown in table XI, application no. 35,
columns 5 or 7 or a portion thereof and encodes a protein having
above-mentioned activity, e.g. conferring an increase of the fine
chemical, and optionally, the activity of YDR513W, e.g. of a
protein as indicated in Table XII, column 5, application no. 35 or
being encoded by a nucleic acid molecule indicated in Table XI,
column 5, application no. 35, or of their homologs, e.g. as
indicated in Table XI or XII, column 7, application no. 35.
[14738] [00149.1.35.35] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table
XI, columns 5 or 7, application no. 35, preferably of Table XI B,
columns 5 or 7, application no. 35 has further one or more of the
activities annotated or known for the a protein as indicated in
Table XII, column 3, application no. 35.
[14739] [0150.0.35.35] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences shown in table XI, application no. 35, columns 5
or 7 for example a fragment which can be used as a probe or primer
or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of gadoleic acid if its
activity is increased. The nucleotide sequences determined from the
cloning of the present protein-according-to-the-invention-encoding
gene allows for the generation of probes and primers designed for
use in identifying and/or cloning its homologues in other cell
types and organisms. The probe/primer typically comprises
substantially purified oligonucleotide. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 15 preferably
about 20 or 25, more preferably about 40, 50 or 75 consecutive
nucleotides of a sense strand of one of the sequences set forth,
e.g., in table XI, application no. 35, columns 5 or 7, an
anti-sense sequence of one of the sequences, e.g., set forth in
table XI, application no. 35, columns 5 or 7, or naturally
occurring mutants thereof. Primers based on a nucleotide of
invention can be used in PCR reactions to clone homologues of the
polypeptide of the invention or of the polypeptide used in the
process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primers pairs shown in Table XIII, application no. 35,
column 7 will result in a fragment of gene product with the
activity of a YDR513W protein, e.g. of a gene encoding of a protein
as indicated in Table XII, column 5, application no. 35 or
expressing a nucleic acid molecule indicated in Table XI, column 5,
application no. 35 or of its homologs, e.g. as indicated in Table
XII, column 7, application no. 35.
[14740] [0151.0.32.35] to [0230.0.32.35] see [0151.0.0.32] to
[0230.0.0.32]
[14741] [0231.0.35.35] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a gadoleic acid degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[14742] [0232.0.32.35] to [0552.0.32.35]: see [0232.0.0.32] to
[0552.0.0.32]
[14743] [0553.0.35.35] We claim: [14744] 1. A process for the
production of gadoleic acid, which comprises [14745] (a) increasing
or generating the activity of a protein as indicated in Table XII,
application no. 35, columns 5 or 7, or a functional equivalent
thereof in a non-human organism, or in one or more parts thereof;
and [14746] (b) growing the organism under conditions which permit
the production of gadoleic acid in said organism. [14747] 2. A
process for the production of gadoleic acid, comprising the
increasing or generating in an organism or a part thereof the
expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[14748] a) nucleic acid molecule encoding of a polypeptide as
indicated in Table XII, application no. 35, columns 5 or 7, or a
fragment thereof, which confers an increase in the amount of
gadoleic acid in an organism or a part thereof; [14749] b) nucleic
acid molecule comprising of a nucleic acid molecule as indicated in
Table XI, application no. 35, columns 5 or 7; [14750] c) nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as a
result of the degeneracy of the genetic code and conferring an
increase in the amount of gadoleic acid in an organism or a part
thereof; [14751] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of gadoleic
acid in an organism or a part thereof; [14752] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of gadoleic acid in an organism or a part
thereof; [14753] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table XIII, application no.
35, columns 7, and conferring an increase in the amount of gadoleic
acid in an organism or a part thereof; [14754] g) nucleic acid
molecule encoding a polypeptide which is isolated with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (f) and conferring an increase in
the amount of gadoleic acid in an organism or a part thereof;
[14755] h) nucleic acid molecule encoding a polypeptide comprising
a consensus as indicated in Table XIV, application no. 35, column
7, and conferring an increase in the amount of gadoleic acid in an
organism or a part thereof; and [14756] i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of gadoleic acid in an organism or a part thereof. [14757]
or comprising a sequence which is complementary thereto. [14758] 3.
The process of claim 1 or 2, comprising recovering of the free or
bound gadoleic acid. [14759] 4. The process of any one of claims 1
to 3, comprising the following steps: [14760] (a) selecting an
organism or a part thereof expressing a polypeptide encoded by the
nucleic acid molecule characterized in claim 2; [14761] (b)
mutagenizing the selected organism or the part thereof; [14762] (c)
comparing the activity or the expression level of said polypeptide
in the mutagenized organism or the part thereof with the activity
or the expression of said polypeptide of the selected organisms or
the part thereof; [14763] (d) selecting the mutated organisms or
parts thereof, which comprise an increased activity or expression
level of said polypeptide compared to the selected organism or the
part thereof; [14764] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [14765] (f) recovering, and
optionally isolating, the free or bound gadoleic acid produced by
the selected mutated organisms or parts thereof. [14766] 5. The
process of any one of claims 1 to 4, wherein the activity of said
protein or the expression of said nucleic acid molecule is
increased or generated transiently or stably. [14767] 6. An
isolated nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [14768] a) nucleic acid
molecule encoding of a polypeptide as indicated in Table XII,
application no. 35, columns 5 or 7, or a fragment thereof, which
confers an increase in the amount of gadoleic acid in an organism
or a part thereof; [14769] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table XI, application no. 35,
columns 5 or 7; [14770] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of gadoleic acid in
an organism or a part thereof; [14771] d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of gadoleic acid in an organism or a part thereof; [14772]
e) nucleic acid molecule which hybridizes with a nucleic acid
molecule of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of gadoleic acid in an
organism or a part thereof; [14773] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table XIII,
application no. 35, column 7, and conferring an increase in the
amount of gadoleic acid in an organism or a part thereof; [14774]
g) nucleic acid molecule encoding a polypeptide which is isolated
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (f) and conferring
an increase in the amount of gadoleic acid in an organism or a part
thereof; [14775] h) nucleic acid molecule encoding a polypeptide
comprising a consensus as indicated in Table XIV, application no.
35, column 7, and conferring an increase in the amount of gadoleic
acid in an organism or a part thereof; and [14776] i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of gadoleic acid in an organism or a part thereof.
[14777] whereby the nucleic acid molecule distinguishes over the
sequence as indicated in Table XI, application no. 35, columns 5 or
7, by one or more nucleotides. [14778] 7. A nucleic acid construct
which confers the expression of the nucleic acid molecule of claim
6, comprising one or more regulatory elements. [14779] 8. A vector
comprising the nucleic acid molecule as claimed in claim 6 or the
nucleic acid construct of claim 7. [14780] 9. The vector as claimed
in claim 8, wherein the nucleic acid molecule is in operable
linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [14781] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [14782] 11. The host cell of claim 10, which is a
transgenic host cell. [14783] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [14784] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [14785] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table XII,
application no. 35, columns 5 or 7, by one or more amino acids
[14786] 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. [14787] 16. A plant tissue,
propagation material, harvested material or a plant comprising the
host cell as claimed in claim 12 which is plant cell or an
Agrobacterium. [14788] 17. A method for screening for agonists and
antagonists of the activity of a polypeptide encoded by the nucleic
acid molecule of claim 6 conferring an increase in the amount of
gadoleic acid in an organism or a part thereof comprising: [14789]
(a) contacting cells, tissues, plants or microorganisms which
express the a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of gadoleic acid in an
organism or a part thereof with a candidate compound or a sample
comprising a plurality of compounds under conditions which permit
the expression the polypeptide; [14790] (b) assaying the gadoleic
acid level or the polypeptide expression level in the cell, tissue,
plant or microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [14791] (c)
identifying a agonist or antagonist by comparing the measured
gadoleic acid level or polypeptide expression level with a standard
gadoleic acid or polypeptide expression level measured in the
absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. [14792] 18. A process for
the identification of a compound conferring increased gadoleic acid
production in a plant or microorganism, comprising the steps:
[14793] (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of gadoleic acid in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
gadoleic acid in an organism or a part thereof; [14794] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [14795] 19. A method for the identification of a
gene product conferring an increase in gadoleic acid production in
a cell, comprising the following steps: [14796] (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in gadoleic
acid after expression with the nucleic acid molecule of claim 6;
[14797] (b) identifying the nucleic acid molecules, which hybridise
under relaxed stringent conditions with the nucleic acid molecule
of claim 6; [14798] (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing gadoleic acid;
[14799] (d) expressing the identified nucleic acid molecules in the
host cells; [14800] (e) assaying the gadoleic acid level in the
host cells; and [14801] (f) identifying nucleic acid molecule and
its gene product which expression confers an increase in the
gadoleic acid level in the host cell in the host cell after
expression compared to the wild type. [14802] 20. A method for the
identification of a gene product conferring an increase in gadoleic
acid production in a cell, comprising the following steps: [14803]
(a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the gadoleic acidamount or level
in an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; [14804]
(b) introducing the candidate nucleic acid molecules in host cells
appropriate for producing gadoleic acid; [14805] (c) expressing the
identified nucleic acid molecules in the host cells; [14806] (d)
assaying the gadoleic acidlevel in the host cells; and [14807] (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the gadoleic acid level in the
host cell after expression compared to the wild type. [14808] 21. A
method for the production of an agricultural composition comprising
the steps of the method of any one of claims 17 to 20 and
formulating the compound identified in any one of claims 17 to 20
in a form acceptable for an application in agriculture. [14809] 22.
A composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [14810] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
gadoleic acid after expression. [14811] 24. Use of the polypeptide
of claim 14 or the nucleic acid construct claim 7 or the gene
product identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of
gadoleic acid levels in an organism. [14812] 25. Cosmetical,
pharmaceutical, food or feed composition comprising the nucleic
acid molecule of claim 6, the polypeptide of claim 14, the nucleic
acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20.
[14813] 26. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the protection of vegetable fats,
oils or waxes.
[14814] 27. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the production of industrial oils,
fats or waxes.
[14815] 28. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the production of detergents,
cleaning agents, cosmetics, dye additives, lubricating agents,
hydraulic oils, preservation agents, flavoring agents, plastic
softeners, formulation agents, flotation agents, wetting agents,
emulsifiers or lubricating agents. [14816] 29. The plant of claim
16, which has a low level of erucic acid. [14817] 30. The plant of
claim 16, which has a high level or erucic acid. [14818] 31. The
plant of any one of claim 16, 29 or 30, which has a low level of
gluconsinolate.
[14819] [0554.0.0.35] Abstract: see [0554.0.0.27]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[14820] [0000.0.36.36] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and corresponding embodiments as
described herein as follows.
[14821] [0001.0.0.36] to [0002.0.0.36]: see [0001.0.0.27] to
[0002.0.0.27]
[14822] [0002.1.36.36] L-alanine is used in various pharmaceutical
and veterinary applications. For example, it is included, together
with other amino acids, in preparations for infusion solutions or
preparations for parenteral administration as clinical preoperative
and postoperative foods, as well as an animal feed supplement.
Furthermore, alanine is used as a food additive on account of its
sweet taste. L-phenylalanine and L-aspartic acid have very
important markets as key components in the manufacture of the
sweetener aspartame. Aspartame (C.sub.14H.sub.18N.sub.2O.sub.5),
L-aspartyl-L-phenylalanine methyl ester, is a compound of three
components, which are methanol, aspartic acid and phenylalanine.
L-aspartic acid is further used as a flavoring agent.
[14823] The amino acid L-citrulline is a metabolite in the urea
cycle. Other amino acids in this cycle are L-arginine and
L-ornithine.L-citrulline is involved in liver detoxification of
ammonia, and has been shown to speed recover from fatigue. It has
also been utilized in the treatment of Ornithine Transcarbamylase
Deficiency and other Urea Cycle disorders. In cell metabolism,
L-arginine and L-citrulline might serve as endogenous N sources
(Ludwig et al., PLANT PHYSIOLOGY, Vol 101, Issue 2 429-434, 1993).
Glycineis a valuable compound of wide use as food additives for
processed foodstuffs and raw materials for agricultural chemicals
and medicines. Glycineis the simplest amino acid, and is used in
crop production as a chelating agent for micronutrients and has
been used as a nitrogen fertilizer, at least on an experimental
basis. As such, it is representative of amino acids used in crop
production. Practically all commercial glycine is produced by
synthetic processes such as the Strecker Synthesis, the reaction of
formaldehyde, ammonia, and hydrogen cyanide, and hydrolysis of the
resulting aminonitrile. Glycineis used as chelating/complexing
agent for cation nutrients, plant growth regulators, substrate for
microbiological products, fertilizer source of nitrogen. Serine is
a primary intermediate in the biosynthesis of a wide variety of
cellular metabolites including such economically important
compounds as choline, glycine, cysteine and tryptophan. In
addition, serine acts as a single carbon donor and is responsible
for 60% to 75% of the total need of the cell for Cl units through
the production of 5,10-methylenetetrahydrofolate from
tetrahydrofolate. These Cl units are used in a wide variety of
biosynthetic pathways including the synthesis of methionine,
inosine monophosphate, other purines and some pyrimidines (e.g.,
thymidine and hydroxymethyl cytidine). The glycine-serine
interconversion, catalysed by glycine decarboxylase and serine
hydroxymethyltransferase, is an important reaction of primary
metabolism in all organisms including plants, by providing
one-carbon units for many biosynthetic reactions. In plants, in
addition, it is an integral part of the photorespiratory metabolic
pathway and produces large amounts of photorespiratory CO2 within
mitochondria (Bauwe et al., Journal of Experimental Botany, Vol.
54, No. 387, pp. 1523-1535, Jun. 1, 2003.) The enzymatic conversion
of phenylalanine to tyrosine is known in eukaryotes. Human
phenylalanine hydroxylase is specifically expressed in the liver to
convert L-phenylalanine to L-tyrosine (Wang et al. J. Biol. Chem.
269 (12): 9137-46 (1994)). Deficiency of the PAH enzyme causes
classic phenylketonurea, a common genetic disorder. Tyrosine and
and their derivatives are also used in organic synthesis. For
example, tyrosine is starting material in the synthesis of
chatecolamines or DOPA (dihydroxy-phenyl-alanine) as well as a
precursor of adrenaline, dopamine and norepinepherine. A variety of
beta-amino-gamma-keto acids can be prepared from commercially
available 5-Oxoproline, also named as pyroglutamic acid PCA and
slats like sodium-PCA, is used as cosmetic ingredient, such as hair
and skin conditioning agent. One optical isomer of PCA (the L form)
is a naturally occurring component of mammalian tissue.
5-Oxoproline is further used as templates in the synthesis of
homochiral glutamate antagonists.
[14824] [0003.0.0.36] to [0008.0.0.36]: see [0003.0.0.27] to
[0008.0.0.27]
[14825] [0008.1.36.36]U.S. Pat. No. 5,498,532 disclose the
production of various L-amino acids like glutamic acid, glutamine,
lysine, threonine, isoleucine, valine, leucine, tryptophan,
phenylalanine, tyrosine, histidine, arginine, ornithine, citrulline
and proline by direct fermentation using, coryneform bacteria
belonging to the genus Corynbacterium or Brevibacterium, which are
inherently unable to assimilate lactose, but due to recombinant DNA
technology able to assimilate lactose, which represent the carbon
source.
[14826] The coproduction of glutamic acid and other amino acids
including lysine, aspartic acid, alanine by an auxotroph of
Bacillus methanolicus is described in U.S. Pat. No. 6,110,713.
[14827] According to the teaching of U.S. Pat. No. 5,677,156
L-aspartic acid can be efficiently produced from maleic acid or
fumaric acid by adding the aspartase-containing microorganism, like
Brevibacterium flavum AB-41 strain (FERM BP-1498) and Eschirichia
coli ATCC 11303.
[14828] U.S. Pat. No. 5,354,672 discloses a method of producing
tyrosine, methionine, or phenylalanine by transiently incorporating
a DNA inversion gene into the host cell, Escherichia coli cells,
which induce hypersecretion of amino acids. Known is also the
production of citrulline in the small intestine as a product of
glutamine metabolism, or in the arginine biosynthetic pathway,
where ornithine carbamoyltransferases catalyse the production of
citrulline from carbamoyl-phosphate and ornithine. Benninghoff et
al. disclose the production of citrulline and ornithine by
interferon-gamma treated macrophages (International Immunology, Vol
3, 413-417, 1991).
[14829] There disclosed is a method for producing glycine in US
20030040085, which comprises subjecting an aqueous solution of
glycinonitrile to a hydrolysis reaction in a hydrolysis reaction
system under the action of a microbial enzyme, thereby converting
the glycinonitrile to glycine while by-producing ammonia. US
20040157290 discloses a process for preparing a serine-rich foreign
protein comprising culturing a bacterium containing the cysteine
synthase (cysK) gene and a gene encoding the foreign protein. US
20030079255 disclose the production of Para-hydroxycinnamic acid by
introducing genes encoding phenylalanine ammonia-lyase from C.
violaceum or R. glutinis tyrosine into a host microorganism and as
intermediates, tyrosine and cinnamic acid are also produced.
[14830] Production of single cell protein and selected amino acids
by microbial fermentation is known, e.g., U.S. Pat. No. 4,652,527.
One amino acid which has been produced on an industrial scale is
lysine, see Tosaka et al., Trends in Biotechnology, 1: 70-74
(1983), Tosaka and Takinami, Progress in Industrial Microbiology,
Ch. 24, pp. 152-172 (Aida et al., 1986). Another example is
glutamic acid which has been produced using bacteria of the genera
Corynebacterium, Brevibacterium, Microbacterium, and Arothrobacter
by fermentation on molasses and starch hydrozylates. Aspartic acid
and alanine are produced by enzymatic means from fumaric acid and
ammonia. Bacillus species have been used in fermentation processes
to produce amino acids, Tosaka et al.; Tosaka and Takinami, as
named above.
[14831] [0009.0.36.36] As described above, the amino acids are
necessary for humans and many mammals, for example for livestock or
other applications in the health care area. L-aspartic acid is one
of the amino acids that is difficult to produce directly by
fermentation. Consequently today the enzyme catalyzed addition of
ammonia to fumaric acid is employed commercially in the production
of aspartic acid. As chelating agents, amino acids increase the
biological availability of different metals. Chelating agents in
general can enhance nutrient uptake, but may also increase the
uptake of toxic metals if those are also present. If cation
impurities are present in micronutrient sources (e.g. Cadmium),
chelation of those metals would make those contaminants more
readily assimilated by plants than in less available forms.
[14832] [0010.0.0.36] to [0011.0.0.36]: see [0010.0.0.27] to
[0011.0.0.27]
[14833] [0012.0.36.36] It is an object of the present invention to
develop an inexpensive process for the synthesis of 5-oxoproline,
alanine, aspartic acid, citrulline, glycine, phenylalanine, serine
and/or tyrosine. Amino acids are (depending on the organism) one of
the most frequently limiting components of food or feed.
[14834] [0013.0.0.36] see [0013.0.0.27]
[14835] [0014.0.36.36] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine. Accordingly, in the present invention, the term "the fine
chemical" as used herein relates to "5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine". Further, the term "the fine chemicals" as used herein
also relates to fine chemicals comprising 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine.
[14836] [0015.0.36.36] In one embodiment, the term "the fine
chemical" or "the respective fine chemical" means 5-oxoproline,
alanine, aspartic acid, citrulline, glycine, phenylalanine, serine
and/or tyrosine, preferably the amino acid of the present invention
in the L configuration, meaning L-5-oxoproline, L-alanine,
L-aspartic acid, L-citrulline, L-glycine, L-phenylalanine, L-serine
and/or L-tyrosine. Throughout the specification the term "the fine
chemical" means 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, phenylalanine, serine and/or tyrosine, preferably the
amino acid of the present invention in the L configuration, its
salts, ester or amids in free form or bound to proteins. In a
preferred embodiment, the term "the fine chemical" means
L-5-oxoproline, L-alanine, L-aspartic acid, L-citrulline,
L-glycine, L-phenylalanine, L-serine and/or L-tyrosine in free form
or its salts or bound to proteins.
[14837] [0016.0.36.36] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [14838] (a) increasing or generating the activity of one
or more of a protein as shown in table XII, application no. 36,
column 3 encoded by the nucleic acid sequences as shown in table
XI, application no. 36, column 5, in a non-human organism or in one
or more parts thereof or [14839] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
amino acids of the invention or fine chemicals comprising amino
acids of the invention, in said organism or in the culture medium
surrounding the organism.
[14840] [0016.1.36.36] Accordingly, the term "the fine chemical"
means in one embodiment "5-oxoproline" in relation to all sequences
listed in Table XI to XIV, application no. 36 line 24 or homologs
thereof and
means in one embodiment "alanine" in relation to all sequences
listed in Tables XI to XIV, lines 25 and/or 26 or homologs thereof
and means in one embodiment "aspartic acid" in relation to all
sequences listed in Table XI to XIV, lines 27 and/or 28, or
homologs thereof and means in one embodiment "citrulline" in
relation to all sequences listed in Table XI to XIV, lines 22
and/or 29 to 30 or homologs thereof and means in one embodiment
"glycine" in relation to all sequences listed in Table XI to XIV,
line 31 or homologs thereof and means in one embodiment
"phenylalanine" in relation to all sequences listed in Table XI to
XIV, lines 32 and/or 33 or homologs thereof and means in one
embodiment "serine" in relation to all sequences listed in Table XI
to XIV, line 23 or homologs thereof and means in one embodiment
"tyrosine" in relation to all sequences listed in Table XI to XIV,
line 34 or homologs thereof.
[14841] Accordingly, in one embodiment the term "the fine chemical"
means "5-oxoproline" and "alanine", "5-oxoproline" and "aspartic
acid", "aspartic acid" and "alanine" and/or "5-oxoproline" and
"alanine" and "aspartic acid" in relation to all sequences listed
in Table XI to XIV, lines 24, 25 and/or 27; in one embodiment the
term "the fine chemical" means "phenylalanineand "tyrosine" in
relation to all sequences listed in Table XI to XIV, lines 33
and/or 34; in one embodiment the term "the fine chemical" means one
fine chemical or any combination of two or three fine chemicals
selected from the group consisting of "glycine", and
"phenylalanine" and "alanine" and "aspartic acid", in relation to
all sequences listed in Table XI to XIV, lines 26, 28, 31 and/or
32;
[14842] Accordingly, the term "the fine chemical" can mean
"5-oxoproline", "alanine", "aspartic acid", "citrulline",
"glycine", " ", "phenylalanine", "serine" and/or "tyrosine", owing
to circumstances and the context. In order to illustrate that the
meaning of the term "the fine chemical" means "5-oxoproline",
"alanine", "aspartic acid", "citrulline", "glycine", " ",
"phenylalanine", "serine" and/or "tyrosine" the term "the
respective fine chemical" is also used.
[14843] [0017.0.0.36] to [0019.0.0.36]: see [0017.0.0.27] to
[0019.0.0.27]
[14844] [0020.0.36.36] Surprisingly it was found, that the
transgenic expression of the Zea mays protein as indicated in Table
XII, column 5, line 22 in a plant conferred an increase in
citrulline content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of citrulline.
[14845] Surprisingly it was found, that the transgenic expression
of the Zea mays protein as indicated in Table XII, column 5, line
23 in plant conferred an increase in serine content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of serine.
[14846] Surprisingly it was found, that the transgenic expression
of the Linum usitatissimum protein as indicated in Table XII,
column 5, line 24, 25 and/or 27 in a plant conferred an increase in
5-oxoplroline, alanine and/or aspartic acid content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of 5-oxoproline in one
embodiment, said protein or its homologs are used for the
production of alanine, in one embodiment, said protein or its
homologs are used for the production of aspartic acid, in one
embodiment, said protein or its homologs are used for the
production of one or more fine chemical selected from the group
consisting of: 5-oxoproline, alanine and/or aspartic acid.
[14847] Surprisingly it was found, that the transgenic expression
of the Hordeum vulgare protein as indicated in Table XII, column 5,
lines 26, 28, 31 and/or 32 in a plant conferred an increase in
alanine, aspartic acid, glycine and/or phenylalanine. content of
the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of alaninein one
embodiment, said protein or its homologs are used for the
production of aspartic aicd, in one embodiment, said protein or its
homologs are used for the production of glycine, in one embodiment,
said protein or its homologs are used for the production of
phenylalanine, in one embodiment, said protein or its homologs are
used for the production of one or more fine chemical selected from
the group consisting of: alanine, aspartic acid, glycine and/or
phenylalanine.
[14848] Surprisingly it was found, that the transgenic expression
of the Brassica napus protein as indicated in Table XII, column 5,
line 29 in Arabidopsis thaliana conferred an increase in citrulline
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of
citrulline.
[14849] Surprisingly it was found, that the transgenic expression
of the Helianthus annuus protein as indicated in Table XII, column
5, lines 30 in thaliana plant conferred an increase in citrulline
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of
citrulline.
[14850] Surprisingly it was found, that the transgenic expression
of the Hordeum vulgare protein as indicated in Table XII, column 5,
lines 33 and 34 in thaliana plant conferred an increase in
phenylalanine and/or tyrosine (or the respective fine chemical)
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of
phenylalanine; in one embodiment, said protein or its homologs are
used for the production of tyrosine, in one embodiment, said
protein or its homologs are used for the production of one or more
fine chemical selected from the group consisting of: phenylalanine
and/or tyrosine.
[14851] [0021.0.0.36] see [0021.0.0.27]
[14852] [0022.0.36.36] The sequence of YOR245C from Saccharomyces
cerevisiae has been published in Dujon, et al., Nature 387 (6632
Suppl), 98-102 (1997), and Goffeau, Science 274 (5287), 546-547,
1996 and its activity is being defined as a Acyl-CoA:diacylglycerol
acyltransferase, preferably of the Caenorhabditis elegans
hypothetical protein KO7B1.4 superfamily. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a gene product with an activity of Caenorhabditis elegans
hypothetical protein KO7B1.4 superfamily, preferably such protein
is having a Acyl-CoA:diacylglycerol acyltransferase activity or its
homolog, e.g. as shown herein, for the production of the the
respective fine chemical, meaning of citrulline, in particular for
increasing the amount of citrulline in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of a
Acyl-CoA:diacylglycerol acyltransferase is increased or generated,
e.g. from Saccharomyces cerevisiae or a plant or a homolog thereof.
The sequence of YKR057W from Saccharomyces cerevisiae has been
published in Goffeau, Science 274 (5287), 546-547 (1996) and Dujon,
Nature 369 (6479), 371-378 (1994) and its activity is being defined
as a ribosomal protein, similar to S21 ribosomal proteins, involved
in ribosome biogenesis and translation, preferably of the rat
ribosomal protein S21 superfamily. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of the rat ribosomal protein S21
superfamily, preferably such protein is having a ribosomal protein,
similar to S21 ribosomal proteins, involved in ribosome biogenesis
and translation activity or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, meaning of serine,
in particular for increasing the amount of serine, preferably of
serine in free or bound form in an organism or a part thereof, as
mentioned. In one embodiment, in the process of the present
invention the activity of a ribosomal protein, similar to S21
ribosomal proteins, involved in ribosome biogenesis and translation
is increased or generated, e.g. from Saccharomyces cerevisiae or a
plant or a homolog thereof.
[14853] The sequence of b1343 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a ATP-dependent RNA helicase,
stimulated by 23S rRNA. Accordingly, in one embodiment, the process
of the present invention comprises the use of a gene product with
an activity of the ATP-dependent RNA helicase, stimulated by 23S
rRNA from E. coli or from a plant or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, meaning
of alanine, 5-oxoproline and/or aspartic acid, in particular for
increasing the amount of alanine, in particular for increasing the
amount of 5-oxoproline, in particular for increasing the amount of
aspartic acid, in particular for increasing the amount of alanine
and 5-oxoproline, in particular for increasing the amount of
alanine and aspartic acid, in particular for increasing the amount
of 5-oxoproline and aspartic acid, in particular for increasing the
amount of alanine and 5-oxoproline and aspartic acid, preferably of
alanine, 5-oxoproline and/or aspartic acid in free or bound form in
an organism or a part thereof, as mentioned.
[14854] The sequence of b2426 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a putative oxidoreductase with
NAD(P)-binding domain, preferably of the ribitol dehydrogenase,
short-chain alcohol dehydrogenase homology superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of a
putative oxidoreductase with NAD(P)-binding domain from E. coli or
from a plant or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of citrulline,
in particular for increasing the amount of citrulline, preferably
citrulline in free or bound form in an organism or a part thereof,
as mentioned.
[14855] The sequence of b2576 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a ATP-dependent RNA helicase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the ATP-dependent RNA helicase from E. coli or from a plant or its
homolog, e.g. as shown herein, for the production of the respective
fine chemical, meaning of alanine, glycine, aspartic acid and/or
phenylalanine, in particular for increasing the amount of alanine,
in particular for increasing the amount of glycine, in particular
for increasing the amount of aspartic acid, in particular for
increasing the amount of phenylalanine, in particular for
increasing the amount of alanine and glycine, in particular for
increasing the amount of alanine and aspartic acid, in particular
for increasing the amount of alanine and phenylalanine, in
particular for increasing the amount of glycine and aspartic acid,
in particular for increasing the amount of glycine and
phenylalanine, in particular for increasing the amount of aspartic
acid and phenylalanine, in particular for increasing the amount of
alanine and glycine and aspartic acid, in particular for increasing
the amount of alanine and glycine and phenylalanine, in particular
for increasing the amount of alanine and aspartic acid and
phenylalanine, in particular for increasing the amount of glycine
and aspartic acid and phenylalanine, in particular for increasing
the amount of alanine and glycine and aspartic acid and
phenylalanine, preferably of alanine, glycine, aspartic acid and/or
phenylalanine in free or bound form in an organism or a part
thereof, as mentioned.
[14856] The sequence of b3983 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity has been defined as a 50S ribosomal subunit
protein L12, preferably of the Escherichia coli ribosomal protein
L11 superfamily. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of the 50S ribosomal subunit protein L12 from E. coli or
from a plant or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of
phenylalanine and/or tyrosine, in particular for increasing the
amount of phenylalanine, in particular for increasing the amount of
tyrosine, in particular for increasing the amount of phenylalanine
and tyrosine, preferably of phenylalanine and/or tyrosine in free
or bound form in an organism or a part thereof, as mentioned.
[14857] The sequence of b4269 from Escherichia coli K12 has been
published in Blattner et al., Science 277 (5331), 1453-1474, 1997,
and its activity is being defined as a putative alcohol
dehydrogenase with NAD(P)-binding and GroES domains. Accordingly,
in one embodiment, the process of the present invention comprises
the use of a gene product with an activity of a putative alcohol
dehydrogenase with NAD(P)-binding and GroES domains from E. coli or
from a plant or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, meaning of citrulline,
in particular for increasing the amount of citrulline, preferably
citrulline in free or bound form in an organism or a part thereof,
as mentioned.
[14858] [0022.1.0.36] to [0023.0.0.36] see [0022.1.0.27] to
[0023.0.0.27]
[14859] [0023.1.36.36] Homologs of the polypeptide disclosed in
table XII, application no. 36, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 36, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 36, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 36,
column 7, resp.
[14860] [0024.0.0.36] see [0024.0.0.27]
[14861] [0025.0.36.36] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 36, column 3 "if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 36, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said organism.
Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 36,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 36,
column 3 of of a E. coli or
[14862] Saccharomyces cerevisae, respectively, protein as mentioned
in table XI to XIV, column 3 respectively and as disclosed in
paragraph [0022] of the respective invention.
[14863] [0025.1.0.36] to [0033.0.0.36]: see [0025.1.0.27] to
[0033.0.0.27]
[14864] [0034.0.36.36] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 36, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[14865] [0035.0.0.36] to [0044.0.0.36]: see [0035.0.0.27] to
[0044.0.0.27]
[14866] [0045.0.36.36] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
36, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line,
between 10% and 50%, 10% and 100%, 10% and 200%, 10% and 300%, 10%
and 400%, 10% and 500%, 20% and 50%, 20% and 250%, 20% and 500%,
50% and 100%, 50% and 250%, 50% and 500%, 500% and 1000% or
more
[14867] [0046.0.36.36] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
36, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 36, column 6 of
the respective line confers an increase of the respective fine
chemical and of further amino acids or their precursors.
[14868] [0047.0.0.36] to [0048.0.0.36]: see [0047.0.0.27] to
[0048.0.0.27]
[14869] [0049.0.36.36] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 36, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 36, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 36, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[14870] [0050.0.36.36] For the purposes of the present invention
"the respective fine chemical" also encompass the corresponding
salts, such as, for example, the potassium, amonium or sodium salts
of the respective fine chemical or the respective amino acid
hydrochloride or sulfateof 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, phenylalanine, serine and/or tyrosine.
[14871] [0051.0.0.36] to [0052.0.0.36]: see [0051.0.0.27] to
[0052.0.0.27]
[14872] [0053.0.36.36] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[14873] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 36, columns 5 and 7 or its homologs activity
having herein-mentioned amino acids of the invention increasing
activity; and/or [14874] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 36,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 36, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned amino acids of the invention increasing
activity; and/or [14875] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned amino acid increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 36, columns 5 and 7 or its
homologs activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [14876] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned amino acids of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 36, columns 5
and 7 or its homologs activity; and/or [14877] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned amino acids of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 36, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [14878] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned amino acids of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 36, columns 5 and 7 or its
homologs activity, and/or [14879] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned amino acids of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 36, columns 5 and 7 or its
homologs activity; and/or [14880] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 36, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[14881] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [14882] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[14883] [0054.0.36.36] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 36 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 36, column 3 or its homologs.
[14884] [0055.0.0.36] to [0071.0.0.36]: see [0055.0.0.27] to
[0071.0.0.27]
[14885] [0072.0.36.36] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, phenylalanine, serine and/or tyrosine further amino acids
or the respective precursors.
[14886] [0073.0.36.36] Accordingly, in one embodiment, the process
according to the invention relates to a process which comprises:
[14887] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [14888] b) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table XII, application no. 36, columns 5 or 7, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, i.e. conferring an increase
of the respective fine chemical in the organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [14889] c) growing the organism,
preferably a microorganism, a non-human animal, a plant or animal
cell, a plant or animal tissue or a plant, under conditions which
permit the production of the respective fine chemical in the
organism, preferably the microorganism, the plant cell, the plant
tissue or the plant; and [14890] d) if desired, recovering,
optionally isolating, the free and/or bound the respective fine
chemical as indicated in any one of Tables XI to XIV, application
no. 36, column 6 "metabolite" and, optionally further free and/or
bound amino acids synthetized by the organism, the microorganism,
the non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[14891] [0074.0.0.36] to [0084.0.0.36]: see [0074.0.0.27] to
[0084.0.0.27]
[14892] [0085.0.36.36] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [14893] a) a nucleic acid sequence as
indicated in Table XI, application no. 36, columns 5 or 7, or a
derivative thereof, or [14894] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 36, columns
5 or 7, or a derivative thereof, or [14895] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[14896] [0086.0.0.36] to [0088.1.0.36]: see [0086.0.0.27] to
[0088.1.0.27]
[14897] [0089.0.0.36] to [0097.0.0.36]: see [0089.0.0.27] to
[0097.0.0.27]
[14898] [0098.0.36.36] In a preferred embodiment, the respective
fine chemical (5-oxoproline, alanine, aspartic acid, citrulline,
glycine, phenylalanine, serine and/or tyrosine) are produced in
accordance with the invention and, if desired, are isolated. The
production of further amino acids and of amino acid mixtures or
mixtures with other compounds by the process according to the
invention is advantageous.
[14899] [0099.0.0.36] to [0102.0.0.36]: see [0099.0.0.27] to
[0102.0.0.27]
[14900] [0103.0.36.36] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [14901] a) nucleic acid molecule encoding,
preferably at least the mature form, of a polypeptide having a
sequence as indicated in Table XII, application no. 36, columns 5
or 7, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [14902] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table XI, application no. 36, columns 5 or 7.;
[14903] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [14904] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[14905] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [14906]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [14907] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [14908] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers pairs
having a sequence as indicated in Table XIII, application no. 36,
columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [14909]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [14910] nucleic acid
molecule which encodes a polypeptide comprising the consensus
columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [14911]
k) nucleic acid molecule comprising one or more of the nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domaine of a polypeptide indicated in Table XII, application no.
36, columns 5 or 7 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[14912] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[14913] [00103.1.36.36.] In one embodiment, the nucleic acid
molecule used in the process of the invention distinguishes over
the sequence indicated in Table XI, application no. 36, columns 5
or 7 by one or more nucleotides. In one embodiment, the nucleic
acid molecule used in the process of the invention does not consist
of the sequence shown in indicated in Table XI, application no. 36,
columns 5 or 7. In one embodiment, the nucleic acid molecule used
in the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI,
application no. 36, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table XII, application no. 36, columns 5 or 7.
[14914] [0104.0.36.36] In one embodiment, the nucleic acid molecule
of the invention distinguishes over the sequence indicated in Table
XI, application no. 36, columns 5 or 7, by one or more nucleotides.
In one embodiment, the nucleic acid molecule of the invention does
not consist of the sequence shown in indicated in Table XI,
application no. 36, columns 5 or 7. In one embodiment, the nucleic
acid molecule of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to a sequence indicated in Table XI,
application no. 36, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table XII, application no. 36, columns 5 or 7.
[14915] [0105.0.0.36] to [0107.0.0.36]: see [0105.0.0.27] to
[0107.0.0.27]
[14916] [0108.0.36.36] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 36, columns 5 or 7, nucleic
acid molecules which are derived from a amino acid sequences as
indicated in Table XII, application no. 36, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 36, columns 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of a polypeptide as indicated in Table XI, application no.
36, column 3, 5 or 7, or e.g. conferring a increase of the
respective fine chemical after increasing its expression or
activity are advantageously increased in the process according to
the invention.
[14917] [0109.0.0.36]: see [0109.0.0.27]
[14918] [0110.0.36.36] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
used in the process of the invention, e.g. of a protein as
indicated in Table XII, application no. 36, column 5, being encoded
by a nucleic acid molecule indicated in Table XI, application no.
36, column 5, or of its homologs, e.g. as indicated in Table XII,
application no. 36, column 7 can be determined from generally
accessible databases.
[14919] [0111.0.0.36]: see [0111.0.0.27]
[14920] [0112.0.36.36] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XI, application no. 36,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 36, columns 5 and 7, and conferring a
5-oxoproline and/or alanine and/or aspartic acid and/or citrulline
and/or glycine and/or phenylalanine and/or serine and/or tyrosine
increase.
[14921] [0113.0.0.36] to [0120.0.0.36]: see [0113.0.0.27] to
[0120.0.0.27]
[14922] [0121.0.36.36] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 36, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a increase of the respective fine
chemical after increasing its activity.
[14923] [0122.0.0.36] to [0127.0.0.36]: see [0122.0.0.27] to
[0127.0.0.27]
[14924] [0128.0.36.36] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 36, column 7, by means of polymerase chain reaction
can be generated on the basis of a sequence shown herein, for
example the sequence as indicated in Table XI, application no. 36,
columns 5 or 7, or the sequences derived from sequences as
indicated in Table XII, application no. 36, columns 5 or 7.
[14925] [0129.0.36.36] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequences indicated in Table XIV,
application no. 36, column 7, are derived from said alignments.
[14926] [0130.0.36.36] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of amino
acids, e.g. of 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, serine and/or tyrosine after increasing its expression or
activity of the protein comprising said fragment.
[14927] [0131.0.0.36] to [0138.0.0.36]: see [0131.0.0.27] to
[0138.0.0.27]
[14928] [0139.0.36.36] Polypeptides having above-mentioned
activity, i.e. conferring an increase of the respective fine
chemical level, derived from other organisms, can be encoded by
other DNA sequences which hybridize to a sequences indicated in
Table XI, application no. 36, columns 5 or 7, under relaxed
hybridization conditions and which code on expression for peptides
having the respective fine chemical, in particular, of 5-oxoproline
and/or alanine and/or aspartic acid and/or citrulline and/or
glycine and/or phenylalanine and/or serine and/or tyrosine resp.,
increasing activity.
[14929] [0140.0.0.36] to [0146.0.0.36]: see [0140.0.0.27] to
[0146.0.0.27]
[14930] [0147.0.36.36] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 36, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences s
such that it can hybridize to one of said nucleotide sequences
thereby forming a stable duplex. Preferably, the hybridisation is
performed under stringent hybridization conditions. However, a
complement of one of the herein disclosed sequences is preferably a
sequence complement thereto according to the base pairing of
nucleic acid molecules well known to the skilled person. For
example, the bases A and G undergo base pairing with the bases T
and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[14931] [0148.0.36.36] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 36,
columns 5 or 7 or a portion thereof and preferably has above
mentioned activity, in particular, of arginine and/or glutamate
and/or proline and/or glutamine increasing activity after
increasing the activity or an activity of a product of a gene
encoding said sequences or their homologs.
[14932] [0149.0.36.36] The nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 36,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity and as indicated in indicated in Table
XII.
[14933] [0150.0.36.36] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table XI, application no. 36, columns
5 or 7, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of arginine and/or glutamate
and/or proline and/or glutamine, resp., if its activity is
increased. The nucleotide sequences determined from the cloning of
the present protein-according-to-the-invention-encoding gene allows
for the generation of probes and primers designed for use in
identifying and/or cloning its homologues in other cell types and
organisms. The probe/primer typically comprises substantially
purified oligonucleotide. The oligonucleotide typically comprises a
region of nucleotide sequence that hybridizes under stringent
conditions to at least about 12, 15 preferably about 20 or 25, more
preferably about 40, 50 or 75 consecutive nucleotides of a sense
strand of one of the sequences set forth, e.g., as indicated in
Table XI, application no. 36, columns 5 or 7, anti-sense sequence
of one of the sequences, e.g., as indicated in Table XI,
application no. 36, columns 5 or 7, or naturally occurring mutants
thereof. Primers based on a nucleotide of invention can be used in
PCR reactions to clone homologues of the polypeptide of the
invention or of the polypeptide used in the process of the
invention, e.g. as the primers described in the examples of the
present invention, e.g. as shown in the examples. A PCR with the
primer pairs indicated in Table XIII, application no. 36, column 7,
will result in a fragment of a polynucleotide sequence as indicated
in Table XI, application no. 36, columns 5 or 7 or its gene
product.
[14934] [0151.0.0.36] see [0151.0.0.27]
[14935] [0152.0.36.36] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 36, columns 5
or 7, such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular an activity increasing the level of 5-oxoproline
and/or alanine and/or aspartic acid and/or citrulline and/or
glycine and/or phenylalanine and/or serine and/or tyrosine as
mentioned above or as described in the examples in plants or
microorganisms is comprised.
[14936] [0153.0.36.36] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XI, application no. 36, columns
5 or 7, such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 36, columns 5 or 7, has for
example an activity of a polypeptide indicated in Table XII,
application no. 36, column 3.
[14937] [0154.0.36.36] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 36, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[14938] [0155.0.0.36] to [0156.0.0.36]: see [0155.0.0.27] to
[0156.0.0.27]
[14939] [0157.0.36.36] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences
indicated in Table XI, application no. 36, columns 5 or 7 (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides comprising the consensus sequences as indicated
in Table XIV, application no. 36, column 7 or of the polypeptide as
indicated in Table XII, application no. 36, columns 5 or 7 or their
functional homologues. Advantageously, the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, a consensus sequences as indicated in Table XIV,
application no. 36, column 7, or of the polypeptide as indicated in
Table XII, application no. 36, columns 5 or 7, or the functional
homologues. In a still further embodiment, the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention encodes a full length protein which is
substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table XIV, application no. 36,
column 7, or of a polypeptide as indicated in Table XII,
application no. 36, columns 5 or 7, or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 36, columns 5 or 7.
Preferably the nucleic acid molecule of the invention is a
functional homologue or identical to a nucleic acid molecule
indicated in Table XI, application no. 36, columns 5 or 7.
[14940] [0158.0.0.36] to [0160.0.0.36]: see [0158.0.0.27] to
[0160.0.0.27]
[14941] [0161.0.36.36] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 36, columns 5 or
7. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[14942] [0162.0.0.36] see [0162.0.0.27]
[14943] [0163.0.36.36] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 36, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of an protein of the invention or used in
the process of the invention.
[14944] [0164.0.0.36] see [0164.0.0.27]
[14945] [0165.0.36.36] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 36, columns 5 or 7.
[14946] [0166.0.0.36] to [0167.0.0.36]: see [0166.0.0.27] to
[0167.0.0.27]
[14947] [0168.0.36.36] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 36, columns 5 or 7, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 36,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table XII, application no. 36, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 36, columns 5
or 7, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table XII, application no.
36, columns 5 or 7, and most preferably at least about 96%, 97%,
98%, or 99% identical to the sequence as indicated in Table XII,
application no. 36, columns 5 or 7.
[14948] [0169.0.0.36] to [0172.0.0.36]: see [0169.0.0.27] to
[0172.0.0.27]
[14949] [0173.0.36.36] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108476 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108476 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[14950] [0174.0.0.36] see [0174.0.0.27]
[14951] [0175.0.36.36] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108477 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108477 by the above program algorithm with the
above parameter set, has a 80% homology.
[14952] [0176.0.36.36] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 36,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 36, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 36, columns
5 or 7.
[14953] [0177.0.36.36] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 36,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptide as indicated in Table XII,
application no. 36, columns 5 or according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 36, columns
5 or 7.
[14954] [0178.0.0.36]: see [0178.0.0.27]
[14955] [0179.0.36.36] A nucleic acid molecule encoding an
homologous to a protein sequence as indicated in Table XII,
application no. 36, columns 5 or 7, can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table XI, application no.
36, columns 5 or 7, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences of a
sequences as indicated in Table XI, application no. 36, columns 5
or 7, by standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[14956] [0180.0.0.36] to [0183.0.0.36]: see [0180.0.0.27] to
[0183.0.0.27]
[14957] [0184.0.36.36] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 36,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 36, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 36,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[14958] [0185.0.36.36] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 36, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table XI, application no. 36, columns 5 or 7. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequences as indicated in Table XI, application no. 36, columns 5
or 7,
[14959] Also preferred is that one or more nucleic acid molecule(s)
used in the process of the invention encodes a polypeptide
comprising a sequence as indicated in Table XII, application no.
36, columns 5 or 7. In one embodiment, the nucleic acid molecule
encodes less than 150, 130, 100, 80, 60, 50, 40 or 30 further amino
acids. In a further embodiment, the encoded polypeptide comprises
less than 20, 15, 10, 9, 8, 7, 6 or 5 further amino acids. In one
embodiment, the encoded polypeptide used in the process of the
invention is identical to the sequences as indicated in Table XII,
application no. 36, columns 5 or 7.
[14960] [0187.0.36.36] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table XII, application no.
36, columns 5 or 7, comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence as
indicated in Table XII, application no. 36, columns 5 or 7.
[14961] [0188.0.36.36] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 36, columns 5 or 7 and is expressed under
identical conditions.
[14962] [0189.0.36.36] Homologues of a sequences as indicated in
Table XI, application no. 36, columns 5 or 7, or of a derived
sequences as indicated in Table XII, application no. 36, columns 5
or 7 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[14963] [0190.0.0.36] to [0203.0.0.36]: see [0190.0.0.27] to
[0203.0.0.27]
[14964] [0204.0.36.36] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule which comprises a
nucleic acid molecule selected from the group consisting of:
[14965] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 36, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII, application no. 36,
column 6 in an organism or a part thereof [14966] b) nucleic acid
molecule comprising, preferably at least the mature form, of a
nucleic acid molecule as indicated in Table XI, application no. 36,
columns 5 or 7, or a fragment thereof conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [14967] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [14968] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[14969] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [14970] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [14971] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to [14972]
(c) and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [14973] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table XIII, application no. 36, column 7, and
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII B, application no.
36, column 6 in an organism or a part thereof; [14974] i) nucleic
acid molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[14975] j) nucleic acid molecule which encodes a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 36, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical, in particularaccording
to table XII, application no. 36, column 6 in an organism or a part
thereof; [14976] k) nucleic acid molecule encoding the amino acid
sequence of a polypeptide encoding a domaine of a polypeptide as
indicated in Table XII, application no. 36, columns 5 or 7, and
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII, application no. 36,
column 6 in an organism or a part thereof; and [14977] l) nucleic
acid molecule which is obtainable by screening a suitable nucleic
acid library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment of at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (h) or of a nucleic acid molecule as
indicated in Table XI, application no. 36, columns 5 or 7, or a
nucleic acid molecule encoding, preferably at least the mature form
of, a polypeptide as indicated in Table XII, application no. 36,
columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical according to table XII B, application no.
36, column 6 in an organism or a part thereof; or which encompasses
a sequence which is complementary thereto; whereby, preferably, the
nucleic acid molecule according to (a) to (l) distinguishes over
the sequence indicated in Table XI, application no. 36, columns 5
or 7, by one or more nucleotides. In one embodiment, the nucleic
acid molecule does not consist of the sequence shown and indicated
in Table XI, application no. 36, columns 5 or 7, In one embodiment,
the nucleic acid molecule is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence indicated in Table XI, application
no. 36, columns 5 or 7.
[14978] In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table XII,
application no. 36, columns 5 or 7.
[14979] In an other embodiment, the nucleic acid molecule of the
present invention is at least 30%, 40%, 50%, or 60% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 36, columns 5 or
7.
[14980] In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table XII,
application no. 36, columns 5 or 7.
[14981] Accordingly, in one embodiment, the nucleic acid molecule
of the differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 36, columns 5 or
7.
[14982] Accordingly, in one embodiment, the nucleic acid molecule
of the present invention encodes a polypeptide, which differs at
least in one or more amino acids from a polypeptide indicated in
Table XII, application no. 36, columns 5 or 7.
[14983] In another embodiment, a nucleic acid molecule indicated in
Table XI, application no. 36, columns 5 or 7.
[14984] Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table XII, application no.
36, columns 5 or 7.
[14985] In a further embodiment, the protein of the present
invention is at least 30%, 40%, 50%, or 60% identical to a protein
sequence indicated in Table XII, application no. 36, more
preferably less than 99%, 985, 97%, 96% or 95% identical to a
sequence as indicated in Table XI, columns 5 or 7.
[14986] [0205.0.0.36] to [0226.0.0.36]: see [0205.0.0.27] to
[0226.0.0.27]
[14987] [0227.0.36.36] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[14988] In addition to a sequence indicated in Table XI,
application no. 36, columns 5 or 7, or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the amino acid biosynthetic pathway such as for
amino acid precursors is expressed in the organisms such as plants
or microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the amino acids desired since, for example, feedback
regulations no longer exist to the same extent or not at all. In
addition it might be advantageously to combine a sequence as
indicated in Table XI, application no. 36, columns 5 or 7, with
genes which generally support or enhances to growth or yield of the
target organisms, for example genes which lead to faster growth
rate of microorganisms or genes which produces stress-, pathogen,
or herbicide resistant plants.
[14989] [0228.0.0.36] to [0230.0.0.36]: see [0228.0.0.27] to
[0230.0.0.27]
[14990] [00231.0.36.36] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a protein degrading 5-oxoproline
and/or alanine and/or aspartic acid and/or citrulline and/or
glycine and/or phenylalanine and/or serine and/or tyrosine resp.,
is attenuated, in particular by reducing the rate of expression of
the corresponding gene.
[14991] [0232.0.0.36] to [0282.0.0.36]: see [0232.0.0.27] to
[0282.0.0.27]
[14992] [0283.0.36.36] Moreover, a native polypeptide conferring
the increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table
XII, application no. 36, column 3, or an antibody against a
polypeptide as indicated in Table XII, application no. 36, columns
5 or 7, which can be produced by standard techniques utilizing
polypeptides comprising or consisting of above mentioned sequences,
e.g. the polypeptid of the present invention or fragment thereof.
Preferred are monoclonal antibodies.
[14993] [0284.0.0.36] see [0284.0.0.27]
[14994] [0285.0.36.36] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 36, columns 5 or 7, or as coded by a nucleic
acid molecule as indicated in Table XI, application no. 36, columns
5 or 7, or functional homologues thereof.
[14995] [0286.0.36.36] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 36, column 7.
[14996] In another embodiment, the present invention relates to a
polypeptide comprising or consisting of a consensus sequence as
indicated in Table XIV, application no. 36, column 7, whereby 20 or
less, preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred
5 or 4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table XIV, application no. 36, column 7.
[14997] [0287.0.0.36] to [0290.0.0.36]: see [0287.0.0.27] to
[0290.0.0.27]
[14998] [0291.0.36.36] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[14999] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table XII,
application no. 36, columns 5 or 7, by one or more amino acids.
[15000] In one embodiment, polypeptide distinguishes form a
sequence as indicated in Table XII, application no. 36, columns 5
or 7, by more than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, evenmore
preferred are more than 40, 50, or 60 amino acids and, preferably,
the sequence of the polypeptide of the invention distinguishes from
a sequence as indicated in Table XII, application no. 36, columns 5
or 7, by not more than 80% or 70% of the amino acids, preferably
not more than 60% or 50%, more preferred not more than 40% or 30%,
even more preferred not more than 20% or 10%. In an other
embodiment, said polypeptide of the invention does not consist of a
sequence as indicated in Table XII A, application no. 36, columns 5
or 7.
[15001] [0292.0.0.36]: see [0292.0.0.27]
[15002] [0293.0.36.36] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, the polypeptide of
the invention has a sequence which distinguishes from a sequence as
indicated in Table XII, application no. 36, columns 5 or 7, by one
or more amino acids. In an other embodiment, said polypeptide of
the invention does not consist of the sequence as indicated in
Table XII, application no. 36, columns 5 or 7. In a further
embodiment, said polypeptide of the present invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical. In one embodiment,
said polypeptide does not consist of the sequence encoded by a
nucleic acid molecules as indicated in Table XI, application no.
36, columns 5 or 7.
[15003] [0294.0.36.36] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 36, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 36, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[15004] [0295.0.0.36] to [0297.0.0.36]: see [0295.0.0.27] to
[0297.0.0.27]
[15005] [0297.1.36.36] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table XII, application no. 36, columns 3, 5 or 7.
[15006] [0298.0.36.36] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 36, columns 5 or 7, such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table XII, application no. 36, columns 5 or 7.
[15007] [0299.0.36.36] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 36,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 36, columns 5 or 7, or which is
homologous thereto, as defined above.
[15008] [0300.0.36.36] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table XII,
application no. 36, columns 5 or 7, in amino acid sequence due to
natural variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprise an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence of a sequence as indicated in Table XII,
application no. 36, columns 5 or 7.
[15009] [0301.0.0.36] see [0301.0.0.27]
[15010] [0302.0.36.36] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 36, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[15011] [0303.0.0.36] see [0303.0.0.27]
[15012] [0304.0.36.36] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 36, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[15013] [0305.0.0.36] to [0308.0.0.36]: see [0305.0.0.27] to
[0308.0.0.27]
[15014] [0309.0.36.36] In one embodiment, an reference to a
"protein (=polypeptide)" of the invention or as indicated in Table
XII, application no. 36, columns 5 or 7, refers to a polypeptide
having an amino acid sequence corresponding to the polypeptide of
the invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table XII, application no. 36, columns 5 or 7, refers
to a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous a polypeptide of the
invention, preferably which is not substantially homologous to a as
indicated in Table XII, application no. 36, columns 5 or 7, e.g., a
protein which does not confer the activity described herein or
annotated or known for as indicated in Table XII, application no.
36, column 3, and which is derived from the same or a different
organism. In one embodiment, a "non-polypeptide of the invention"
or "other polypeptide" not being indicated in Table XII,
application no. 36, columns 5 or 7, does not confer an increase of
the respective fine chemical in an organism or part thereof.
[15015] [0310.0.0.36] to [0334.0.0.36]: see [0310.0.0.27] to
[0334.0.0.27]
[15016] [0335.0.36.36] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XI, application
no. 36, columns 5 or 7, and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
application no. 36, columns 5 or 7, and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of an
protein encoded by a nucleic acid sequence of one of the sequences
as indicated in Table Xl, application no. 36, columns 5 or 7,
and/or homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[15017] [0336.0.0.36] to [0342.0.0.36]: see [0336.0.0.27] to
[0342.0.0.27]
[15018] [0343.0.36.36] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 36, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table XI, application no. 36, columns 5 or 7, or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[15019] [0344.0.0.36] to [0361.0.0.36]: see [0344.0.0.27] to
[0361.0.0.27]
[15020] [0362.0.36.36] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 36, columns 5 or 7, e.g. encoding a
polypeptide having protein activity, as indicated in Table XII,
application no. 36, columns 3. Due to the above mentioned activity
the respective fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity of the polypeptide of the invention or nucleic
acid molecule of the invention is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a polypeptide as
indicated in Table XII, application no. 36, columns 5 or 7, means
herein that due to modulation or manipulation of the genome, an
activity as annotated for a polypeptide as indicated in Table XII,
application no. 36, column 3, e.g. having a sequence as indicated
in Table XII, application no. 36, columns 5 or 7 is increased in a
cell or an organism or a part thereof. Examples are described above
in context with the process of the invention
[15021] [0363.0.0.36] to [0384.0.0.36]: see [0363.0.0.27] to
[0384.0.0.27]
[15022] [0385.0.36.36] The fermentation broths obtained in this
way, containing in particular 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, phenylalanine, serine and/or tyrosine,
normally have a dry matter content of from 7.5 to 25% by weight.
Sugar-limited fermentation is additionally advantageous, at least
at the end, but especially over at least 30% of the fermentation
time. This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 3 g/l during
this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed entirely or
partly by separation methods, such as, for example, centrifugation,
filtration, decantation or a combination of these methods, from the
fermentation broth or left completely in it. The fermentation broth
can then be thickened or concentrated by known methods, such as,
for example, with the aid of a rotary evaporator, thin-film
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. This concentrated fermentation broth can then be
worked up by freeze-drying, spray drying, spray granulation or by
other processes.
[15023] An other method for purification the amino acids of the
invention, in particular 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, phenylalanine, serine and/or tyrosine is a
process by means of electrodialysis as described in U.S. Pat. No.
6,551,803.
[15024] [0386.0.0.36] to [0392.0.0.36]: see [0386.0.0.27] to
[0392.0.0.27]
[15025] [0393.0.36.36] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [15026] (a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [15027] (b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table XI, application no. 36, columns 5 or 7, and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[15028] (c) introducing the candidate nucleic acid molecules in
host cells, preferably in a plant cell or a microorganism,
appropriate for producing the fine chemical; [15029] (d) expressing
the identified nucleic acid molecules in the host cells; [15030]
(e) assaying the the fine chemical level in the host cells; and
[15031] (f) identifying the nucleic acid molecule and its gene
product which expression confers an increase in the the fine
chemical level in the host cell after expression compared to the
wild type.
[15032] [0394.0.0.36] to [0460.0.0.36]: see [0394.0.0.27] to
[0460.0.0.27]
[0461.0.36.36] Example 10
Cloning SEQ ID NO: 108476 for the Expression in Plants
[15033] [0462.0.0.36] see [0462.0.0.27]
[15034] [0463.0.36.36] SEQ ID NO: 108476 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[15035] [0464.0.0.36] to [0466.0.0.36]: see [0464.0.0.27] to
[0466.0.0.27]
[15036] [0467.0.36.36] The following primer sequences were selected
for the gene SEQ ID NO: 108476: [15037] i) forward primer SEQ ID
NO: 108478 [15038] ii) reverse primer SEQ ID NO: 108479
[15039] [0468.0.0.36] to [0479.0.0.36]: see [0468.0.0.27] to
[0479.0.0.27]
[0480.0.36.36] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 108476
[15040] [0481.0.0.36] to [0552.0.0.36]: see [0481.0.0.27] to
[0552.0.0.27]
[15041] [0553.0.36.36]
1. A process for the production of 5-oxoproline, alanine, aspartic
acid, citrulline, glycine, phenylalanine, serine and/or tyrosine
resp., which comprises [15042] (a) increasing or generating the
activity of a protein as indicated in Table XII, columns 5 or 7,
application no. 36 resp., or a functional equivalent thereof in a
non-human organism, or in one or more parts thereof; and [15043]
(b) growing the organism under conditions which permit the
production of 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, phenylalanine, serine and/or tyrosine resp. in said
organism. 2. A process for the production of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine resp., comprising the increasing or generating in an
organism or a part thereof the expression of at least one nucleic
acid molecule comprising a nucleic acid molecule selected from the
group consisting of: [15044] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table XII, columns 5 or 7, application
no. 36 resp., or a fragment thereof, which confers an increase in
the amount of ccchemical, e.g of 5-oxoproline, anine, aspartic
acid, citrulline, glycine, phenylalanine, serine and/or tyrosine
resp., in an organism or a part thereof; [15045] b) nucleic acid
molecule comprising of a nucleic acid molecule as indicated in
Table XI, application no. 36, columns 5 or 7, application no. 36
resp., [15046] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof; [15047] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical as indicated in
table XII, column 6, e.g of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, phenylalanine, serine and/or tyrosine resp.,
in an organism or a part thereof; [15048] e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
under stringent hybridisation conditions and conferring an increase
in the amount of the fine chemical as indicated in table XII,
column 6, e.g of 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, phenylalanine, serine and/or tyrosine resp., in an
organism or a part thereof; [15049] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table XIII,
application no. 36, column 7, and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g ofthe 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, phenylalanine, serine and/or tyrosine resp., in an
organism or a part thereof; [15050] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
the fine chemical as indicated in table XII, column 6, e.g of
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
phenylalanine, serine and/or tyrosine resp., in an organism or a
part thereof; [15051] h) nucleic acid molecule encoding a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 36, column 7, resp., and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, phenylalanine, serine and/or tyrosine resp.,
in an organism or a part thereof; and [15052] i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, phenylalanine, serine and/or tyrosine resp., in an
organism or a part thereof. [15053] or comprising a sequence which
is complementary thereto. 3. The process of claim 1 or 2,
comprising recovering of the free or bound 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine resp. 4. The process of any one of claims 1 to 3,
comprising the following steps: [15054] (a) selecting an organism
or a part thereof expressing a polypeptide encoded by the nucleic
acid molecule characterized in claim 2; [15055] (b) mutagenizing
the selected organism or the part thereof; [15056] (c) comparing
the activity or the expression level of said polypeptide in the
mutagenized organism or the part thereof with the activity or the
expression of said polypeptide of the selected organisms or the
part thereof; [15057] (d) selecting the mutated organisms or parts
thereof, which comprise an increased activity or expression level
of said polypeptide compared to the selected organism or the part
thereof; [15058] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [15059] (f) recovering, and
optionally isolating, the free or bound 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine resp., produced by the selected mutated organisms or parts
thereof. 5. The process of any one of claims 1 to 4, wherein the
activity of said protein or the expression of said nucleic acid
molecule is increased or generated transiently or stably. 6. An
isolated nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [15060] a) nucleic acid
molecule encoding of a polypeptide as indicated in Table XII,
application no. 36, columns 5 or 7., or a fragment thereof, which
confers an increase in the amount of the fine chemical as indicated
in table XII, column 6, e.g of 5-oxoproline, alanine, aspartic
acid, citrulline, glycine, phenylalanine, serine and/or tyrosine
resp., in an organism or a part thereof; [15061] b) nucleic acid
molecule comprising of a nucleic acid molecule as indicated in
Table XI, application no. 36, columns 5 or 7, resp., [15062] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof; [15063] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical as indicated in
table XII, column 6, e.g of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, phenylalanine, serine and/or tyrosine resp.,
in an organism or a part thereof; [15064] e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
under stringent hybridisation conditions and conferring an increase
in the amount of the fine chemical as indicated in table XII,
column 6, e.g of 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, phenylalanine, serine and/or tyrosine resp., in an
organism or a part thereof; [15065] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table XIII,
application no. 36, column 7, resp., and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, phenylalanine, serine and/or tyrosine resp., in an
organism or a part thereof; [15066] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
the fine chemical as indicated in table XII, column 6, e.g of
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
phenylalanine, serine and/or tyrosine resp., in an organism or a
part thereof; [15067] h) nucleic acid molecule encoding a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 36, column 7, resp., and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, phenylalanine, serine and/or tyrosine resp.,
in an organism or a part thereof; and [15068] i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, phenylalanine, serine and/or tyrosine resp., in an
organism or a part thereof. whereby the nucleic acid molecule
distinguishes over the sequence as indicated in Table XI,
application no. 36, columns 5 or 7 resp., by one or more
nucleotides. 7. A nucleic acid construct which confers the
expression of the nucleic acid molecule of claim 6, comprising one
or more regulatory elements. 8. A vector comprising the nucleic
acid molecule as claimed in claim 6 or the nucleic acid construct
of claim 7. 9. The vector as claimed in claim 8, wherein the
nucleic acid molecule is in operable linkage with regulatory
sequences for the expression in a prokaryotic or eukaryotic, or in
a prokaryotic and eukaryotic, host. 10. A host cell, which has been
transformed stably or transiently with the vector as claimed in
claim 8 or 9 or the nucleic acid molecule as claimed in claim 6 or
the nucleic acid construct of claim 7 or produced as described in
claim any one of claims 2 to 5. 11. The host cell of claim 10,
which is a transgenic host cell. 12. The host cell of claim 10 or
11, which is a plant cell, an animal cell, a microorganism, or a
yeast cell, a fungus cell, a prokaryotic cell, an eukaryotic cell
or an archaebacterium. 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. 14. A polypeptide produced by the
process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table XII,
application no. 36, columns 5 or 7, resp., by one or more amino
acids 15. An antibody, which binds specifically to the polypeptide
as claimed in claim 14. 16. A plant tissue, propagation material,
harvested material or a plant comprising the host cell as claimed
in claim 12 which is plant cell or an Agrobacterium. 17. A method
for screening for agonists and antagonists of the activity of a
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof comprising:
[15069] (a) contacting cells, tissues, plants or microorganisms
which express the a polypeptide encoded by the nucleic acid
molecule of claim 5 conferring an increase in the amount of
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
phenylalanine, serine and/or tyrosine resp., in an organism or a
part thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; [15070] (b) assaying the 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine resp., level or the polypeptide expression level in the
cell, tissue, plant or microorganism or the media the cell, tissue,
plant or microorganisms is cultured or maintained in; and [15071]
(c) identifying a agonist or antagonist by comparing the measured
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
phenylalanine, serine and/or tyrosine resp., level or polypeptide
expression level with a standard 5-oxoproline, alanine, aspartic
acid, citrulline, glycine, phenylalanine, serine and/or tyrosine
resp., or polypeptide expression level measured in the absence of
said candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. 18. A process for the identification
of a compound conferring increased 5-oxoproline, alanine, aspartic
acid, citrulline, glycine, phenylalanine, serine and/or tyrosine
resp., production in a plant or microorganism, comprising the
steps: [15072] (a) culturing a plant cell or tissue or
microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, phenylalanine, serine and/or tyrosine resp.,
in an organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with dais readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine resp., in an organism or a part thereof; [15073] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system.
19. A method for the identification of a gene product conferring an
increase in 5-oxoproline, alanine, aspartic acid, citrulline,
glycine, phenylalanine, serine and/or tyrosine resp., production in
a cell, comprising the following steps: [15074] (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
phenylalanine, serine and/or tyrosine resp., after expression with
the nucleic acid molecule of claim 6; [15075] (b) identifying the
nucleic acid molecules, which hybridise under relaxed stringent
conditions with the nucleic acid molecule of claim 6; [15076] (c)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, phenylalanine, serine and/or tyrosine resp.;
[15077] (d) expressing the identified nucleic acid molecules in the
host cells; [15078] (e) assaying the 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine resp., level in the host cells; and [15079] (f)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosine resp., level in the host cell in the host cell after
expression compared to the wild type. 20. A method for the
identification of a gene product conferring an increase in
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
phenylalanine, serine and/or tyrosine resp., production in a cell,
comprising the following steps: [15080] (a) identifying in a data
bank nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the 5-oxoproline, alanine, aspartic acid, citrulline, glycine,
phenylalanine, serine and/or tyrosine resp., amount or level in an
organism or a part thereof after expression, and which are at least
20% homolog to the nucleic acid molecule of claim 6; [15081] (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, phenylalanine, serine and/or tyrosine resp.;
[15082] (c) expressing the identified nucleic acid molecules in the
host cells; [15083] (d) assaying the 5-oxoproline, alanine,
aspartic acid, citrulline, glycine, phenylalanine, serine and/or
tyrosineresp., level in the host cells; and [15084] (e) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the 5-oxoproline, alanine, aspartic acid,
citrulline, glycine, phenylalanine, serine and/or tyrosine resp.,
level in the host cell after expression compared to the wild type.
21. A method for the production of an agricultural composition
comprising the steps of the method of any one of claims 17 to 20
and formulating the compound identified in any one of claims 17 to
20 in a form acceptable for an application in agriculture. 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. 23. Use of the
nucleic acid molecule as claimed in claim 6 for the identification
of a nucleic acid molecule conferring an increase of 5-oxoproline,
alanine, aspartic acid, citrulline, glycine, phenylalanine, serine
and/or tyrosine resp., after expression. 24. Use of the polypeptide
of claim 14 or the nucleic acid construct claim 7 or the gene
product identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of
5-oxoproline, alanine, aspartic acid, citrulline, glycine,
phenylalanine, serine and/or tyrosine resp., levels in an organism.
25. Food or feed composition comprising the nucleic acid molecule
of claim 6, the polypeptide of claim 14, the nucleic acid construct
of claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the harvested material of claim
16, the host cell of claim 10 to 12 or the gene product identified
according to the method of claim 19 or 20. 26. Use of the nucleic
acid molecule of claim 6, the polypeptide of claim 14, the nucleic
acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20 for the protection of a plant against a 5-oxoproline and/or
alanine and/or aspartic acid and/or citrulline and/or glycine
and/or homoserine and/or phenylalanine and/or serine and/or
tyrosine synthesis inhibiting herbicide.
[15085] [0554.0.0.36] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[15086] [0000.0.37.37] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[15087] [0001.0.0.37] see [0001.0.0.27]
[15088] [0002.0.37.37] Oils and fats, which chemically are glycerol
esters of fatty acids (triacylglycerols (TAGs)), play a major role
in nutrition but more and more in nonfood applications such as
lubricants, hydraulic oil, biofuel, or oleochemicals for coatings,
plasticizer, soaps, and detergents (W. Lohs and W. Friedt, in
Designer Oil Crops, D. J. Murphy, Ed. (VCH, Weinheirn, Gerrnany,
1993)). The ideal oil for industrial application would consist of a
particular type of fatty acid that could be supplied constantly at
a competitively low price as compared with raw materials based on
mineral oil products. Furthermore, such a fatty acid may have a
reactive group in addition to the carboxyl function to provide an
additional target for chemical modifications (Topfer et al.,
Science, Vol. 268, 681-686, 1995).
[15089] [0003.0.32.37] to [0004.0.32.37] see paragraph
[0003.0.32.32] to [0004.0.32.32] above
[15090] [0005.0.37.37] Further sources of fatty acids are membrane
lipids of organisms. Preferably lipids are phopholipids and/or
glycolipids, more preferably glycerophospholipids, galactolipids
and/or sphingolipids.
[15091] [0006.0.37.37.] Margaric acid was first mentioned in the
early 1800s. 1813 M. E. Chevreul discovered that fats are composed
of fatty acids and named one of these "margaric acid" because it
glistened with lustrous pearly drops that reminded him of the Greek
word for pearl, margaron or margarites. In the middle of the 1800s
W. H. Heintz showed that "margaric acid" discovered by Chevreul was
an indefinite mixture of palmitic and stearic acids.
[15092] Today, the term "margaric acid" is the trivial name for
heptadecanoic acid (17:0), which is naturally occurring in minor
amounts.
[15093] The fatty acid with odd number of carbon atoms is present
in trace amounts in plants, in triglycerides from Brazil-nut oil,
Dracocephalum moldavica oil, Poppy-seed, Palm, Almond, Sunflower or
Soyabean. Margaric acid can be isolated from tallow (1%), specially
from subcutaneous adipose tissue in subcutaneous fat from
lambs.
[15094] Margaric acid can be ingredient of satiety agents or
fungicide composition. It is further used as ingredient in
cosmetics, pharmaceuticals and in feed and food, like baking
adjuvants as disclosed in US 20030143312 or accordind to US
20040097392 as component in surfactant systems.
[15095] The heptadecanoic acid is mainly used as an internal
standard in quantification of fatty acids. It can be further useful
in treatment of neurological diseases which may be caused by yeast,
fungi or prions based on yeast or fungal etiology (U.S. Pat. No.
6,652,866) or in antikeratolytic-wound healing compositions (U.S.
Pat. No. 5,641,814). Heptadecanoic acid was produced up to now in
higher amount primarily by organic synthesis.
[15096] [0007.0.37.37.] ./.
[15097] [0008.0.32.37] see [0008.0.32.32]
[15098] [0009.0.37.37.] Further poly unsaturated .omega.-3- and/or
.omega.-6-fatty acids important part of animal feed and human food
are delta 7,10 hexadecadienic acid (16:2(n-6)) and delta 7,10, 13
hexadecatrienic acid (16:3(n-3)). Hexadecadienic acid is a minor
component of some seed and fish oils, and of plant leaves but the
precursor of hexadecatrienic acid 16:3(n-3), which is a common
constituent of leaf lipids. This acid is known to occur in
photosynthetic leaves, such as for example Arabidopsis thaliana,
rape leaves, fern lipid, ginko leaves, potato leaves, tomato leaves
and spinach. It may also occur in the leaves of Brassicaceae
plants, such as horse radish, cabbage, turnip, Chinese mustard,
cauliflower and watercress.
[15099] In higher plants, the galactolipids contain a high
proportion of polyunsaturated fatty acids, up to 95% of which can
be linolenic acid (18:3(n-3)). In this instance, the most abundant
molecular species of mono- and digalactosyldiacylglycerols must
have 18:3 at both sn-I and sn-2 positions of the glycerol backbone.
Plants such as the pea, which have 18:3 as almost the only fatty
acid in the monogalactosyldiacylglycerols, have been termed "18:3
plants". Other species, and Arabidopsis thaliana is an example,
contain appreciable amounts of hexadecatrienoic acid (16:3(n-3)) in
the monogalactosyldiacylglycerols, and they are termed "16:3
plants".
[15100] As mentioned, polyunsaturated fatty acid are further used
in the cosmetic industry. The application US 20030039672 discloses
a cosmetic method for treating aged, sensitive, dry, flaky,
wrinkled and/or photodamaged skin through topical application of a
composition which comprises an unsaturated C16 fatty acid having at
least three double bonds, which may be preferably hexadecatrienoic
acid.
[15101] [0010.0.32.37.] to [0011.0.32.37] see [0010.0.32.32] to
[0011.0.32.32] above
[15102] [0012.0.37.37] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes which
participate in the biosynthesis of fatty acids and make it possible
to produce certain fatty acids specifically on an industrial scale
without unwanted byproducts forming. In the selection of genes for
biosynthesis two characteristics above all are particularly
important. On the one hand, there is as ever a need for improved
processes for obtaining the highest possible contents of
polyunsaturated fatty acids on the other hand as less as possible
byproducts should be produced in the production process.
[15103] [0013.0.0.37] see [0013.0.0.27]
[15104] [0014.0.37.37] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is heptadecanoic acid (C17:0,
margaric acid) and/or and/or hexadecatrienoic acid, preferably
delta 7,10,13 hexadecatrienoic acid (C16:3 (n-3), cis 7-cis 10-cis
13-hexadecatrienoic acid, hiragonic acid) or tryglycerides, lipids,
oils or fats containing heptadecanoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid. Accordingly,
in the present invention, the term "the fine chemical" as used
herein relates to "heptadecanoic acid and/or hexadecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid and/or
tryglycerides, lipids, oils and/or fats containing heptadecanoic
acid and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid". Further, the term "the fine chemicals" as
used herein also relates to fine chemicals comprising heptadecanoic
acid and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or triglycerides, lipids, oils and/or
fats containing heptadecanoic acid and/or hexadecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid.
[15105] [0015.0.37.37] In one embodiment, the term "the fine
chemical" means heptadecanoic acid and/or hexadecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid and/or
tryglycerides, lipids, oils and/or fats containing heptadecanoic
acid and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid. Throughout the specification the term "the
fine chemical" means heptadecanoic acid and/or hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid and/or
tryglycerides, lipids, oils and/or fats containing heptadecanoic
acid and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic and its salts, ester, thioester or heptadecanoic
acid and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid in free form or bound to other compounds such
as triglycerides, glycolipids, phospholipids etc. In a preferred
embodiment, the term "the fine chemical" means heptadecanoic acid
and/or 2-hydroxy palmitic acid and/or 2-hydroxy-tetracosenoic-acid
and/or hexadecadienoic acid, preferably delta 7,10 hexadecadienoic
acid and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid, in free form or its salts or bound to a
glycerol backbone or to glycerol-3-phosphate backbone or to a
sphingosine-phosphate backbone or to sphingosine-mono- or
oligosaccharide backbone or to a glycerol-3-mono- or disaccharide
backbone. Triglycerides, lipids, oils, fats or lipid mixture
thereof shall mean any triglyceride, lipid, oil and/or fat
containing any bound or free heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid for example sphingolipids, phosphoglycerides, lipids,
glycerophospholipids, galactolipids, glycolipids such as
glycosphingolipids, phospholipids such as phosphatidylethanolamine,
phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol or diphosphatidylglycerol, or as
monoacylglyceride, diacylglyceride or triacylglyceride or other
fatty acid esters such as acetyl-Coenzym A thioester, which contain
further saturated or unsaturated fatty acids in the fatty acid
molecule.
[15106] [0016.0.37.37] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [15107] (a) increasing or generating the activity of one
or more of a protein as shown in table XII, application no. 37,
column 3 encoded by the nucleic acid sequences as shown in table
XI, application no. 37, column 5, in a non-human organism or in one
or more parts thereof or [15108] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
fatty acid of the invention or fine chemicals comprising the fatty
acids of the invention, in said organism or in the culture medium
surrounding the organism.
[15109] [0017.0.0.37] to [0019.0.0.37] see [0017.0.0.27] to
[0019.0.0.27]
[15110] [0020.0.37.37] Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, application no. 37, column 5, line 35 in a plant
conferred an increase in Margaric Acid (C17:0) content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of Margaric Acid (C17:0).
[15111] Surprisingly it was found, that the transgenic expression
of the Brassica napus protein as indicated in Table XII,
application no. 37, column 5, line 36 in thaliana plant conferred
an increase in Hexadeca-trienoic Acid (C16:3) content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of Hexadeca-trienoic Acid
(C16:3).
[15112] [0021.0.0.37] see [0021.0.0.27]
[15113] [0022.0.37.37] The sequence of YDR513W from Saccharomyces
cerevisiae has been published in Jacq et al., Nature 387 (6632
Suppl), 75-78 (1997) and in Goffeau et al., Science 274 (5287),
546-547, 1996 and its cellular activity has been characterized as a
glutathione reductase, preferably of the glutaredoxin superfamily.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the glutaredoxin superfamily, preferably a protein with a
glutathione reductase activity or its homolog, e.g. as shown
herein, from Saccharomyces cerevisiae or a plant or its homolog,
e.g. as shown herein, for the production of the fine chemical,
meaning of heptadecanoic acid and/or tryglycerides, lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing heptadecanoic acid, in
particular for increasing the amount of heptadecanoic acid and/or
tryglycerides, lipids, preferably glycerophospholipids,
sphingolipids and/or galactolipids, oils and/or fats containing
margaric acid, preferably heptadecanoic acid in free or bound form
in an organism or a part thereof, as mentioned. In one further
embodiment the YDR513W protein expression is increased together
with the increase of another gene of the lipid biosynthesis
pathway, preferably with a gene encoding a protein being involved
in the production of fatty acids or encoding a fatty acid
transporter protein or a compound, which functions as a sink for
the respective fatty acid. In one embodiment, in the process of the
present invention the activity of a protein of the glutaredoxin
superfamily, preferably of a glutathione reductase is increased or
generated, e.g. from Saccharomyces cerevisiae or a plant or a
homolog thereof.
[15114] The sequence of b3430 from Escherichia coli K12 has been
published in Blattner, Science 277(5331), 1453-1474, 1997, and its
activity is being defined as a glucose-1-phosphate
adenylyltransferase, preferably of the a glucose-1-phosphate
adenylyltransferase superfamily. Accordingly, in one embodiment,
the process of the present invention comprises the use of a gene
product with an activity of the a glucose-1-phosphate
adenylyltransferase superfamily, preferably such protein is having
a a glucose-1-phosphate adenylyltransferase activity from E. coli
or a plant or its homolog, e.g. as shown herein, for the production
of the respective fine chemical, meaning of hexydecatrienoic acid
(C16:3, preferably C16:3 delta 7,10, 13) and/or tryglycerides,
lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing hexydecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid, in particular for
increasing the amount of hexydecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid and/or tryglycerides, lipids,
preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing hexydecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid in free or bound
form in an organism or a part thereof, as mentioned. In one further
embodiment the b3430 protein expression is increased together with
the increase of another gene of the lipid biosynthesis pathway,
preferably with a gene encoding a protein being involved in the
production of galactolipids, preferably of
monogalactosyldiacylglycerol In one embodiment, in the process of
the present invention the activity of a a glucose-1-phosphate
adenylyltransferase is increased or generated, e.g. from E. coli or
a plant or a homolog thereof.
[15115] [0022.1.0.37] to [0023.0.0.37] see [0022.1.0.27] to
[0023.0.0.27]
[15116] [0023.1.37.37] Homologs of the polypeptide disclosed in
table XII, application no. 37, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 37, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 37, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 37,
column 7, resp.
[15117] [0024.0.0.37] see [0024.0.0.27]
[15118] [0025.0.37.37] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 37, column 3 "if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 37, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[15119] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 37,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 37,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[15120] [0026.0.0.37] to [0033.0.0.37] see [0026.0.0.27] to
[0033.0.0.27]
[15121] [0034.0.37.37] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 37, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[15122] [0035.0.0.37] to [0036.0.0.37] see [0035.0.0.27] to
[0036.0.0.27]
[15123] [0037.0.37.37] A series of mechanisms exists via which a
modification of the a protein, e.g. the polypeptide of the
invention can directly or indirectly affect the yield, production
and/or production efficiency of the fatty acid.
[15124] [0038.0.0.37] to [0044.0.0.37] see [0038.0.0.27] to
[0044.0.0.27]
[15125] [0045.0.37.37] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
37, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 37, column 6 of
the respective line, between 10% and 50%, 10% and 100%, 10% and
200%, 10% and 300%, 10% and 400%, 10% and 500%, 20% and 50%, 20%
and 250%, 20% and 500%, 50% and 100%, 50% and 250%, 50% and 500%,
500% and 1000% or more
[15126] [0046.0.37.37] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
37, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 37, column 6 of
the respective line confers an increase of the respective fine
chemical and of further fatty acid or their precursors.
[15127] [0047.0.0.37] and [0048.0.0.37] see [0047.0.0.27] and
[0048.0.0.27]
[15128] [0049.0.37.37] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 37, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 37, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 37, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[15129] [0050.0.37.37] For the purposes of the present invention,
the term "heptadecanoic acid", and/or "hexadecatrienoic acid",
preferably "delta 7,10,13 hexadecatrienoic acid" and/or C24:1 fatty
acid also encompasses the corresponding salts, such as, for
example, the potassium or sodium salts of the above named fatty
acids or the salts of the above named fatty acids with amines such
as diethylamine or the esters the above named fatty acids.
[15130] [0051.0.37.37] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
fine chemical, i.e. an increased amount of the free chemical free
or bound, e.g fatty acid compositions. Depending on the choice of
the organism used for the process according to the present
invention, for example a microorganism or a plant, compositions or
mixtures of various fatty acids can be produced.
[15131] [0052.0.0.37] see [0052.0.0.27]
[15132] [0053.0.37.37] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[15133] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 37, columns 5 and 7 or its homologs activity
having herein-mentioned fatty acid of the invention increasing
activity; and/or [15134] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 37,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 37, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned fatty acid of the invention increasing
activity; and/or [15135] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned fatty acid increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 37, columns 5 and 7 or its
homologs activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [15136] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned fatty acid of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 37, columns 5
and 7 or its homologs activity; and/or [15137] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned fatty acid of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 37, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [15138] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned fatty acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 37, columns 5 and 7 or its
homologs activity, and/or [15139] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned fatty acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 37, columns 5 and 7 or its
homologs activity; and/or [15140] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 37, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[15141] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [15142] k) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[15143] [0054.0.37.37] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein according to Table XII, application no. 37,
column 3, or its homologs activity, e.g. as indicated in Table XII,
application no. 37, columns 5 or 7.
[15144] [0055.0.0.37] to [0069.0.0.37] see [0055.0.0.27] to
[0069.0.0.27]
[15145] [0070.0.37.37] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below into an organism
alone or in combination with other genes, it is possible not only
to increase the biosynthetic flux towards the end product, but also
to increase, modify or create de novo an advantageous, preferably
novel metabolites composition in the organism, e.g. an advantageous
fatty acid composition comprising a higher content of (from a
viewpoint of nutritonal physiology limited) fatty acids, like
heptadecanoic acid and/or hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid.
[15146] [0071.0.32.37] see [0071.0.32.32]
[15147] [0072.0.37.37] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to heptadecanoic acid and/or hexadecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid, triglycerides,
lipids, preferably glycerophospholipids, sphingolipids and/or
galactolipids, oils and/or fats containing heptadecanoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid compounds like other fatty acids such as
palmitate, stearate, palmitoleate, oleate, linoleate and/or
linoleate or erucic acid and/or, arachidonic acid.
[15148] [0073.0.37.37] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[15149] (a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [15150] (b) increasing an activity of
a polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table XII, application no. 37, columns 5 or 7, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, i.e. conferring an increase
of the respective fine chemical in the organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [15151] (c) growing the organism,
preferably the microorganism, the non-human animal, the plant or
animal cell, the plant or animal tissue or the plant under
conditions which permit the production of the fine chemical in the
organism, preferably the microorganism, the plant cell, the plant
tissue or the plant; and [15152] (d) if desired, recovering,
optionally isolating, the free and/or bound the fine chemical and,
optionally further free and/or bound fatty acids synthesized by the
organism, the microorganism, the non-human animal, the plant or
animal cell, the plant or animal tissue or the plant.
[15153] [0074.0.37.37] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the fine chemical or the free and bound the fine
chemical but as option it is also possible to produce, recover and,
if desired isolate, other free or/and bound fatty acids.
[15154] [0075.0.0.37] to [0077.0.0.37] see [0075.0.0.27] to
[0077.0.0.27]
[15155] [0078.0.0.37] to [0084.0.0.37]: see [0078.0.0.27] to
[0084.0.0.27]
[15156] [0085.0.37.37] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [15157] a) the nucleic acid sequence as
shown in table XI, application no. 37, columns 5 or 7, or a
derivative thereof, or [15158] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as shown table XI, application no. 37, columns 5 or
7, or a derivative thereof, or [15159] c) (a) and (b) is/are not
present in its/their natural genetic environment or has/have been
modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[15160] [0086.0.0.37] to [0087.0.0.37] see [0086.0.0.27] to
[0087.0.0.27]
[15161] [0088.0.37.37] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose fatty acid
content is modified advantageously owing to the nucleic acid
molecule of the present invention expressed. This is important for
plant breeders since, for example, the nutritional value of plants
for animals is dependent on the abovementioned fatty acids and the
general amount of fatty acids as energy source in feed. After the
above mentioned protein activity has been increased or generated,
or after the expression of nucleic acid molecule or polypeptide
according to the invention has been generated or increased, the
transgenic plant generated thus is grown on or in a nutrient medium
or else in the soil and subsequently harvested.
[15162] [0089.0.32.37] to [0097.0.32.37] see [0089.0.0.32] to
[0097.0.0.32]
[15163] [0098.0.37.37] In a preferred embodiment, the fine chemical
(heptadecanoic acid and/or hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid) is produced in accordance with the
invention and, if desired, is isolated. The production of further
fatty acids such as palmitoleic acid, palmitic acid, stearic acid,
oleic acid, linoleic acid, nervonic acid and/or linolenic acid
mixtures thereof or mixtures of other fatty acids by the process
according to the invention is advantageous.
[15164] [0099.0.32.37] to [0102.0.32.37] see [0099.0.32.32] to
[0102.0.32.32]
[15165] [0102.1.37.37] An other analytical method is described by
Summit et al, (Proceedings of the Ocean Drilling Program,
Scientific Results Volume 169, 2000). The fatty acid methyl esters
are analyzed by capillary gas chromatography with flame ionization
detection. Various mono- and polyunsaturated fatty acids can
further be individually distinguished and quantified in one sample
without prior separation by semi-selective HSQC (heteronuclear
single quantum coherence)-NMR (Willker et al., Magn. Reson. Chem.
36, S79-S84 (1998)).
[15166] Fatty acid compositions, preferably of monogalactosyl
diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) can be
investigated using HPLC/ESI-MS combined with in-source (or cone
voltage) fragmentation, negative-ion electrospray ionization (ESI)
mass spectrometry interfaced with high performance liquid
chromatography (HPLC) (Kim et al., Bull. Korean Chem. Soc. 2003,
Vol. 24, No. 8).
[15167] [0103.0.37.37] In a preferred embodiment, the present
invention relates to a process for the production of the fine
chemical comprising or generating in an organism or a part thereof
the expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[15168] a) nucleic acid molecule encoding, preferably at least the
mature form, of the polypeptide shown in Table XII, application no.
37, columns 5 or 7, or a fragment thereof, which confers an
increase in the amount of the fine chemical in an organism or a
part thereof; [15169] b) nucleic acid molecule comprising,
preferably at least the mature form, of the nucleic acid molecule
shown in Table XI, application no. 37, columns 5 or 7; [15170] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [15171] d) nucleic acid molecule encoding a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [15172] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of the fine chemical in an organism or a
part thereof; [15173] f) nucleic acid molecule encoding a
polypeptide, the polypeptide being derived by substituting,
deleting and/or adding one or more amino acids of the amino acid
sequence of the polypeptide encoded by the nucleic acid molecules
(a) to (d), preferably to (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[15174] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [15175] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers shown in Table XIII, application no. 37, column
7, and conferring an increase in the amount of the fine chemical in
an organism or a part thereof; [15176] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from an expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[15177] j) nucleic acid molecule which encodes a polypeptide
comprising the consensus sequence shown in Table XIV, application
no. 37, columns 7, and conferring an increase in the amount of the
fine chemical in an organism or a part thereof; [15178] k) nucleic
acid molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of the polypeptide shown in Table XII, application no. 37, columns
5 or 7, and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; and [15179] l) nucleic
acid molecule which is obtainable by screening a suitable library
under stringent conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k), preferably to
(a) to (c), or with a fragment of at least 15 nt, preferably 20 nt,
30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k), preferably to [15180] (a) to (c), and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; or which comprises a sequence which is
complementary thereto.
[15181] [0104.0.37.37] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence shown in Table
XI, application no. 37, columns 5 or 7, by one or more nucleotides
or does not consist of the sequence shown in Table XI, application
no. 37, columns 5 or 7. In one embodiment, the nucleic acid
molecule of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to the sequence shown in Table XI,
application no. 37, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of the sequence
shown in Table XII, application no. 37, columns 5 or 7.
[15182] [0105.0.0.37] to [0107.0.0.37] see [0105.0.0.27] to
[0107.0.0.27]
[15183] [0108.0.37.37] Nucleic acid molecules with the sequence
shown in Table XI, application no. 37, columns 5 or 7, nucleic acid
molecules which are derived from the amino acid sequences shown in
Table XII, application no. 37, columns 5 or 7, or from polypeptides
comprising the consensus sequence shown in Table XIV, application
no. 37, columns 5 or 7, or their derivatives or homologues encoding
polypeptides with the enzymatic or biological activity of a
polypeptide as indicated in Table XI, application no. 37, column 3,
5 or 7, or e.g. conferring a increase in the respective fine
chemical after increasing its expression or activity are
advantageously increased in the process according to the
invention.
[15184] [0109.0.37.37] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the fatty acids produced in the process according to
the invention.
[15185] [0110.0.0.37] see [0110.0.0.27]
[15186] [0111.0.0.37] see [0111.0.0.27]
[15187] [0112.0.37.37] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XI, application no. 37,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 37, columns 5 and 7, and conferring an
increase of the respective fine chemical.
[15188] [0113.0.0.37] to [0120.0.0.37] see [0113.0.0.27] to
[0120.0.0.27]
[15189] [0121.0.37.37] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 37, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e.
[15190] conferring an increase in the level of heptadecanoic acid
after increasing the activity of the polypeptide sequences
indicated in Table XII, columns 5 or 7, application no. 37;
[15191] or conferring increase in the level of hexadecatrienoic
acid, preferably delta 7,10,13 hexadecatrienoic acid after
increasing the activity of the polypeptide sequences indicated in
Table XII, application no. 37, columns 5 or 7.
[15192] [0122.0.0.37] to [0127.0.0.37] see [0122.0.0.27] to
[0127.0.0.27]
[15193] [0128.0.37.37] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 37, columns 7, resp., by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table XI, application no.
37, columns 5 or 7, resp., or the sequences derived from sequences
as indicated in Table XII, application no. 37, columns 5 or 7,
resp.
[15194] [0129.0.37.37] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequences indicated in Table XIV,
application no. 37, columns 7, resp., are derived from said
alignments.
[15195] [0130.0.37.37] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of fatty
acids, e.g. of heptadecanoic acid and/or hexadecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid after increasing its
expression or activity of the protein comprising said fragment.
[15196] [0131.0.0.37] to [0138.0.0.37] see [0131.0.0.27] to
[0138.0.0.27]
[15197] [0139.0.37.37] Polypeptides having above-mentioned
activity, i.e. conferring an increase of the respective fine
chemical level, derived from other organisms, can be encoded by
other DNA sequences which hybridize to a sequences indicated in
Table XI, columns 5 or 7 resp., under relaxed hybridization
conditions and which code on expression for peptides having the
heptadecanoic acid and/or hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid increasing activity.
[15198] [0140.0.0.37] to [0146.0.0.37]: see [0140.0.0.27] to
[0146.0.0.27]
[15199] [0147.0.37.37] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 37, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences s
such that it can hybridize to one of said nucleotide sequences
thereby forming a stable duplex. Preferably, the hybridisation is
performed under stringent hybridization conditions. However, a
complement of one of the herein disclosed sequences is preferably a
sequence complement thereto according to the base pairing of
nucleic acid molecules well known to the skilled person. For
example, the bases A and G undergo base pairing with the bases T
and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[15200] [0148.0.37.37] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence shown in Table XI, application no. 37,
columns 5 or 7, or a portion thereof and preferably has above
mentioned activity, in particular having a heptadecanoic acid
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid delta 15 fatty acid-increasing activity after increasing its
activity or an activity of a product of a gene encoding said
sequence or its homologs.
[15201] [0149.0.37.37] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 37,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity, e.g. conferring an increase of the
respective fine chemical, e.g. of heptadecanoic acid and/or 2
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and optionally the activity of a protein
indicated in Table XII, application no. 37, column 5,
[15202] [00149.1.37.37] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table
XI, columns 5 or 7, has further one or more of the activities
annotated or known for the a protein as indicated in Table XII,
application no. 37, column 3,
[15203] [0150.0.37.37] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences shown in Table XI, application no. 37, columns 5
or 7, resp., for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid if its activity is increased. The nucleotide sequences
determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., in Table XI, application no. 37,
columns 5 or 7, an anti-sense sequence of one of the sequences,
e.g., set forth in Table XI, application no. 37, columns 5 or 7,
resp., or naturally occurring mutants thereof. Primers based on a
nucleotide of invention can be used in PCR reactions to clone
homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primers shown in Table XIII,
application no. 37, column 7, resp., will result in a fragment of a
polynucleotide sequence as indicated in Table XI, application no.
37, columns 5 or 7.
[15204] [0151.0.0.37] see [0151.0.0.27]
[15205] [0152.0.37.37] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 37, columns 5
or 7, resp., such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular an activity increasing the level of amino acids, in
particular of heptadecanoic acid and/or hexadecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid as mentioned above
or as described in the examples in plants or microorganisms is
comprised.
[15206] [0153.0.37.37] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XI, application no. 37, columns
5 or 7, such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 37, columns 5 or 7, has for
example an activity of a polypeptide indicated in Table XII,
application no. 37, column 3.
[15207] [0154.0.37.37] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 37, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[15208] [0155.0.0.37] to [0156.0.0.37]: see [0155.0.0.27] to
[0156.0.0.27]
[15209] [0157.0.37.37] The invention further relates to nucleic
acid molecules that differ from one of a nucleotide sequences as
indicated in Table XI, application no. 37, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides encoded by the sequences as indicated in Table
XIV, application no. 37, columns, or of the polypeptide as
indicated in Table XII, application no. 37, columns 5 or 7, or
their functional homologues. Advantageously, the nucleic acid
molecule of the invention comprises, or in an other embodiment has,
a nucleotide sequence encoding a protein comprising, or in an other
embodiment having, a consensus sequences as indicated in Table XIV,
application no. 37, columns 7, or of the polypeptide as as
indicated in Table XII, columns 5 or 7, or the functional
homologues. In a still further embodiment, the nucleic acid
molecule of the invention encodes a full length protein which is
substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table XIV, application no. 37,
columns 5 or 7, or of a polypeptide as indicated in Table XII,
application no. 37, columns 5 or 7, or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 37, columns 5 or 7.
[15210] [0158.0.0.37] to [0160.0.0.37]: see [0158.0.0.27] to
[0160.0.0.27]
[15211] [0161.0.37.37] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 37, columns 5 or
7, The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[15212] [0162.0.0.37] see [0162.0.0.27]
[15213] [0163.0.37.37] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, columns 5 or 7, corresponds to a
naturally-occurring nucleic acid molecule of the invention. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein). Preferably, the nucleic
acid molecule encodes a natural protein having above-mentioned
activity, e.g. conferring the respective fine chemical increase
after increasing the expression or activity thereof or the activity
of a protein of the invention or used in the process of the
invention.
[15214] [0164.0.0.37] see [0164.0.0.27]
[15215] [0165.0.37.37] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 37, columns 5 or 7.
[15216] [0166.0.0.37] to [0167.0.0.37] see [0166.0.0.27] to
[0167.0.0.27]
[15217] [0168.0.37.37] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
columns 5 or 7, yet retain said activity described herein. The
nucleic acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table XII, application no. 37, columns 5 or 7, and is
capable of participation in the increase of production of the
respective fine chemical after increasing its activity, e.g. its
expression. Preferably, the protein encoded by the nucleic acid
molecule is at least about 60% identical to a sequence as indicated
in Table XII, application no. 37, columns 5 or 7 more preferably at
least about 70% identical to one of the sequences as indicated in
Table XII, application no. 37, columns 5 or 7, even more preferably
at least about 80%, 90%, or 95% homologous to a sequence as
indicated in Table XII, application no. 37, columns 5 or 7 and most
preferably at least about 96%, 97%, 98%, or 99% identical to the
sequence as indicated in Table XII, application no. 37, columns 5
or 7.
[15218] [0169.0.0.37] to [0172.0.0.37] see [0169.0.0.27] to
[0172.0.0.27]
[15219] [0173.0.32.37] to [0175.0.32.37] see [0173.0.32.32] to
[0175.0.32.32]
[15220] [0176.0.37.37] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 37,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 37, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 37, columns
5 or 7.
[15221] [0177.0.37.37] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 37,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptides as indicated in Table XII,
application no. 37, columns 5 or 7 according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 37, columns
5 or 7
[15222] [0178.0.0.37] see [0178.0.0.27]
[15223] [0179.0.37.37] A nucleic acid molecule encoding a
homologous to a protein sequence of as indicated in Table XII,
application no. 37, columns 5 or 7, can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table XIB, application no.
37, columns 5 or 7 such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences of a
sequences as indicated in Table XI, application no. 37, columns 5
or 7, by standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[15224] [0180.0.0.37] to [0183.0.0.37] see [0180.0.0.27] to
[0183.0.0.27]
[15225] [0184.0.37.37] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 37,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, columns 5 or 7, comprise also
allelic variants with at least approximately 30%, 35%, 40% or 45%
homology, by preference at least approximately 50%, 60% or 70%,
more preferably at least approximately 90%, 91%, 92%, 93%, 94% or
95% and even more preferably at least approximately 96%, 97%, 98%,
99% or more homology with one of the nucleotide sequences shown or
the abovementioned derived nucleic acid sequences or their
homologues, derivatives or analogues or parts of these. Allelic
variants encompass in particular functional variants which can be
obtained by deletion, insertion or substitution of nucleotides from
the sequences shown, preferably from a sequence as indicated in
Table XI, columns 5 or 7, or from the derived nucleic acid
sequences, the intention being, however, that the enzyme activity
or the biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[15226] [0185.0.37.37] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, columns 5 or 7, In one embodiment, it is preferred that
the nucleic acid molecule comprises as little as possible other
nucleotide sequences not shown in any one of sequences as indicated
in Table XI, application no. 37, columns 5 or 7, In one embodiment,
the nucleic acid molecule comprises less than 500, 400, 300, 200,
100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a further
embodiment, the nucleic acid molecule comprises less than 30, 20 or
10 further nucleotides. In one embodiment, a nucleic acid molecule
used in the process of the invention is identical to a sequences as
indicated in Table XI, application no. 37, columns 5 or 7,
[15227] [0186.0.37.37] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encode a
polypeptide comprising a sequence as indicated in Table XII,
application no. 37, columns 5 or 7, In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 37, columns 5 or 7.
[15228] [0187.0.37.37] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising a sequence as indicated in Table XII, application no.
37, columns 5 or 7, comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence
encoding a sequences as indicated in Table XII, application no. 37,
columns 5 or 7.
[15229] [0188.0.37.37] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 37, columns 5 or 7, preferably compared to a
sequence as indicated in Table XII, application no. 37, column 5,
and expressed under identical conditions.
[15230] [0189.0.37.37] Homologues of a sequences as indicated in
Table XI, application no. 37, columns 5 or 7, or of a derived
sequences as indicated in Table XII, application no. 37, columns 5
or 7, also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives which comprise
noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[15231] [0190.0.0.37] to [0203.0.0.37] see [0190.0.0.27] to
[0203.0.0.27]
[15232] [0204.0.37.37] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule, which comprises a
nucleic acid molecule selected from the group consisting of:
[15233] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 37, columns 5 or 7 or a fragment thereof conferring
an increase in the amount of the respective fine chemical, i.e.
heptadecanoic acid and/or and/or hexadecatrienoic acid, preferably
delta 7,10,13 hexadecatrienoic acid in an organism or a part
thereof; [15234] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule as indicated in
Table XI, application no. 37, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [15235] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[15236] d) nucleic acid molecule encoding a polypeptide whose
sequence has at least 50% identity with the amino acid sequence of
the polypeptide encoded by the nucleic acid molecule of (a) to (c)
and conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [15237] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[15238] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [15239] g)
nucleic acid molecule encoding a fragment or an epitope of a
polypeptide which is encoded by one of the nucleic acid molecules
of (a) to (e), preferably to (a) to (c) and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; [15240] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying a cDNA library or a
genomic library using primers or primer pairs as indicated in Table
XIII, application no. 37, column 7, and conferring an increase in
the amount of the respective fine chemical, i.e. heptadecanoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid in an organism or a part thereof; [15241] i)
nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from a expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (g), preferably to (a) to (c) and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [15242] j) nucleic acid molecule which encodes a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 37, column 7, and conferring an increase in
the amount of the respective fine chemical i.e. heptadecanoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid preferably C24:1 delta 15 fatty acid in an
organism or a part thereof; [15243] k) nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a
domaine of a polypeptide as indicated in Table XII, application no.
37, columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical i.e. heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid in an organism or a part thereof; and [15244] l) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment of at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (h) or of a nucleic acid molecule as
indicated in Table XI, application no. 37, columns 5 or 7, or a
nucleic acid molecule encoding, preferably at least the mature form
of, the polypeptide as indicated in Table XII, application no. 37,
columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; whereby,
preferably, the nucleic acid molecule according to (a) to (l)
distinguishes over a sequence depicted in as indicated in Table XI,
application no. 37, columns 5 or 7, by one or more nucleotides. In
one embodiment, the nucleic acid molecule of the invention does not
consist of a sequence as indicated in Table XI, application no. 37,
columns 5 or 7, In an other embodiment, the nucleic acid molecule
of the present invention is at least 30% identical and less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table XI, application no. 37, columns 5 or 7, In a
further embodiment the nucleic acid molecule does not encode a
polypeptide sequence as indicated in Table XII, application no. 37,
columns 5 or 7, Accordingly, in one embodiment, the nucleic acid
molecule of the present invention encodes in one embodiment a
polypeptide which differs at least in one or more amino acids from
a polypeptide indicated in Table XII, application no. 37, columns 5
or 7, In another embodiment, a nucleic acid molecule indicated in
Table XI, application no. 37, columns 5 or 7, does not encode a
protein of a sequence indicated in Table XII, application no. 37,
columns 5 or 7, Accordingly, in one embodiment, the protein encoded
by a sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table XII, application no.
37, columns 5 or 7, In a further embodiment, the protein of the
present invention is at least 30% identical to a protein sequence
indicated in Table XII, application no. 37, columns 5 or 7, and
less than 100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 985, 97%, 96% or 95% identical to a
sequence as indicated in Table XI, application no. 37, columns 5 or
7.
[15245] [0205.0.0.37] to [0206.0.0.37]: see [0205.0.0.27] to
[0206.0.0.27]
[15246] [0207.0.37.37] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes are genes of the fatty acid metabolism, amino
acid metabolism, of glycolysis, of the tricarboxylic acid
metabolism, of triacylglycerol or lipid, preferably
glycerophospholipids, sphingolipids and/or galactolipids
biosynthesis or their combinations. As described herein, regulator
sequences or factors can have a positive effect on preferably the
gene expression of the genes introduced, thus increasing it. Thus,
an enhancement of the regulator elements may advantageously take
place at the transcriptional level by using strong transcription
signals such as promoters and/or enhancers. In addition, however,
an enhancement of translation is also possible, for example by
increasing mRNA stability or by inserting a translation enhancer
sequence.
[15247] [0208.0.0.37] to [0226.0.0.37] see [0208.0.0.27] to
[0226.0.0.27]
[15248] [0227.0.37.37] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[15249] In addition to the sequence mentioned in Table XI,
application no. 37, columns 5 or 7, or its derivatives, it is
advantageous additionally to express and/or mutate further genes in
the organisms. Especially advantageously, additionally at least one
further gene of the fatty acid biosynthetic pathway such as for
palmitate, stearate, palmitoleate, oleate, linoleate and/or
linolenate or of the of triacylglycerol or lipid, preferably
glycerophospholipids, sphingolipids and/or galactolipids
biosynthetic pathway is expressed in the organisms such as plants
or microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the amino acids desired since, for example, feedback
regulations no longer exist to the same extent or not at all. In
addition it might be advantageously to combine the sequences shown
in Table XI, application no. 37, columns 5 or 7, with genes which
generally support or enhances to growth or yield of the target
organism, for example genes which lead to faster growth rate of
microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[15250] [0228.0.32.37] to [0229.0.32.37] see [0228.0.32.32]
[0229.0.32.32]
[15251] [0229.1.37.37] Further nucleic acid sequences which can be
expressed in combination with the sequences used in the process
and/or the abovementioned biosynthesis genes are the sequences
encoding further genes of the saturated, unsaturated,
polyunsaturated and/or hydroxylated fatty acid biosynthesis such as
the FA2H gene encoding a fatty acid 2-hydroxylase (Alderson et al.,
J. Biol. Chem. Vol. 279, No. 47, Issue of November 19, pp.
48562-48568, 2004), .omega.-3-desaturases encoded by FAD2 and FAD3
(US 20030221217), delta-12 fatty acid desaturase, delta-15 fatty
acid desaturase as disclosed by Okuley, et al., Plant Cell
6:147-158 (1994), Lightner et al., WO94/11516, Yadav, N., et al.,
Plant Physiol., 103:467-476 (1993), WO 93/11245 and Arondel, V. et
al., Science, 258:1353-1355 (1992), US 20040083503.
[15252] [0230.0.0.37] see [0230.0.0.27]
[15253] [0231.0.37.37] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a heptadecanoic acid and/or and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid, preferably C24:1 delta 15 fatty acid degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[15254] [0232.0.0.37] to [0276.0.0.37] see [0232.0.0.27] to
[0276.0.0.27]
[15255] [0277.0.32.37] see paragraph [0277.0.32.32]
[15256] [0278.0.0.37] to [0282.0.0.37] see [0278.0.0.27] to
[0282.0.0.27]
[15257] [0283.0.37.37] Moreover, a native polypeptide conferring
the increase of the fine chemical in an organism or part thereof
can be isolated from cells (e.g., endothelial cells), for example
using the antibody of the present invention as described below, in
particular, an antibody against a protein as indicated in Table
XII, application no. 37, column 3, e.g. an antibody against a
polypeptide as indicated in Table XII, application no. 37, columns
5 or 7, which can be produced by standard techniques utilizing
polypeptides comprising or consisting of above mentioned sequences,
e.g. the polypeptide of the present invention or fragment thereof.
Preferred are monoclonal antibodies.
[15258] [0284.0.0.37] see [0284.0.0.27]
[15259] [0285.0.37.37] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 37, columns 5 or 7, or as coded by a nucleic
acid molecule as indicated in Table XI, application no. 37, columns
5 or 7, or functional homologues thereof.
[15260] [0286.0.37.37] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 37, column 7, and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 37, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid, or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table XIV, application no. 37, column 7.
[15261] [0287.0.0.37] to [0290.0.0.37] see [0287.0.0.27] to
[0290.0.0.27]
[15262] [0291.0.37.37] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[15263] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table XII,
application no. 37, columns 5 or 7, by one or more amino acids. In
one embodiment, polypeptide distinguishes form a sequence as
indicated in Table XII, application no. 37, columns 5 or 7, by more
than 5, 6, 7, 8 or 9 amino acids, preferably by more than 10, 15,
20, 25 or 30 amino acids, even more preferred are more than 40, 50,
or 60 amino acids and, preferably, the sequence of the polypeptide
of the invention distinguishes from a sequence as indicated in
Table XII, application no. 37, columns 5 or 7, by not more than 80%
or 70% of the amino acids, preferably not more than 60% or 50%,
more preferred not more than 40% or 30%, even more preferred not
more than 20% or 10%. In an other embodiment, said polypeptide of
the invention does not consist of a sequence as indicated in
Table.
[15264] [0292.0.0.37] see [0292.0.0.27]
[15265] [0293.0.37.37] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or by a nucleic acid molecule used in the process
of the invention. In one embodiment, the polypeptide of the
invention has a sequence which distinguishes from a sequence as
indicated in Table XII, application no. 37, column 5 and/or 7, by
one or more amino acids. In an other embodiment, said polypeptide
of the invention does not consist of the sequence as indicated in
Table XII, application no. 37, columns 5 or 7. In a further
embodiment, said polypeptide of the present invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical. In one embodiment,
said polypeptide does not consist of the sequence encoded by a
nucleic acid molecules as indicated in Table XI, application no.
37, columns 5 or 7.
[15266] [0294.0.37.37] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 37, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 37, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[15267] [0295.0.0.37] to [0297.0.0.37] see [0295.0.0.27] to
[0297.0.0.27]
[15268] [00297.1.37.37] Non-polypeptide of the
invention-chemicalsare e.g. polypeptides having not the activity of
a polypeptide indicated in Table XII, application no. 37, columns 5
or 7.
[15269] [0298.0.37.37] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 37, columns 5 or 7, such
that the protein or portion thereof maintains the ability to confer
the activity of the present invention. The portion of the protein
is preferably a biologically active portion as described herein.
Preferably, the polypeptide used in the process of the invention
has an amino acid sequence identical to a sequence as indicated in
Table XII, application no. 37, columns 5 or 7.
[15270] [0299.0.37.37] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences as shown in Table XII, application no. 37,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence of Table XI,
application no. 37, columns 5 or 7, or which is homologous thereto,
as defined above.
[15271] [0300.0.37.37] Accordingly the polypeptide of the present
invention can vary from the sequences shown in Table XII,
application no. 37, columns 5 or 7, in amino acid sequence due to
natural variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprise an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence shown in table XII, application no. 37, columns
5 or 7.
[15272] [0301.0.0.37] see [0301.0.0.27]
[15273] [0302.0.37.37] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., the amino acid sequence shown in Table
XII, columns 5 or 7, or the amino acid sequence of a protein
homologous thereto, which include fewer amino acids than a full
length polypeptide of the present invention or used in the process
of the present invention or the full length protein which is
homologous to an polypeptide of the present invention or used in
the process of the present invention depicted herein, and exhibit
at least one activity of polypeptide of the present invention or
used in the process of the present invention.
[15274] [0303.0.0.37] see [0303.0.0.27]
[15275] [0304.0.37.37] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention, may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table,
application no. 37, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[15276] [0305.0.0.37] to [0308.0.0.37] see [0305.0.0.27] to
[0308.0.0.27]
[15277] [0309.0.37.37] In one embodiment, an reference to a
"protein (=polypeptide)" of the invention or as indicated in Table
XII, application no. 37, columns 5 or 7, refers to a polypeptide
having an amino acid sequence corresponding to the polypeptide of
the invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table XII, application no. 37, columns 5 or 7, refers
to a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous a polypeptide of the
invention, preferably which is not substantially homologous to a as
indicated in Table XII, application no. 37, columns 5 or 7, e.g., a
protein which does not confer the activity described herein or
annotated or known for as indicated in Table XII, application no.
37, column 3, and which is derived from the same or a different
organism. In one embodiment, a "non-polypeptide of the invention"
or "other polypeptide" not being indicated in Table XII,
application no. 37, columns 5 or 7, does not confer an increase of
the respective fine chemical in an organism or part thereof.
[15278] [0310.0.0.37] to [0334.0.0.37] see [0310.0.0.27] to
[0334.0.0.27]
[15279] [0335.0.37.37] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of the nucleic acid sequences of the table XI, application no. 37,
columns 5 or 7 and/or homologs thereof. As described inter alia in
WO 99/32619, dsRNAi approaches are clearly superior to traditional
antisense approaches. The invention therefore furthermore relates
to double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived therefrom), bring about altered
metabolic activity by the reduction in the expression of the
nucleic acid sequences of the Table XI, application no. 37, columns
5 or 7, and/or homologs thereof. In a double-stranded RNA molecule
for reducing the expression of an protein encoded by a nucleic acid
sequence of one of the Table XI, application no. 37, columns 5 or
7, and/or homologs thereof, one of the two RNA strands is
essentially identical to at least part of a nucleic acid sequence,
and the respective other RNA strand is essentially identical to at
least part of the complementary strand of a nucleic acid
sequence.
[15280] [0336.0.0.37] to [0342.0.0.37] see [0336.0.0.27] to
[0342.0.0.27]
[15281] [0343.0.37.37] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence of one of the sequences shown in Table XI, application no.
37, columns 5 or 7, or its homolog is not necessarily required in
order to bring about effective reduction in the expression. The
advantage is, accordingly, that the method is tolerant with regard
to sequence deviations as may be present as a consequence of
genetic mutations, polymorphisms or evolutionary divergences. Thus,
for example, using the dsRNA, which has been generated starting
from a sequence of one of sequences shown in Table XI, application
no. 37, columns 5 or 7, or homologs thereof of the one organism,
may be used to suppress the corresponding expression in another
organism.
[15282] [0344.0.0.37] to [0361.0.0.37] see [0344.0.0.27] to
[0361.0.0.27]
[15283] [0362.0.37.37] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 37, columns 5 or 7. Due to the above
mentioned activity the fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention is increased,
e.g. due to an increased expression or specific activity of the
subject matters of the invention in a cell or an organism or a part
thereof. In one embodiment, transgenic for a polypeptide having an
activity of a polypeptide as indicated in Table XII, application
no. 37, columns 5 or 7, means herein that due to modulation or
manipulation of the genome, an activity as annotated for a
polypeptide as indicated in Table XII, application no. 37, column
3, e.g. having a sequence as indicated in Table XII, application
no. 37, columns 5 or 7, is increased in a cell or an organism or a
part thereof. Examples are described above in context with the
process of the invention.
[15284] [0363.0.0.37] to [0373.0.0.37] see [0363.0.0.27] to
[0373.0.0.27]
[15285] [0374.0.32.37] see [0374.0.32.32]
[15286] [0375.0.0.37] to [0376.0.0.37] see [0375.0.0.27] to
[0376.0.0.27]
[15287] [0377.0.32.37] see [0377.0.32.32]
[15288] [0378.0.0.37] to [0379.0.0.37] see [0378.0.0.27] to
[0379.0.0.27]
[15289] [0379.1.37.37] In one embodiment, the amino acid, in
particular, heptadecanoic acid and/or hexadecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid resp., is a mixture
comprising of one or more the respective fine chemicals. In one
embodiment, the respective fine chemical means here amino acid, in
particular heptadecanoic acid and/or hexadecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid. In one embodiment,
amino acid means here a mixture of the respective fine
chemicals.
[15290] [0380.0.32.37] to [0392.0.32.37] see [0380.0.32.32] to
[0392.0.0.32]
[15291] [0393.0.37.37] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [15292] (a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [15293] (b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence shown in table
XI, application no. 37, columns 5 or 7, and, optionally, isolating
the full length cDNA clone or complete genomic clone; [15294] (c)
introducing the candidate nucleic acid molecules in host cells,
preferably in a plant cell or a microorganism, appropriate for
producing the fine chemical; [15295] (d) expressing the identified
nucleic acid molecules in the host cells; [15296] (e) assaying the
fine chemical level in the host cells; and [15297] (f) identifying
the nucleic acid molecule and its gene product which expression
confers an increase in the fine chemical level in the host cell
after expression compared to the wild type.
[15298] [0394.0.32.37] to [0552.0.32.37] see [0394.0.0.32] to
[0552.0.0.32]
[15299] [0553.0.37.37] [15300] 1. A process for the production of
heptadecanoic acid and/or hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid which comprises (a) increasing or
generating the activity of a protein as indicated in Table XII
application no. 37, columns 5 or 7, or a functional equivalent
thereof in a non-human organism, or in one or more parts thereof;
and (b) growing the organism under conditions which permit the
production of heptadecanoic acid and/or hexadecatrienoic acid,
preferably delta 7,10, 13 hexadecatrienoic acid in said organism.
[15301] 2. A process for the production of heptadecanoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid, comprising the increasing or generating in
an organism or a part thereof the expression of at least one
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: [15302] a) nucleic acid molecule
encoding of the polypeptide shown in table XII, application no. 37,
columns 5 or 7, or a fragment thereof, which confers an increase in
the amount of heptadecanoic acid and/or hexadecatrienoic acid,
preferably delta 7,10,13 hexadecatrienoic acid in an organism or a
part thereof; [15303] b) nucleic acid molecule comprising of the
nucleic acid molecule shown in table XI, application no. 37,
columns 5 or 7; [15304] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of heptadecanoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid in an organism or a part thereof; [15305] d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid in an organism or a part thereof; [15306] e) nucleic acid
molecule which hybridizes with a nucleic acid molecule of (a) to
(c) under under stringent hybridisation conditions and conferring
an increase in the amount of heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid in an organism or a part thereof; [15307] f) nucleic acid
molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers as shown in Table XIII,
application no. 37, column 7, and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[15308] g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of heptadecanoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid in an organism or a part thereof; [15309] h)
nucleic acid molecule encoding a polypeptide comprising the
consensus sequence shown in table XIV, application no. 37, column
7., and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; and [15310] i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof.
or comprising a sequence which is complementary thereto. [15311] 3.
The process of claim 1 or 2, comprising recovering of the free or
bound heptadecanoic acid and/or hexadecatrienoic acid, preferably
delta 7,10,13 hexadecatrienoic acid. [15312] 4. The process of any
one of claims 1 to 3, comprising the following steps: [15313] (a)
selecting an organism or a part thereof expressing a polypeptide
encoded by the nucleic acid molecule characterized in claim 2;
[15314] (b) mutagenizing the selected organism or the part thereof;
[15315] (c) comparing the activity or the expression level of said
polypeptide in the mutagenized organism or the part thereof with
the activity or the expression of said polypeptide of the selected
organisms or the part thereof; [15316] (d) selecting the mutated
organisms or parts thereof, which comprise an increased activity or
expression level of said polypeptide compared to the selected
organism or the part thereof; [15317] (e) optionally, growing and
cultivating the organisms or the parts thereof; and [15318] (f)
recovering, and optionally isolating, the free or bound
heptadecanoic acid and/or hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid produced by the selected mutated
organisms or parts thereof. [15319] 5. The process of any one of
claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. [15320] 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [15321] a) nucleic acid molecule encoding of the
polypeptide shown in table XII, application no. 37, columns 5 or 7,
or a fragment thereof, which confers an increase in the amount of
heptadecanoic acid and/or hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid in an organism or a part thereof;
[15322] b) nucleic acid molecule comprising of the nucleic acid
molecule shown in table XI, application no. 37, columns 5 or 7;
[15323] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid in an organism or a part thereof; [15324] d) nucleic acid
molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of heptadecanoic acid and/or hexadecatrienoic acid,
preferably delta 7,10, 13 hexadecatrienoic acid in an organism or a
part thereof; [15325] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under stringent
hybridisation conditions and conferring an increase in the amount
of heptadecanoic acid and/or hexadecatrienoic acid, preferably
delta 7,10,13 hexadecatrienoic acid in an organism or a part
thereof; [15326] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers as shown in Table XIII, application no. 37, column 7, and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [15327] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
heptadecanoic acid and/or hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid in an organism or a part thereof;
[15328] h) nucleic acid molecule encoding a polypeptide comprising
the consensus sequence shown in table XIV, application no. 37,
column 7, and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; and [15329] i) nucleic
acid molecule which is obtainable by screening a suitable nucleic
acid library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof.
[15330] whereby the nucleic acid molecule distinguishes over the
sequence as shown in table XI, application no. 37, columns 5 or 7,
by one or more nucleotides. [15331] 7. A nucleic acid construct
which confers the expression of the nucleic acid molecule of claim
6, comprising one or more regulatory elements. [15332] 8. A vector
comprising the nucleic acid molecule as claimed in claim 6 or the
nucleic acid construct of claim 7. [15333] 9. The vector as claimed
in claim 8, wherein the nucleic acid molecule is in operable
linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [15334] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [15335] 11. The host cell of claim 10, which is a
transgenic host cell. [15336] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [15337] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [15338] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over the sequence as shown in table XII, application
no. 37, columns 5 or 7, by one or more amino acids [15339] 15. An
antibody, which binds specifically to the polypeptide as claimed in
claim 14. [15340] 16. A plant tissue, propagation material,
harvested material or a plant comprising the host cell as claimed
in claim 12 which is plant cell or an Agrobacterium. [15341] 17. A
method for screening for agonists and antagonists of the activity
of a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid in an organism or a part thereof comprising: [15342] (a)
contacting cells, tissues, plants or microorganisms which express
the a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of heptadecanoic acid and/or
2-hydroxy palmitic acid and/or hexadecatrienoic acid, preferably
delta 7,10,13 hexadecatrienoic acid in an organism or a part
thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; [15343] (b) assaying the palmitic acid level or
the polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [15344] (c)
identifying a agonist or antagonist by comparing the measured
palmitic acid level or polypeptide expression level with a standard
palmitic acid or polypeptide expression level measured in the
absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. [15345] 18. A process for
the identification of a compound conferring increased heptadecanoic
acid and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid and/or 24:1 fatty acid, preferably C24:1
delta 15 fatty acid production in a plant or microorganism,
comprising the steps: [15346] (a) culturing a plant cell or tissue
or microorganism or maintaining a plant expressing the polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid in an organism or a part thereof and a readout system capable
of interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with dais readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of heptadecanoic acid and/or
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid in an organism or a part thereof; [15347] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [15348] 19. A method for the identification of a
gene product conferring an increase in heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid production in a cell, comprising the following steps: [15349]
(a) contacting the nucleic acid molecules of a sample, which can
contain a candidate gene encoding a gene product conferring an
increase in heptadecanoic acid hexadecatrienoic acid, preferably
delta 7,10,13 hexadecatrienoic acid after expression with the
nucleic acid molecule of claim 6; [15350] (b) identifying the
nucleic acid molecules, which hybridise under relaxed stringent
conditions with the nucleic acid molecule of claim 6; [15351] (c)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid; [15352] (d) expressing the identified nucleic acid molecules
in the host cells; [15353] (e) assaying the heptadecanoic acid
and/or and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid level in the host cells; and [15354] (f)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid level in the host cell in the host cell after expression
compared to the wild type. [15355] 20. A method for the
identification of a gene product conferring an increase in
heptadecanoic acid and/or and/or hexadecatrienoic acid, preferably
delta 7,10,13 hexadecatrienoic acid production in a cell,
comprising the following steps: [15356] (a) identifying in a data
bank nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the heptadecanoic acid and/or hexadecatrienoic acid, preferably
delta 7,10, 13 hexadecatrienoic acid amount or level in an organism
or a part thereof after expression, and which are at least 20%
homolog to the nucleic acid molecule of claim 6;
[15357] (b) introducing the candidate nucleic acid molecules in
host cells appropriate for producing heptadecanoic acid acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid; [15358] (c) expressing the identified nucleic acid molecules
in the host cells; [15359] (d) assaying the heptadecanoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid level in the host cells; and [15360] (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the heptadecanoic acid and/or
hexadecatrienoic acid, preferably delta 7,10,13 hexadecatrienoic
acid level in the host cell after expression compared to the wild
type. [15361] 21. A method for the production of an agricultural
composition comprising the steps of the method of any one of claims
17 to 20 and formulating the compound identified in any one of
claims 17 to 20 in a form acceptable for an application in
agriculture. [15362] 22. A composition comprising the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of any one of claim 8 or 9, an
antagonist or agonist identified according to claim 17, the
compound of claim 18, the gene product of claim 19 or 20, the
antibody of claim 15, and optionally an agricultural acceptable
carrier. [15363] 23. Use of the nucleic acid molecule as claimed in
claim 6 for the identification of a nucleic acid molecule
conferring an increase of heptadecanoic acid and/or 2-hydroxy
palmitic acid and/or hexadecatrienoic acid, preferably delta
7,10,13 hexadecatrienoic acid after expression. [15364] 24. Use of
the polypeptide of claim 14 or the nucleic acid construct claim 7
or the gene product identified according to the method of claim 19
or 20 for identifying compounds capable of conferring a modulation
of heptadecanoic acid and/or hexadecatrienoic acid, preferably
delta 7,10,13 hexadecatrienoic acid levels in an organism. [15365]
25. Food or feed composition comprising the nucleic acid molecule
of claim 6, the polypeptide of claim 14, the nucleic acid construct
of claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the harvested material of claim
16, the host cell of claim 10 to 12 or the gene product identified
according to the method of claim 19 or 20. [15366] 26. Use of the
nucleic acid molecule of claim 6, the polypeptide of claim 14, the
nucleic acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20 for the protection of a plant against a heptadecanoic acid
and/or hexadecatrienoic acid, preferably delta 7,10,13
hexadecatrienoic acid synthesis inhibiting herbicide.
[15367] [0554.0.0.37] Abstract: see [0554.0.0.27]
PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[15368] [0000.0.38.38] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[15369] [0001.0.0.38] see [0001.0.0.27]
[15370] [0002.0.38.38]
[15371] The malic acid oxaloacetate shuttle is characteristic for
plant cells. It transports redox equivalents intracellularly.
[15372] Malic acid is not only a central metabolite in intermediary
flow of carbon in organisms. In higher plants, vacuolar malic acid
accumulation, and hence, transtonoplast malic acid transport, also
plays a paramount role in many physiological functions. These
include adjustment of osmotic and turgor potentials in extension
growth and movements of stomata and pulvini, pH-regulation, e.g.
during nitrate reduction, and others (for review, see Luttge et al,
Plant Physiol, 124(2000), 1335-1348).
[15373] Osawa and Matsumoto, Plant Physiol, 126(2001), 411-420
discuss the involvement malic acid in aluminium resistance in
plants.
[15374] Malic acid is a common constituent of all plants, and its
formation is controlled by an enzyme (protein catalyst) called
malic acid dehydrogenase (MDH).
[15375] Malic acid occupies a central role in plant metabolism. Its
importance in plant mineral nutrition is reflected by the role it
plays in symbiotic nitrogen fixation, phosphorus acquisition, and
aluminum tolerance.
[15376] During phosphorus deficiency, malic acid is frequently
secreted from roots to release unavailable forms of phosphorus.
[15377] In nitrogen-fixing root nodules, malic acid is the primary
substrate for bacteroid respiration, thus fueling nitrogenase.
[15378] Trihydroxibutanoic acid, trihydroxybutyric acid lacton can
be regarded as hypothetic precusors of pheromone like metabolites
in plants.
[15379] Due to these interesting physiological roles and
agrobiotechnological potential of malic acid, trihydroxybutyric
acid or trihydroxybutanoic acid there is a need to identify the
genes of enzymes and other proteins involved in malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid or trihydroxybutanoic
acid metabolism, and to generate mutants or transgenic plant lines
with which to modify the malic acid, or trihydroxybutanoic acid
content.
[15380] [0003.0.38.38] to [0007.0.38.38] -/-
[15381] [0008.0.38.38] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce malic acid, or trihydroxybutanoic
acid in plants. That type of production permits control over
quality, quantity and selection of the most suitable and efficient
producer organisms. The latter is especially important for
commercial production economics and therefore availability to
consumers. In addition it is desirable to produce malic acid, or
trihydroxybutanoic acid in plants in order to increase plant
productivity and resistance against biotic and abiotic stress as
discussed before.
[15382] Methods of recombinant DNA technology have been used for
some years to improve the production of fine chemicals in
microorganisms and plants by amplifying individual biosynthesis
genes and investigating the effect on production of fine chemicals.
It is for example reported, that the xanthophyll astaxanthin could
be produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Agrobacterium
tumefaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. Those results indicated
that about 75 percent of the carotenoids found in the flower of the
transformed plant contained a keto group.
[15383] [0009.0.38.38] Thus, it would be advantageous if an algae,
plant or other microorganism were available which produce large
amounts malic acid, ortrihydroxybutanoic acid. The invention
discussed hereinafter relates in some embodiments to such
transformed prokaryotic or eukaryotic microorganisms.
[15384] It would also be advantageous if plants were available
whose roots, leaves, stem, fruits or flowers produced large amounts
of glyceric malic acid, or trihydroxybutanoic acid. The invention
discussed hereinafter relates in some embodiments to such
transformed plants.
[15385] [0010.0.38.38] Therefore improving the quality of
foodstuffs and animal feeds is an important task of the
food-and-feed industry. This is necessary since, for example malic
acid, or trihydroxybutanoic acid, as mentioned above, which occur
in plants and some microorganisms are limited with regard to the
supply of mammals. Especially advantageous for the quality of
foodstuffs and animal feeds is as balanced as possible a specific
malic acid, or trihydroxybutanoic acid profile in the diet since an
excess of malic acid, or trihydroxybutanoic acid above a specific
concentration in the food has a positive effect. A further increase
in quality is only possible via addition of further malic acid,
and/or trihydroxybutanoic acid, which are limiting.
[15386] [0011.0.38.38] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add malic acid, and/or
trihydroxybutanoic acid in a balanced manner to suit the
organism.
[15387] [0012.0.38.38] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
proteins which participate in the biosynthesis of, malic acid,
and/or trihydroxybutanoic acid and make it possible to produce them
specifically on an industrial scale without unwanted by-products
forming. In the selection of genes for biosynthesis two
characteristics above all are particularly important. On the one
hand, there is as ever a need for improved processes for obtaining
the highest possible contents of, malic acid, and/or
trihydroxybutanoic acid; on the other hand as less as possible
by-products should be produced in the production process.
[15388] [0013.0.0.38] see [0013.0.0.27]
[15389] [0014.0.38.38] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a malic acid, and/or
trihydroxybutanoic acid. Accordingly, in the present invention, the
term "the fine chemical" as used herein relates to a malic acid,
and/or trihydroxybutanoic acid.
[15390] Further, the term "the fine chemicals" as used herein also
relates to fine chemicals comprising, malic acid, and/or
trihydroxybutanoic acid.
[15391] [0015.0.38.38] In one embodiment, the term "the fine
chemical" or "the respective fine chemical" means at least one
chemical compound with malic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid activity.
[15392] In one embodiment, the term "the fine chemical" or "the
respective fine chemical" means malic acid. In one embodiment, the
term "the fine chemical" or "the respective fine chemical" means
trihydroxybutanoic acid depending on the context in which the term
is used. Throughout the specification the term "the fine chemical"
or "the respective fine chemical" means malic acid and/or
trihydroxybutanoic acid, its salts, ester, thioester or in free
form or bound to other compounds such sugars or sugar polymers,
like glucoside, e.g. diglucoside.
[15393] In particular it is known to the skilled that anionic
compounds as acids are present in an equilibrium of the acid and
its salts according to the pH present in the respective compartment
of the cell or organism and the pK of the acid. Thus, the term "the
fine chemical", the term "the respective fine chemical", the term
"acid" or the use of a demonination referring to a neutralized
anionic compound respectivley relates the anionic form as well as
the neutralised status of that compound. Thus, malic acid also
relates to malate, or trihydroxybutanoic acid relates also to
trihydroxybutanoic.
[15394] In one embodiment, the term "the fine chemical" and the
term "the respective fine chemical" mean at least one chemical
compound with an activity of the above mentioned fine chemical
[15395] [0016.0.38.38] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [15396] (a) increasing or generating the activity of one
or more [15397] of a protein as shown in table XII, application no.
38, column 3 encoded by the nucleic acid sequences as shown in
table XI, application no. 38, column 5, in a non-human organism or
in one or more parts thereof or [15398] (b) growing the organism
under conditions which permit the production of the fine chemical,
thus organic acid of the invention or fine chemicals comprising the
organic acids of the invention, in said organism or in the culture
medium surrounding the organism.
[15399] [0017.0.0.38] to [0018.0.0.38]: see [0017.0.0.27] to
[0018.0.0.27]
[15400] [0019.0.38.38] Advantageously the process for the
production of the respective fine chemical leads to an enhanced
production of the fine respective chemical. The terms "enhanced" or
"increase" mean at least a 10%, 20%, 30%, 40% or 50%, preferably at
least 60%, 70%, 80%, 90% or 100%, more preferably 150%, 200%, 300%,
400% or 500% or more percent higher production of the respective
fine chemical in comparison to the reference as defined below, e.g.
that means in comparison to an organism without the aforementioned
modification of the activity of a protein having the activity of a
protein indicated in Table XII, column 3, application 14 or encoded
by nucleic acid molecule indicated in Table XI, columns 5 or 7,
application 14.
[15401] [0020.0.38.38] Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, application no. 38, column 5, line 37 in a plant
conferred an increase in Malic Acid content of the transformed
plants. Thus, in one embodiment, said protein or its homologs are
used for the production of Malic Acid.
[15402] Surprisingly it was found, that the transgenic expression
of the Llnum usitatissimum protein as indicated in Table XII,
application no. 38, column 5, line 38 in thaliana plant conferred
an increase in Trihydroxybutanoic acid content of the transformed
plants. Thus, in one embodiment, said protein or its homologs are
used for the production of Trihydroxybutanoic acid.
[15403] [0021.0.0.38] see [0021.0.0.27]
[15404] [0022.0.38.38] The sequence of b1343 from Escherichia coli
K12 has been published in Blattner, Science 277(5331), 1453-1474,
1997, and its activity is being defined as a ATP-dependent RNA
helicase, stimulated by 23S rRNA. Accordingly, in one embodiment,
the process of the present invention comprises the use of a
"ATP-dependent RNA helicase, stimulated by 23S rRNA" from E. coli
or a plant or its homolog, e.g. as shown herein, for the production
of the fine chemical, meaning of trihydroxybutanoic acid and/or
compositions containing trihydroxybutanoic acid, in particular for
increasing the amount of trihydroxybutanoic acid in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a protein having an activity in rRNA processing or translation is
increased or generated, e.g. from E. coli or a plant or a homolog
thereof. Accordingly, in one embodiment, in the process of the
present invention the activity of a ATP-dependent RNA helicase,
stimulated by 23S rRNA or its homolog is increased for the
production of the fine chemical, meaning of trihydroxybutanoic
acid, in particular for increasing the amount of trihydroxybutanoic
acid in free or bound form in an organism or a part thereof, as
mentioned.
[15405] The sequence of YFR007W from Saccharomyces cerevisiae has
been published in Goffeau, A. et al. Science 274 (5287), 546-547
(1996) and its activity is being defined as an unclassified
protein. Accordingly, in one embodiment, the process of the present
invention comprises the use of said unclassified protein from
Saccharomyces cerevisiae or a plant or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, in
particular for increasing the amount of fumaric acid preferably in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of the unclassified protein YFR007W is increased.
[15406] [0022.1.0.38] to [0023.0.0.38] see [0022.1.0.38] to
[0023.0.0.38]
[15407] [0023.1.38.38] Homologs of the polypeptide disclosed in
table XII, application no. 38, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 38, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 38, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 38,
column 7, resp.
[15408] [0024.0.0.38] see [0024.0.0.27]
[15409] [0025.0.38.38] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 38, column 3 "if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 38, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[15410] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 38,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 38,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[15411] [0025.1.0.38] and [0025.2.0.38] see [0025.1.0.27] and
[0025.2.0.27]
[15412] [0025.3.38.38] In one embodiment, the polypeptide of the
invention or used in the method of the invention confers said
activity, e.g. the increase of the respective fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table XI,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table XI,
column 4 are derived from the same families, orders, classes or
phylums.
[15413] [0026.0.0.38] to [0033.0.0.38]: see [0026.0.0.27] to
[0033.0.0.27]
[15414] [0034.0.38.38] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 38, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[15415] [0035.0.0.38] to [0044.0.0.38]: see [0035.0.0.27] to
[0044.0.0.27]
[15416] [0045.0.38.38] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
38, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 38, column 6 of
the respective line, between 10% and 50%, 10% and 100%, 10% and
200%, 10% and 300%, 10% and 400%, 10% and 500%, 20% and 50%, 20%
and 250%, 20% and 500%, 50% and 100%, 50% and 250%, 50% and 500%,
500% and 1000% or more
[15417] [0046.0.38.38] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
38, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 38, column 6 of
the respective line confers an increase of the respective fine
chemical and of further organic acid or their precursors.
[15418] [0047.0.0.38] to [0048.0.0.38]: see [0047.0.0.27] to
[0048.0.0.27]
[15419] [0049.0.38.38] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 38, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 38, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 38, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[15420] [0050.0.38.38] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of malic acid, and/or trihydroxybutanoic acid, resp., or
their ester, or glucoside thereof, e.g the diglucoside thereof.
[15421] [0051.0.38.38] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g. compositions comprising malic acid,
and/or trihydroxybutanoic acid. Depending on the choice of the
organism used for the process according to the present invention,
for example a microorganism or a plant, compositions or mixtures of
organic acids or there salts, e.g. malic acid, and/or
trihydroxybutanoic acid can be produced.
[15422] [0052.0.0.38] see [0052.0.0.27]
[15423] [0053.0.38.38] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[15424] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 38, columns 5 and 7 or its homologs activity
having herein-mentioned organic acid of the invention increasing
activity; and/or [15425] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 38,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 38, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned organic acid of the invention increasing
activity; and/or [15426] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned organic acid
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 38, columns 5
and 7 or its homologs activity, or decreasing the inhibiitory
regulation of the polypeptide of the invention; and/or [15427] d)
generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned organic acid of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 38, columns 5
and 7 or its homologs activity; and/or [15428] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned organic acid of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 38, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [15429] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned organic acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 38, columns 5 and 7 or its
homologs activity, and/or [15430] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned organic acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 38, columns 5 and 7 or its
homologs activity; and/or [15431] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 38, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[15432] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [15433] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[15434] [0054.0.38.38] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table XII, application no.
38, columns 3 or 5, resp., or its homologs activity, e.g. as
indicated in Table XII, application no. 38, columns 5 or 7,
resp.
[15435] [0055.0.0.38] to [0067.0.0.38]: see [0055.0.0.27] to
[0067.0.0.27]
[15436] [0068.0.38.38] The mutation is introduced in such a way
that the production of malic acid, and/or trihydroxybutanoic acid
is not adversely affected.
[15437] [0069.0.0.38] see [0069.0.0.27]
[15438] [0070.0.38.38] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or used in the process of the invention
or the polypeptide of the invention or used in the process of the
invention, for example the nucleic acid construct mentioned below,
or encoding a protein of the invention or used in the process of
the invention into an organism alone or in combination with other
genes, it is possible not only to increase the biosynthetic flux
towards the end product, but also to increase, modify or create de
novo an advantageous, preferably novel metabolite composition in
the organism, e.g. an advantageous composition of malic acid,
and/or trihydroxybutanoic acid or their biochemical derivatives,
e.g. comprising a higher content of (from a viewpoint of
nutritional physiology limited) malic acid, and/or
trihydroxybutanoic acid or their derivatives.
[15439] [0071.0.0.38] see [0071.0.0.27]
[15440] [0072.0.38.38] %
[15441] [0073.0.38.38] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[15442] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; and [15443] b) increasing an activity
of a polypeptide of the invention or used in the process of the
invention or a homolog thereof, e.g. as indicated in Table XII,
application no. 38, columns 5 or 7, or of a polypeptide being
encoded by the nucleic acid molecule of the present invention and
described below, e.g. conferring an increase of the respective fine
chemical in an organism, preferably in a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a
plant, and [15444] c) growing an organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant under conditions which permit the
production of the respective fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and [15445] d) if desired, recovering, optionally
isolating, the free and/or bound the respective fine chemical
synthesized by the organism, the microorganism, the non-human
animal, the plant or animal cell, the plant or animal tissue or the
plant.
[15446] [0074.0.38.38] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound respective fine chemical.
[15447] [0075.0.0.38] to [0077.0.0.38]: see [0075.0.0.27] to
[0077.0.0.27]
[15448] [0078.0.38.38] The organism such as microorganisms or
plants or the recovered, and if desired isolated, the respective
fine chemical can then be processed further directly into
foodstuffs or animal feeds or for other applications. The
fermentation broth, fermentation products, plants or plant products
can be purified with methods known to the person skilled in the
art. Products of these different work-up procedures are malic acid
and/or trihydroxybutanoic acid or comprising compositions of malic
acid, and/or trihydroxybutanoic acid still comprising fermentation
broth, plant particles and cell components in different amounts,
advantageously in the range of from 0 to 99% by weight, preferably
below 80% by weight, especially preferably below 50% by weight.
[15449] [0079.0.0.38] to [0084.0.0.38]: see [0079.0.0.27] to
[0084.0.0.27]
[15450] [0084.0.38.38] -/-
[15451] [0085.0.38.38] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [15452] a) a nucleic acid sequence as
indicated in Table XI, application no. 38, columns 5 or 7, or a
derivative thereof, or [15453] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 38, columns
5 or 7, or a derivative thereof, or [15454] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[15455] [0086.0.0.38] to [0087.0.0.38]: see [0086.0.0.27] to
[0087.0.0.27]
[15456] [0088.0.38.38] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned fine
chemicals or the plants are more resistant to biotic and abiotic
stress and the yield is increased.
[15457] [0088.1.0.38] see [0088.1.0.27]
[15458] [0089.0.0.38] to [0094.0.0.38]: see [0089.0.0.27] to
[0094.0.0.27]
[15459] [0095.0.38.38] It may be advantageous to increase the pool
of malic acid, and/or trihydroxybutanoic acid in the transgenic
organisms by the process according to the invention in order to
isolate high amounts of the pure respective fine chemical and/or to
obtain increased resistance against biotic and abiotic stresses and
to obtain higher yield.
[15460] [0096.0.38.38] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention or used in the
process of the invention together with the transformation of a
protein or polypeptid or a compound, which functions as a sink for
the desired fine chemical, for example in the organism, is useful
to increase the production of the respective fine chemical.
[15461] [0097.0.38.38] -/-
[15462] [0098.0.38.38] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated.
[15463] [0099.0.38.38] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[15464] [0100.0.38.38] Transgenic plants which comprise the fine
chemicals such as malic acid, and/or trihydroxybutanoic acid
synthesized in the process according to the invention can
advantageously be marketed directly without there being any need
for the fine chemicals synthesized to be isolated. Plants for the
process according to the invention are listed as meaning intact
plants and all plant parts, plant organs or plant parts such as
leaf, stem, seeds, root, tubers, anthers, fibers, root hairs,
stalks, embryos, calli, cotelydons, petioles, harvested material,
plant tissue, reproductive tissue and cell cultures which are
derived from the actual transgenic plant and/or can be used for
bringing about the transgenic plant. In this context, the seed
comprises all parts of the seed such as the seed coats, epidermal
cells, seed cells, endosperm or embryonic tissue.
[15465] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, as extracts, e.g. ether, alcohol,
or other organic solvents or water containing extract and/or free
fine chemicals. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pre-treated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[15466] Because malic acid, and/or trihydroxybutanoic acid in
microorganisms are localized intracellular, their recovery
essentially comes down to the isolation of the biomass.
Well-established approaches for the harvesting of cells include
filtration, centrifugation and coagulation/flocculation as
described herein. Of the residual hydrocarbon, adsorbed on the
cells, has to be removed. Solvent extraction or treatment with
surfactants have been suggested for this purpose.
[15467] [0101.0.38.38] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[15468] [0102.0.38.38] Malic acid and/or trihydroxybutanoic acid
can for example be detected advantageously via HPLC, LC or GC
separation methods. The unambiguous detection for the presence of
malic acidand/or trihydroxybutanoic acid containing products can be
obtained by analyzing recombinant organisms using analytical
standard methods: LC, LC-MS, MS or TLC. The material to be analyzed
can be disrupted by sonication, grinding in a glass mill, liquid
nitrogen and grinding, cooking, or via other applicable
methods.
[15469] [0103.0.38.38] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [15470] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide having a
sequence as indicated in Table XII, application no. 38, columns 5
or 7, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [15471] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table XI, application no. 38, columns 5 or 7,
[15472] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [15473] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[15474] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [15475]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [15476] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[15477] h) nucleic acid molecule comprising a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers pairs having a
sequence as indicated in Table XIII, application no. 38, columns 7,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [15478] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from an
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [15479] j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence having a sequences as
indicated in Table XIV, application no. 38, columns 7, and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [15480] k) nucleic acid
molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of the polypeptide indicated in Table XII, application no. 38,
columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[15481] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[15482] [00103.1.38.38] In one embodiment, the nucleic acid
molecule used in the process of the invention distinguishes over
the sequence indicated in Table XI, application no. 38, columns 5
or 7, by one or more nucleotides. In one embodiment, the nucleic
acid molecule used in the process of the invention does not consist
of the sequence shown in indicated in Table XI, application no. 38,
columns 5 or 7: In one embodiment, the nucleic acid molecule used
in the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI, columns
5 or 7. In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table XII,
application no. 38, columns 5 or 7.
[15483] [0104.0.38.38] In one embodiment, the nucleic acid molecule
used in the process of the present invention or used in the the
process of the invention distinguishes over the sequence indicated
in Table XI, application no. 38, columns 5 or 7, by one or more
nucleotides. In one embodiment, the nucleic acid molecule of the
invention or used in the process of the invention does not consist
of the sequence indicated in Table XI, application no. 38, columns
5 or 7. In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence indicated in Table XI, application no.
38, columns 5 or 7. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table XII, application no. 38, columns 5 or 7.
[15484] [0105.0.0.38] to [0107.0.0.38]: see [0105.0.0.27] to
[0107.0.0.27]
[15485] [0108.0.38.38] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 38, columns 5 or 7, nucleic
acid molecules which are derived from an amino acid sequences as
indicated in Table XII, application no. 38, columns 5 or 7 or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 38, column 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of an activity of a polypeptide as indicated in Table XII,
application no. 38, column 3, 5 or 7, e.g. conferring the increase
of the respective fine chemical, meaning malic acid, and/or
trihydroxybutanoic acid, resp., after increasing its expression or
activity, are advantageously increased in the process according to
the invention.
[15486] [0109.0.38.38] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular malic
acid and/or trihydroxybutanoic acid, produced in the process
according to the invention.
[15487] [0110.0.0.38] see [0110.0.0.27]
[15488] [0111.0.0.38] see [0111.0.0.27]
[15489] [0112.0.38.38] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XII, application no. 38,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 38, columns 5 and 7 and conferring an
increase in the malic acid and/or trihydroxybutanoic acid
level.
[15490] [0113.0.0.38] to [0114.0.0.38]: see [0113.0.0.27] to
[0114.0.0.27]
[15491] [0115.0.0.38] see [0115.0.0.27]
[15492] [0116.0.0.38] to [0120.0.0.38] see [0116.0.0.27] to
[0120.0.0.27]
[15493] [0120.1.38.38]: -/-
[15494] [0121.0.38.38] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 38, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a malic acid level or conferring a
trihydroxybutanoic acid increase after increasing the activity of
the polypeptide sequences indicated in Table XII, application no.
38, columns 5 or 7,
[15495] [0122.0.0.38] to [0127.0.0.38]: see [0122.0.0.27] to
[0127.0.0.27]
[15496] [0128.0.38.38] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 38, columns 7, by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table XI, application no.
38, columns 5 or 7, or the sequences derived from a sequence as
indicated in Table XII, application no. 38, columns 5 or 7.
[15497] [0129.0.38.38] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention or used in the process of the invention, from which
conserved regions, and in turn, degenerate primers can be derived.
Conserved region for the polypeptide of the invention or used in
the process of the invention are indicated in the alignments shown
in the figures. Conserved regions are those, which show a very
little variation in the amino acid in one particular position of
several homologs from different origin. The consensus sequence
indicated in Table XIV, application no. 38, columns 7 is derived
from said alignments.
[15498] [0130.0.38.38] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of malic
acid, and/or trihydroxybutanoic acid after increasing the
expression or activity the protein comprising said fragment.
[15499] [0131.0.0.38] to [0138.0.0.38]: see [0131.0.0.27] to
[0138.0.0.27]
[15500] [0139.0.38.38] Polypeptides having above-mentioned
activity, i.e. conferring the respective fine chemical level
increase, derived from other organisms, can be encoded by other DNA
sequences which hybridise to a sequence indicated in Table XI,
application no. 38, columns 5 or 7, for trihydroxybutanoic acid
under relaxed hybridization conditions and which code on expression
for peptides having the respective fine chemical, i.e. malic acid,
and/or trihydroxybutanoic acid, resp., increasing-activity.
[15501] [0140.0.0.38] to [0146.0.0.38]: see [0140.0.0.27] to
[0146.0.0.27]
[15502] [0147.0.38.38] Further, the nucleic acid molecule of the
invention or used in the process of the invention comprises a
nucleic acid molecule, which is a complement of one of the
nucleotide sequences of above mentioned nucleic acid molecules or a
portion thereof. A nucleic acid molecule which is complementary to
one of the nucleotide sequences indicated in Table XI, application
no. 38, columns 5 or 7, is one which is sufficiently complementary
to one of said nucleotide sequences such that it can hybridise to
one of said nucleotide sequences, thereby forming a stable duplex.
Preferably, the hybridisation is performed under stringent
hybridization conditions. However, a complement of one of the
herein disclosed sequences is preferably a sequence complement
thereto according to the base pairing of nucleic acid molecules
well known to the skilled person. For example, the bases A and G
undergo base pairing with the bases T and U or C, resp. and visa
versa. Modifications of the bases can influence the base-pairing
partner.
[15503] [0148.0.38.38] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 38,
columns 5 or 7, or a portion thereof and preferably has above
mentioned activity, in particular having a malic acid, and/or
trihydroxybutanoic acid increasing activity after increasing the
activity or an activity of a product of a gene encoding said
sequences or their homologs.
[15504] [0149.0.38.38] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 38,
columns 5 or 7 or a portion thereof and encodes a protein having
above-mentioned activity, e.g. conferring a of glyceric acid,
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid and/or trihydroxybutanoic acid
increase, resp., and optionally, the activity of protein indicated
in Table XII, column 5, lines 190 to 226 or lines 564 to 594,
preferably in Table XII, application no. 38, columns 7.
[15505] [00149.1.38.38] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table XI, application no. 38, columns 5 or
7, has further one or more of the activities annotated or known for
a protein as indicated in Table XII, application no. 38, column
3.
[15506] [0150.0.38.38] Moreover, the nucleic acid molecule of the
invention or used in the process of the invention can comprise only
a portion of the coding region of one of the sequences indicated in
Table XI, application no. 38, columns 5 or 7, for example a
fragment which can be used as a probe or primer or a fragment
encoding a biologically active portion of the polypeptide of the
present invention or of a polypeptide used in the process of the
present invention, i.e. having above-mentioned activity, e.g.
conferring an increase of malic acid, and/or trihydroxybutanoic
acid, resp., if its activity is increased. The nucleotide sequences
determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table XI,
application no. 38, columns 5 or 7, an anti-sense sequence of one
of the sequences, e.g., as indicated in Table XI, application no.
38, columns 5 or 7, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table XIII, application no. 38, column 7 will result in a fragment
of a polynucleotide sequence as indicated in Table XI, application
no. 38, columns 5 or 7 or its gene product. [0151.0.0.38]: see
[0151.0.0.27]
[15507] [0152.0.38.38] The nucleic acid molecule of the invention
or used in the process of the invention encodes a polypeptide or
portion thereof which includes an amino acid sequence which is
sufficiently homologous to an amino acid sequence as indicated in
Table XII, application no. 38, columns 5 or 7, such that the
protein or portion thereof maintains the ability to participate in
the respective fine chemical production, in particular a malic acid
or trihydroxybutanoic acid increasing activity as mentioned above
or as described in the examples in plants or microorganisms is
comprised.
[15508] [0153.0.38.38] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 38,
columns 5 or 7 such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 38, columns 5 or 7, has for
example an activity of a polypeptide indicated in Table XII,
application no. 38, column 3.
[15509] [0154.0.38.38] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 38, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[15510] [0155.0.0.38] to [0156.0.0.38]: see [0155.0.0.27] to
[0156.0.0.27]
[15511] [0157.0.38.38] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences as
indicated in Table XI, application no. 38, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 38, columns 7, or as polypeptides
depicted in Table XII, application no. 38, columns 5 or 7, or the
functional homologues. Advantageously, the nucleic acid molecule of
the invention or used in the process of the invention comprises, or
in an other embodiment has, a nucleotide sequence encoding a
protein comprising, or in an other embodiment having, an amino acid
sequence of a consensus sequences as indicated in Table XIV,
application no. 38, columns 7, or of the polypeptide as indicated
in Table XII, application no. 38, columns 5 or 7, resp., or the
functional homologues. In a still further embodiment, the nucleic
acid molecule of the invention encodes a full length protein which
is substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table XIV, application no. 38,
columns 5 or 7, or of a polypeptide as indicated in Table XII,
application no. 38, columns 5 or 7, or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 38, columns 5 or 7
[15512] [0158.0.0.38] to [0160.0.0.38]: see [0158.0.0.27] to
[0160.0.0.27]
[15513] [0161.0.38.38] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 38, columns 5 or
7. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[15514] [0162.0.0.38] see [0162.0.0.27]
[15515] [0163.0.38.38] Preferably, a nucleic acid molecule of the
invention or used in the process of the invention that hybridizes
under stringent conditions to a sequence as indicated in Table XI,
application no. 38, columns 5 or 7 corresponds to a
naturally-occurring nucleic acid molecule of the invention. As used
herein, a "naturally-occurring" nucleic acid molecule refers to an
RNA or DNA molecule having a nucleotide sequence that occurs in
nature (e.g., encodes a natural protein). Preferably, the nucleic
acid molecule encodes a natural protein having above-mentioned
activity, e.g. conferring the increase of the amount of the
respective fine chemical in a organism or a part thereof, e.g. a
tissue, a cell, or a compartment of a cell, after increasing the
expression or activity thereof or the activity of a protein of the
invention or used in the process of the invention.
[15516] [0164.0.0.38] see [0164.0.0.27]
[15517] [0165.0.38.38] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 38, columns 5 or 7 resp.
[15518] [0166.0.0.38] to [0167.0.0.38]: see [0166.0.0.27] to
[0167.0.0.27]
[15519] [0168.0.38.38] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 38, columns 5 or 7, preferably in Table XII,
columns 7, resp., yet retain said activity described herein. The
nucleic acid molecule can comprise a nucleotide sequence encoding a
polypeptide, wherein the polypeptide comprises an amino acid
sequence at least about 50% identical to an amino acid sequence as
indicated in Table XII, application no. 38, columns 5 or 7, resp.,
and is capable of participation in the increase of production of
the respective fine chemical after increasing its activity, e.g.
its expression. Preferably, the protein encoded by the nucleic acid
molecule is at least about 60% identical to a sequence as indicated
in Table XII, application no. 38, columns 5 or 7, resp., more
preferably at least about 70% identical to one of the sequences as
indicated in Table XII, application no. 38, columns 5 or 7, resp.,
even more preferably at least about 80%, 90%, 95% homologous to a
sequence as indicated in Table XII, application no. 38, columns 5
or 7, resp., and most preferably at least about 96%, 97%, 98%, or
99% identical to the sequence as indicated in Table XII,
application no. 38, columns 5 or 7.
[15520] [0169.0.0.38] to [0172.0.0.38]: see [0169.0.0.27] to
[0172.0.0.27]
[15521] [0173.0.38.38] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 108417 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108417 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[15522] [0174.0.0.38]: see [0174.0.0.27]
[15523] [0175.0.38.38] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108418 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108418 by the above program algorithm with the
above parameter set, has a 80% homology.
[15524] [0176.0.38.38] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 38,
columns 5 or 7, resp., according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 38, columns 5 or 7, resp., according to the
invention and are distinguished by essentially the same properties
as a polypeptide as indicated in Table XII, application no. 38,
columns 5 or 7 resp.
[15525] [0177.0.38.38] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 38,
columns 5 or 7, resp., according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 38, columns 5 or 7, resp., according to the
invention and encode polypeptides having essentially the same
properties as a polypeptide as indicated in Table XII, application
no. 38, columns 5 or 7, resp.
[15526] [0178.0.0.38] see [0178.0.0.27]
[15527] [0179.0.38.38] A nucleic acid molecule encoding a
homologous to a protein sequence as indicated in Table XII,
application no. 38, columns 5 or 7, resp., can be created by
introducing one or more nucleotide substitutions, additions or
deletions into a nucleotide sequence of the nucleic acid molecule
of the present invention, in particular as indicated in Table XI,
application no. 38, columns 5 or 7, resp., such that one or more
amino acid substitutions, additions or deletions are introduced
into the encoded protein. Mutations can be introduced into the
encoding sequences for example into sequences of nucleic acid
molecules as indicated in Table XI, application no. 38, columns 5
or 7, resp., by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis.
[15528] [0180.0.0.38] to [0183.0.0.38]: see [0180.0.0.27] to
[0183.0.0.27]
[15529] [0184.0.38.38] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 38,
columns 5 or 7, resp., or of the nucleic acid sequences derived
from a sequences as indicated in Table XII, application no. 38,
columns 5 or 7, resp., comprise also allelic variants with at least
approximately 30%, 35%, 40% or 45% homology, by preference at least
approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
XI, application no. 38, columns 5 or 7, resp., or from the derived
nucleic acid sequences, the intention being, however, that the
enzyme activity or the biological activity of the resulting
proteins synthesized is advantageously retained or increased.
[15530] [0185.0.38.38] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 38, columns 5 or 7, resp. In one
embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotides not shown in any
one of sequences as indicated in Table XI, application no. 38,
columns 5 or 7, resp. In one embodiment, the nucleic acid molecule
comprises less than 500, 400, 300, 200, 100, 90, 80, 70, 60, 50 or
40 further nucleotides. In a further embodiment, the nucleic acid
molecule comprises less than 30, 20 or 10 further nucleotides. In
one embodiment, a nucleic acid molecule used in the process of the
invention is identical to a sequence as indicated in Table XI,
application no. 38, columns 5 or 7, resp.
[15531] [0186.0.38.38] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table XII,
application no. 38, columns 5 or 7, resp. In one embodiment, the
nucleic acid molecule encodes less than 150, 130, 100, 80, 60, 50,
40 or 30 further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 38, columns 5 or 7 resp.
[15532] [0187.0.38.38] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table XII, application no.
38, columns 5 or 7, resp., comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a sequences as indicated in Table XII,
application no. 38, columns 5 or 7 resp.
[15533] [0188.0.38.38] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 38, columns 5 or 7 resp., preferably compared
to a sequence as indicated in preferably in Table XII, application
no. 38, columns 5 or 7, and is expressed under identical
conditions.
[15534] [0189.0.38.38] Homologues of a sequence as indicated in
Table XI, application no. 38, columns 5 or 7 resp., or of a derived
sequences as indicated in Table XII, application no. 38, columns 5
or 7, resp., also mean truncated sequences, cDNA, single-stranded
DNA or RNA of the coding and noncoding DNA sequence. Homologues of
said sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[15535] [0190.0.0.38]: see [0190.0.0.27]
[15536] [0191.0.0.38] see [0191.0.0.27]
[15537] [0191.0.38.38] The organisms or part thereof produce
according to the herein mentioned process of the invention an
increased level of free and/or bound the respective fine chemical
compared to said control or selected organisms or parts
thereof.
[15538] [0192.0.0.38] to [0203.0.0.38]: see [0192.0.0.27] to
[0203.0.0.27]
[15539] [0204.0.38.38] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule, which comprises a
nucleic acid molecule selected from the group consisting of:
[15540] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 38, columns 5 or 7, resp.; or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, i.e. malic acid or trihydroxybutanoic acid, resp., in an
organism or a part thereof [15541] b) nucleic acid molecule
comprising, preferably at least the mature form, of a nucleic acid
molecule as indicated in Table XI, application no. 38, columns 5 or
7, resp., or a fragment thereof conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [15542] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [15543] d) nucleic
acid molecule encoding a polypeptide whose sequence has at least
50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [15544] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; [15545] f) nucleic acid molecule encoding a polypeptide,
the polypeptide being derived by substituting, deleting and/or
adding one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[15546] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [15547] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table XIII, application no. 38, columns 5 or 7, and
conferring an increase in the amount of the respective fine
chemical, i.e. malic acid or trihydroxybutanoic acid, resp., in an
organism or a part thereof; [15548] i) nucleic acid molecule
encoding a polypeptide which is isolated, e.g. from a expression
library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [15549] j) nucleic acid molecule which encodes a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 38, columns 7, and conferring an increase in
the amount of the respective fine chemical, or trihydroxybutanoic
acidresp., in an organism or a part thereof; [15550] k) nucleic
acid molecule encoding the amino acid sequence of a polypeptide
encoding a domain of a polypeptide as indicated in Table XII,
application no. 38, columns 5 or 7, resp., and conferring an
increase in the amount of the respective fine chemical, i.e. malic
acid or trihydroxybutanoic acid resp., in an organism or a part
thereof; and [15551] l) nucleic acid molecule which is obtainable
by screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt,
200 nt or 500 nt of the nucleic acid molecule characterized in (a)
to (h) or of a nucleic acid molecule as indicated in Table XI,
application no. 38, columns 5 or 7, resp., or a nucleic acid
molecule encoding, preferably at least the mature form of, a
polypeptide as indicated in Table XII, application no. 38, columns
5 or 7, resp., and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
encompasses a sequence which is complementary thereto; whereby,
preferably, the nucleic acid molecule according to (a) to (l)
distinguishes over a sequence as indicated in Table XI, application
no. 38, columns 5 or 7, resp., by one or more nucleotides. In one
embodiment, the nucleic acid molecule of the invention does not
consist of the sequence as indicated in Table XI, application no.
38, columns 5 or 7, resp. In an other embodiment, the nucleic acid
molecule of the present invention is at least 30% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence as indicated in Table XI, application no. 38, columns 5 or
7, resp. In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table XII,
application no. 38, columns 5 or 7, resp. Accordingly, in one
embodiment, the nucleic acid molecule of the present invention
encodes in one embodiment a polypeptide which differs at least in
one or more amino acids from a polypeptide indicated in Table XII,
application no. 38, columns 5 or 7, does not encode a protein of a
sequence as indicated in Table XII, application no. 38, columns 5
or 7. Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table XII, application no.
38, columns 5 or 7. In a further embodiment, the protein of the
present invention is at least 30% identical to a protein sequence
indicated in Table XII, application no. 38, columns 5 or 7, and
less than 100%, preferably less than 99.999%, 99.99% or 99.9%, more
preferably less than 99%, 985, 97%, 96% or 95% identical to a
sequence as indicated in Table XII, application no. 38, columns 5
or 7.
[15552] [0205.0.0.38] to [0206.0.0.38]: see [0205.0.0.27] to
[0206.0.0.27]
[15553] [0207.0.38.38] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the glutamic acid metabolism, the
phosphoenolpyruvate metabolism, the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[15554] [0208.0.0.38] to [0226.0.0.38]: see [0208.0.0.27] to
[0226.0.0.27]
[15555] [0227.0.38.38] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms. In addition
to a sequence indicated in Table XI, application no. 38, columns 5
or 7 or its derivatives, it is advantageous to express and/or
mutate further genes in the organisms. Especially advantageously,
additionally at least one further gene of the glutamic acid or
phosphoenolpyruvate metabolic pathway, is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the fine
chemicals desired since, for example, feedback regulations no
longer exist to the same extent or not at all. In addition it might
be advantageously to combine one or more of the sequences indicated
in Table XI, application no. 38, columns 5 or 7, resp., with genes
which generally support or enhances to growth or yield of the
target organism, for example genes which lead to faster growth rate
of microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[15556] [0228.0.38.38] In a further embodiment of the process of
the invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins directly or indirectly involved
in the glutamic acid or phosphoenolpyruvate metabolism.
[15557] [0229.0.38.38] %
[15558] [0230.0.0.38] see [230.0.0.27]
[15559] [0231.0.38.38] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a malic acid, and/or
trihydroxybutanoic acid degrading protein is attenuated, in
particular by reducing the rate of expression of the corresponding
gene.
[15560] [0232.0.0.38] to [0276.0.0.38]: see [0232.0.0.27] to
[0276.0.0.27]
[15561] [0277.0.38.38] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously.
[15562] [0278.0.0.38] to [0282.0.0.38]: see [0278.0.0.27] to
[0282.0.0.27]
[15563] [0283.0.38.38] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells, for example using the antibody
of the present invention as described below, e.g. an antibody
against a protein as indicated in Table XII, application no. 38,
column 3, resp., or an antibody against a polypeptide as indicated
in Table XII, application no. 38, columns 5 or 7, resp., or an
antigenic part thereof which can be produced by standard techniques
utilizing polypeptides comprising or consisting of above mentioned
sequences, e.g. the polypeptid of the present invention or fragment
thereof. Preferred are monoclonal antibodies specifically binding
to polypeptide as indicated in Table XII, application no. 38,
columns 5 or 7.
[15564] [0284.0.0.38] see [0284.0.0.27]
[15565] [0285.0.38.38] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 38, columns 5 or 7, resp., or as coded by a
nucleic acid molecule as indicated in Table XI, application no. 38,
columns 5 or 7, resp., or functional homologues thereof.
[15566] [0286.0.38.38] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 38, columns 7, and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 38, columns 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a poylpeptide or to a polypeptide comprising more than
one consensus sequences as indicated in Table XIV, application no.
38, column 7.
[15567] [0287.0.0.38] to [0289.0.0.38]: see [0287.0.0.27] to
[0289.0.0.27]
[15568] [00290.0.38.38] The alignment was performed with the
Software AlignX (Sep. 25, 2002) a component of Vector NTI Suite
8.0, InforMax.TM., Invitrogen.TM. life science software, U.S. Main
Office, 7305 Executive Way, Frederick, Md. 21704, USA with the
following settings: For pair wise alignments: gap opening penalty:
10.0; gap extension penalty 0,1. For multiple alignments: Gap
opening penalty: 10.0; Gap extension penalty: 0.1; Gap separation
penalty range: 8; Residue substitution matrix: blosum62;
Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0,5;
Consensus calculation options: Residue fraction for consensus: 0.9.
Presettings were selected to allow also for the alignment of
conserved amino acids
[15569] [0291.0.38.38] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. Accordingly, in one embodiment, the present
invention relates to a polypeptide comprising or consisting of
plant or microorganism specific consensus sequences.
[15570] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table XII,
application no. 38, columns 5 or 7, esp., by one or more amino
acids. In one embodiment, polypeptide distinguishes from a sequence
as indicated in Table XII, application no. 38, columns 5 or 7,
resp., by more than 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, even more preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table XII, application no. 38, columns 5 or 7, resp.,
by not more than 80% or 70% of the amino acids, preferably not more
than 60% or 50%, more preferred not more than 40% or 30%, even more
preferred not more than 20% or 10%. In an other embodiment, said
polypeptide of the invention does not consist of a sequence as
indicated in Table XII, application no. 38, columns 5 or 7.
[15571] [0292.0.0.38] see [0292.0.0.27]
[15572] [0293.0.38.38] In one embodiment, the invention relates to
a polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or by a nucleic acid molecule used in the process of the
invention. In one embodiment, the polypeptide of the invention has
a sequence which distinguishes from a sequence as indicated in
Table XII, application no. 38, columns 5 or 7, resp., by one or
more amino acids. In an other embodiment, said polypeptide of the
invention does not consist of the sequence as indicated in Table
XII, columns 5 or 7 resp. In a further embodiment, said polypeptide
of the present invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical. In one embodiment, said polypeptide does not
consist of the sequence encoded by a nucleic acid molecules as
indicated in Table XI, application no. 38, columns 5 or 7 resp.
[15573] [0294.0.38.38] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 38, column 3, resp., which
distinguishes over a sequence as indicated in Table XII,
application no. 38, columns 5 or 7, resp., by one or more amino
acids, preferably by more than 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, even more
preferred are more than 40, 50, or 60 amino acids but even more
preferred by less than 70% of the amino acids, more preferred by
less than 50%, even more preferred my less than 30% or 25%, more
preferred are 20% or 15%, even more preferred are less than
10%.
[15574] [0295.0.0.38] to [0296.0.0.38]: see [0295.0.0.27] to
[0296.0.0.27]
[15575] [0297.0.0.38] see [0297.0.0.27]
[15576] [00297.1.38.38] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity and/or amino acid
sequence of a polypeptide indicated in Table XII, application no.
38, columns 3, 5 or 7
[15577] [0298.0.38.38] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 38, columns 5 or 7,
resp. The portion of the protein is preferably a biologically
active portion as described herein. Preferably, the polypeptide
used in the process of the invention has an amino acid sequence
identical to a sequence as indicated in Table XII, application no.
38, columns 5 or 7 resp.
[15578] [0299.0.38.38] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 38,
columns 5 or 7, resp. The preferred polypeptide of the present
invention preferably possesses at least one of the activities
according to the invention and described herein. A preferred
polypeptide of the present invention includes an amino acid
sequence encoded by a nucleotide sequence which hybridizes,
preferably hybridizes under stringent conditions, to a nucleotide
sequence as indicated in Table XI, application no. 38, columns 5 or
7 resp., or which is homologous thereto, as defined above.
[15579] [0300.0.38.38] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table XII,
application no. 38, columns 5 or 7, resp., in amino acid sequence
due to natural variation or mutagenesis, as described in detail
herein. Accordingly, the polypeptide comprise an amino acid
sequence which is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%
or 70%, preferably at least about 75%, 80%, 85% or 90, and more
preferably at least about 91%, 92%, 93%, 94% or 95%, and most
preferably at least about 96%, 97%, 98%, 99% or more homologous to
an entire amino acid sequence of as indicated in Table XII,
application no. 38, columns 5 or 7
[15580] [0301.0.0.38] see [0301.0.0.27]
[15581] [0302.0.38.38] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 38, columns 5 or 7, resp., or the amino
acid sequence of a protein homologous thereto, which include fewer
amino acids than a full length polypeptide of the present invention
or used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[15582] [0303.0.0.38] see [0303.0.0.27]
[15583] [0304.0.38.38] Manipulation of the nucleic acid molecule of
the invention or the nucleic acid molecule used in the method of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 38, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[15584] [0305.0.0.38] to [0308.0.0.38]: see [0305.0.0.27] to
[0308.0.0.27]
[15585] [00306.1.27.27] In one embodiment, the compound is a
composition comprising the repective fine chemical, i.e. said
organic acids, or a recovered fine chemical, i.e. said organic acid
free or in protein-bound form.
[15586] [0309.0.38.38] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table XII,
application no. 38, columns 5 or 7, resp., refers to a polypeptide
having an amino acid sequence corresponding to the polypeptide of
the invention or used in the process of the invention, whereas an
"other polypeptide" not being indicated in Table XII, application
no. 38, columns 5 or 7, resp., refers to a polypeptide having an
amino acid sequence corresponding to a protein which is not
substantially homologous to a polypeptide of the invention,
preferably which is not substantially homologous to a polypeptide
as indicated in Table XII, application no. 38, columns 5 or 7,
resp., e.g., a protein which does not confer the activity described
herein or annotated or known for as indicated in Table XII,
application no. 38, column 3 resp., and which is derived from the
same or a different organism. In one embodiment, an "other
polypeptide" not being indicated in Table XII, application no. 38,
columns 5 or 7, resp., does not confer an increase of the
respective fine chemical in an organism or part thereof. In one
embodiment, a "non-polypeptide of the invention" or "other
polypeptide" not being indicated in Table XII, application no. 38,
columns 5 or 7 resp., does not confer an increase of the respective
fine chemical in an organism or part thereof.
[15587] [0310.0.0.38] to [0334.0.0.38]: see [0310.0.0.27] to
[0334.0.0.27]
[15588] [0335.0.38.38] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XI, application
no. 38, columns 5 or 7, resp., and/or homologs thereof. As
described inter alia in WO 99/32619, dsRNAi approaches are clearly
superior to traditional antisense approaches. The invention
therefore furthermore relates to double-stranded RNA molecules
(dsRNA molecules) which, when introduced into an organism,
advantageously into a plant (or a cell, tissue, organ or seed
derived there from), bring about altered metabolic activity by the
reduction in the expression of a nucleic acid sequences as
indicated in Table XI, application no. 38, columns 5 or 7, resp.,
and/or homologs thereof. In a double-stranded RNA molecule for
reducing the expression of a protein encoded by a nucleic acid
sequence as indicated in Table XI, application no. 38, columns 5 or
7, resp., and/or homologs thereof, one of the two RNA strands is
essentially identical to at least part of a nucleic acid sequence,
and the respective other RNA strand is essentially identical to at
least part of the complementary strand of a nucleic acid
sequence.
[15589] [0336.0.0.38] to [0342.0.0.38]: see [0336.0.0.27] to
[0342.0.0.27]
[15590] [0343.0.38.38] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 38, columns 5 or
7, resp., or its homolog is not necessarily required in order to
bring about effective reduction in the expression. The advantage
is, accordingly, that the method is tolerant with regard to
sequence deviations as may be present as a consequence of genetic
mutations, polymorphisms or evolutionary divergences. Thus, for
example, using the dsRNA, which has been generated starting from a
sequence as indicated in Table XI, application no. 38, columns 5 or
7, resp., or homologs thereof of the one organism, may be used to
suppress the corresponding expression in another organism.
[15591] [0344.0.0.38] to [0361.0.0.38]: see [0344.0.0.27] to
[0361.0.0.27]
[15592] [0362.0.38.38] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 38, columns 5 or 7, resp., e.g. encoding
a polypeptide having protein activity, as indicated in Table XII,
application no. 38, columns 3, resp., Due to the abovementioned
activity the respective fine chemical content in a cell or an
organism is increased. For example, due to modulation or
manipulation, the cellular activity of the polypeptide of the
invention or nucleic acid molecule of the invention or the nucleic
acid molecule or polypeptide used in the method of the invention is
increased, e.g. due to an increased expression or specific activity
of the subject matters of the invention in a cell or an organism or
a part thereof. Transgenic for a polypeptide having an activity of
a polypeptide as indicated in Table XII, application no. 38,
columns 5 or 7, resp., means herein that due to modulation or
manipulation of the genome, an activity as annotated for a
polypeptide as indicated in Table XII, application no. 38, column
3, e.g. having a sequence as indicated in Table XII, application
no. 38, columns 5 or 7, resp., is increased in a cell or an
organism or a part thereof. Examples are described above in context
with the process of the invention.
[15593] [0363.0.0.38] see [0363.0.0.27]
[15594] [0364.0.38.38] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table XII, application no. 38, column 3, resp. with
the corresponding protein-encoding sequence as indicated in Table
XI, application no. 38, column 5, resp., becomes a transgenic
expression cassette when it is modified by non-natural, synthetic
"artificial" methods such as, for example, mutagenization. Such
methods have been described (U.S. Pat. No. 5,565,350; WO 00/15815;
also see above).
[15595] [0365.0.0.38] to [0373.0.0.38]: see [0365.0.0.27] to
[0373.0.0.27]
[15596] [0374.0.38.38] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. malic acid, and/or
trihydroxybutanoic acid, in particular the respective fine
chemical, produced in the process according to the invention may,
however, also be isolated from the plant in the form of their free
malic acid, and/or trihydroxybutanoic acid, in particular the free
respective fine chemical, or bound in or to compounds or moieties,
like glucosides, e.g. diglucosides.
[15597] The respective fine chemical produced by this process can
be harvested by harvesting the organisms either from the culture in
which they grow or from the field. This can be done via expressing,
grinding and/or extraction, salt precipitation and/or ion-exchange
chromatography or other chromatographic methods of the plant parts,
preferably the plant seeds, plant fruits, plant tubers and the
like.
[15598] [0375.0.0.38] to [0376.0.0.38]: see [0375.0.0.27] to
[0376.0.0.27]
[15599] [0377.0.38.38] Accordingly, the present invention relates
also to a process whereby the produced malic acid, and/or
trihydroxybutanoic acid is isolated.
[15600] [0378.0.38.38] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the malic acid,
and/or trihydroxybutanoic acid produced in the process can be
isolated. The resulting malic acid, and/or trihydroxybutanoic acid
can, if appropriate, subsequently be further purified, if desired
mixed with other active ingredients such as vitamins, amino acids,
carbohydrates, antibiotics and the like, and, if appropriate,
formulated.
[15601] [0379.0.38.38] In one embodiment, gamma-aminobutyric acid
and shikimate are a mixture of the respective fine chemicals.
[15602] [0380.0.38.38] The malic acid, and/or trihydroxybutanoic
acid obtained in the process are suitable as starting material for
the synthesis of further products of value. For example, they can
be used in combination with each other or alone for the production
of pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the malic acid, and/or
trihydroxybutanoic acid composition produced or the respective fine
chemical produced if desired and formulating the product with a
pharmaceutical acceptable carrier or formulating the product in a
form acceptable for an application in agriculture. A further
embodiment according to the invention is the use of the malic acid
and/or trihydroxybutanoic acid produced in the process or of the
transgenic organisms in animal feeds, foodstuffs, medicines, food
supplements, cosmetics or pharmaceuticals or for the production of
malic acid, and/or trihydroxybutanoic acid e.g. after isolation of
the respective fine chemical or without, e.g. in situ, e.g. in the
organism used for the process for the production of the respective
fine chemical.
[15603] [0381.0.0.38] to [0382.0.0.38]: see [0381.0.0.27] to
[0382.0.0.27]
[15604] [0383.0.38.38] -/-
[15605] [0384.0.0.38] see [0384.0.0.27]
[15606] [0385.0.38.38] The fermentation broths obtained in this
way, containing in particular malic acid, and/or trihydroxybutanoic
acid in mixtures with other organic acids, amino acids,
polypeptides or polysaccarides, normally have a dry matter content
of from 1 to 70% by weight, preferably 7.5 to 25% by weight.
Sugar-limited fermentation is additionally advantageous, e.g. at
the end, for example over at least 30% of the fermentation time.
This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 10 g/l,
preferably to 0 to 3 g/I during this time. The fermentation broth
is then processed further. Depending on requirements, the biomass
can be removed or isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth or left completely in it.
[15607] The fermentation broth can then be thickened or
concentrated by known methods, such as, for example, with the aid
of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[15608] [0386.0.38.38] Accordingly, it is possible to purify the
glyceric acid, citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid produced according to the invention
further. For this purpose, the product-containing composition is
subjected for example to separation via e.g. an open column
chromatography or HPLC in which case the desired product or the
impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use.
[15609] [0387.0.0.38] to [0392.0.0.38]: see [0387.0.0.27] to
[0392.0.0.27]
[15610] [0393.0.38.38] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [15611] a) contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [15612] b) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table XI, application no. 38,
columns 5 or 7, resp., and, optionally, isolating the full length
cDNA clone or complete genomic clone; [15613] c) introducing the
candidate nucleic acid molecules in host cells, preferably in a
plant cell or a microorganism, appropriate for producing the
respective fine chemical; [15614] d) expressing the identified
nucleic acid molecules in the host cells; [15615] e) assaying the
respective fine chemical level in the host cells; and [15616] f)
identifying the nucleic acid molecule and its gene product which
expression confers an increase in the respective fine chemical
level in the host cell after expression compared to the wild
type.
[15617] [0394.0.0.38] to [0398.0.0.38]: see [0394.0.0.27] to
[0398.0.0.27]
[15618] [0399.0.38.38] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increase of the respective fine chemical production in a
plant or microorganism, comprising the steps:
[15619] culturing a cell or tissue or microorganism or maintaining
a plant expressing the polypeptide according to the invention or a
nucleic acid molecule encoding said polypeptide and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and the polypeptide of the present invention or used
in the process of the invention; and
[15620] identifying if the compound is an effective agonist by
detecting the presence or absence or increase of a signal produced
by said readout system.
[15621] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in respective fine chemical
production.
[15622] [00399.1.0.38] see [0399.1.0.27]
[15623] [0400.0.0.38] to [0415.0.0.38]: see [0400.0.0.27] to
[0415.0.0.27]
[15624] [0416.0.0.38] see [0416.0.0.27]
[15625] [0417.0.38.38] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the glyceric acid, citramalic acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid biosynthesis
pathways. In particular, the overexpression of the polypeptide of
the present invention may protect an organism such as a
microorganism or a plant against inhibitors, which block the
glyceric acid, citramalic acid, fumaric acid, malic acid, pyruvic
acid, succinic acid, trihydroxybutyric acid and/or
trihydroxybutanoic acid synthesis.
[15626] [0418.0.0.38] to [0423.0.0.38]: see [0418.0.0.27] to
[0423.0.0.27]
[15627] [0424.0.38.38] Accordingly, the nucleic acid of the
invention or used in the method of the invention, the polypeptide
of the invention or used in the method of the invention, the
nucleic acid construct of the invention, the organisms, the host
cell, the microorganisms, the plant, plant tissue, plant cell, or
the part thereof of the invention, the vector of the invention, the
agonist identified with the method of the invention, the nucleic
acid molecule identified with the method of the present invention,
can be used for the production of the respective fine chemical or
of the respective fine chemical and one or more other organic
acids. Accordingly, the nucleic acid of the invention, or the
nucleic acid molecule identified with the method of the present
invention or the complement sequences thereof, the polypeptide of
the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the respective fine chemical in a organism or part
thereof, e.g. in a cell.
[15628] [0425.0.0.38] to [0434.0.0.38]: see [0425.0.0.27] to
[0434.0.0.27]
[0435.0.38.38] Example 3
In-Vivo and In-Vitro Mutagenesis
[15629] [0436.0.38.38] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of glyceric acid,
citramalic acid, fumaric acid, malic acid, pyruvic acid, succinic
acid, trihydroxybutyric acid and/or trihydroxybutanoic acid can be
carried out by passing a plasmid DNA (or another vector DNA)
containing the desired nucleic acid sequence or nucleic acid
sequences, e.g. the nucleic acid molecule of the invention or the
vector of the invention, through E. coli and other microorganisms
(for example Bacillus spp. or yeasts such as Saccharomyces
cerevisiae) which are not capable of maintaining the integrity of
its genetic information. Usual mutator strains have mutations in
the genes for the DNA repair system [for example mutHLS, mutD, mutT
and the like; for comparison, see Rupp, W. D. (1996) DNA repair
mechanisms in Escherichia coli and Salmonella, pp. 2277-2294, ASM:
Washington]. The skilled worker knows these strains. The use of
these strains is illustrated for example in Greener, A. and
Callahan, M. (1994) Strategies 7; 32-34.
[15630] In-vitro mutation methods such as increasing the
spontaneous mutation rates by chemical or physical treatment are
well known to the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widely used as
chemical agents for random in-vitro mutagenesis. The most common
physical method for mutagenesis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[15631] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[15632] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.38.38] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpina
[15633] [0438.0.38.38] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology", VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[15634] [0439.0.38.38] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
Saccharomyces can be achieved for example by LiCI or sheroplast
transformation (Bishop et al., Mol. Cell. Biol., 6, 1986:
3401-3409; Sherman et al., Methods in Yeasts in Genetics, [Cold
Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et al.,
Technical Tips Online 1998, 1:51: P01525 or Gietz et al., Methods
Mol. Cell. Biol. 5, 1995: 255f) or electroporation (Delorme E.,
Appl. Environ. Microbiol., vol. 55, no. 9, 1989: 2242-2246).
[15635] [0440.0.38.38] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002) teaches a homolog recombination system dependent on
rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this system
for homologous recombination are vectors derived from the Ylp
series. Plasmid vectors derived for example from the 2.mu.-Vector
are known by the skilled worker and used for the expression in
yeasts. Other preferred vectors are for example pART1, pCHY21 or
pEVP11 as they have been described by McLeod et al. (EMBO J. 1987,
6:729-736) and Hoffman et al. (Genes Dev. 5, 1991: :561-571.) or
Russell et al. (J. Biol. Chem. 258, 1983: 143-149.). Other
beneficial yeast vectors are plasmids of the REP, REP-X, pYZ or RIP
series.
[15636] [0441.0.0.38] to [0443.0.0.38] see [0441.0.0.27] to
[0443.0.0.27]
[0444.0.38.38] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[15637] [0445.0.38.38] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[15638] [0446.0.0.38] to [0450.0.0.38]: see [0446.0.0.27] to
[0450.0.0.27]
[15639] [0451.0.0.38] see [0451.0.5.5]
[15640] [0452.0.0.38] to [0453.0.0.38]: see [0452.0.0.27] to
[0453.0.0.27]
[15641] [0454.0.38.38] Analysis of the effect of the nucleic acid
molecule on the production of glyceric acid, citramalic acid,
fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid and/or trihydroxybutanoic acid
[15642] [0455.0.38.38] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a glyceric acid, citramalic acid, fumaric acid,
malic acid, pyruvic acid, succinic acid, trihydroxybutyric acid
and/or trihydroxybutanoic acid) can be determined by growing the
modified microorganisms or the modified plant under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the elevated production of
desired product (i.e. of glyceric acid, citramalic acid, fumaric
acid, malic acid, pyruvic acid, succinic acid, trihydroxybutyric
acid and/or trihydroxybutanoic acid). These analytical techniques
are known to the skilled worker and comprise spectroscopy,
thin-layer chromatography, various types of staining methods,
enzymatic and microbiological methods and analytical chromatography
such as high-performance liquid chromatography (see, for example,
Ullman, Encyclopedia of Industrial Chemistry, Vol. A2, p. 89-90 and
p. 443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987)
"Applications of HPLC in Biochemistry" in: Laboratory Techniques in
Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993)
Biotechnology, Vol. 3, Chapter III: "Product recovery and
purification", p. 469-714, VCH: Weinheim; Better, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[15643] [0456.0.0.38]: see [0456.0.0.27]
[0457.0.38.38] Example 9
Purification of Glyceric Acid, Citramalic Acid, Fumaric Acid, Malic
Acid, Pyruvic Acid, Succinic Acid, Trihydroxybutyric Acid or
Trihydroxybutanoic Acid
[15644] [0458.0.38.38] Abbreviations; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[15645] The unambiguous detection for the presence of glyceric
acid, citramalic acid, fumaric acid, malic acid, pyruvic acid,
succinic acid, trihydroxybutyric acid or trihydroxybutanoic acid
can be obtained by analyzing recombinant organisms using analytical
standard methods: LC, LC-MSMS or TLC, as described. The total
amount produced in the organism for example in yeasts used in the
inventive process can be analysed for example according to the
following procedure:
[15646] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods.
[15647] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[15648] A typical sample pre-treatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography.
[15649] For analysis, solvent delivery and aliquot removal can be
accomplished with a robotic system comprising a single injector
valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000
W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of
50% potassium hydroxide hydro-ethanolic solution (4 water--1
ethanol) can be added to each vial, followed by the addition of 3
ml of octanol. The saponification treatment can be conducted at
room temperature with vials maintained on an IKA HS 501 horizontal
shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours
at 250 movements/minute, followed by a stationary phase of
approximately one hour.
[15650] Following saponification, the supernatant can be diluted
with 0 0.17 ml of methanol. The addition of methanol can be
conducted under pressure to ensure sample homogeneity. Using a 0.25
ml syringe, a 0.1 ml aliquot can be removed and transferred to HPLC
vials for analysis.
[15651] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete
with a quaternary pump, vacuum degassing system, six-way injection
valve, temperature regulated autosampler, column oven and
Photodiode Array detector can be used [Agilent Technologies
available through Ultra Scientific Inc., 250 Smith Street, North
Kingstown, R.I.]. The column can be a Waters YMC30, 5-micron,
4.6.times.250 mm with a guard column of the same material [Waters,
34 Maple Street, Milford, Mass.]. The solvents for the mobile phase
can be 81 methanol: 4 water: 15 tetrahydrofuran (THF) stabilized
with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injections were
20 l. Separation can be isocratic at 30.degree. C. with a flow rate
of 1.7 ml/minute. The peak responses can be measured by absorbance
at 447 nm.
[15652] [0459.0.38.38] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the glyceric acid, citramalic acid, fumaric acid,
malic acid, pyruvic acid, succinic acid, trihydroxybutyric acid
and/or trihydroxybutanoic acid can be further purified with a
so-called RTHPLC. As eluent acetonitrile/water or
chloroform/acetonitrile mixtures can be used. If necessary, these
chromatography steps may be repeated, using identical or other
chromatography resins. The skilled worker is familiar with the
selection of suitable chromatography resin and the most effective
use for a particular molecule to be purified.
[15653] [0460.0.0.38] see [0460.0.0.27]
[0461.0.38.38] Example 10
Cloning SEQ ID NO: 108417 for the Expression in Plants
[15654] [0462.0.0.38] see [0462.0.0.27]
[15655] [0463.0.38.38] SEQ ID NO: 108417 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[15656] [0464.0.0.0.38] to [0466.0.0.38]: see [0464.0.0.27] to
[0466.0.0.27]
[15657] [0466.1.38.38] In case the Herculase enzyme can be used for
the amplification, the PCR amplification cycles were as follows: 1
cycle of 2-3 minutes at 94.degree. C., followed by 25-30 cycles of
in each case 30 seconds at 94.degree. C., 30 seconds at
55-60.degree. C. and 5-10 minutes at 72.degree. C., followed by 1
cycle of 10 minutes at 72.degree. C., then 4.degree. C.
[15658] [0467.0.38.38] The following primer sequences were selected
for the gene SEQ ID NO: 108417:
i) forward primer (SEQ ID NO: 108419) ii) reverse primer (SEQ ID
NO: 108420)
[15659] [0468.0.0.38] to [0470.0.0.38]: see [0468.0.0.27] to
[0470.0.0.27]
[15660] [0470.1.38.38] The PCR-products, produced by Pfu Turbo DNA
polymerase, were phosphorylated using a T4 DNA polymerase using a
standard protocol (e.g. MBI Fermentas) and cloned into the
processed binary vector.
[15661] [0471.0.0.38] see [0471.0.0.27]
[15662] [0471.1.38.38] The DNA termini of the PCR-products,
produced by Herculase DNA polymerase, were blunted in a second
synthesis reaction using Pfu Turbo DNA polymerase. The composition
for the protocol of the blunting the DNA-termini was as follows:
0.2 mM blunting dTTP and 1.25 u Pfu Turbo DNA polymerase. The
reaction was incubated at 72.degree. C. for 30 minutes. Then the
PCR-products were phosphorylated using a T4 DNA polymerase using a
standard protocol (e.g. MBI Fermentas) and cloned into the
processed vector as well.
[15663] [0472.0.0.38] to [0479.0.0.38]: see [0472.0.0.27] to
[0479.0.0.27]
[0480.0.38.38] Example 11
Generation of Transgenic Plants which Express SEQ ID NO:108417
[15664] [0481.0.0.38] to [0513.0.0.38]: see [0481.0.0.27] to
[0513.0.0.27]
[15665] [0514.0.38.38] As an alternative, the organic acids can be
detected as described in Farre E. et al., Plant Physiol, 2001, Vol.
127, pp. 685-700.
[15666] [0515.0.0.38] to [0552.0.0.38]: see [0515.0.0.27] to
[0552.0.0.27]
[15667] [0553.0.38.38] [15668] 1. A process for the production of
malic acid, or trihydroxybutanoic acid, which comprises a)
increasing or generating the activity of a protein as indicated in
Table XII, application no. 38, columns 5 or 7, or a functional
equivalent thereof in a non-human organism or in one or more parts
thereof; and b) growing the organism under conditions which permit
the production of malic acid or trihydroxybutanoic acid in said
organism. [15669] 2. A process for the production of malic acid, or
trihydroxybutanoic acid, comprising the increasing or generating in
an organism or a part thereof the expression of at least one
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: [15670] a) nucleic acid molecule
encoding of a polypeptide as indicated in Table XII, application
no. 38, columns 5 or 7, or a fragment thereof, which confers an
increase in the amount of malic acid or trihydroxybutanoic acid in
an organism or a part thereof; [15671] b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table XI,
application no. 38, columns 5 or 7; [15672] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of malic acid, or trihydroxybutanoic acid in an organism or
a part thereof; [15673] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of malic acid
or trihydroxybutanoic acid in an organism or a part thereof;
[15674] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of malic acid
or trihydroxybutanoic acid in an organism or a part thereof;
[15675] f) nucleic acid molecule which encompasses a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers or
primer pairs as indicated in Table XIII, application no. 38,
columns 5 or 7 and conferring an increase in the amount of malic
acid, or trihydroxybutanoic acid in an organism or a part thereof;
[15676] g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of malic acid, or
trihydroxybutanoic acid in an organism or a part thereof; [15677]
h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table XIV, application no. 38, columns 5
or 7, and conferring an increase in the amount of malic acid, or
trihydroxybutanoic acid in an organism or a part thereof; and
[15678] i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of malic acid, or
trihydroxybutanoic acid in an organism or a part thereof. [15679]
or comprising a sequence which is complementary thereto. [15680] 3.
The process of claim 1 or 2, comprising recovering of the free or
bound malic acid, or trihydroxybutanoic acid. [15681] 4. The
process of any one of claims 1 to 3, comprising the following
steps: [15682] a) selecting an organism or a part thereof
expressing a polypeptide encoded by the nucleic acid molecule
characterized in claim 2; [15683] b) mutagenizing the selected
organism or the part thereof; [15684] c) comparing the activity or
the expression level of said polypeptide in the mutagenized
organism or the part thereof with the activity or the expression of
said polypeptide of the selected organisms or the part thereof;
[15685] d) selecting the mutated organisms or parts thereof, which
comprise an increased activity or expression level of said
polypeptide compared to the selected organism or the part thereof;
[15686] e) optionally, growing and cultivating the organisms or the
parts thereof; and [15687] f) recovering, and optionally isolating,
the free or bound malic acid, trihydroxybutanoic acid produced by
the selected mutated organisms or parts thereof. [15688] 5. The
process of any one of claims 1 to 4, wherein the activity of said
protein or the expression of said nucleic acid molecule is
increased or generated transiently or stably. [15689] 6. An
isolated nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [15690] a) nucleic acid
molecule encoding of a polypeptide as indicated in Table XII,
application no. 38, columns 5 or 7 or a fragment thereof, which
confers an increase in the amount of glyceric acid. citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid in an organism or
a part thereof; [15691] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table XI, application no. 38,
columns 5 or 7; [15692] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of malic acid, or
trihydroxybutanoic acid in an organism or a part thereof; [15693]
d) nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of malic acid, or trihydroxybutanoic acid
in an organism or a part thereof; [15694] e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of malic acid, or trihydroxybutanoic acid in an organism
or a part thereof; [15695] f) nucleic acid molecule which
encompasses a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers or primer pairs as indicated in Table XIII,
application no. 38, column 7, and conferring an increase in the
amount of glyceric acid. citramalic acid, fumaric acid, malic acid,
pyruvic acid, succinic acid, trihydroxybutyric acid or
trihydroxybutanoic acid in an organism or a part thereof; [15696]
g) nucleic acid molecule encoding a polypeptide which is isolated
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (f) and conferring
an increase in the amount of malic acid, or trihydroxybutanoic acid
in an organism or a part thereof; [15697] h) nucleic acid molecule
encoding a polypeptide comprising a consensus as indicated in Table
XIV, application no. 38, column 7, and conferring an increase in
the amount of in an organism or a part thereof; and [15698] i)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment thereof having at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (k) and
conferring an increase in the amount of malic acid, or
trihydroxybutanoic acid in an organism or a part thereof. [15699]
whereby the nucleic acid molecule distinguishes over the sequence
as indicated in Table XI, columns 5 or 7, by one or more
nucleotides. [15700] 7. A nucleic acid construct which confers the
expression of the nucleic acid molecule of claim 6, comprising one
or more regulatory elements. [15701] 8. A vector comprising the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7. [15702] 9. The vector as claimed in claim 8,
wherein the nucleic acid molecule is in operable linkage with
regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic, host. [15703] 10. A
host cell, which has been transformed stably or transiently with
the vector as claimed in claim 8 or 9 or the nucleic acid molecule
as claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 2 to 5. [15704]
11. The host cell of claim 10, which is a transgenic host cell.
[15705] 12. The host cell of claim 10 or 11, which is a plant cell,
an animal cell, a microorganism, or a yeast cell, a fungus cell, a
prokaryotic cell, an eukaryotic cell or an archaebacterium. [15706]
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. [15707] 14. A polypeptide produced by the process as claimed in
claim 13 or encoded by the nucleic acid molecule as claimed in
claim 6 whereby the polypeptide distinguishes over a sequence as
indicated in Table XII, application no. 38, columns 5 or 7, by one
or more amino acids. [15708] 15. An antibody, which binds
specifically to the polypeptide as claimed in claim 14. [15709] 16.
A plant tissue, propagation material, harvested material or a plant
comprising the host cell as claimed in claim 12 which is plant cell
or an Agrobacterium. [15710] 17. A method for screening for
agonists and antagonists of the activity of a polypeptide encoded
by the nucleic acid molecule of claim 6 conferring an increase in
the amount of malic acid, or trihydroxybutanoic acid in an organism
or a part thereof comprising: (a) contacting cells, tissues, plants
or microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of malic acid or trihydroxybutanoic acid in an organism or a
part thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; (b) assaying the malic acid, or trihydroxybutanoic
acid level or the polypeptide expression level in the cell, tissue,
plant or microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and (c) identifying a
agonist or antagonist by comparing the malic acid, or
trihydroxybutanoic acid level or polypeptide expression level with
a standard malic acid, or trihydroxybutanoic acid or polypeptide
expression level measured in the absence of said candidate compound
or a sample comprising said plurality of compounds, whereby an
increased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an agonist and
a decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[15711] 18. A process for the identification of a compound
conferring malic acid, or trihydroxybutanoic acid production in a
plant or microorganism, comprising the steps: (a) culturing a plant
cell or tissue or microorganism or maintaining a plant expressing
the polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of malic acid, or
trihydroxybutanoic acid in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of malic
acid or trihydroxybutanoic acid in an organism or a part thereof;
(b) identifying if the compound is an effective agonist by
detecting the presence or absence or increase of a signal produced
by said readout system. [15712] 19. A method for the identification
of a gene product conferring an increase in malic acid, or
trihydroxybutanoic acid production in a cell, comprising the
following steps: (a) contacting the nucleic acid molecules of a
sample, which can contain a candidate gene encoding a gene product
conferring an increase in malic acid or trihydroxybutanoic acid
after expression with the nucleic acid molecule of claim 6; (b)
identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; (c) introducing the candidate nucleic acid molecules in
host cells appropriate for producing malic acid, or
trihydroxybutanoic acid; (d) expressing the identified nucleic acid
molecules in the host cells; (e) assaying the malic acid or
trihydroxybutanoic acid level in the host cells; and (f)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the malic acid, or
trihydroxybutanoic acid level in the host cell in the host cell
after expression compared to the wild type. [15713] 20. A method
for the identification of a gene product conferring an increase in
malic acid or trihydroxybutanoic acid production in a cell,
comprising the following steps: [15714] (a) identifying in a data
bank nucleic acid molecules of an organism; which can contain a
candidate gene encoding a gene product conferring an increase in
the malic acid, or trihydroxybutanoic acid amount or level in an
organism or a part thereof after expression, and which are at least
20% homolog to the nucleic acid molecule of claim 6; [15715] (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing malic acid or trihydroxybutanoic acid;
[15716] (c) expressing the identified nucleic acid molecules in the
host cells; [15717] (d) assaying the malic acid, or
trihydroxybutanoic acid level in the host cells; and [15718] (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the glyceric acid. citramalic
acid, fumaric acid, malic acid, pyruvic acid, succinic acid,
trihydroxybutyric acid or trihydroxybutanoic acid level in the host
cell after expression compared to the wild type. [15719] 21. A
method for the production of an agricultural composition comprising
the steps of the method of any one of claims 17 to 20 and
formulating the compound identified in any one of claims 17 to 20
in a form acceptable for an application in agriculture. [15720] 22.
A composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier.
[15721] 23. Use of the nucleic acid molecule as claimed in claim 6
for the identification of a nucleic acid molecule conferring an
increase of malic acid or trihydroxybutanoic acid after expression.
[15722] 24. Use of the polypeptide of claim 14 or the nucleic acid
construct claim 7 or the gene product identified according to the
method of claim 19 or 20 for identifying compounds capable of
conferring a modulation of malic acid or trihydroxybutanoic acid
levels in an organism. [15723] 25. Agrochemical, pharmaceutical,
food or feed composition comprising the nucleic acid molecule of
claim 6, the polypeptide of claim 14, the nucleic acid construct of
claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the harvested material of claim
16, the host cell of claim 10 to 12 or the gene product identified
according to the method of claim 19 or 20. [15724] 26. The method
of any one of claims 1 to 5, the nucleic acid molecule of claim 6,
the polypeptide of claim 14, the nucleic acid construct of claim 7,
the vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20, wherein the fine chemical is malic
acid or trihydroxybutanoic acid.
[15725] [0554.0.0.38] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[15726] [0000.0.39.39] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[15727] [0001.0.0.39] see [0001.0.0.27]
[15728] [0002.0.39.39] Gamma-aminobutyric acid is used to enhance
growth of specified plants, prevent development of powdery mildew
on grapes, and suppress certain other plant diseases. Humans and
animals normally ingest and metabolize gamma-aminobutyric acid in
variable amounts. Gamma-aminobutyric acid was registered (licensed
for sale) as growth enhancing pesticidal active ingredient in 1998.
Gamma-aminobutyric acid is an important signal which helps to
regulate mineral availability in plants. Minerals support the
biochemical pathways governing growth and reproduction as well as
the pathways that direct plant's response to a variety of biotic
and abiotic stresses. Mineral needs are especially high during
times of stress and at certain stages of plant growth.
Gamma-aminobutyric acid levels in plants naturally increase at
these times.
[15729] Gamma-Aminobutyric acid (GABA), a nonprotein amino acid, is
often accumulated in plants following environmental stimuli that
can also cause ethylene production. Exogenous GABA causes up to a
14-fold increase in the ethylene production rate after about 12 h.
GABA causes increases in ACC synthase mRNA accumulation, ACC
levels, ACC oxidase mRNA levels and in vitro ACC oxidase activity.
Possible roles of GABA as a signal transducer are suggested, see
Plant Physiol. 115(1):129-35(1997)
[15730] Gamma-aminobutyric acid (GABA), a four-carbon non-protein
amino acid, is a significant component of the free amino acid pool
in most prokaryotic and eukaryotic organisms. In plants, stress
initiates a signal-transduction pathway, in which increased
cytosolic Ca.sup.2+ activates Ca.sup.2+/calmodulin-dependent
glutamate decarboxylase activity and GABA synthesis. Elevated H+
and substrate levels can also stimulate glutamate decarboxylase
activity. GABA accumulation probably is mediated primarily by
glutamate decarboxylase. Experimental evidence supports the
involvement of GABA synthesis in pH regulation, nitrogen storage,
plant development and defence, as well as a compatible osmolyte and
an alternative pathway for glutamate utilization, see Trends Plant
Sci. 4(11):446-452(1999).
[15731] Gamma-aminobutyric acid enhances nutrient uptake by roots
and leaves so that plant nutrient levels are higher than those
achieved by using nutrients alone. When plants are stressed and
nutrient uptake is limited, it is believed that gamma-aminobutyric
acid facilitates nutrient utilization, thereby enhancing growth
during stress.
[15732] Rapid GABA accumulation in response to wounding may play a
role in plant defense against insects (Ramputh and Brown, Plant
Physiol. 111(1996): 1349-1352). The development of gamma
aminobutyrate (GABA) as a potential control agent in
plant-invertebrate pest systems has been reviewed in She1p et al.,
Canadien Journal of Botany (2003) 81, 11, 1045-1048. The authors
describe that available evidence indicates that GABA accumulation
in plants in response to biotic and abiotic stresses is mediated
via the activation of glutamate decarboxylase. More applied
research, based on the fact that GABA acts as an inhibitory
neurotransmitter in invertebrate pests, indicates that ingested
GABA disrupts nerve functioning and causes damage to oblique-banded
leaf roller larvae, and that walking or herbivory by tobacco
budworm and oblique-banded leaf roller larvae stimulate GABA
accumulation in soybean and tobacco, respectively. In addition,
elevated levels of endogenous GABA in genetically engineered
tobacco deter feeding by tobacco budworm larvae and infestation by
the northern root-knot nematode. Therefore the author concluded
that genetically engineered crop species having high GABA-producing
potential may be an alternative strategy to chemical pesticides for
the management of invertebrate pests.
[15733] During angiosperm reproduction, pollen grains form a tube
that navigates through female tissues to the micropyle, delivering
sperm to the egg. In vitro, GABA stimulates pollen tube growth. The
Arabidopsis POP2 gene encodes a transaminase that degrades GABA and
contributes to the formation of a gradient leading up to the
micropyle, see Cell. 114(1):47-59(2003).
[15734] Due to these interesting physiological roles and
agrobiotechnological potential of GABA there is a need to identify
the genes of enzymes and other proteins involved in GABA
metabolism, and to generate mutants or transgenic plant lines with
which to modify the GABA content in plants.
[15735] [0003.0.39.39] %
[15736] [0004.0.39.39] %
[15737] [0005.0.39.39] %
[15738] [0006.0.39.39] %
[15739] [0007.0.39.39] %
[15740] [0008.0.39.39] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce gamma-aminobutyric acid in plants.
That type of production permits control over quality, quantity and
selection of the most suitable and efficient producer organisms.
The latter is especially important for commercial production
economics and therefore availability to consumers. In addition it
is desireable to produce gamma-aminobutyric acid or shikimate in
plants in order to increase plant productivity and resistance
against biotic and abiotic stress as discussed before.
[15741] Methods of recombinant DNA technology have been used for
some years to improve the production of fine chemicals in
microorganisms and plants by amplifying individual biosynthesis
genes and investigating the effect on production of fine chemicals.
It is for example reported, that the xanthophyll astaxanthin could
be produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. Those results indicated
that about 75 percent of the carotenoids found in the flower of the
transformed plant contained a keto group.
[15742] [0009.0.39.39] Thus, it would be advantageous if an algae,
plant or other microorganism were available which produce large
amounts gamma-aminobutyric acid. The invention discussed
hereinafter relates in some embodiments to such transformed
prokaryotic or eukaryotic microorganisms.
[15743] It would also be advantageous if plants were available
whose roots, leaves, stem, fruits or flowers produced large amounts
of gamma-aminobutyric acid. The invention discussed hereinafter
relates in some embodiments to such transformed plants.
[15744] [0010.0.39.39] Therefore improving the quality of
foodstuffs and animal feeds is an important task of the
food-and-feed industry. This is necessary since, for example
gamma-aminobutyric acid, as mentioned above, which occur in plants
and some microorganisms are limited with regard to the supply of
mammals. Especially advantageous for the quality of foodstuffs and
animal feeds is as balanced as possible a specific
gamma-aminobutyric acid profile in the diet since an excess of
gamma-aminobutyric acid above a specific concentration in the food
has a positive effect. A further increase in quality is only
possible via addition of further gamma-aminobutyric acid, which are
limiting.
[15745] [0011.0.39.39] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add gamma-aminobutyric acid in
a balanced manner to suit the organism.
[15746] [0012.0.39.39] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
proteins which participate in the biosynthesis of
gamma-aminobutyric acid and make it possible to produce them
specifically on an industrial scale without unwanted byproducts
forming. In the selection of genes for biosynthesis two
characteristics above all are particularly important. On the one
hand, there is as ever a need for improved processes for obtaining
the highest possible contents of gamma-aminobutyric acid, on the
other hand as less as possible byproducts should be produced in the
production process.
[15747] [0013.0.0.39] see [0013.0.0.27]
[15748] [0014.0.39.39] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a gamma-aminobutyric acid.
Accordingly, in the present invention, the term "the fine chemical"
as used herein relates to a gamma-aminobutyric acid. Further, the
term "the fine chemicals" as used herein also relates to fine
chemicals comprising gamma-aminobutyric acid.
[15749] [0015.0.39.39] In one embodiment, the term "the fine
chemical" or "the respective fine chemical" means at least one
chemical compound with gamma-aminobutyric acid.
[15750] In one embodiment, the term "the fine chemical" means a
gamma-aminobutyric acid. Throughout the specification the term "the
fine chemical" means gamma-aminobutyric acid, its salts, ester,
thioester or in free form or bound to other compounds such sugars
or sugarpolymers, like glucoside, e.g. diglucoside.
[15751] [0016.0.39.39] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [15752] (a) increasing or generating the activity of one
or more of a protein as shown in table XII, application no. 39,
column 3 encoded by the nucleic acid sequences as shown in table
XI, application no. 39, column 5, in a non-human organism or in one
or more parts thereof or [15753] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
GABA of the invention or fine chemicals comprising amino acids of
the invention, in said organism or in the culture medium
surrounding the organism
[15754] [0017.0.0.39] to [0019.0.0.39]: see [0017.0.0.27] to
[0019.0.0.27]
[15755] [0020.0.39.39] Surprisingly it was found, that the
transgenic expression of the Helianthus annuus protein as indicated
in Table XII, application no. 39, column 5, line 39 in a plant
conferred an increase in gamma-Aminobutyric acid (GABA) content of
the transformed plants. Thus, in one embodiment, said protein or
its homologs are used for the production of gamma-Aminobutyric acid
(GABA).
[15756] [0021.0.0.39] see [0021.0.0.27]
[15757] [0022.0.39.39] The sequence of b0651 from Escherichia coli
K12 has been published in Blattner et al., Science 277 (5331),
1453-1474, 1997, and its activity is being defined as a pyrimidine
specific nucleoside hydrolase. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
with pyrimidine specific nucleoside hydrolase activity from
Escherichia coli K12 or a plant or its homolog, e.g. as shown
herein, for the production of the respective fine chemical, in
particular for increasing the amount of gamma-aminobutyric acid
preferably in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention said activity, e.g. the activity of a protein with
pyrimidine specific nucleoside hydrolase activity, is
increased.
[15758] [0022.1.0.39] to [0023.0.0.39] see [0022.1.0.27]
[0023.0.0.27]
[15759] [0023.1.39.39] Homologs of the polypeptide disclosed in
table XII, application no. 39, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 39, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 39, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 39,
column 7, resp.
[15760] [0024.0.0.39] see [0024.0.0.27]
[15761] [0025.0.39.39] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 39, column 3 "if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 39, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[15762] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 39,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 39,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[15763] [0025.1.0.39] see [0025.1.0.27]
[15764] [0026.0.0.39] to [0033.0.0.39]: see [0026.0.0.27] to
[0033.0.0.27]
[15765] [0034.0.39.39] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 39, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[15766] [0035.0.0.39] to [0038.0.0.39]: see [0035.0.0.27] to
[0038.0.0.27]
[15767] [0039.0.0.39]: see [0039.0.0.27]
[15768] [0040.0.0.39] to [0044.0.0.39]: see [0040.0.0.27] to
[0044.0.0.27]
[15769] [0045.0.39.39] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
39, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 39, column 6 of
the respective line, between 10% and 50%, 10% and 100%, 10% and
200%, 10% and 300%, 10% and 400%, 10% and 500%, 20% and 50%, 20%
and 250%, 20% and 500%, 50% and 100%, 50% and 250%, 50% and 500%,
500% and 1000% or more
[15770] [0046.0.39.39] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
39, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 39, column 6 of
the respective line confers an increase of the respective fine
chemical and of further GABA or their precursors.
[15771] [0047.0.0.39] to [0048.0.0.39]: see [0047.0.0.27] to
[0048.0.0.27]
[15772] [0049.0.39.39] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 39, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 39, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 39, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[15773] [0050.0.39.39] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of gamma-aminobutyric acid, or their ester, or glucoside
thereof, e.g the diglucoside thereof.
[15774] [0051.0.39.39] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g compositions comprising
gamma-aminobutyric acid. Depending on the choice of the organism
used for the process according to the present invention, for
example a microorganism or a plant, compositions or mixtures of
gamma-aminobutyric acid can be produced.
[15775] [0052.0.0.39] see [0052.0.0.27]
[15776] [0053.0.39.39] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[15777] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 39, columns 5 and 7 or its homologs activity
having herein-mentioned GABA of the invention increasing activity;
and/or [15778] b) stabilizing a mRNA conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention as shown in table XI, application no. 39, columns 5 and
7, e.g. a nucleic acid sequence encoding a polypeptide having the
activity of a protein as indicated in table XII, application no.
39, columns 5 and 7 or its homologs activity or of a mRNA encoding
the polypeptide of the present invention having herein-mentioned
GABAof the invention increasing activity; and/or [15779] c)
increasing the specific activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention or of the polypeptide of the present
invention having herein-mentioned GABA increasing activity, e.g. of
a polypeptide having the activity of a protein as indicated in
table XII, application no. 39, columns 5 and 7 or its homologs
activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [15780] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned GABA of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 39, columns 5 and 7 or its
homologs activity; and/or [15781] e) stimulating activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the present invention or a polypeptide
of the present invention having herein-mentioned GABA of the
invention increasing activity, e.g. of a polypeptide having the
activity of a protein as indicated in table XII, application no.
39, columns 5 and 7 or its homologs activity, by adding one or more
exogenous inducing factors to the organisms or parts thereof;
and/or [15782] f) expressing a transgenic gene encoding a protein
conferring the increased expression of a polypeptide encoded by the
nucleic acid molecule of the present invention or a polypeptide of
the present invention, having herein-mentioned GABA of the
invention increasing activity, e.g. of a polypeptide having the
activity of a protein as indicated in table XII, application no.
39, columns 5 and 7 or its homologs activity, and/or [15783] g)
increasing the copy number of a gene conferring the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or the
polypeptide of the invention having herein-mentioned GABA of the
invention increasing activity, e.g. of a polypeptide having the
activity of a protein as indicated in table XII, application no.
39, columns 5 and 7 or its homologs activity; and/or [15784] h)
increasing the expression of the endogenous gene encoding the
polypeptide of the invention, e.g. a polypeptide having the
activity of a protein as indicated in table XII, application no.
39, columns 5 and 7 or its homologs activity, by adding positive
expression or removing negative expression elements, e.g.
homologous recombination can be used to either introduce positive
regulatory elements like for plants the 35S enhancer into the
promoter or to remove repressor elements form regulatory regions.
Further gene conversion methods can be used to disrupt repressor
elements or to enhance to activity of positive elements. Positive
elements can be randomly introduced in plants by T-DNA or
transposon mutagenesis and lines can be identified in which the
positive elements have be integrated near to a gene of the
invention, the expression of which is thereby enhanced; and/or
[15785] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [15786] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[15787] [0054.0.39.39] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of the respective fine
chemical after increasing the expression or activity of the encoded
polypeptide or having the activity of a polypeptide having an
activity of a protein as indicated in Table XII, application no.
39, columns 3 or 5, or its homologs activity, e.g. as indicated in
Table XII, application no. 39, columns 5 or 7.
[15788] [0055.0.0.39] to [0067.0.0.39]: see [0055.0.0.27] to
[0067.0.0.27]
[15789] [0068.0.39.39] The mutation is introduced in such a way
that the production of gamma-aminobutyric acid or shikimate or
putrescine is not adversely affected.
[15790] [0069.0.0.39] see [0069.0.0.27]
[15791] [0070.0.39.39] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolite composition in the organism, e.g. an advantageous
composition ofgamma-aminobutyric acid and shikimate or their
biochemical derivatives, e.g. comprising a higher content of (from
a viewpoint of nutritional physiology limited) gamma-aminobutyric
acid and shikimate or putrescine or their derivatives.
[15792] [0071.0.0.39] see [0071.0.0.27]
[15793] [0072.0.39.39] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to shikimate their biochemical derivatives like
chorismate, prephenate, anthranilate, phenylpyruvate,
phenylalanine, 4-hydroxyphenylbutyrate, tyrosine, tryptophan,
vanillin, salicylic acid, lawsone or scopletin.
[15794] [0073.0.39.39] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[15795] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [15796] b) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table XII, application no. 39, columns 5 or 7, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the respective fine chemical in an organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [15797] c) growing an organism,
preferably a microorganism, a non-human animal, a plant or animal
cell, a plant or animal tissue or a plant under conditions which
permit the production of the respective fine chemical in the
organism, preferably the microorganism, the plant cell, the plant
tissue or the plant; and [15798] d) if desired, recovering,
optionally isolating, the free and/or bound the respective fine
chemical synthesized by the organism, the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[15799] [0074.0.39.39] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound respective fine chemical.
[15800] [0075.0.0.39] to [0077.0.0.39]: see [0075.0.0.27] to
[0077.0.0.27]
[15801] [0078.0.39.39] The organism such as microorganisms or
plants or the recovered, and if desired isolated, the respective
fine chemical can then be processed further directly into
foodstuffs or animal feeds or for other applications. The
fermentation broth, fermentation products, plants or plant products
can be purified with methods known to the person skilled in the
art. Products of these different work-up procedures are
gamma-aminobutyric acid or shikimate or putrescine or comprising
compositions of gamma-butyric acid or shikimate or putrescine still
comprising fermentation broth, plant particles and cell components
in different amounts, advantageously in the range of from 0 to 99%
by weight, preferably below 80% by weight, especially preferably
below 50% by weight.
[15802] [0079.0.0.39] to [0084.0.0.39]: see [0079.0.0.27] to
[0084.0.0.27]
[15803] [0084.0.39.39] %
[15804] [0085.0.39.39] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [15805] a) a nucleic acid sequence as
indicated in Table XI, application no. 39, columns 5 or 7, or a
derivative thereof, or [15806] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 39, columns
5 or 7, or a derivative thereof, or [15807] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[15808] [0086.0.0.39] to [0087.0.0.39]: see [0086.0.0.27] to
[0087.0.0.27]
[15809] [0088.0.39.39] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned fine
chemicals or the plants are more resistant to biotic and abiotic
stress and the yield is increased.
[15810] [0088.1.0.39] see [0088.1.0.27]
[15811] [0089.0.0.39] to [0090.0.0.39]: see [0089.0.0.27] to
[0090.0.0.27]
[15812] [0091.0.0.39] see [0091.0.0.27]
[15813] [0092.0.0.39] to [0094.0.0.39]: see [0092.0.0.27] to
[0094.0.0.27]
[15814] [0095.0.39.39] It may be advantageous to increase the pool
of gamma-aminobutyric acid or shikimate or putrescine in the
transgenic organisms by the process according to the invention in
order to isolate high amounts of the pure respective fine chemical
and/or to obtain increased resistance against biotic and abiotic
stresses and to obtain higher yield.
[15815] [0096.0.39.39] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example in
the organism, is useful to increase the production of the
respective fine chemical.
[15816] [0097.0.39.39] %
[15817] [0098.0.39.39] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated.
[15818] [0099.0.39.39] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells.
[15819] If microorganisms are used in the process according to the
invention, the fermentation broth can be processed after the
cultivation. Depending on the requirement, all or some of the
biomass can be removed from the fermentation broth by separation
methods such as, for example, centrifugation, filtration, decanting
or a combination of these methods, or else the biomass can be left
in the fermentation broth. The fermentation broth can subsequently
be reduced, or concentrated, with the aid of known methods such as,
for example, rotary evaporator, thin-layer evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. Afterwards
advantageously further compounds for formulation can be added such
as corn starch or silicates. This concentrated fermentation broth
advantageously together with compounds for the formulation can
subsequently be processed by lyophilization, spray drying, and
spray granulation or by other methods. Preferably the respective
fine chemical comprising compositions are isolated from the
organisms, such as the microorganisms or plants or the culture
medium in or on which the organisms have been grown, or from the
organism and the culture medium, in the known manner, for example
via extraction, distillation, crystallization, chromatography or a
combination of these methods. These purification methods can be
used alone or in combination with the aforementioned methods such
as the separation and/or concentration methods.
[15820] [0100.0.39.39] Transgenic plants which comprise the fine
chemicals such as gamma-aminobutyric acid or shikimate or
putrescine synthesized in the process according to the invention
can advantageously be marketed directly without there being any
need for the fine chemicals synthesized to be isolated. Plants for
the process according to the invention are listed as meaning intact
plants and all plant parts, plant organs or plant parts such as
leaf, stem, seeds, root, tubers, anthers, fibers, root hairs,
stalks, embryos, calli, cotelydons, petioles, harvested material,
plant tissue, reproductive tissue and cell cultures which are
derived from the actual transgenic plant and/or can be used for
bringing about the transgenic plant. In this context, the seed
comprises all parts of the seed such as the seed coats, epidermal
cells, seed cells, endosperm or embryonic tissue.
[15821] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, as extracts, e.g. ether, alcohol,
or other organic solvents or water containing extract and/or free
fine chemicals. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pretreated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[15822] Because gamma-aminobutyric acid or shikimate or putrescine
in microorganisms are localized intracellular, their recovery
essentially comes down to the isolation of the biomass.
Well-established approaches for the harvesting of cells include
filtration, centrifugation and coagulation/flocculation as
described herein. Of the residual hydrocarbon, adsorbed on the
cells, has to be removed. Solvent extraction or treatment with
surfactants have been suggested for this purpose.
[15823] [0101.0.39.39] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[15824] [0102.0.39.39] Gamma-aminobutyric acid or shikimate or
putrescine can for example be detected advantageously via HPLC, LC
or GC separation methods. The unambiguous detection for the
presence of gamma-aminobutyric acid or shikimate containing or
putrescine products can be obtained by analyzing recombinant
organisms using analytical standard methods: LC, LC-MS, MS or TLC).
The material to be analyzed can be disrupted by sonication,
grinding in a glass mill, liquid nitrogen and grinding, cooking, or
via other applicable methods.
[15825] [0103.0.39.39] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [15826] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide having a
sequence as indicated in Table XII, application no. 39, columns 5
or 7, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [15827] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table XI, application no. 39, columns 5 or 7,
[15828] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [15829] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[15830] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [15831]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [15832] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [15833] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers pairs
having a sequence as indicated in Table XIII, application no. 39,
columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [15834]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [15835] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequences as indicated in Table XIV, application
no. 39, columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [15836]
k) nucleic acid molecule comprising one or more of the nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domain of the polypeptide indicated in Table XII, application no.
39, columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[15837] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[15838] [0104.0.39.39] In one embodiment, the nucleic acid molecule
used in the process of the present invention distinguishes over the
sequence indicated in Table XI, application no. 39, columns 5 or 7,
by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence indicated in Table XI, application no. 39, columns 5
or 7. In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence indicated in Table XI, application no.
39, columns 5 or 7. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table XII, application no. 39, columns 5 or 7.
[15839] [0105.0.0.39] to [0107.0.0.39]: see [0105.0.0.27] to
[0107.0.0.27]
[15840] [0108.0.39.39] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 39, columns 5 or 7, nucleic
acid molecules which are derived from an amino acid sequences as
indicated in Table XII, application no. 39, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 39, column 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of an activity of a polypeptide as indicated in Table XII,
application no. 39, column 3, 5 or 7, e.g. conferring the increase
of the respective fine chemical, meaning gamma-aminobutyric acid or
shikimate or putrescine, resp., after increasing its expression or
activity, are advantageously increased in the process according to
the invention.
[15841] [0109.0.39.39] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular
gamma-aminobutyric acid or shikimate or putrescine, produced in the
process according to the invention.
[15842] [0110.0.0.39] see [0110.0.0.27]
[15843] [0111.0.0.39] see [0111.0.0.27]
[15844] [0112.0.39.39] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XII, application no. 39,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 39, columns 5 and 7, and conferring an
increase in the gamma-aminobutyric acid or shikimate or putrescine
level.
[15845] [0113.0.0.39] to [0114.0.0.39]: see [0113.0.0.27] to
[0114.0.0.27]
[15846] [0115.0.0.39] see [0115.0.0.27]
[15847] [0116.0.0.39] to [0120.0.0.39] see [0116.0.0.27] to
[0120.0.0.27]
[15848] [0120.1.39.39]: %
[15849] [0121.0.39.39] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 39, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a gamma-aminobutyric acid level increase
after increasing the activity of the polypeptide sequences
indicated in Table XII, columns 5 or 7,
[15850] [0122.0.0.39] to [0127.0.0.39]: see [0122.0.0.27] to
[0127.0.0.27]
[15851] [0128.0.39.39] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 39, columns 7, by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table XI, application no.
39, columns 5 or 7, or the sequences derived from a sequences as
indicated in Table XII, application no. 39, columns 5 or 7.
[15852] [0129.0.39.39] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence indicated in Table XIV,
application no. 39, columns 7, is derived from said alignments.
[15853] [0130.0.39.39] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g.
[15854] conferring the increase of gamma-aminobutyric acid or
shikimate after increasing the expression or activity the protein
comprising said fragment.
[15855] [0131.0.0.39] to [0138.0.0.39]: see [0131.0.0.27] to
[0138.0.0.27]
[15856] [0139.0.39.39] Polypeptides having above-mentioned
activity, i.e. conferring the respective fine chemical level
increase, derived from other organisms, can be encoded by other DNA
sequences which hybridise to a sequence indicated in Table XI,
application no. 39, columns 5 or 7, under relaxed hybridization
conditions and which code on expression for peptides having the
respective fine chemical, i.e. gamma-aminobutyric acid
increasing-activity.
[15857] [0140.0.0.39] to [0146.0.0.39]: see [0140.0.0.27] to
[0146.0.0.27]
[15858] [0147.0.39.39] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 39, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences such
that it can hybridise to one of said nucleotide sequences, thereby
forming a stable duplex. Preferably, the hybridisation is performed
under stringent hybridization conditions. However, a complement of
one of the herein disclosed sequences is preferably a sequence
complement thereto according to the base pairing of nucleic acid
molecules well known to the skilled person. For example, the bases
A and G undergo base pairing with the bases T and U or C, resp. and
visa versa. Modifications of the bases can influence the
base-pairing partner.
[15859] [0148.0.39.39] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 39,
columns 5 or 7, or a portion thereof and preferably has above
mentioned activity, in particular having a gamma-aminobutyric acid
or shikimate or putrescine increasing activity after increasing the
activity or an activity of a product of a gene encoding said
sequences or their homologs.
[15860] [0149.0.39.39] The nucleic acid molecule of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 39,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity, e.g. conferring a of gamma-aminobutyric
acid or shikimate or putrescine increase, resp., and optionally,
the activity of protein indicated in Table XII, application no. 39,
column 5.
[15861] [00149.1.39.39] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table XI, application no. 39, columns 5 or
7, has further one or more of the activities annotated or known for
a protein as indicated in Table XII, application no. 39, column
3.
[15862] [0150.0.39.39] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table XI, application no. 39, columns
5 or 7, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of gamma-aminobutyric acid if
its activity is increased. The nucleotide sequences determined from
the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table XI,
application no. 39, columns 5 or 7, an anti-sense sequence of one
of the sequences, e.g., as indicated in Table XI, application no.
39, columns 5 or 7, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table XIII, application no. 39, column 7, will result in a fragment
of a polynucleotide sequence as indicated in Table XI, application
no. 39, columns 5 or 7, or its gene product.
[15863] [0151.0.0.39]: see [0151.0.0.27]
[15864] [0152.0.39.39] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 39, columns 5
or 7, such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular a gamma-aminobutyric acid increasing activity as
mentioned above or as described in the examples in plants or
microorganisms is comprised.
[15865] [0153.0.39.39] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 39,
columns 5 or 7 such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 39, columns 5 or 7, has for
example an activity of a polypeptide indicated in Table XII,
application no. 39, column 3.
[15866] [0154.0.39.39] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 39, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[15867] [0155.0.0.39] to [0156.0.0.39]: see [0155.0.0.27] to
[0156.0.0.27]
[15868] [0157.0.39.39] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences as
indicated in Table XI, application no. 39, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
polypeptides comprising the sequence as indicated in Table XIV,
application no. 39, columns 7, or as polypeptides depicted in Table
XII, application no. 39, columns 5 or 7, or the functional
homologues. Advantageously, the nucleic acid molecule of the
invention comprises, or in an other embodiment has, a nucleotide
sequence encoding a protein comprising, or in an other embodiment
having, an amino acid sequence of a consensus sequences as
indicated in Table XIV, application no. 39, columns 7, or of the
polypeptide as indicated in Table XII, application no. 39, columns
5 or 7, or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
XIV, application no. 39, columns 5 or 7, or of a polypeptide as
indicated in Table XII, application no. 39, columns 5 or 7, or the
functional homologues. However, in a preferred embodiment, the
nucleic acid molecule of the present invention does not consist of
a sequence as indicated in Table XI, application no. 39, columns 5
or 7.
[15869] [0158.0.0.39] to [0160.0.0.39]: see [0158.0.0.27] to
[0160.0.0.27]
[15870] [0161.0.39.39] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 39, columns 5 or
7. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[15871] [0162.0.0.39] see [0162.0.0.27]
[15872] [0163.0.39.39] Preferably, a nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 39, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the increase of
the amount of the respective fine chemical in a organism or a part
thereof, e.g. a tissue, a cell, or a compartment of a cell, after
increasing the expression or activity thereof or the activity of a
protein of the invention or used in the process of the
invention.
[15873] [0164.0.0.39] see [0164.0.0.27]
[15874] [0165.0.39.39] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 39, columns 5 or 7.
[15875] [0166.0.0.39] to [0167.0.0.39]: see [0166.0.0.27] to
[0167.0.0.27]
[15876] [0168.0.39.39] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 39, columns 5 or 7, resp., yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 39,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table XII, application no. 39, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 39, columns 5
or 7, even more preferably at least about 80%, 90%, 95% homologous
to a sequence as indicated in Table XII, application no. 39,
columns 5 or 7, and most preferably at least about 96%, 97%, 98%,
or 99% identical to the sequence as indicated in Table XII,
application no. 39, columns 5 or 7.
[15877] [0169.0.0.39] to [0172.0.0.39]: see [0169.0.0.27] to
[0172.0.0.27]
[15878] [0173.0.39.39] For example a sequence, which has 80%
homology with sequence SEQ ID NO: 108175 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108175 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[15879] [0174.0.0.39]: see [0174.0.0.27]
[15880] [0175.0.39.39] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108176 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108176 by the above program algorithm with the
above parameter set, has a 80% homology.
[15881] [0176.0.39.39] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 39,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 39, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 39, columns
5 or 7.
[15882] [0177.0.39.39] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 39,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 39, columns 5 or 7, according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 39, columns
5 or 7.
[15883] [0178.0.0.39] see [0178.0.0.27]
[15884] [0179.0.39.39] A nucleic acid molecule encoding a
homologous to a protein sequence as indicated in Table XII,
application no. 39, columns 5 or 7 can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table XI, application no.
39, columns 5 or 7, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences as
indicated in Table XI, application no. 39, columns 5 or 7, by
standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[15885] [0180.0.0.39] to [0183.0.0.39]: see [0180.0.0.27] to
[0183.0.0.27]
[15886] [0184.0.39.39] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 39,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 39, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 39,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[15887] [0185.0.39.39] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 39, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table XI, application no. 39, columns 5 or 7. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequence as indicated in Table XI, columns 5 or 7.
[15888] [0186.0.39.39] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table XII,
application no. 39, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 39, columns 5 or 7.
[15889] [0187.0.39.39] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table XII, application no.
39, columns 5 or 7., comprises less than 100 further nucleotides.
In a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence
encoding a sequences as indicated in Table XII, application no. 39,
columns 5 or 7.
[15890] [0188.0.39.39] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 39, columns 5 or 7, and is expressed under
identical conditions.
[15891] [0189.0.39.39] Homologues of a sequences as indicated in
Table XI, application no. 39, columns 5 or 7, or of a derived
sequences as indicated in Table XII, application no. 39, columns 5
or 7, also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[15892] [0190.0.0.39]: see [0190.0.0.27]
[15893] [0191.0.39.39] The organisms or part thereof produce
according to the herein mentioned process of the invention an
increased level of free and/or bound the respective fine chemical
compared to said control or selected organisms or parts
thereof.
[15894] [0192.0.0.39] to [0203.0.0.39]: see [0192.0.0.27] to
[0203.0.0.27]
[15895] [0204.0.39.39] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule, which comprises a
nucleic acid molecule selected from the group consisting of:
[15896] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 39, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, i.e. gamma-aminobutyric in an organism or a part thereof
[15897] b) nucleic acid molecule comprising, preferably at least
the mature form, of a nucleic acid molecule as indicated in Table
XI, application no. 39, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [15898] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [15899] d) nucleic acid molecule encoding a polypeptide
whose sequence has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [15900]
e) nucleic acid molecule which hybridizes with a nucleic acid
molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [15901]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c),
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [15902] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[15903] h) nucleic acid molecule comprising a nucleic acid molecule
which is obtained by amplifying a cDNA library or a genomic library
using primers or primer pairs as indicated in Table XIII,
application no. 39, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical, i.e. gamma-aminobutyric
acid in an organism or a part thereof; [15904] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [15905] j) nucleic acid molecule which encodes a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 39, columns 5 or 7, and conferring an increase
in the amount of the respective fine chemical, i.e.
gamma-aminobutyric acid in an organism or a part thereof; [15906]
k) nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domaine of a polypeptide as indicated in
Table XII, application no. 39, columns 5 or 7, and conferring an
increase in the amount of the respective fine chemical, i.e.
gamma-aminobutyric acid in an organism or a part thereof; and
[15907] l) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment of at least
15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of
the nucleic acid molecule characterized in (a) to (h) or of a
nucleic acid molecule as indicated in Table XI, application no. 39,
columns 5 or 7, or a nucleic acid molecule encoding, preferably at
least the mature form of, a polypeptide as indicated in Table XII,
application no. 39, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof; or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over a sequence as indicated in Table
XI, application no. 39, columns 5 or 7, by one or more nucleotides.
In one embodiment, the nucleic acid molecule of the invention does
not consist of the sequence as indicated in Table XI, application
no. 39, columns 5 or 7. In an other embodiment, the nucleic acid
molecule of the present invention is at least 30% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence as indicated in Table XI, application no. 39, columns 5 or
7, resp. In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table XII,
application no. 39, columns 5 or 7. Accordingly, in one embodiment,
the nucleic acid molecule of the present invention encodes in one
embodiment a polypeptide which differs at least in one or more
amino acids from a polypeptide indicated in Table XII, application
no. 39, columns 5 or 7, does not encode a protein of a sequence as
indicated in Table XII, application no. 39, columns 5 or 7.
Accordingly, in one embodiment, the protein encoded by a sequences
of a nucleic acid according to (a) to (l) does not consist of a
sequence as indicated in Table XII, application no. 39, columns 5
or 7. In a further embodiment, the protein of the present invention
is at least 30% identical to a protein sequence indicated in Table
XII, application no. 39, columns 5 or 7, and less than 100%,
preferably less than 99.999%, 99.99% or 99.9%, more preferably less
than 99%, 98%, 97%, 96% or 95% identical to a sequence as indicated
in Table XII, application no. 39, columns 5 or 7.
[15908] [0205.0.0.39] to [0206.0.0.39]: see [0205.0.0.27] to
[0206.0.0.27]
[15909] [0207.0.39.39] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the glutamic acid metabolism, the
phosphoenolpyruvate metabolism, the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[15910] [0208.0.0.39] to [0226.0.0.39]: see [0208.0.0.27] to
[0226.0.0.27]
[15911] [0227.0.39.39] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[15912] In addition to a sequence indicated in Table XI,
application no. 39, columns 5 or 7, or its derivatives, it is
advantageous to express and/or mutate further genes in the
organisms. Especially advantageously, additionally at least one
further gene of the glutamic acid or phosphoenolpyruvate metabolic
pathway, is expressed in the organisms such as plants or
microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the fine chemicals desired since, for example,
feedback regulations no longer exist to the same extent or not at
all. In addition it might be advantageously to combine one or more
of the sequences indicated in Table XI, application no. 39, columns
5 or 7, with genes which generally support or enhances to growth or
yield of the target organisms, for example genes which lead to
faster growth rate of microorganisms or genes which produces
stress-, pathogen, or herbicide resistant plants.
[15913] [0228.0.39.39] In a further embodiment of the process of
the invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins directly or indirectly involved
in the glutamic acid or phosphoenolpyruvate metabolism.
[15914] [0229.0.39.39] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences indicated
in Table XI, application no. 39, columns 5 or 7, used in the
process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the aromatic amino acid
pathway, such as tryptophan, phenylalanine or tyrosine. These genes
can lead to an increased synthesis of the essential amino acids
tryptophan, phenylalanine or tyrosine.
[15915] [0230.0.0.39] see [230.0.0.27]
[15916] [0231.0.39.39] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a gamma-aminobutyric acid degrading
protein is attenuated, in particular by reducing the rate of
expression of the corresponding gene. A person skilled in the art
knows for example, that the inhibition of an enzyme or a gene of
the biosynthesis of the essential aromatic amino acids tryptophan,
tyrosine or phenylalanine may increase the amount of shikimate
accumulating in an organism, in particular in plants. Furthermore
the inhibition of tryptophan, phenylalanine or tyrosine degrading
enzymes also may result in an increased shikimate accumulation in
the organism.
[15917] [0232.0.0.39] to [0276.0.0.39]: see [0232.0.0.27] to
[0276.0.0.27]
[15918] [0277.0.39.39] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously.
[15919] [0278.0.0.39] to [0282.0.0.39]: see [0278.0.0.27] to
[0282.0.0.27]
[15920] [0283.0.39.39] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells, for example using the antibody
of the present invention as described below, e.g. an antibody
against a protein as indicated in Table XII, application no. 39,
column 3, or an antibody against a polypeptide as indicated in
Table XII, application no. 39, columns 5 or 7, which can be
produced by standard techniques utilizing the polypeptid of the
present invention or fragment thereof. Preferred are monoclonal
antibodies.
[15921] [0284.0.0.39] see [0284.0.0.27]
[15922] [0285.0.39.39] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 39, columns 5 or 7, or as coded by a nucleic
acid molecule as indicated in Table XI, application no. 39, columns
5 or 7, or functional homologues thereof.
[15923] [0286.0.39.39] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 39, column 7, and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 39, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table XIV, application no. 39, column 7.
[15924] [0287.0.0.39] to [0289.0.0.39]: see [0287.0.0.27] to
[0289.0.0.27]
[15925] [00290.0.39.39] The alignment was performed with the
Software AlignX (Sep. 25, 2002) a component of Vector NTI Suite
8.0, InforMax.TM., Invitrogen.TM. life science software, U.S. Main
Office, 7305 Executive Way, Frederick, Md. 21704, USA with the
following settings: For pairwise alignments: gap opening penalty:
10.0; gap extension penalty 0,1. For multiple alignments: Gap
opening penalty: 10.0; Gap extension penalty: 0.1; Gap separation
penalty range: 8; Residue substitution matrix: blosum62;
Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0,5;
Consensus calculation options: Residue fraction for consensus: 0.9.
Presettings were selected to allow also for the alignment of
conserved amino acids
[15926] [0291.0.39.39] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[15927] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table XII,
application no. 39, columns 5 or 7, by one or more amino acids. In
one embodiment, polypeptide distinguishes from a sequence as
indicated in Table XII, application no. 39, columns 5 or 7, by more
than 5, 6, 7, 8 or 9 amino acids, preferably by more than 10, 15,
20, 25 or 30 amino acids, even more preferred are more than 40, 50,
or 60 amino acids and, preferably, the sequence of the polypeptide
of the invention distinguishes from a sequence as indicated in
Table XII, application no. 39, columns 5 or 7, by not more than 80%
or 70% of the amino acids, preferably not more than 60% or 50%,
more preferred not more than 40% or 30%, even more preferred not
more than 20% or 10%.
[15928] [0292.0.0.39] see [0292.0.0.27]
[15929] [0293.0.39.39] In one embodiment, the invention relates to
a polypeptide conferring an increase in the fine chemical in an
organism or part being encoded by the nucleic acid molecule of the
invention or by a nucleic acid molecule used in the process of the
invention. In one embodiment, the polypeptide of the invention has
a sequence which distinguishes from a sequence as indicated in
Table XII, application no. 39, columns 5 or 7, by one or more amino
acids. In an other embodiment, said polypeptide of the invention
does not consist of the sequence as indicated in Table XII,
application no. 39, columns 5 or 7. In a further embodiment, said
polypeptide of the present invention is less than 100%, 99.999%,
99.99%, 99.9% or 99% identical. In one embodiment, said polypeptide
does not consist of the sequence encoded by a nucleic acid
molecules as indicated in Table XI, application no. 39, columns 5
or 7.
[15930] [0294.0.39.39] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 39, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 39, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[15931] [0295.0.0.39] to [0296.0.0.39]: see [0295.0.0.27] to
[0296.0.0.27]
[15932] [0297.0.0.39] see [0297.0.0.27]
[15933] [00297.1.39.39] %
[15934] [0298.0.39.39] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 39, columns 5 or 7. The
portion of the protein is preferably a biologically active portion
as described herein. Preferably, the polypeptide used in the
process of the invention has an amino acid sequence identical to a
sequence as indicated in Table XII, application no. 39, columns 5
or 7.
[15935] [0299.0.39.39] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 39,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 39, columns 5 or 7, which is homologous
thereto, as defined above.
[15936] [0300.0.39.39] Accordingly the polypeptide of the present
invention can vary from a sequence as indicated in Table XII,
application no. 39, columns 5 or 7 in amino acid sequence due to
natural variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprise an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence of as indicated in Table XII, application no.
39, columns 5 or 7.
[15937] [0301.0.0.39] see [0301.0.0.27]
[15938] [0302.0.39.39] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 39, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[15939] [0303.0.0.39] see [0303.0.0.27]
[15940] [0304.0.39.39] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 39, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[15941] [0305.0.0.39] to [0308.0.0.39]: see [0305.0.0.27] to
[0308.0.0.27]
[15942] [0309.0.39.39] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table XII,
application no. 39, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas an
"other polypeptide" not being indicated in Table XII, application
no. 39, columns 5 or 7, refers to a polypeptide having an amino
acid sequence corresponding to a protein which is not substantially
homologous to a polypeptide of the invention, preferably which is
not substantially homologous to a polypeptide as indicated in Table
XII, application no. 39, columns 5 or 7, --e.g., a protein which
does not confer the activity described herein or annotated or known
for as indicated in Table XII, application no. 39, column 3, and
which is derived from the same or a different organism. In one
embodiment, an "other polypeptide" not being indicated in Table
XII, application no. 39, columns 5 or 7, does not confer an
increase of the respective fine chemical in an organism or part
thereof.
[15943] [0310.0.0.39] to [0334.0.0.39]: see [0310.0.0.27] to
[0334.0.0.27]
[15944] [0335.0.39.39] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XI, columns 5 or
7, and/or homologs thereof. As described inter alia in WO 99/32619,
dsRNAi approaches are clearly superior to traditional antisense
approaches. The invention therefore furthermore relates to
double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived there from), bring about
altered metabolic activity by the reduction in the expression of a
nucleic acid sequences as indicated in Table XI, application no.
39, columns 5 or 7, and/or homologs thereof. In a double-stranded
RNA molecule for reducing the expression of aprotein encoded by a
nucleic acid sequence as indicated in Table XI, application no. 39,
columns 5 or 7, and/or homologs thereof, one of the two RNA strands
is essentially identical to at least part of a nucleic acid
sequence, and the respective other RNA strand is essentially
identical to at least part of the complementary strand of a nucleic
acid sequence.
[15945] [0336.0.0.39] to [0342.0.0.39]: see [0336.0.0.27] to
[0342.0.0.27]
[15946] [0343.0.39.39] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 39, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table XI, application no. 39, columns 5 or 7, or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[15947] [0344.0.0.39] to [0350.0.0.39]: see [0344.0.0.27] to
[0350.0.0.27]
[15948] [0351.0.0.39] to [0361.0.0.39]: see [0351.0.0.27] to
[0361.0.0.27]
[15949] [0362.0.39.39] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 39, columns 5 or 7, e.g. encoding a
polypeptide having protein activity, as indicated in Table XII,
application no. 39, columns 3. Due to the abovementioned activity
the respective fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity of the polypeptide of the invention or nucleic
acid molecule of the invention is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a polypeptide as
indicated in Table XII, application no. 39, columns 5 or 7, means
herein that due to modulation or manipulation of the genome, an
activity as annotated for a polypeptide as indicated in Table XII,
application no. 39, column 3, e.g. having a sequence as indicated
in Table XII, application no. 39, columns 5 or 7, is increased in a
cell or an organism or a part thereof. Examples are described above
in context with the process of the invention.
[15950] [0363.0.0.39] see [0363.0.0.27]
[15951] [0364.0.39.39] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table XII, application no. 39, column 3, with the
corresponding protein-encoding sequence as indicated in Table XI,
application no. 39, column 5, becomes a transgenic expression
cassette when it is modified by non-natural, synthetic "artificial"
methods such as, for example, mutagenization. Such methods have
been described (U.S. Pat. No. 5,565,350; WO 00/15815; also see
above).
[15952] [0365.0.0.39] to [0373.0.0.39]: see [0365.0.0.27] to
[0373.0.0.27]
[15953] [0374.0.39.39] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. gamma-aminobutyric acid or
shikimate, in particular the respective fine chemical, produced in
the process according to the invention may, however, also be
isolated from the plant in the form of their free
gamma-aminobutyric acid, in particular the free respective fine
chemical, or bound in or to compounds or moieties, like glucosides,
e.g. diglucosides. The respective fine chemical produced by this
process can be harvested by harvesting the organisms either from
the culture in which they grow or from the field. This can be done
via expressing, grinding and/or extraction, salt precipitation
and/or ion-exchange chromatography or other chromatographic methods
of the plant parts, preferably the plant seeds, plant fruits, plant
tubers and the like.
[15954] [0375.0.0.39] to [0376.0.0.39]: see [0375.0.0.27] to
[0376.0.0.27]
[15955] [0377.0.39.39] Accordingly, the present invention relates
also to a process whereby the produced gamma-aminobutyric acid or
shikimate is isolated.
[15956] [0378.0.39.39] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the
gamma-aminobutyric acid or shikimate produced in the process can be
isolated. The resulting gamma-aminobutyric acid or shikimate can,
if appropriate, subsequently be further purified, if desired mixed
with other active ingredients such as vitamins, amino acids,
carbohydrates, antibiotics and the like, and, if appropriate,
formulated.
[15957] [0379.0.39.39] In one embodiment, gamma-aminobutyric acid
and shikimate are a mixture of the respective fine chemicals.
[15958] [0380.0.39.39] The gamma-aminobutyric acid or shikimate
obtained in the process are suitable as starting material for the
synthesis of further products of value. For example, they can be
used in combination with each other or alone for the production of
pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the gamma-aminobutyric acid
or shikimate composition produced or the respective fine chemical
produced if desired and formulating the product with a
pharmaceutical acceptable carrier or formulating the product in a
form acceptable for an application in agriculture. A further
embodiment according to the invention is the use of the
gamma-aminobutyric acid or shikimate produced in the process or of
the transgenic organisms in animal feeds, foodstuffs, medicines,
food supplements, cosmetics or pharmaceuticals or for the
production of gamma-aminobutyric acid or shikimate e.g. after
isolation of the respective fine chemical or without, e.g. in situ,
e.g in the organism used for the process for the production of the
respective fine chemical.
[15959] [0381.0.0.39] to [0382.0.0.39]: see [0381.0.0.27] to
[0382.0.0.27]
[15960] [0383.0.39.39] %
[15961] [0384.0.0.39] see [0384.0.0.27]
[15962] [0385.0.39.39] The fermentation broths obtained in this
way, containing in particular gamma-aminobutyric acid or shikimate
in mixtures with other organic acids, aminoacids, polypeptides or
polysaccarides, normally have a dry matter content of from 1 to 70%
by weight, preferably 7.5 to 25% by weight. Sugar-limited
fermentation is additionally advantageous, e.g. at the end, for
example over at least 30% of the fermentation time. This means that
the concentration of utilizable sugar in the fermentation medium is
kept at, or reduced to, 0 to 10 g/l, preferably to 0 to 3 g/I
during this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed or isolated
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation, coagulation/flocculation
or a combination of these methods, from the fermentation broth or
left completely in it.
[15963] The fermentation broth can then be thickened or
concentrated by known methods, such as, for example, with the aid
of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[15964] [0386.0.39.39] Accordingly, it is possible to purify the
gamma-aminobutyric acid or shikimate produced according to the
invention further. For this purpose, the product-containing
compositions subjected for example to separation via e.g. an open
column chromatography or HPLC in which case the desired product or
the impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use.
[15965] [0387.0.0.39] to [0392.0.0.39]: see [0387.0.0.27] to
[0392.0.0.27]
[15966] [0393.0.39.39] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps: [15967] a. contacting
e.g. hybridising, the nucleic acid molecules of a sample, e.g.
cells, tissues, plants or microorganisms or a nucleic acid library,
which can contain a candidate gene encoding a gene product
conferring an increase in the respective fine chemical after
expression, with the nucleic acid molecule of the present
invention; [15968] b. identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence as indicated in Table XI, application no. 39,
columns 5 or 7, and, optionally, isolating the full length cDNA
clone or complete genomic clone; [15969] c. introducing the
candidate nucleic acid molecules in host cells, preferably in a
plant cell or a microorganism, appropriate for producing the
respective fine chemical; [15970] d. expressing the identified
nucleic acid molecules in the host cells; [15971] e. assaying the
respective fine chemical level in the host cells; and [15972] f.
identifying the nucleic acid molecule and its gene product which
expression confers an increase in the respective fine chemical
level in the host cell after expression compared to the wild
type.
[15973] [0394.0.0.39] to [0398.0.0.39]: see [0394.0.0.27] to
[0398.0.0.27]
[15974] [0399.0.39.39] Furthermore, in one embodiment, the present
invention relates to process for the identification of a compound
conferring increase of the respective fine chemical production in a
plant or microorganism, comprising the steps:
a) culturing a cell or tissue or microorganism or maintaining a
plant expressing the polypeptide according to the invention or a
nucleic acid molecule encoding said polypeptide and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and the polypeptide of the present invention or used
in the process of the invention; and b) identifying if the compound
is an effective agonist by detecting the presence or absence or
increase of a signal produced by said readout system.
[15975] The screen for a gene product or an agonist conferring an
increase in the respective fine chemical production can be
performed by growth of an organism for example a microorganism in
the presence of growth reducing amounts of an inhibitor of the
synthesis of the respective fine chemical. Better growth, e.g.
higher dividing rate or high dry mass in comparison to the control
under such conditions would identify a gene or gene product or an
agonist conferring an increase in respective fine chemical
production.
[15976] [00399.1.39.39] One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table XII,
application no. 39, columns 5 or 7 or a homolog thereof, e.g.
comparing the phenotype of nearly identical organisms with low and
high activity of a protein as indicated in Table XII, application
no. 39, columns 5 or 7, after incubation with the drug.
[15977] [0400.0.0.39] to [0415.0.0.39]: see [0400.0.0.27] to
[0415.0.0.27]
[15978] [0416.0.0.39] see [0416.0.0.27]
[15979] [0417.0.39.39] The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the gamma-aminobutyric acid or shikimate
or putrescine biosynthesis pathways. In particular, the
overexpression of the polypeptide of the present invention may
protect an organism such as a microorganism or a plant against
inhibitors, which block the gamma-aminobutyric acid or shikimate or
putrescine synthesis.
[15980] [0418.0.0.39] to [0423.0.0.39]: see [0418.0.0.27] to
[0423.0.0.27]
[15981] [0424.0.39.39] Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other organic acids.
Accordingly, the nucleic acid of the invention, or the nucleic acid
molecule identified with the method of the present invention or the
complement sequences thereof, the polypeptide of the invention, the
nucleic acid construct of the invention, the organisms, the host
cell, the microorganisms, the plant, plant tissue, plant cell, or
the part thereof of the invention, the vector of the invention, the
antagonist identified with the method of the invention, the
antibody of the present invention, the antisense molecule of the
present invention, can be used for the reduction of the respective
fine chemical in a organism or part thereof, e.g. in a cell.
[15982] [0425.0.0.39] to [0434.0.0.39]: see [0425.0.0.27] to
[0434.0.0.27]
[0435.0.39.39] Example 3
In-Vivo and In-Vitro Mutagenesis
[15983] [0436.0.39.39] An in vivo mutagenesis of organisms such as
Saccharomyces, Mortierella, Escherichia and others mentioned above,
which are beneficial for the production of gamma-aminobutyric acid
or shikimate or putrescine can be carried out by passing a plasmid
DNA (or another vector DNA) containing the desired nucleic acid
sequence or nucleic acid sequences, e.g. the nucleic acid molecule
of the invention or the vector of the invention, through E. coli
and other microorganisms (for example Bacillus spp. or yeasts such
as Saccharomyces cerevisiae) which are not capable of maintaining
the integrity of its genetic information. Usual mutator strains
have mutations in the genes for the DNA repair system [for example
mutHLS, mutD, mutT and the like; for comparison, see Rupp, W. D.
(1996) DNA repair mechanisms in Escherichia coli and Salmonella,
pp. 2277-2294, ASM: Washington]. The skilled worker knows these
strains. The use of these strains is illustrated for example in
Greener, A. and Callahan, M. (1994) Strategies 7; 32-34.
[15984] In-vitro mutation methods such as increasing the
spontaneous mutation rates by chemical or physical treatment are
well known to the skilled person. Mutagens like 5-bromo-uracil,
N-methyl-N-nitro-N-nitrosoguanidine (=NTG), ethyl methanesulfonate
(=EMS), hydroxylamine and/or nitrous acid are widly used as
chemical agents for random in-vitro mutagensis. The most common
physical method for mutagensis is the treatment with UV
irradiation. Another random mutagenesis technique is the
error-prone PCR for introducing amino acid changes into proteins.
Mutations are deliberately introduced during PCR through the use of
error-prone DNA polymerases and special reaction conditions known
to a person skilled in the art. For this method randomized DNA
sequences are cloned into expression vectors and the resulting
mutant libraries screened for altered or improved protein activity
as described below.
[15985] Site-directed mutagensis method such as the introduction of
desired mutations with an M13 or phagemid vector and short
oligonucleotides primers is a well-known approach for site-directed
mutagensis. The clou of this method involves cloning of the nucleic
acid sequence of the invention into an M13 or phagemid vector,
which permits recovery of single-stranded recombinant nucleic acid
sequence. A mutagenic oligonucleotide primer is then designed whose
sequence is perfectly complementary to nucleic acid sequence in the
region to be mutated, but with a single difference: at the intended
mutation site it bears a base that is complementary to the desired
mutant nucleotide rather than the original. The mutagenic
oligonucleotide is then allowed to prime new DNA synthesis to
create a complementary full-length sequence containing the desired
mutation. Another site-directed mutagensis method is the PCR
mismatch primer mutagensis method also known to the skilled person.
Dpnl site-directed mutagensis is a further known method as
described for example in the Stratagene Quickchange.TM.
site-directed mutagenesis kit protocol. A huge number of other
methods are also known and used in common practice.
[15986] Positive mutation events can be selected by screening the
organisms for the production of the desired respective fine
chemical.
[0437.0.39.39] Example 4
DNA Transfer Between Escherichia coli, Saccharomyces cerevisiae and
Mortierella alpina
[15987] [0438.0.39.39] Shuttle vectors such as pYE22m, pPAC-ResQ,
pClasper, pAUR224, pAMH10, pAML10, pAMT10, pAMU10, pGMH10, pGML10,
pGMT10, pGMU10, pPGAL1, pPADH1, pTADH1, pTAex3, pNGA142, pHT3101
and derivatives thereof which allow the transfer of nucleic acid
sequences between Escherichia coli, Saccharomyces cerevisiae and/or
Mortierella alpina are available to the skilled worker. An easy
method to isolate such shuttle vectors is disclosed by Soni R. and
Murray J. A. H. [Nucleic Acid Research, vol. 20 no. 21, 1992:
5852]: If necessary such shuttle vectors can be constructed easily
using standard vectors for E. coli (Sambrook, J. et al., (1989),
"Molecular Cloning: A Laboratory Manual", Cold Spring Harbor
Laboratory Press or Ausubel, F. M. et al. (1994) "Current Protocols
in Molecular Biology", John Wiley & Sons) and/or the
aforementioned vectors, which have a replication origin for, and
suitable marker from, Escherichia coli, Saccharomyces cerevisiae or
Mortierella alpina added. Such replication origins are preferably
taken from endogenous plasmids, which have been isolated from
species used in the inventive process. Genes, which are used in
particular as transformation markers for these species are genes
for kanamycin resistance (such as those which originate from the
Tn5 or Tn-903 transposon) or for chloramphenicol resistance
(Winnacker, E. L. (1987) "From Genes to Clones--Introduction to
Gene Technology", VCH, Weinheim) or for other antibiotic resistance
genes such as for G418, gentamycin, neomycin, hygromycin or
tetracycline resistance.
[15988] [0439.0.39.39] Using standard methods, it is possible to
clone a gene of interest into one of the above-described shuttle
vectors and to introduce such hybrid vectors into the microorganism
strains used in the inventive process. The transformation of
[15989] Saccharomyces can be achieved for example by LiCI or
sheroplast transformation (Bishop et al., Mol. Cell. Biol., 6,
1986: 3401-3409; Sherman et al., Methods in Yeasts in Genetics,
[Cold Spring Harbor Lab. Cold Spring Harbor, N. Y.] 1982, Agatep et
al., Technical Tips Online 1998, 1:51: P01525 or Gietz et al.,
Methods Mol. Cell. Biol. 5, 1995: 255f) or electroporation (Delorme
E., Appl. Environ. Microbiol., vol. 55, no. 9, 1989:
2242-2246).
[15990] [0440.0.39.39] If the transformed sequence(s) is/are to be
integrated advantageously into the genome of the microorganism used
in the inventive process for example into the yeast or fungi
genome, standard techniques known to the skilled worker also exist
for this purpose. Solinger et al. (Proc Natl Acad Sci USA., 2001
(15): 8447-8453) and Freedman et al. (Genetics, Vol. 162, 15-27,
September 2002) teaches a homolog recombination system dependent on
rad 50, rad51, rad54 and rad59 in yeasts. Vectors using this system
for homologous recombination are vectors derived from the Ylp
series. Plasmid vectors derived for example from the 2.mu.-Vector
are known by the skilled worker and used for the expression in
yeasts. Other preferred vectors are for example pART1, pCHY21 or
pEVP11 as they have been described by McLeod et al. (EMBO J. 1987,
6:729-736) and Hoffman et al. (Genes Dev. 5, 1991: :561-571.) or
Russell et al. (J. Biol. Chem. 258, 1983: 143-149.). Other
beneficial yeast vectors are plasmids of the REP, REP-X, pYZ or RIP
series.
[15991] [0441.0.0.39] see [0441.0.0.27]
[15992] [0442.0.0.39] see [0442.0.0.27]
[15993] [0443.0.0.39] see [0443.0.0.27]
[0444.0.39.39] Example 6
Growth of Genetically Modified Organism: Media and Culture
Conditions
[15994] [0445.0.39.39] Genetically modified Yeast, Mortierella or
Escherichia coli are grown in synthetic or natural growth media
known by the skilled worker. A number of different growth media for
Yeast, Mortierella or Escherichia coli are well known and widely
available. A method for culturing Mortierella is disclosed by Jang
et al. [Bot. Bull. Acad. Sin. (2000) 41:41-48]. Mortierella can be
grown at 20.degree. C. in a culture medium containing: 10 g/l
glucose, 5 g/l yeast extract at pH 6.5. Furthermore Jang et al.
teaches a submerged basal medium containing 20 g/l soluble starch,
5 g/l Bacto yeast extract, 10 g/l KNO.sub.3, 1 g/l
KH.sub.2PO.sub.4, and 0.5 g/l MgSO.sub.4.7H.sub.2O, pH 6.5.
[15995] [0446.0.0.39] to [0450.0.0.39]: see [0446.0.0.27] to
[0450.0.0.27]
[15996] [0451.0.0.39] see [0451.0.5.5]
[15997] [0452.0.0.39] to [0453.0.0.39]: see [0452.0.0.27] to
[0453.0.0.27]
[15998] [0454.0.39.39] Analysis of the effect of the nucleic acid
molecule on the production of gamma-aminobutyric acid or shikimate
or putrescine
[15999] [0455.0.39.39] The effect of the genetic modification in
plants, fungi, algae, ciliates or on the production of a desired
compound (such as a gamma-aminobutyric acid) can be determined by
growing the modified microorganisms or the modified plant under
suitable conditions (such as those described above) and analyzing
the medium and/or the cellular components for the elevated
production of desired product (i.e. of gamma-aminobutyric acid or
shikimate or putrescine). These analytical techniques are known to
the skilled worker and comprise spectroscopy, thin-layer
chromatography, various types of staining methods, enzymatic and
microbiological methods and analytical chromatography such as
high-performance liquid chromatography (see, for example, Ullman,
Encyclopedia of Industrial Chemistry, Vol. A2, p. 89-90 and p.
443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987)
"Applications of HPLC in Biochemistry" in: Laboratory Techniques in
Biochemistry and Molecular Biology, Vol. 17; Rehm et al. (1993)
Biotechnology, Vol. 3, Chapter III: "Product recovery and
purification", p. 469-714, VCH: Weinheim; Belter, P. A., et al.
(1988) Bioseparations: downstream processing for Biotechnology,
John Wiley and Sons; Kennedy, J. F., and Cabral, J. M. S. (1992)
Recovery processes for biological Materials, John Wiley and Sons;
Shaeiwitz, J. A., and Henry, J. D. (1988) Biochemical Separations,
in: Ullmann's Encyclopedia of Industrial Chemistry, Vol. B3;
Chapter 11, p. 1-27, VCH: Weinheim; and Dechow, F. J. (1989)
Separation and purification techniques in biotechnology, Noyes
Publications).
[16000] [0456.0.0.39]: see [0456.0.0.27]
[0457.0.39.39] Example 9
Purification of Gamma-Aminobutyric Acid
[16001] [0458.0.39.39] Abbreviations; GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[16002] The unambiguous detection for the presence of
gamma-aminobutyric acid or shikimate or putrescine can be obtained
by analyzing recombinant organisms using analytical standard
methods: LC, LC-MSMS or TLC, as described. The total amount
produced in the organism for example in yeasts used in the
inventive process can be analysed for example according to the
following procedure:
[16003] The material such as yeasts, E. coli or plants to be
analyzed can be disrupted by sonication, grinding in a glass mill,
liquid nitrogen and grinding or via other applicable methods.
[16004] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[16005] A typical sample pretreatment consists of a total lipid
extraction using such polar organic solvents as acetone or alcohols
as methanol, or ethers, saponification, partition between phases,
separation of non-polar epiphase from more polar hypophasic
derivatives and chromatography.
[16006] For analysis, solvent delivery and aliquot removal can be
accomplished with a robotic system comprising a single injector
valve Gilson 232XL and a 402 2S1V diluter [Gilson, Inc. USA, 3000
W. Beltline Highway, Middleton, Wis.]. For saponification, 3 ml of
50% potassium hydroxide hydro-ethanolic solution (4 water--1
ethanol) can be added to each vial, followed by the addition of 3
ml of octanol. The saponification treatment can be conducted at
room temperature with vials maintained on an IKA HS 501 horizontal
shaker [Labworld-online, Inc., Wilmington, N.C.] for fifteen hours
at 250 movements/minute, followed by a stationary phase of
approximately one hour.
[16007] Following saponification, the supernatant can be diluted
with 0.17 ml of methanol. The addition of methanol can be conducted
under pressure to ensure sample homogeneity. Using a 0.25 ml
syringe, a 0.1 ml aliquot can be removed and transferred to HPLC
vials for analysis.
[16008] For HPLC analysis, a Hewlett Packard 1100 HPLC, complete
with a quaternary pump, vacuum degassing system, six-way injection
valve, temperature regulated autosampler, column oven and
Photodiode Array detector can be used [Agilent Technologies
available through Ultra Scientific Inc., 250 Smith Street, North
Kingstown,
[16009] RI]. The column can be a Waters YMC30, 5-micron,
4.6.times.250 mm with a guard column of the same material [Waters,
34 Maple Street, Milford, Mass.]. The solvents for the mobile phase
can be 81 methanol: 4 water: 15 tetrahydrofuran (THF) stabilized
with 0.2% BHT (2,6-di-tert-butyl-4-methylphenol). Injections were
20 l. Separation can be isocratic at 30.degree. C. with a flow rate
of 1.7 ml/minute. The peak responses can be measured by absorbance
at 447 nm.
[16010] [0459.0.39.39] If required and desired, further
chromatography steps with a suitable resin may follow.
Advantageously, the gamma-aminobutyric acid can be further purified
with a so-called RTHPLC. As eluent acetonitrile/water or
chloroform/acetonitrile mixtures can be used. If necessary, these
chromatography steps may be repeated, using identical or other
chromatography resins. The skilled worker is familiar with the
selection of suitable chromatography resin and the most effective
use for a particular molecule to be purified.
[16011] [0460.0.0.39] see [0460.0.0.27]
[0461.0.39.39] Example 10
Cloning SEQ ID NO: 108175 for the Expression in Plants
[16012] [0462.0.0.39] see [0462.0.0.27]
[16013] [0463.0.39.39] SEQ ID NO: 108175 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[16014] [0464.0.0.0.39] to [0466.0.0.39]: see [0464.0.0.27] to
[0466.0.0.27]
[16015] [0466.1.39.39] In case the Herculase enzyme can be used for
the amplification, the PCR amplification cycles were as follows: 1
cycle of 2-3 minutes at 94.degree. C., followed by 25-30 cycles of
in each case 30 seconds at 94.degree. C., 30 seconds at
55-60.degree. C. and 5-10 minutes at 72.degree. C., followed by 1
cycle of 10 minutes at 72.degree. C., then 4.degree. C.
[16016] [0467.0.39.39] The following primer sequences were selected
for the gene SEQ ID NO: 108175:
i) forward primer SEQ ID NO: 108177 ii) reverse primer SEQ ID NO:
108178
[16017] [0468.0.0.39] to [0470.0.0.39]: see [0468.0.0.27] to
[0470.0.0.27]
[16018] [0470.1.39.39] The PCR-products, produced by Pfu Turbo DNA
polymerase, were phosphorylated using a T4 DNA polymerase using a
standard protocol (e.g. MBI Fermentas) and cloned into the
processed binary vector.
[16019] [0471.0.39.39] see [0471.0.0.27]
[16020] [0471.1.39.39] The DNA termini of the PCR-products,
produced by Herculase DNA polymerase, were blunted in a second
synthesis reaction using Pfu Turbo DNA polymerase. The composition
for the protocol of the blunting the DNA-termini was as follows:
0.2 mM blunting dTTP and 1.25 u Pfu Turbo DNA polymerase. The
reaction was incubated at 72.degree. C. for 30 minutes. Then the
PCR-products were phosphorylated using a T4 DNA polymerase using a
standard protocol (e.g. MBI Fermentas) and cloned into the
processed vector as well.
[16021] [0472.0.39.39] to [0479.0.39.39]: see [0472.0.0.27] to
[0479.0.0.27]
[0480.0.39.39] Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 108175
[16022] [0481.0.0.39] to [0513.0.0.39]: see [0481.0.0.27] to
[0513.0.0.27]
[16023] [0514.0.39.39] As an alternative, gamma-aminobutyric acid
can be detected as described in Haak and Reineke, Antimicrob.
Agents Chemother. 19(3): 493(1981)
[16024] As an alternative, shikimate can be detected as described
in Gould and Erickson, J Antibiot 41(5), 688-9 (1988).
[16025] As an alternative, putrescine can be detected as described
in Endo Y., Anal Biochem. 89(1):235-46(1978).
[16026] [0515.0.0.39] to [0552.0.0.39]: see [0515.0.0.27] to
[0552.0.0.27]
[16027] [0553.0.39.39] [16028] 1. A process for the production of
gamma-aminobutyric acid, which comprises [16029] a) increasing or
generating the activity of a protein as indicated in Table XII,
application no. 39, columns 5 or 7, or a functional equivalent
thereof in a non-human organism or in one or more parts thereof;
and [16030] b) growing the organism under conditions which permit
the production of gamma-aminobutyric acid in said organism. [16031]
2. A process for the production of gamma-aminobutyric acid or
shikimate or putrescine, comprising the increasing or generating in
an organism or a part thereof the expression of at least one
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: [16032] a) nucleic acid molecule
encoding of a polypeptide as indicated in Table XII, application
no. 39, columns 5 or 7 or a fragment thereof, which confers an
increase in the amount of gamma-aminobutyric acid in an organism or
a part thereof; [16033] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table XI, application no. 39,
columns 5 or 7; [16034] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of gamma-aminobutyric
acid in an organism or a part thereof; [16035] d) nucleic acid
molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of gamma-aminobutyric acid in an organism or a part
thereof; [16036] e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of gamma-aminobutyric acid in an organism or a part thereof;
[16037] f) nucleic acid molecule which encompasses a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers or
primer pairs as indicated in Table XIII, application no. 39,
columns 5 or 7, and conferring an increase in the amount of
gamma-aminobutyric acid in an organism or a part thereof; [16038]
g) nucleic acid molecule encoding a polypeptide which is isolated
with the aid of monoclonal antibodies against a polypeptide encoded
by one of the nucleic acid molecules of (a) to (f) and conferring
an increase in the amount of gamma-aminobutyric acid in an organism
or a part thereof; [16039] h) nucleic acid molecule encoding a
polypeptide comprising a consensus as indicated in Table XIV,
application no. 39, column 7, and conferring an increase in the
amount of gamma-aminobutyric acid in an organism or a part thereof;
and [16040] i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of gamma-aminobutyric acid in an organism or a part thereof.
[16041] or comprising a sequence which is complementary thereto.
[16042] 3. The process of claim 1 or 2, comprising recovering of
the free or bound gamma-aminobutyric acid. [16043] 4. The process
of any one of claims 1 to 3, comprising the following steps:
[16044] a) selecting an organism or a part thereof expressing a
polypeptide encoded by the nucleic acid molecule characterized in
claim 2; [16045] b) mutagenizing the selected organism or the part
thereof; [16046] c) comparing the activity or the expression level
of said polypeptide in the mutagenized organism or the part thereof
with the activity or the expression of said polypeptide of the
selected organisms or the part thereof; [16047] d) selecting the
mutated organisms or parts thereof, which comprise an increased
activity or expression level of said polypeptide compared to the
selected organism or the part thereof; [16048] e) optionally,
growing and cultivating the organisms or the parts thereof; and
[16049] f) recovering, and optionally isolating, the free or bound
gamma-aminobutyric acid produced by the selected mutated organisms
or parts thereof. [16050] 5. The process of any one of claims 1 to
4, wherein the activity of said protein or the expression of said
nucleic acid molecule is increased or generated transiently or
stably. [16051] 6. An isolated nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[16052] a) nucleic acid molecule encoding of a polypeptide as
indicated in Table XII, application no. 39, columns 5 or 7 or a
fragment thereof, which confers an increase in the amount of
gamma-aminobutyric acid in an organism or a part thereof; [16053]
b) nucleic acid molecule comprising of a nucleic acid molecule as
indicated in Table XI, application no. 39, columns 5 or 7; [16054]
c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of gamma-aminobutyric acid in
an organism or a part thereof; [16055] d) nucleic acid molecule
which encodes a polypeptide which has at least 50% identity with
the amino acid sequence of the polypeptide encoded by the nucleic
acid molecule of (a) to (c) and conferring an increase in the
amount of gamma-aminobutyric acid in an organism or a part thereof;
[16056] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridisation
conditions and conferring an increase in the amount of
gamma-aminobutyric acid in an organism or a part thereof; [16057]
f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table XIII, application no. 39, column 7, and
conferring an increase in the amount of gamma-aminobutyric acid in
an organism or a part thereof; [16058] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
gamma-aminobutyric acid in an organism or a part thereof; [16059]
h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table XIV, application no. 39, column 7,
and conferring an increase in the amount of gamma-aminobutyric acid
in an organism or a part thereof; and [16060] i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of gamma-aminobutyric acid in an organism or a part
thereof. [16061] whereby the nucleic acid molecule distinguishes
over the sequence as indicated in Table XI, application no. 39,
columns 5 or 7, by one or more nucleotides. [16062] 7. A nucleic
acid construct which confers the expression of the nucleic acid
molecule of claim 6, comprising one or more regulatory elements.
[16063] 8. A vector comprising the nucleic acid molecule as claimed
in claim 6 or the nucleic acid construct of claim 7. [16064] 9. The
vector as claimed in claim 8, wherein the nucleic acid molecule is
in operable linkage with regulatory sequences for the expression in
a prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [16065] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [16066] 11. The host cell of claim 10, which is a
transgenic host cell. [16067] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [16068] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [16069] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table XII,
application no. 39, columns 5 or 7, by one or more amino acids.
[16070] 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. [16071] 16. A plant tissue,
propagation material, harvested material or a plant comprising the
host cell as claimed in claim 12 which is plant cell or an
Agrobacterium. [16072] 17. A method for screening for agonists and
antagonists of the activity of a polypeptide encoded by the nucleic
acid molecule of claim 6 conferring an increase in the amount of
gamma-aminobutyric acid in an organism or a part thereof
comprising: (a) contacting cells, tissues, plants or microorganisms
which express the a polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
gamma-aminobutyric acid in an organism or a part thereof with a
candidate compound or a sample comprising a plurality of compounds
under conditions which permit the expression the polypeptide; (b)
assaying the gamma-aminobutyric acid or shikimate or putrescine
level or the polypeptide expression level in the cell, tissue,
plant or microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and (c) identifying a
agonist or antagonist by comparing the measured gamma-aminobutyric
acid or shikimate or putrescine level or polypeptide expression
level with a standard gamma-aminobutyric acid or polypeptide
expression level measured in the absence of said candidate compound
or a sample comprising said plurality of compounds, whereby an
increased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an agonist and
a decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
[16073] 18. A process for the identification of a compound
conferring increased gamma-aminobutyric acid or production in a
plant or microorganism, comprising the steps: (a) culturing a plant
cell or tissue or microorganism or maintaining a plant expressing
the polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of gamma-aminobutyric acid in
an organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with said readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of gamma-aminobutyric acid in
an organism or a part thereof; (b) identifying if the compound is
an effective agonist by detecting the presence or absence or
increase of a signal produced by said readout system. [16074] 19. A
method for the identification of a gene product conferring an
increase in gamma-aminobutyric acid production in a cell,
comprising the following steps: (a) contacting the nucleic acid
molecules of a sample, which can contain a candidate gene encoding
a gene product conferring an increase in gamma-aminobutyric acid
after expression with the nucleic acid molecule of claim 6; (b)
identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; (c) introducing the candidate nucleic acid molecules in
host cells appropriate for producing gamma-aminobutyric acid; (d)
expressing the identified nucleic acid molecules in the host cells;
(e) assaying the gamma-aminobutyric acid level in the host cells;
and (f) identifying nucleic acid molecule and its gene product
which expression confers an increase in the gamma-aminobutyric acid
level in the host cell in the host cell after expression compared
to the wild type. [16075] 20. A method for the identification of a
gene product conferring an increase in gamma-aminobutyric acid
production in a cell, comprising the following steps: [16076] (a)
identifying in a data bank nucleic acid molecules of an organism;
which can contain a candidate gene encoding a gene product
conferring an increase in the gamma-aminobutyric acid amount or
level in an organism or a part thereof after expression, and which
are at least 20% homolog to the nucleic acid molecule of claim 6;
[16077] (b) introducing the candidate nucleic acid molecules in
host cells appropriate for producing gamma-aminobutyric acid;
[16078] (c) expressing the identified nucleic acid molecules in the
host cells; [16079] (d) assaying the gamma-aminobutyric acid level
in the host cells; and [16080] (e) identifying nucleic acid
molecule and its gene product which expression confers an increase
in the gamma-aminobutyric acid level in the host cell after
expression compared to the wild type. [16081] 21. A method for the
production of an agricultural composition comprising the steps of
the method of any one of claims 17 to 20 and formulating the
compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. [16082] 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [16083] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
gamma-aminobutyric acid after expression. [16084] 24. Use of the
polypeptide of claim 14 or the nucleic acid construct claim 7 or
the gene product identified according to the method of claim 19 or
20 for identifying compounds capable of conferring a modulation of
gamma-aminobutyric acid levels in an organism. [16085] 25.
Agrochemical, pharmaceutical, food or feed composition comprising
the nucleic acid molecule of claim 6, the polypeptide of claim 14,
the nucleic acid construct of claim 7, the vector of claim 8 or 9,
the antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20.
[16086] 26. The method of any one of claims 1 to 5, the nucleic
acid molecule of claim 6, the polypeptide of claim 14, the nucleic
acid construct of claim 7, the vector of claim 8 or 9, the
antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20, wherein the fine chemical is gamma-aminobutyric acid.
[16087] [0554.0.0.39] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[16088] [0000.0.40.40] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[16089] [0001.0.40.40] The present invention relates to a process
for the production of the fine chemical in a microorganism, a plant
cell, a plant, a plant tissue or in one or more parts thereof. The
invention furthermore relates to nucleic acid molecules,
polypeptides, nucleic acid constructs, vectors, antisense
molecules, antibodies, host cells, plant tissue, propagation
material, harvested material, plants, microorganisms as well as
agricultural compositions and to their use.
[16090] [0002.0.40.40] Coenzymes are molecules that cooperate in
the catalytic action of an enzyme. Like enzymes, coenzymes are not
irreversibly changed during catalysis; they are either unmodified
or regenerated. Each kind of coenzyme has a particular chemical
function. Coenzymes may either be attached by covalent bonds to a
particular enzyme or exist freely in solution, but in either case
they participate intimately in the chemical reactions catalyzed by
the enzyme.
[16091] [0003.0.40.40] Coenzyme Q10 (CoQ 10) or ubiquinone is
essentially a vitamin or vitamin-like substance. Disagreements on
nomenclature notwithstanding, vitamins are defined as organic
compounds essential in minute amounts for normal body function
acting as coenzymes or precursors to coenzymes. Coenzyme Q10 or
CoQ10 belongs to a family of substances called ubiquinones.
Ubiquinones, also known as coenzymes Q and mitoquinones, are
lipophilic, water-insoluble substances involved in electron
transport and energy production in mitochondria. The basic
structure of ubiquinones consists of a benzoquinone "head" and a
terpinoid "tail." The "head" structure participates in the redox
activity of the electron transport chain. The major difference
among the various coenzymes Q is in the number of isoprenoid units
(5-carbon structures) in the "tail." Coenzymes Q contain one to 12
isoprenoid units in the "tail"; 10 isoprenoid units are common in
animals. Coenzymes Q occur in the majority of aerobic organisms,
from bacteria to plants and animals. Two numbering systems exist
for designation of the number of isoprenoid units in the terpinoid
"tail": coenzyme Qn and coenzyme Q(x). N refers to the number of
isoprenoid side chains, and x refers to the number of carbons in
the terpinoid "tail" and can be any multiple of five. Thus,
coenzyme Q10 refers to a coenzyme Q having 10 isoprenoid units in
the "tail." Since each isoprenoid unit has five carbons, coenzyme
Q10 can also be designated coenzyme Q(50). The structures of
coenzymes Q are analogous to those of vitamin K2. Coenzyme Q10 is
also known as Coenzyme Q(50), CoQ10, CoQ(50), ubiquinone (50),
ubiquinol-10 and ubidecarerone.
[16092] They are present naturally in foods and sometimes are also
synthesized in the body. CoQ10 likewise is found in small amounts
in a wide variety of foods and is synthesized in all tissues. The
biosynthesis of CoQ10 from the amino acid tyrosine is a multistage
process requiring at least eight vitamins and several trace
elements. Coenzymes are cofactors upon which the comparatively
large and complex enzymes absolutely depend for their function.
Coenzyme Q10 is the coenzyme for at least three mitochondrial
enzymes (complexes I, II and III) as well as enzymes in other parts
of the cell. Mitochondrial enzymes of the oxidative phosphorylation
pathway are essential for the production of the high-energy
phosphate, adenosine triphosphate (ATP), upon which all cellular
functions depend. The electron and proton transfer functions of the
quinone ring are of fundamental importance to all life forms;
ubiquinone in the mitochondria of animals, plastoquinone in the
chloroplast of plants, and menaquinone in bacteria. The term
"bioenergetics" has been used to describe the field of biochemistry
looking specifically at cellular energy production. In the related
field of free radical chemistry, CoQ10 has been studied in its
reduced form as a potent antioxidant. The bioenergetics and free
radical chemistry of CoQ10 are reviewed in Gian Paolo Littarru's
book, Energy and Defense, published in 1994. The precise chemical
structure of CoQ10 is 2,3 dimethoxy-5 methyl-6 decaprenyl
benzoquinone
[16093] [0004.0.40.40] Discovered in 1957, CoQ-10 is also called
ubiquinone because it belongs to a class of compounds called
quinones, and because it's ubiquitous in living organisms,
especially in the heart, liver, and kidneys. It plays a crucial
role in producing energy in cells. And it acts as a powerful
antioxidant, meaning that it helps neutralize cell-damaging
molecules called free radicals. Manufactured by all cells in the
body, CoQ-10 is also found in small amounts in foods, notably meat
and fish. By the mid-1970's, the industrial technology to produce
pure CoQ10 in quantities sufficient for larger clinical trials was
established. Principally CoQ10 can be isolated from microorganisms
or plants or algae; in particular mitochondria are a common source
for CoQ10. Alternatively, they are obtained advantageously from
animals or fish.
[16094] [0005.0.40.40] Since the actions of supplemental CoQ10 have
yet to be clarified, the mechanism of these actions is a matter of
speculation. However, much is known about the biochemistry of
CoQ10. CoQ10 is an essential cofactor in the mitochondrial electron
transport chain, where it accepts electrons from complex I and II,
an activity that is vital for the production of ATP. CoQ10 has
antioxidant activity in mitochondria and cellular membranes,
protecting against peroxidation of lipid membranes. It also
inhibits the oxidation of LDL-cholesterol. LDL-cholesterol
oxidation is believed to play a significant role in the
pathogenesis of atherosclerosis. CoQ10 is biosynthesized in the
body and shares a common synthetic pathway with cholesterol.
[16095] CoQ10 levels decrease with aging in humans. Why this occurs
is not known but may be due to decreased synthesis and/or increased
lipid peroxidation which occurs with aging. Significantly decreased
levels of CoQ10 have been noted in a wide variety of diseases in
both animal and human studies. CoQ10 deficiency may be caused by
insufficient dietary CoQ10, impairment in CoQ10 biosynthesis,
excessive utilization of CoQ10 by the body, or any combination of
the three. Decreased dietary intake is presumed in chronic
malnutrition and cachexia.
[16096] [0006.0.40.40] The relative contribution of CoQ10
biosynthesis versus dietary CoQ10 is under investigation. Karl
Folkers takes the position that the dominant source of CoQ10 in man
is biosynthesis. This complex, 17 step process, requiring at least
seven vitamins (vitamin B2-riboflavin, vitamin B3-niacinamide,
vitamin B6, folic acid, vitamin B12, vitamin C, and pantothenic
acid) and several trace elements, is, by its nature, highly
vulnerable. Karl Folkers argues that suboptimal nutrient intake in
man is almost universal and that there is subsequent secondary
impairment in CoQ10 biosynthesis. This would mean that average or
"normal" levels of CoQ10 are really suboptimal and the very low
levels observed in advanced disease states represent only the tip
of a deficiency "ice berg".
[16097] Supplemental CoQ10 may have cardioprotective,
cytoprotective and neuroprotective activities. There are claims
that it has positive effects in cancer, muscular dystrophy and
immune dysfunction. Similarly, it may inhibit obesity or enhance
athletic performance.
[16098] [0007.0.40.40] HMG-CoA reductase inhibitors used to treat
elevated blood cholesterol levels by blocking cholesterol
biosynthesis also block CoQ10 biosynthesis. The resulting lowering
of blood CoQ10 level is due to the partially shared biosynthetic
pathway of CoQ10 and cholesterol. In patients with heart failure
this is more than a laboratory observation. It has a significant
harmful effect which can be negated by oral CoQ10
supplementation.
[16099] Increased body consumption of CoQ10 is the presumed cause
of low blood CoQ10 levels seen in excessive exertion,
hypermetabolism, and acute shock states. It is likely that all
three mechanisms (insufficient dietary CoQ10, impaired CoQ10
biosynthesis, and excessive utilization of CoQ10) are operable to
varying degrees in most cases of observed CoQ10 deficiency.
[16100] [0008.0.40.40] In nature, Coenzymes Q0 to Q9 are found as
well. E.g. Coenzyme Q9 is a derivative of CoQ10 found e.g. in the
chloroplast of plants. Coenzyme Q9 has a shorter aliphatic group
bound to the ring structure. Due to the high structural homology of
Coenzymes Q0 to Q9 are expected to provide the same or very similar
activities as CoQ10 in cells or organisms. However, Matsura et al.,
Biochim Biophys Acta, 1992, 1123(3) pp. 309-15 concluded from their
study that CoQ9 constantly acts as a potential antioxidant in
hepatocytes whereas CoQ10 manly exhibit its antioxidant activity in
cells containing CoQ10 as the predominate CoQ homolog. Coenzyme Q10
is actual a very common ingredient in different types of cosmetics,
due to its protective role against radicals and its predicted
function in skin tautening.
[16101] [0009.0.40.40] Thus, Coenzymes, in particular CoQ10 or CoQ9
can be used in a lot of different applications, for example in
cosmetics, pharmaceuticals and in feed and food.
[16102] [0010.0.40.40] Therefore improving the productivity of said
Coenzymes and improving the quality of cosmetics, pharmaceuticals,
foodstuffs and animal feeds, in particular of nutrition
supplements, is an important task of the different industries.
[16103] [0011.0.40.40] To ensure a high productivity of said
Coenzymes in plants or microorganism, it is necessary to manipulate
the natural biosynthesis of said Coenzymes in said organisms.
[16104] [0012.0.40.40] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
regulators which participate in the biosynthesis of said Coenzymes
and make it possible to produce certain said Coenzymes specifically
on an industrial scale without unwanted byproducts forming. In the
selection of genes for biosynthesis two characteristics above all
are particularly important. On the one hand, there is as ever a
need for improved processes for obtaining the highest possible
contents of said Coenzymes on the other hand as less as possible
byproducts should be produced in the production process.
[16105] [0013.0.0.40] It was now found that this object is achieved
by providing the process according to the invention described
herein and the embodiments characterized in the claims.
[16106] [0014.0.40.40] It was found that the overexpression of the
nucleic acid molecules characterized herein confers an increase in
the content of Coenzyme Q10 or Coenzyme Q9 in plants. Accordingly,
in a first embodiment, the invention relates to a process for the
production of Coenzyme Q10 and/or Coenzyme Q9. Accordingly, in a
further embodiment, the invention relates to a process for the
production of a fine chemical, whereby the fine chemical is
Coenzyme Q10 and/or Coenzyme Q9. Accordingly, in the present
invention, the term "the fine chemical" as used herein relates to
"Coenzyme Q10 and/or Coenzyme Q9". Further, in another embodiment
the term "the fine chemicals" as used herein also relates to
compositions of the fine chemicals comprising Coenzyme Q10 and/or
Coenzyme Q9.
[16107] [0015.0.40.40] In one embodiment, the term "the respective
fine chemicals" means Coenzyme Q10 and/or Coenzyme Q9, depending on
the context. For example, the increase of the gene product of a
gene with activity of the Gene/ORF Locus name or its homologs as
mentioned confers the increase of the level of coenzymes CoQ9 in
plants or parts thereof. For example, the increase of the gene
product of a gene with the ORF name b2426 from E. coli or a plant
or its homologs confer the increase of the level of coenzymes CoQ9
and CoQ10 in plants or parts thereof.
[16108] Accordingly, in one embodiment, the term "the respective
fine chemicals" means Coenzyme Q10 and Coenzyme Q9. In one
embodiment, the term "the respective fine chemical" means Coenzyme
Q10 or Coenzyme Q9. In a further embodiment, the term "the
respective fine chemical" means Coenzyme Q10 and/or Coenzyme Q9 and
their salts, ester, or thioester or Coenzyme Q10 and/or Coenzyme Q9
in free form or bound to protein(s), e.g. enzyme(s), or peptide(s),
e.g. polypeptide(s) or to membranes or parts thereof, e.g. in the
form of oils or waxes or in compositions with lipids, oils, fats or
lipid mixture, as well as Coenzyme Q10 and/or Coenzyme Q9 in its
reduced or oxidized form. In a preferred embodiment, the term "the
respective fine chemical" means Coenzyme Q10 or CoQ9, in free form
or its salts or bound to peptide(s) or protein(s). Lipids, oils,
waxes, fats or lipid mixture shall mean any, lipid, oil, wax and/or
fat containing any bound or free Coenzyme Q10 and/or Coenzyme
Q9.
[16109] In one embodiment, the term "the fine chemical" and the
term "the respective fine chemical" mean at least one chemical
compound with an activity of the above mentioned fine chemical.
[16110] [0016.0.40.40] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [16111] (a) increasing or generating the activity of one
or more [16112] of a protein as shown in table XII, application no.
40, column 3 encoded by the nucleic acid sequences as shown in
table XI, application no. 40, column 5, in a non-human organism or
in one or more parts thereof or [16113] (b) growing the organism
under conditions which permit the production of the fine chemical,
thus Coenzyme of the invention or fine chemicals comprising
coenzyme of the invention, in said organism or in the culture
medium surrounding the organism
[16114] [0016.1.40.40] In one embodiment, the method of the present
invention confers the increase of the content of more than one of
the respective fine chemicals, i.e. of Coenzyme Q9 and/or Coenzyme
Q10.
[16115] Accordingly, the term "the fine chemical" can mean
"Coenzyme Q9", and/or "Coenzyme Q10", owing to circumstances and
the context. In order to illustrate that the meaning of the term
"the fine chemical" means "Coenzyme Q9" and/or "Coenzyme Q10" the
term "the respective fine chemical" is also used.
[16116] [0017.0.0.40] to [0019.0.0.40] see [0017.0.0.27] to
[0019.0.0.27]
[16117] [0019.0.40.40]
[16118] [0020.0.40.40] Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, application no. 40, column 5, line 40 in a plant
conferred an increase in Coenzyme Q9 content of the transformed
plants.
[16119] Thus, in one embodiment, said protein or its homologs are
used for the production of Coenzyme Q9.
[16120] Surprisingly it was found, that the transgenic expression
of the Brassica napus protein as indicated in Table XII,
application no. 40, column 5, line 41 in a plant conferred an
increase in Coenzyme Q9 content of the transformed plants. Thus, in
one embodiment, said protein or its homologs are used for the
production of Coenzyme Q9.
[16121] [0021.0.0.40] see [0021.0.0.27]
[16122] [0022.0.40.40] The sequence of b2426 (Accession number
NP.sub.--416921) from
[16123] Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a putative oxidoreductase with NAD(P)-binding domain.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a gene product with an activity of
the "ribitol dehydrogenase, short-chain alcohol dehydrogenase
homology" superfamily, preferably being involved in C-compound and
carbohydrate utilization, metabolism of vitamins, cofactors and
prosthetic groups, biosynthesis of secondary products derived from
primary amino acids, aerobic aromate catabolism, anaerobic aromate
catabolism, and/or catabolism of secondary metabolites,
biosynthesis of the cysteine-aromatic group and/or degradation of
amino acids of the cysteine-aromatic group, aromate anabolism, more
preferred a protein with the activity of a putative oxidoreductase
with NAD(P)-binding domain from E. coli or a plant or its homolog,
e.g. as shown herein in Table XII, column 5 or 7, line 40 or as
encoded by the nucleic acid molecule shown in Table XI, column 5 or
7, line 40, for the production of the respective fine chemical,
meaning of Coenzyme Q9, in particular for increasing the amount of
Coenzyme Q9, preferably Coenzyme Q9 in free or bound form in an
organism or a part thereof, as mentioned.
[16124] The sequence of YDR513W (ACCESSION NP.sub.--010801) from
Saccharomyces cerevisiae has been published in Jacq et al., Nature
387 (6632 Suppl), 75-78 (1997) and in Goffeau et al., Science 274
(5287), 546-547, 1996 and its cellular activity has characterized
as glutathione reductase. Accordingly, in one embodiment, the
process of the present invention comprises the use of glutathione
reductase or a protein of the glutaredoxin superfamily, in
particular of a protein having a deoxyribonucleotide metabolism,
cytoplasm, stress response, detoxification, and/or electron
transport and membrane-associated energy conservation activity for
the production of Coenzyme Q9. Accordingly, in one embodiment, the
process of the present invention comprises the use of protein with
tha activity of YDR513W from Saccharomyces cerevisiaeor a plant,
e.g. as indicated herein in Table XII, line 41, columns 5, or its
homologue, e.g. as shown herein in Table XII, line 41, column 7,
for the production of the respective fine chemical, meaning of
Coenzyme Q9, in particular for increasing the amount of reduced
and/or oxidized Coenzyme Q9 and/or Coenzyme Q9 in free or bound
form in an organism or a part thereof, as mentioned. In one
embodiment, in the process of the present invention the activity of
a glutathione reductase is increased or generated, e.g. from
Saccharomyces cerevisiae or a plant or a homolog thereof.
[16125] [0022.1.0.40] to [0023.0.40.40] see [0022.1.0.27] to
[0023.0.0.27]
[16126] [0023.1.40.40] Homologs of the polypeptide disclosed in
table XII, application no. 40, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 40, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 40, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 40,
column 7, resp.
[16127] [0024.0.0.40] see [0024.0.0.27]
[16128] [0025.0.40.40] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 40, column 3 "if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 40, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[16129] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 40,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 40,
column 3 of E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[16130] [0025.1.40.40] In one embodiment, the polypeptide of the
invention or used in the method of the invention confers said
activity, e.g. the increase of the respective fine chemical in an
organism or a part thereof, if it is derived from an organism,
which is evolutionary close to the organism indicated in Table XI,
column 4 and is expressed in an organism, which is evolutionary
distant to the origin organism. For example origin and expressing
organism are derived from different families, orders, classes or
phylums whereas origin and the organism indicated in Table XI,
column 4 are derived from the same families, orders, classes or
phylums.
[16131] [0025.2.0.40] see [0025.2.0.27]
[16132] [0025.1.0.40] see [0025.1.0.27]
[16133] [0026.0.0.40] to [0033.0.0.40] see[0026.0.0.27] to
[0033.0.0.27]
[16134] [0034.0.40.40] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 40, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[16135] [0035.0.0.40] to [0044.0.0.40] see [0035.0.0.27] to
[0044.0.0.27]
[16136] [0045.0.40.40] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
40, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 40, column 6 of
the respective line, between 10% and 50%, 10% and 100%, 10% and
200%, 10% and 300%, 10% and 400%, 10% and 500%, 20% and 50%, 20%
and 250%, 20% and 500%, 50% and 100%, 50% and 250%, 50% and 500%,
500% and 1000% or more
[16137] [0046.0.40.40] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
40, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, application no. 40, column 6 of
the respective line confers an increase of the respective fine
chemical and of further Coenzyme or their precursors.
[16138] [0047.0.0.40] to [0048.0.0.40] see [0047.0.0.27] to
[0048.0.0.27]
[16139] [0049.0.40.40] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 40, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 40, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 40, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[16140] [0050.0.40.40] ./.
[16141] [0051.0.40.40] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g. hydrophobic or lipophilic
compositions. Depending on the choice of the organism used for the
process according to the present invention, for example a
microorganism or a plant, compositions or mixtures of the
respective fine chemical and various other coenzymes, vitamins
and/or antioxidants, e.g. vitamin B6 or vitamin E, can be
produced.
[16142] [0052.0.0.40] see [0052.0.0.27]
[16143] [0053.0.40.40] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[16144] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 40, columns 5 and 7 or its homologs activity
having herein-mentioned Coenzyme of the invention increasing
activity; and/or [16145] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 40,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 40, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned Coenzyme of the invention increasing
activity; and/or [16146] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned Coenzyme increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 40, columns 5 and 7 or its
homologs activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [16147] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned Coenzyme of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 40, columns 5
and 7 or its homologs activity; and/or [16148] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned Coenzyme of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 40, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [16149] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned Coenzyme of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 40, columns 5 and 7 or its
homologs activity, and/or [16150] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned Coenzyme of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 40, columns 5 and 7 or its
homologs activity; and/or [16151] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 40, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[16152] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [16153] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[16154] [0054.0.40.40] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity is the polypeptide of the present
invention, e.g. conferring the increase of Coenzyme Q10 and/or
Coenzyme Q9 after increasing the expression or activity of the
encoded polypeptide or having the activity of a polypeptide having
an, e.g. of a protein as indicated in Table XII, application no.
40, column 5, encoded by a nucleic acid molecule indicated in Table
XI, application no. 40, column 5, or of their homologs, e.g. as
indicated in Table XI or XII, application no. 40, column 7.
[16155] [0055.0.0.40] to [0067.0.0.40] see [0055.0.0.27] to
[0067.0.0.27]
[16156] [0068.0.40.40] The mutation is introduced in such a way
that the production of the Coenzyme Q9 and/or Coenzyme Q10 is not
adversely affected.
[16157] [0069.0.0.40] see [0069.0.0.27]
[16158] [0070.0.40.40] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or used in the method of the invention or
the polypeptide of the invention or used in the method of the
invention, for example the nucleic acid construct mentioned below,
or encoding the protein as indicated in Table XII, application no.
40, column 5, or being encoded by a nucleic acid molecule indicated
in Table XI, application no. 40, column 5, or of its homologs, e.g.
as indicated in Table XII, application no. 40, column 7, into an
organism alone or in combination with other genes, it is possible
not only to increase the biosynthetic flux towards the end product,
but also to increase, modify or create de novo an advantageous,
preferably novel metabolites composition in the organism, e.g. an
advantageous hydrophopic composition comprising a higher content of
(from a viewpoint of nutritional physiology limited) coenzymes,
vitamins and/or antioxidants, e.g.
[16159] vitamin B6 and/or vitamin E. In one embodiment, the
expression of a protein as indicated in Table XII, application no.
40, column 5, or being encoded by a nucleic acid molecule indicated
in Table XI, application no. 40, column 5, or of its homologs, e.g.
as indicated in Table XII, application no. 40, column 7, conferring
an increase of coenzymes, in particular of Coenzyme Q9 and/or
Coenzyme Q10, and preferably of further coenzymes, vitamins, and/or
antioxidants.
[16160] [0071.0.40.40] Preferably the composition further comprises
higher amounts of metabolites positively affecting or lower amounts
of metabolites negatively affecting the nutrition or health of
animals or humans provided with said compositions or organisms of
the invention or parts thereof. Likewise, the number or activity of
further genes which are required for the import or export of
nutrients or metabolites, including amino acids, fatty acids,
vitamins, coenzymes, antioxidants etc. or any one of their
precursors, required for the cell's biosynthesis of the respective
fine chemical may be increased so that the concentration of
necessary or relevant precursors, e.g. of isoprenoids, acetyl CoA,
HMG-CoA, mevalonate, Isopentenyl pyrophosphate, Geranyl
pyrophosphate, Farnesyl Pyrophosphate, or other cofactors or
intermediates within the organelle, e.g. in mitochondria or
plastids, resp., within (a) cell(s) or within the corresponding
storage compartments is increased. Owing to the increased or novel
generated activity of the polypeptide of the invention or used in
the method of the invention or owing to the increased number of
nucleic acid sequences of the invention or used in the method of
the invention and/or to the modulation of further genes which are
involved in the biosynthesis of the respective fine chemical, e.g.
by increasing the activity of enzymes synthesizing precursors, e.g.
Lovastatin, HMG-CoA Reductase, Mevalonate Kinase, or by destroying
the activity of one or more genes which are involved in the
breakdown of the respective fine chemical, it is possible to
increase the yield, production and/or production efficiency of the
respective fine chemical in the host organism, such as plants or
the microorganisms.
[16161] [0072.0.40.40] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are
antioxidants, further coenzymes, vitamins, e.g. vitamin B6 or
vitamin E, or triglycerides, fatty acids, lipids, oils and/or fats
containing Coenzyme Q10 and/or Coenzyme Q9.
[16162] [0073.0.40.40] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[16163] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, an
organelle, a plant or animal tissue or a plant; [16164] b)
increasing the protein activity or of a polypeptide being encoded
by the nucleic acid molecule of the present invention and described
below, e.g. of a protein as indicated in Table XII, application no.
40, column 5 or being encoded by a nucleic acid molecule indicated
in Table XI, application no. 40, column 5 or of its homologs, e.g.
as indicated in Table XII, application no. 40, column 7, and
conferring an increase of the respective fine chemical in the
organism, preferably in the microorganism, the non-human animal,
the plant or animal cell, the plant or animal tissue, the organelle
or the plant, [16165] c) growing the organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant under conditions which permit the
production of the respective fine chemical in the organism,
preferably the microorganism, the plant cell, the plant tissue or
the plant; and [16166] d) if desired, recovering, optionally
isolating, the free and/or bound the respective fine chemical and,
optionally further free and/or bound vitamins, coenzymes or
antioxidants synthesized by the organism, the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[16167] [0074.0.40.40] The organism, in particular the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue, the organelle or the plant is
advantageously grown in such a way that it is not only possible to
recover, if desired isolate the free or bound the respective fine
chemical or the free and bound the respective fine chemical but as
option it is also possible to produce, recover and, if desired
isolate, other free or/and bound antioxidants, vitamins or
coenzymes and mixtures thereof.
[16168] [0075.0.0.40] to [0077.0.0.40] see [0075.0.0.27] to
[0077.0.0.27]
[16169] [0078.0.40.40] The organism such as microorganisms or
plants or the recovered, and if desired isolated, fine chemical can
then be processed further directly into foodstuffs or animal feeds
or for other applications, for example according to the disclosures
made in the following US patent publications: U.S. Pat. No.
6,380,252: Use of L-acetylcarnitine, L-isovalerylcarnitine,
L-propionylcarnitine for increasing the levels of IGF-1, U.S. Pat.
No. 6,372,198: Dentifrice for the mineralization and
remineralization of teeth, U.S. Pat. No. 6,368,617: Dietary
supplement, U.S. Pat. No. 6,350,473: Method for treating
hypercholesterolemia, hyperlipidemia, and atherosclerosis, U.S.
Pat. No. 6,335,361: Method of treating benign forgetfulness, U.S.
Pat. No. 6,329,432: Mesozeaxanthin formulations for treatment of
retinal disorders, U.S. Pat. No. 6,328,987: Cosmetic skin care
compositions containing alpha interferon, U.S. Pat. No. 6,312,703:
Compressed lecithin preparations, U.S. Pat. No. 6,306,392:
Composition comprising a carnitine and glutathione, useful to
increase the absorption of glutathione and synergize its effects,
U.S. Pat. No. 6,303,586: Supportive therapy for diabetes,
hyperglycemia and hypoglycemia, U.S. Pat. No. 6,297,281:
Association of no syntase inhibitors with trappers of oxygen
reactive forms, U.S. Pat. No. 6,294,697: Discrete-length
polyethylene glycols, U.S. Pat. No. 6,277,842: Dietary supplemental
method for fat and weight reduction, U.S. Pat. No. 6,261,250:
Method and apparatus for enhancing cardiovascular activity and
health through rhythmic limb elevation, U.S. Pat. No. 6,258,855:
Method of retarding and ameliorating carpal tunnel syndrome, U.S.
Pat. No. 6,258,848: Methods and compositions for increasing insulin
sensitivity, U.S. Pat. No. 6,258,847: Use of 2-mercaptoethanolamine
(2-MEA) and related aminothiol compounds and copper(II)-3,5
di-isopropyl salicylates and related compounds in the prevention
and treatment of various diseases, U.S. Pat. No. 6,255,354:
Preparation of a pulmonary surfactant for instillation and oral
application, U.S. Pat. No. 6,254,547: Breath methylated alkane
contour: a new marker of oxidative stress and disease, U.S. Pat.
No. 6,248,552: Enzyme-based assay for determining effects of
exogenous and endogenous factors on cellular energy production,
U.S. Pat. No. 6,248,363: Solid carriers for improved delivery of
active ingredients in pharmaceutical compositions, U.S. Pat. No.
6,245,800: Method of preventing or treating statin-induced toxic
effects using L-carnitine or an alkanoyl L-carnitine, U.S. Pat. No.
6,245,378: Nutritional supplement for facilitating skeletal muscle
adaptation to strenuous exercise and counteracting defatigation in
asthenic individuals, U.S. Pat. No. 6,242,491: Use of creatine or
creatine compounds for skin preservation, U.S. Pat. No. 6,232,346:
Composition for improvement of cellular nutrition and mitochondrial
energetics, U.S. Pat. No. 6,231,836: Folic acid dentifrice, U.S.
Pat. No. 6,228,891: Use of
2,3-dimethoxy-5-methyl-6-decaprenyl-1,4-benzoquinone, U.S. Pat. No.
6,228,402: Xylitol-containing non-human foodstuff and method, U.S.
Pat. No. 6,228,347: Antioxidant gel for gingival conditions, U.S.
Pat. No. 6,218,436: Pharmaceutically active carotenoids, U.S. Pat.
No. 6,203,818: Nutritional supplement for cardiovascular health,
U.S. Pat. No. 6,200,550: Oral care compositions comprising coenzyme
Q10, U.S. Pat. No. 6,191,172: Water-soluble compositions of
bioactive lipophilic compounds, U.S. Pat. No. 6,184,255:
Pharmaceutical composition comprising coenzyme Q10, U.S. Pat. No.
6,166,077: Use of L-acetylcarnitine, L-isovalerylcarnitine,
L-propionylcarnitine for increasing the levels of IGF-1, U.S. Pat.
No. 6,162,419: Stabilized ascorbyl compositions, U.S. Pat. No.
6,159,508: Xylitol-containing non-human foodstuff and method, U.S.
Pat. No. 6,159,476: Herbal supplement for increased muscle strength
and endurance for athletes, U.S. Pat. No. 6,153,582: Defined
serumfree medical solution for ophthalmology, U.S. Pat. No.
6,136,859: Pharmaceutical formulation for treating liver disorders,
U.S. Pat. No. 6,107,281: Compounds and their combinations for the
treatment of influenza infection, U.S. Pat. No. 6,106,286: Method
and device for administering medicine to the periodontium, U.S.
Pat. No. 6,099,854: Dry composition containing flavonol useful as a
food supplement, U.S. Pat. No. 6,086,910: Food supplements, U.S.
Pat. No. 6,080,788: Composition for improvement of cellular
nutrition and mitochondrial energetics, U.S. Pat. No. 6,069,167:
Use of antioxidant agents to treat cholestatic liver disease, U.S.
Pat. No. 6,063,820: Medical food for diabetics, U.S. Pat. No.
6,054,261: Coenzyme Q.sub.10 compositions for organ protection
during perfusion, U.S. Pat. No. 6,051,250: Process for the
stabilization of vesicles of amphiphilic lipid(s) and composition
for topical application containing the said stabilized
vesicles,
[16170] The fermentation broth, fermentation products, plants or
plant products can be purified in the customary manner by
hydrolysis with strong bases, extraction and crystallization or via
thin layer chromatography and other methods known to the person
skilled in the art and described herein below. Products of these
different work-up procedures are fatty acids or fatty acid
compositions which still comprise fermentation broth, plant
particles and cell components in different amounts, advantageously
in the range of from 0 to 99% by weight, preferably below 80% by
weight, especially preferably between below 50% by weight.
[16171] [0079.0.0.40] to [0084.0.0.40] see [0079.0.0.27] to
[0084.0.0.27]
[16172] [0084.2.40.40] Coenzyme Q10 production was reported in
Agrobacterium sp., Protaminobacter rubber and Paracoccus
denitrificans. Coenzyme Q9 production was reported in Candida
tropicalis. Production of ubiquiones with side chain length of 6-10
units, e.g. including Coenzyme Q10 and Coenzyme Q9 was reported for
controlled continuous culture of phototrophic bacteria (wild-type
strains of Rhodobacter capsulatus, Rhodobacter sphaeroides,
Thiocapsa roseopersicina and Ectothiorhodospira shaposhnikovii.
Cells mostly contained one main ubiquinone, whereby the content and
composition dependent on growth conditions, substrates and other
factors. Preferred is a production of more than 0,1, preferably
more than 1 to 6 mg/g dry cells in one of said organisms or in any
other microorganism, even more preferred are more than 10 mg/g dry
cells, 20 mg/g dry cells, 50 mg/g dry cells, 100 mg/g dry cells,
200 mg/g dry cells, 300 mg/g dry cells, 500 mg/g dry cells or
more.
[16173] [0084.0.0.40] see [0084.0.0.27]
[16174] [0085.0.40.40] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [16175] a) a nucleic acid sequence as
shown in table XI, application no. 40, columns 5 or 7 or a
derivative thereof, or [16176] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as shown table XI, application no. 40, columns 5 or 7
or a derivative thereof, or [16177] c) (a) and (b) is/are not
present in its/their natural genetic environment or has/have been
modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[16178] [0086.0.0.40] to [0087.0.0.40] see [0086.0.0.27] to
[0087.0.0.27]
[16179] [0088.0.40.40] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose Coenzyme
content, in particular its Coenzyme Q9 and/or Coenzyme Q10 content
is modified advantageously owing to the nucleic acid molecule of
the present invention expressed. This is important for plant
breeders since, for example, the nutritional value of plants for
feed or nutrition is dependent on the abovementioned coenzymes, in
particular on the essential coenzyme Q10, and the general amount of
coenzymes as source in feed or food. After the activityof a protein
as indicated in Table XII, application no. 40, column 5, or being
encoded by a nucleic acid molecule indicated in Table XI,
application no. 40, column 5, or of its homologs, e.g. as indicated
in Table XII, application no. 40, column 7 has been increased or
generated, or after the expression of nucleic acid molecule or
polypeptide according to the invention has been generated or
increased, the transgenic plant generated thus is grown on or in a
nutrient medium or else in the soil and subsequently harvested.
[16180] [0088.1.0.40] see [0088.1.0.27]
[16181] [0089.0.0.40] to [0094.0.0.40] see [0089.0.0.27] to
[0094.0.0.27]
[16182] [0095.0.40.40] It may be advantageous to increase the pool
of the respective fine chemical in the transgenic organisms by the
process according to the invention in order to isolate high amounts
of the essentially pure fine chemical.
[16183] [0096.0.40.40] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid for example a coenzyme
precursor (e.g. isoprenoids, acetyl CoA, HMG-CoA, mevalonate,
isopentyl pyrophosphate, geranyl pyrophosphate, farnesyl
pyrophosphate, etc.) transporter protein or a compound, which
increases the production of Coenzyme precursors is useful to
increase the production of the respective fine chemical (see Bao
and Ohlrogge, Plant Physiol. 1999 August; 120 (4): 1057-1062).
[16184] [0097.0.40.40] In may also be advantageous to increase the
content of the lipid bound respective fine chemical.
[16185] [0098.0.40.40] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated. The production of further antioxidants,
vitamins or coenzymes such as coenzymes Q0, Q1, Q2, Q3, Q4, Q5, Q6,
Q7, and/or Q8 or Q10 or retinal, vitamin E, vitamin B6, or other
fat soluble vitamins or mixtures thereof by the process according
to the invention is advantageous, e.g. for the production of
compositions used in food and/or feed or cosmetic production.
[16186] [0099.0.40.40] In the case of the fermentation of
microorganisms, the abovementioned coenzymes may accumulate in
membrane fragments and/or in the lipophilic or hydrophobic
fraction. If microorganisms are used in the process according to
the invention, the fermentation broth can be processed after the
cultivation. Depending on the requirement, all or some of the
biomass can be removed from the fermentation broth by separation
methods such as, for example, centrifugation, filtration, decanting
or a combination of these methods, or else the biomass can be left
in the fermentation broth. The fermentation broth can subsequently
be reduced, or concentrated, with the aid of known methods such as,
for example, rotary evaporator, thin-layer evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth advantageously together with
compounds for the formulation can subsequently be processed by
lyophilization, spray drying, spray granulation or by other
methods. Preferably the Coenzymes or the lipophilic or hydrophobic
compositions are isolated from the organisms, such as the
microorganisms or plants or the culture medium in or on which the
organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[16187] [0100.0.40.40] Transgenic plants which comprise Coenzyme Q9
and Coenzyme Q10 synthesized in the process according to the
invention can advantageously be marketed directly without there
being any need for the membrane fragments, cells, cell fragments,
organelles, plastids, waxes, oils, lipids or fats comprising the
respective fine chemical to be isolated. For example, as coenzymes
may be isolated from membranes, e.g. mitochondrial or plastid
membranes, it can be sufficient to isolate only cell fractions
comprising said membrane or fragments of said membranes. Plants for
the process according to the invention are listed as meaning intact
plants and all plant parts, plant organs or plant parts such as
leaf, stem, seeds, root, tubers, anthers, fibers, root hairs,
stalks, embryos, calli, cotelydons, petioles, harvested material,
plant tissue, reproductive tissue and cell cultures which are
derived from the actual transgenic plant and/or can be used for
bringing about the transgenic plant. In this context, the seed
comprises all parts of the seed such as the seed coats, epidermal
cells, seed cells, endosperm or embryonic tissue. However, the
respective fine chemical produced in the process according to the
invention can also be isolated from the organisms, advantageously
plants, in the form of their cells, cell fragments, plastids,
organelles, membranes, membrane fragments, waxes oils, fats, lipids
and/or fatty acids. Coenzyme Q9 or Coenzyme Q10 produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts, preferably the plant
seeds. To increase the efficiency of oil extraction it is
beneficial to clean, to temper and if necessary to hull and to
flake the plant material especially the seeds. In this context,
oils, fats, lipids, waxes and/or fatty acids fractions can be
obtained by what is known as cold beating or cold pressing without
applying heat. In the case of microorganisms, the latter are, after
harvesting, for example extracted directly without further
processing steps or else, after disruption, extracted via various
methods with which the skilled worker is familiar. Chemically pure
the fine chemical comprising compositions are advantageous for
applications in the food or feed industry sector, the cosmetic
sector and especially the pharmacological industry sector.
[16188] [0101.0.0.40] see [0101.0.0.27]
[16189] [0102.0.40.40] Coenzymes can for example be detected
advantageously via LC separation methods. The unambiguous detection
for the presence of Coenzymes products can be obtained by analyzing
recombinant organisms using analytical standard methods like LC-MS,
LC-MSMS, or TLC. The material to be analyzed can be disrupted by
sonication, grinding in a glass mill, liquid nitrogen and grinding,
cooking, or via other applicable methods; see also Biotechnology of
Vitamins, Pigments and Growth Factors, Edited by Erik J. Vandamme,
London, 1989, p. 96 to 103.
[16190] [0103.0.40.40] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [16191] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide shown in
table XII, application no. 40, columns 5 or 7 or a fragment
thereof, which confers an increase in the amount of the respective
fine chemical in an organism or a part thereof; [16192] b) nucleic
acid molecule comprising, preferably at least the mature form, of
the nucleic acid molecule shown in table XI, application no. 40,
columns 5 or 7; [16193] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the respective
fine chemical in an organism or a part thereof; [16194] d) nucleic
acid molecule encoding a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [16195] e) nucleic acid molecule which hybridizes
with a nucleic acid molecule of (a) to (c) under stringent
hybridisation conditions and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[16196] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c) and conferring an increase in the amount
of the respective fine chemical in an organism or a part thereof;
[16197] g) nucleic acid molecule encoding a fragment or an epitope
of a polypeptide which is encoded by one of the nucleic acid
molecules of (a) to (e), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [16198] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
nucleic acid molecules from a cDNA library or a genomic library
using the primers shown in Table XIII, application no. 40, column 7
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [16199] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from an
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(h), preferably to (a) to (c), and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [16200] j) nucleic acid molecule which encodes a
polypeptide comprising the consensus sequence shown in table XIV,
application no. 40, column 7 and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [16201] k) nucleic acid molecule comprising one or more of
the nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domain of the polypeptide shown in table
XII, application no. 40, columns 5 or 7 and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; and [16202] l) nucleic acid molecule which is
obtainable by screening a suitable library under stringent
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k), preferably to (a) to (c), or
with a fragment of at least 15 nt, preferably 20 nt, 30 nt, 50 nt,
100 nt, 200 nt or 500 nt of the nucleic acid molecule characterized
in (a) to (k), preferably to (a) to (c), and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; or which comprises a sequence which is complementary
thereto.
[16203] [0103.1.40.40] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table XI, application no. 40, columns 5 or 7,
by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence indicated in Table XI, application no. 40, columns 5
or 7: In one embodiment, the nucleic acid molecule used in the
process of the invention is less than 100%, 99.999%, 99.99%, 99.9%
or 99% identical to a sequence indicated in Table XI, application
no. 40, columns 5 or 7. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table XII, application no. 40, columns 5 or 7.
[16204] [0103.2.40.40] ./.
[16205] [0104.0.40.40] In one embodiment, the nucleic acid molecule
or the invention or used in the process of the invention
distinguishes over the sequence shown in table XI, application no.
40, columns 5 or 7 by one or more nucleotides or does not consist
of the sequence shown in table XI, application no. 40, columns 5 or
7. In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence shown in table XI, application no. 40,
columns 5 or 7. In another embodiment, the nucleic acid molecule
does not encode a polypeptide of the sequence shown in table XII,
application no. 40, columns 5 or 7.
[16206] [0105.0.0.40] to [0107.0.0.40] see [0105.0.0.27] to
[0107.0.0.27]
[16207] [0108.0.40.40] Nucleic acid molecules with the sequence
shown in table XI, application no. 40, columns 5 or 7, nucleic acid
molecules which are derived from the amino acid sequences shown in
table XII, application no. 40, columns 5 or 7 or from polypeptides
comprising the consensus sequence shown in table XIV, application
no. 40, column 7, or their derivatives or homologues encoding
polypeptides with the enzymatic or biological activity of an
protein as shown in table XII, application no. 40, column 5 or 7,
or conferring a Coenzyme Q10 and/or Coenzyme Q9 increase dependent
on its expression or its activity, are advantageously increased in
the process according to the invention.
[16208] [0109.0.0.40] see [0109.0.0.27]
[16209] [0110.0.40.40] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
used in the process of the invention, e.g. of a protein as
indicated in Table XII, application no. 40, column 5, or being
encoded by a nucleic acid molecule indicated in Table XI,
application no. 40, column 5, or of its homologs, e.g. as indicated
in Table XII, application no. 40, column 7, can be determined from
generally accessible databases.
[16210] [0111.0.0.40] see [0111.0.0.27]
[16211] [0112.0.40.40] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with protein
activity as shown in table XI, application no. 40, column 3, and
conferring Coenzyme Q10 and/or Coenzyme Q9 increase, e.g. a protein
as indicated in Table XII, as shown in table XII, application no.
40, column 5 or 7, column 5, or being encoded by a nucleic acid
molecule indicated in Table XI, application no. 40, column 5, or of
their homologs, e.g. as indicated in Table XI or XII, application
no. 40, column 7.
[16212] [0113.0.0.40] to [0117.0.0.40] see [0113.0.0.27] to
[0117.0.0.27]
[16213] [0118.1.40.40] In one embodiment, the nucleic acid molecule
according to the invention or used in the process of the invention
originates from a plant with a high Coenzyme Q10 and/or Coenzyme Q9
content. In one embodiment, the nucleic acid molecule according to
the invention or used in the process of the invention originates
from and/or is transformed into a plant with a high Coenzyme Q10
and/or Coenzyme Q9 content.
[16214] [0118.0.0.40] to [0120.0.0.40] see [0118.0.0.27] to
[0120.0.0.27]
[16215] [0121.0.40.40] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences shown in table XII,
application no. 40, columns 5 or 7 or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a respective fine chemical increase after
increasing its activity, e.g. having the activity of an protein, as
indicated in Table XII, application no. 40, column 5 or 3, or being
encoded by a nucleic acid molecule indicated in Table XI,
application no. 40, column 5, or of its homologs, e.g. as indicated
in Table XII, application no. 40, column 7.
[16216] [0122.0.0.40] to [0127.0.0.40] see [0122.0.0.27] to
[0127.0.0.27]
[16217] [0128.0.40.40] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs shown in Table XIII, application
no. 40, column 7, by means of polymerase chain reaction can be
generated on the basis of a sequence shown herein, for example the
sequence shown in table XI, application no. 40, columns 5 or 7 or
the sequences derived from table XII, application no. 40, columns 5
or 7.
[16218] [0129.0.40.40] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention or used in the method of the invention are indicated in
the alignments shown in the figures. Conserved regions are those,
which show a very little variation in the amino acid in one
particular position of several homologs from different origin. The
consensus sequence shown in table XIV, application no. 40, column 7
is derived from said alignments.
[16219] [0130.0.40.40] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of the
respective fine chemical, in particular of Coenzyme Q10 and/or
Coenzyme Q9 after increasing the expression or activity or having
an activity as shown in table XI, application no. 40, column 3, or
further functional homologs of the polypeptide of the invention,
e.g. homologs from a protein as indicated in Table XII, application
no. 40, column 5, or being encoded by a nucleic acid molecule
indicated in Table XI, application no. 40, column 5, or of its
homologs, e.g. as indicated in Table XII, application no. 40,
column 7, from other organisms.
[16220] [0131.0.0.40] to [0138.0.0.40] see [0131.0.0.27] to
[0138.0.0.27]
[16221] [0139.0.40.40] Polypeptides having above-mentioned
activity, i.e. conferring the respective fine chemical increase,
derived from other organisms, can be encoded by other DNA sequences
which hybridize to the sequences shown in table XI, application no.
40, columns 5 or 7 under relaxed hybridization conditions and which
code on expression for peptides having the respective fine
chemical-increasing activity.
[16222] [0140.0.0.40] to [0146.0.0.40] see [0140.0.0.27] to
[0146.0.0.27]
[16223] [0147.0.40.40] Further, the nucleic acid molecule of the
invention or used in the method of the invention comprises a
nucleic acid molecule, which is a complement of one of the
nucleotide sequences of above mentioned nucleic acid molecules or a
portion thereof. A nucleic acid molecule which is complementary to
one of the nucleotide sequences shown in table XI, application no.
40, columns 5 or 7 is one which is sufficiently complementary to
one of the nucleotide sequences shown in table XI, application no.
40, columns 5 or 7 such that it can hybridize to one of the
nucleotide sequences shown in table XI, application no. 40, columns
5 or 7, thereby forming a stable duplex. Preferably, the
hybridisation is performed under stringent hybridization
conditions. However, a complement of one of the herein disclosed
sequences is preferably a sequence complement thereto according to
the base pairing of nucleic acid molecules well known to the
skilled person. For example, the bases A and G undergo base pairing
with the bases T and U or C, resp. and visa versa. Modifications of
the bases can influence the base-pairing partner.
[16224] [0148.0.40.40] The nucleic acid molecule of the invention
or used in the method of the invention comprises a nucleotide
sequence which is at least about 30%, 35%, 40% or 45%, preferably
at least about 50%, 55%, 60% or 65%, more preferably at least about
70%, 80%, or 90%, and even more preferably at least about 95%, 97%,
98%, 99% or more homologous to a nucleotide sequence shown in table
XI application no.
[16225] 40, columns 5 or 7, or a functional portion thereof and
preferably has above mentioned activity, in particular having a the
respective fine chemical, in particular Coenzyme Q10 and/or
Coenzyme Q9-increasing activity after increasing the activity or an
activity of an gene product as shown in table XI, application no.
40, column 3, e.g. a gene encoding a protein as indicated in Table
XII, application no. 40, column 5, or comprising or expressing a
nucleic acid molecule indicated in Table XI, application no. 40,
column 5, or of their homologs, e.g. as indicated in Table XI or
XII, application no. 40, column 7.
[16226] [0149.0.40.40] The nucleic acid molecule of the invention
or used in the method of the invention comprises a nucleotide
sequence which hybridizes, preferably hybridizes under stringent
conditions as defined herein, to one of the nucleotide sequences
shown in table XI, application no. 40, columns 5 or 7, or a portion
thereof and encodes a protein having above-mentioned activity, e.g.
conferring an increase of the fine chemical, and optionally, the
activity ofa protein e.g. of a protein as indicated in Table XII,
application no. 40, column 5, or being encoded by a nucleic acid
molecule indicated in Table XI, application no. 40, column 5, or of
their homologs, e.g. as indicated in Table XI or XII, application
no. 40, column 7.
[16227] [00149.1.40.40] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table
XI, application no. 40, columns 5 or 7, has further one or more of
the activities annotated or known for the a protein as indicated in
Table XII, application no. 40, column 3.
[16228] [0150.0.40.40] Moreover, the nucleic acid molecule of the
invention or used in the method of the invention can comprise only
a portion of the coding region of one of the sequences shown in
table XI, application no. 40, for example a fragment which can be
used as a probe or primer or a fragment encoding a biologically
active portion of the polypeptide of the present invention or of a
polypeptide used in the process of the present invention, i.e.
having above-mentioned activity, e.g. conferring an increase of
Coenzyme Q10 and/or Coenzyme Q9 if its activity is increased. The
nucleotide sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., in table XI, application no. 40,
an anti-sense sequence of one of the sequences, e.g., set forth in
table XI, application no. 40, columns 5 or 7, or naturally
occurring mutants thereof. Primers based on a nucleotide of
invention can be used in PCR reactions to clone homologues of the
polypeptide of the invention or of the polypeptide used in the
process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primers pairs shown in Table XIII, lines 231 to 234
and/or 235 to 242 and/or 599 to 602, column 7 will result in a
fragment of a gene product, e.g. of a gene encoding of a protein as
indicated in Table XII, application no. 40, column 5, or expressing
a nucleic acid molecule indicated in Table XI, application no. 40,
column 5, or of its homologs, e.g. as indicated in Table XII,
application no. 40, column 7.
[16229] [0151.0.0.40] see [0151.0.0.27]
[16230] [0152.0.40.40] The nucleic acid molecule of the invention
or used in the method of the invention encodes a polypeptide or
portion thereof which includes an amino acid sequence which is
sufficiently homologous to the amino acid sequence shown in table
XII, application no. 40, columns 5 or 7 such that the protein or
portion thereof maintains the ability to participate in the
respective fine chemical production, in particular a Coenzyme Q10
and/or Coenzyme Q9 increasing the activity as mentioned above or as
described in the examples in plants or microorganisms is
comprised.
[16231] [0153.0.40.40] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence shown in table XII, application no. 40, columns 5 or
7 such that the protein or portion thereof is able to participate
in the increase of the respective fine chemical production. In one
embodiment, a protein or portion thereof as indicated in Table XII,
application no. 40, columns 5 or 7, has for example an activity of
a polypeptide indicated in Table XII, application no. 40, column
3.
[16232] [0154.0.40.40] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence of table XII, application no. 40, columns 5 or 7 and
having above-mentioned activity, e.g. conferring preferably the
increase of the respective fine chemical.
[16233] [0155.0.0.40] to [0156.0.0.40] see [0155.0.0.27] to
[0156.0.0.27]
[16234] [0157.0.40.40] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences
shown in table XI, application no. 40, columns 5 or 7 (and portions
thereof) due to degeneracy of the genetic code and thus encode a
polypeptide of the present invention, in particular a polypeptide
having above mentioned activity, e.g. conferring an increase in the
respective fine chemical in a organism, e.g. as that polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 40, column 7, resp., or of the polypeptides encoded
by the sequence shown in table XII, application no. 40, columns 5
or 7 or the functional homologues. Advantageously, the nucleic acid
molecule of the invention or used in the method of the invention
comprises, or in an other embodiment has, a nucleotide sequence
encoding a protein comprising, or in an other embodiment having, an
amino acid sequence as indicated in Table XIV, application no. 40,
column 7, resp., or as shown in table XII, application no. 40,
columns 5 or 7 or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention or used in
the method of the invention encodes a full length protein which is
substantially homologous to an amino acid sequence shown in table
XII, application no. 40, columns 5 or 7 or the functional
homologues. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of the sequence
shown in table XI, application no. 40,
[16235] [0158.0.0.40] to [0160.0.0.40] see [0158.0.0.27] to
[0160.0.0.27]
[16236] [0161.0.40.40] Accordingly, in another embodiment, a
nucleic acid molecule of the invention or used in the method of the
invention is at least 15, 20, 25 or 30 nucleotides in length.
Preferably, it hybridizes under stringent conditions to a nucleic
acid molecule comprising a nucleotide sequence of the nucleic acid
molecule of the present invention or used in the process of the
present invention, e.g. comprising a sequence shown in table XI,
application no. 40, columns 5 or 7. The nucleic acid molecule is
preferably at least 20, 30, 50, 100, 250 or more nucleotides in
length.
[16237] [0162.0.0.40] see [0162.0.0.27]
[16238] [0163.0.40.40] Preferably, a nucleic acid molecule of the
invention or used in the method of the invention that hybridizes
under stringent conditions to a sequence shown in table XI,
application no. 40, columns 5 or 7 corresponds to a
naturally-occurring nucleic acid molecule of the invention or used
in the method of the invention. As used herein, a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA
molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes a natural protein). Preferably, the nucleic acid molecule
encodes a natural protein having above-mentioned activity, e.g.
conferring the respective fine chemical increase after increasing
the expression or activity thereof or the activity of a protein of
the invention or used in the process of the invention.
[16239] [0164.0.0.40] see [0164.0.0.27]
[16240] [0165.0.40.40] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. shown
in table XI, application no. 40.
[16241] [0166.0.0.40] to [0167.0.0.40] see [0166.0.0.27] to
[0167.0.0.27]
[16242] [0168.0.40.40] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in the sequences shown in table XII,
application no. 40, columns 5 or 7 yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence shown in table XII, application no. 40. and is
capable of participation in the increase of production of the
respective fine chemical after increasing its activity, e.g. its
expression. Preferably, the protein encoded by the nucleic acid
molecule is at least about 60% identical to the sequence shown in
table XII, application no. 40, columns 5 or 7 more preferably at
least about 70% identical to one of the sequences shown in table
XII, application no. 40, columns 5 or 7, even more preferably at
least about 80%, 90%, 95% homologous to the sequence shown in table
XII, application no. 40, columns 5 or 7 and most preferably at
least about 96%, 97%, 98%, or 99% identical to the sequence shown
in table XII, application no. 40, columns 5 or 7.
[16243] [0169.0.0.40] to [0172.0.0.40] see [0169.0.0.27] to
[0172.0.0.27]
[16244] [0173.0.32.40] to [0175.0.32.40] see [0173.0.32.32] to
[0175.0.32.32]
[16245] [0176.0.40.40] Functional equivalents derived from one of
the polypeptides as shown in table XII, application no. 40, columns
5 or 7 according to the invention by substitution, insertion or
deletion have at least 30%, 35%, 40%, 45% or 50%, preferably at
least 55%, 60%, 65% or 70% by preference at least 80%, especially
preferably at least 85% or 90%, 91%, 92%, 93% or 94%, very
especially preferably at least 95%, 97%, 98% or 99% homology with
one of the polypeptides as shown in table XII, application no. 40,
columns 5 or 7 according to the invention and are distinguished by
essentially the same properties as a polypeptide as shown in table
XII, application no. 40, columns 5 or 7.
[16246] [0177.0.40.40] Functional equivalents derived from a
nucleic acid sequence as shown in table XI, application no. 40,
columns 5 or 7 according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptides as shown in table XII,
application no. 40, columns 5 or 7 according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as shown in table XII, application no. 40, columns 5 or
7.
[16247] [0178.0.0.40] see [0178.0.0.27]
[16248] [0179.0.40.40] A nucleic acid molecule encoding an
homologous to a protein sequence of table XII, application no. 40,
columns 5 or 7, can be created by introducing one or more
nucleotide substitutions, additions or deletions into a nucleotide
sequence of the nucleic acid molecule of the present invention, in
particular of table XI, application no. 40, columns 5 or 7, such
that one or more amino acid substitutions, additions or deletions
are introduced into the encoded protein. Mutations can be
introduced into the encoding sequences of table XI, application no.
40, columns 5 or 7, by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis.
[16249] [0180.0.0.40] to [0183.0.0.40] see [0180.0.0.27] to
[0183.0.0.27]
[16250] [0184.0.40.40] Homologues of the nucleic acid sequences
used, with a sequence shown in table XI, application no. 40,
columns 5 or 7, comprise also allelic variants with at least
approximately 30%, 35%, 40% or 45% homology, by preference at least
approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from a
sequences shown, preferably from table XI, application no. 40,
columns 5 or 7 or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[16251] [0185.0.40.40] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more of the sequences shown in
any of the table XI, application no. 40, columns 5 or 7 In one
embodiment, it is preferred that the nucleic acid molecule
comprises as little as possible other nucleotides not shown in any
one of table XI, application no. 40, columns 5 or 7. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, the nucleic
acid molecule use in the process of the invention is identical to a
sequences shown in table XI, application no. 40, columns 5 or
7.
[16252] [0186.0.40.40] Also preferred is that one or more nucleic
acid molecule used in the process of the invention encodes a
polypeptide comprising a sequence shown in table XII, application
no. 40, columns 5 or 7. In one embodiment, the nucleic acid
molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment used in the inventive process, the
encoded polypeptide is identical to a sequence shown in table XII,
application no. 40, columns 5 or 7.
[16253] [0187.0.40.40] In one embodiment, the nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising a sequence shown in table XII, application no. 40,
columns 5 or 7, and comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence of a
sequence shown in table XI, application no. 40, columns 5 or 7.
[16254] [0188.0.40.40] Polypeptides (=proteins), which still have
the essential enzymatic activity of the polypeptide of the present
invention conferring an increase of the respective fine chemical
i.e. whose activity is essentially not reduced, are polypeptides
with at least 10% or 20%, by preference 30% or 40%, especially
preferably 50% or 60%, very especially preferably 80% or 90 or more
of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide shown in table XII,
application no. 40, columns 5 or 7, expressed under identical
conditions.
[16255] [0189.0.40.40] Homologues of nucleic acid sequences shown
in table XI, application no. 40, columns 5 or 7 or of the derived
sequences shown in table XII, application no. 40, columns 5 or 7
also mean truncated sequences, cDNA, single-stranded DNA or RNA of
the coding and noncoding DNA sequence. Homologues of said sequences
are also understood as meaning derivatives, which comprise
noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[16256] [0190.0.0.40] to [0191.0.0.40] see [0190.0.0.27] to
[0191.0.0.27]
[16257] [0191.1.40.40] In one embodiment, the organisms or a part
thereof provides according to the herein mentioned process of the
invention an increased level of the fine chemical bound to any wax,
triglycerides, lipid, oil and/or fat or any composition thereof
containing any bound or free Coenzyme Q10 and/or Coenzyme Q9, for
example bound to lipids, lipoproteins, membrane fractions,
sphingolipids, phosphoglycerides, lipids, glycolipids such as
glycosphingolipids, phospholipids such as phosphatidylethanolamine,
phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,
phosphatidylinositol or diphosphatidylglycerol, or as
monoacylglyceride, diacylglyceride or triacylglyceride, compared to
said control or selected organisms or parts thereof.
[16258] [0192.0.0.40] to [0203.0.0.40] see [0192.0.0.27] to
[0203.0.0.27]
[16259] [0204.0.40.40] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule, which comprises a
nucleic acid molecule selected from the group consisting of:
[16260] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide shown in table XII, application no.
40, columns 5 or 7; or a fragment thereof conferring an increase in
the amount of the respective fine chemical in an organism or a part
thereof [16261] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule shown in table
XI, application no. 40, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [16262] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [16263] d) nucleic acid molecule encoding a polypeptide
whose sequence has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [16264]
e) nucleic acid molecule which hybridizes with a nucleic acid
molecule of (a) to (c) under stringent hybridisation conditions and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [16265] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [16266] g) nucleic acid molecule
encoding a fragment or an epitope of a polypeptide which is encoded
by one of the nucleic acid molecules of (a) to (e), preferably to
(a) to [16267] (c) and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [16268]
h) nucleic acid molecule comprising a nucleic acid molecule which
is obtained by amplifying a cDNA library or a genomic library using
the primers or primer pairs indicated in Table XIII, application
no. 40, column 7 and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [16269]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from a expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (g), preferably to (a) to (c) and conferring an
increase in the amount of the respective fine chemical in an
organism or a part thereof; [16270] j) nucleic acid molecule which
encodes a polypeptide comprising a consensus sequence shown in
table XIV, application no. 40, column 7 and conferring an increase
in the amount of the respective fine chemical in an organism or a
part thereof; [16271] k) nucleic acid molecule encoding the amino
acid sequence of a polypeptide encoding a domain of the polypeptide
shown in table XII, application no. 40, columns 5 or 7, and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; and [16272] l) nucleic
acid molecule which is obtainable by screening a suitable nucleic
acid library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment of at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (h) or of the nucleic acid molecule shown
in table XI, application no. 40, columns 5 or 7 or a nucleic acid
molecule encoding, preferably at least the mature form of, a
polypeptide shown in table XII, application no. 40, columns 5 or 7,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; or which encompasses a
sequence which is complementary thereto; whereby, preferably, the
nucleic acid molecule according to (a) to (l) distinguishes over a
sequence depicted in table XI, application no. 40, columns 5 or 7
by one or more nucleotides. In one embodiment, the nucleic acid
molecule of the invention or used in the method of the invention
does not consist of a sequence shown in table XI, columns 5 or 7.
In an other embodiment, the nucleic acid molecule of the present
invention is at least 30% identical and less than 100%, 99.999%,
99.99%, 99.9% or 99% identical to a sequence shown in table XI,
application no. 40, columns 5 or 7. In a further embodiment the
nucleic acid molecule does not encode a polypeptide sequence shown
in table XII, columns 5 or 7. Accordingly, in one embodiment, the
nucleic acid molecule of the present invention encodes in one
embodiment a polypeptide which differs at least in one or more
amino acids from a polypeptide shown in table XII, application no.
40, columns 5 or 7 but does not encode a protein of a sequence
shown in table XII, application no. 40, columns 5 or 7.
Accordingly, in one embodiment, the protein encoded by a sequence
of a nucleic acid according to (a) to (l) does not consist of a
sequence shown in table XII, application no. 40, columns 5 or 7. In
a further embodiment, the protein of the present invention is at
least 30% identical to protein sequence depicted in table XII,
application no. 40, columns 5 or 7 and less than 100%, preferably
less than 99.999%, 99.99% or 99.9%, more preferably less than 99%,
985, 97%, 96% or 95% identical to a sequence shown in table XII,
application no. 40, columns 5 or 7.
[16273] [0205.0.0.40] to [0226.0.0.40] see [0205.0.0.27] to
[0226.0.0.27]
[16274] [0227.0.40.40] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[16275] In addition to a sequence mentioned in table XI,
application no. 40, column 7, columns 5 or 7 or its derivatives, it
is advantageous additionally to express and/or mutate further genes
in the organisms. Especially advantageously, additionally at least
one further gene of the isoprenoid biosynthetic pathway such as for
acetyl CoA, HMG-CoA, Mevalonate, Isopentyl pyrophosphate, Geranyl
pyrophosphate, Farnesyl pyrophosphate e.g. HMG-CoA Reductase,
Mevalonate, Kinase, etc., is expressed in the organisms such as
plants or microorganisms. It is also possible that the regulation
of the natural genes has been modified advantageously so that the
gene and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the isoprenoids, coenzyme precursor or coenzymes,
preferably Q9 and/or Q10, as desired since, for example, feedback
regulations no longer exist to the same extent or not at all. In
addition it might be advantageously to combine one or more
sequences shown in table XI, application no. 40, columns 5 or 7
with genes which generally support or enhances to growth or yield
of the target organism, for example genes which lead to faster
growth rate of microorganisms or genes which produces stress-,
pathogen, or herbicide resistant plants.
[16276] [0227.1.40.40] In addition to the sequence mentioned in
table XI, application no. 40, columns 5 or 7 or its derivatives, it
is advantageous additionally to knock out and/or mutate further
genes in the organisms. E.g. it may be advantageous, if one or more
genes of the catabolic pathway for isoprenoids or quinones are
reduced, deleted or in another way knocked out in the organisms
such as plants or microorganisms.
[16277] [0228.0.40.40] In a further embodiment of the process of
the invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the isoprenoid
metabolism, in particular in Coenzyme Q9 or Coenzyme Q10
synthesis.
[16278] [0229.0.40.40] ./.
[16279] [0230.1.40.40] ./.
[16280] [0230.2.40.40] ./.
[16281] [0230.0.0.40] see [0230.0.0.27]
[16282] [0231.0.40.40] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a Coenzyme Q10 and/or Coenzyme Q9
degrading protein is attenuated, in particular by reducing the rate
of expression of the corresponding gene.
[16283] [0232.0.0.40] to [0276.0.0.40] see [0232.0.0.27] to
[0276.0.0.27]
[16284] [0277.0.40.40] The coenzymes produced can be isolated from
the organism by methods with which the skilled worker is familiar,
for example, via extraction, salt precipitation and/or different
chromatography methods, e.g. as mentioned above. The process
according to the invention can be conducted batchwise,
semibatchwise or continuously. The respective fine chemical
produced in the process according to the invention can be isolated
as mentioned above from the organisms, advantageously plants, in
the form of their waxes, membrane fractions, oils, fats, lipids
and/or fatty acids. Fractions comprising the coenzymes produced by
this process can be obtained by harvesting the organisms, either
from the crop in which they grow, or from the field. This can be
done via pressing or extraction of the plant parts, preferably the
plant seeds.
[16285] [0278.0.0.40] to [0283.0.0.40] see [0278.0.0.27] to
[0283.0.0.27]
[16286] [0283.0.40.40] Moreover, a native polypeptide conferring
the increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, in particular, an anti-protein as shown in table XI,
application no. 40, column 3-antibody or an antibody against a
polypeptide as shown in table XII, application no. 40, columns 5 or
7 or a antigenic part thereof, which can be produced by standard
techniques utilizing the polypeptid of the present invention or
characterized in the process of the present invention or fragment
thereof, i.e., the polypeptide of this invention. Preferred are
monoclonal antibodies specifically binding to a polypeptide as
indicated in Table XII, application no. 40, columns 5 or 7.
[16287] [0284.0.0.40] see [0284.0.0.27]
[16288] [0285.0.40.40] In one embodiment, the present invention
relates to a polypeptide having a sequence shown in table XII,
application no. 40, columns 5 or 7 or as coded by a nucleic acid
molecule shown in table XI, application no. 40, columns 5 or 7 or
functional homologues thereof.
[16289] [0286.0.40.40] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased comprising or consisting of a consensus sequence shown in
table XIV, application no. 40, column 7 and in one another
embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence shown in table
XIV, application no. 40, column 7 whereby 20 or less, preferably 15
or 10, preferably 9, 8, 7, or 6, more preferred 5 or 4, even more
preferred 3, even more preferred 2, even more preferred 1, most
preferred 0 of the amino acids positions indicated can be replaced
by any amino acid or, in an further embodiment, can be replaced
and/or absent. In one embodiment, the present invention relates to
the method of the present invention comprising a polypeptide or to
a polypeptide comprising more than one consensus sequences (of an
individual line) as indicated in Table XIV, application no. 40,
column 7.
[16290] [0287.0.0.40] to [0289.0.0.40] see [0287.0.0.27] to
[0289.0.0.27]
[16291] [0290.0.0.40] see [0290.0.0.27]
[16292] [0291.0.40.40] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[16293] In one embodiment, said polypeptide of the invention or
used in the method of the invention distinguishes over a sequence
shown in table XII, columns 5 or 7 by one or more amino acids. In
one embodiment, polypeptide distinguishes form a sequence shown in
table XII, application no. 40, columns 5 or 7 by more than 5, 6, 7,
8 or 9 amino acids, preferably by more than 10, 15, 20, 25 or 30
amino acids, even more preferred are more than 40, 50, or 60 amino
acids and, preferably, the sequence of the polypeptide of the
invention distinguishes from a sequence shown in table XII,
application no. 40, columns 5 or 7 by not more than 80% or 70% of
the amino acids, preferably not more than 60% or 50%, more
preferred not more than 40% or 30%, even more preferred not more
than 20% or 10%. In an other embodiment, said polypeptide of the
invention does not consist of a sequence shown in table XII,
application no. 40, columns 5 or 7.
[16294] [0292.0.0.40] see [0292.0.0.27]
[16295] [0293.0.40.40] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part being encoded by the nucleic acid molecule
of the invention or used in the process of the invention and having
a sequence which distinguishes from the sequence as shown in table
XII, application no. 40, columns 5 or 7 by one or more amino acids.
In another embodiment, said polypeptide of the invention does not
consist of the sequence shown in table XII, application no. 40,
columns 5 or 7. In a further embodiment, said polypeptide of the
present invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by the nucleic acid molecules shown in table
XI, application no. 40, columns 5 or 7.
[16296] [0294.0.40.40] In one embodiment, the present invention
relates to a polypeptide protein activity as shown in table XI,
application no. 40, column 3, which distinguishes over the sequence
depicted in table XII, application no. 40, columns 5 or 7 by one or
more amino acids, preferably by more than 5, 6, 7, 8 or 9 amino
acids, preferably by more than 10, 15, 20, 25 or 30 amino acids,
even more preferred are more than 40, 50, or 60 amino acids but
even more preferred by less than 70% of the amino acids, more
preferred by less than 50%, even more preferred my less than 30% or
25%, more preferred are 20% or 15%, even more preferred are less
than 10%.
[16297] [0295.0.0.40] to [0296.0.0.40] see [0295.0.0.27] to
[0296.0.0.27]
[16298] [0297.0.40.40] The language "substantially free of cellular
material" includes preparations of the polypeptide of the invention
or used in the method of the invention in which the protein is
separated from cellular components of the cells in which it is
naturally or recombinantly produced. In one embodiment, the
language "substantially free of cellular material" includes
preparations having less than about 30% (by dry weight) of
"contaminating protein", more preferably less than about 20% of
"contaminating protein", still more preferably less than about 10%
of "contaminating protein", and most preferably less than about 5%
"contaminating protein". The term "Contaminating protein" relates
to polypeptides, which are not polypeptides of the present
invention. When the polypeptide of the present invention or
biologically active portion thereof is recombinantly produced, it
is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less
than about 10%, and most preferably less than about 5% of the
volume of the protein preparation. The language "substantially free
of chemical precursors or other chemicals" includes preparations in
which the polypeptide of the present invention is separated from
chemical precursors or other chemicals, which are involved in the
synthesis of the protein. The language "substantially free of
chemical precursors or other chemicals" includes preparations
having less than about 30% (by dry weight) of chemical precursors
of a protein as indicated in Table XII, application no. 40, column
5, or being encoded by a nucleic acid molecule indicated in Table
XI, application no. 40, column 5, or of its homologs, e.g. as
indicated in Table XII, application no. 40, column 7, or chemicals,
more preferably less than about 20% chemical precursors of a
protein as indicated in Table XII, application no. 40, column 5, or
being encoded by a nucleic acid molecule indicated in Table XI,
application no. 40, column 5, or of its homologs, e.g. as indicated
in Table XII, application no. 40, column 7, or of, still more
preferably less than about 10% chemical precursors of a protein as
indicated in Table XII, application no. 40, column 5, or being
encoded by a nucleic acid molecule indicated in Table XI,
application no. 40, column 5, or of its homologs, e.g. as indicated
in Table XII, application no. 40, column 7, or of and most
preferably less than about 5% chemical precursors of a protein as
indicated in Table XII, application no. 40, column 5, I or being
encoded by a nucleic acid molecule indicated in Table XI,
application no. 40, column 5, or of its homologs, e.g. as indicated
in Table XII, application no. 40, column 7, or of. In preferred
embodiments, isolated proteins or biologically active portions
thereof lack contaminating proteins from the same organism from
which the polypeptide of the present invention is derived.
Typically, such proteins are produced by recombinant
techniques.
[16299] [0298.0.40.40] A polypeptide of the invention or used in
the method of the invention can participate in the process of the
present invention. The polypeptide or a portion thereof comprises
preferably an amino acid sequence which is sufficiently homologous
to an amino acid sequence shown in table XII, application no. 40,
columns 5 or 7.
[16300] [0299.0.40.40] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
amino acid sequences as shown in table XII, application no. 40,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence of table XI,
application no. 40, columns 5 or 7 or which is homologous thereto,
as defined above.
[16301] [0300.0.40.40] Accordingly the polypeptide of the present
invention can vary from the sequences shown in table XII,
application no. 40, columns 5 or 7 in amino acid sequence due to
natural variation or mutagenesis, as described in detail herein.
Accordingly, the polypeptide comprise an amino acid sequence which
is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and most preferably at
least about 96%, 97%, 98%, 99% or more homologous to an entire
amino acid sequence shown in table XII, application no. 40, columns
5 or 7.
[16302] [0301.0.0.40] see [0301.0.0.27]
[16303] [0302.0.40.40] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., the amino acid sequence shown in table
XII, application no. 40, columns 5 or 7 or the amino acid sequence
of a protein homologous thereto, which include fewer amino acids
than a full length polypeptide of the present invention or used in
the process of the present invention or the full length protein
which is homologous to an polypeptide of the present invention or
used in the process of the present invention depicted herein, and
exhibit at least one activity of polypeptide of the present
invention or used in the process of the present invention.
[16304] [0303.0.0.40] see [0303.0.0.27]
[16305] [0304.0.40.40] Manipulation of the nucleic acid molecule of
the invention or used in the method of the invention may result in
the production of protein indicated in Table XII, application no.
40, column 5, or being encoded by a nucleic acid molecule indicated
in Table XI, application no. 40, column 5, or of its homologs, e.g.
as indicated in Table XII, application no. 40, column 7, having
differences from the wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[16306] [0305.0.40.40] Any mutagenesis strategies for the
polypeptide of the present invention or the polypeptide used in the
process of the present invention to result in increasing said
activity are not meant to be limiting; variations on these
strategies will be readily apparent to one skilled in the art.
Using such strategies, and incorporating the mechanisms disclosed
herein, the nucleic acid molecule and polypeptide of the invention
or used in the method of the invention may be utilized to generate
plants or parts thereof, expressing wildtype proteins as indicated
in Table XII, application no. 40, column 5, or being encoded by a
nucleic acid molecule indicated in Table XI, application no. 40,
column 5 or of its homologs, e.g. as indicated in Table XII,
application no. 40, column 7, or a mutant of a protein as indicated
in Table XII, application no. 40, column 5 or 7, or being encoded
by a nucleic acid molecule indicated in Table XI, application no.
40, column 5, or of its homologs, e.g. as indicated in Table XII,
application no. 40, column 7, e.g. a mutated, and encoding nucleic
acid molecules and polypeptide molecules of the invention or used
in the method of the invention such that the yield, production,
and/or efficiency of production of a desired compound is
improved.
[16307] [0306.0.0.40] to [0308.0.0.40] see [0306.0.0.27] to
[0308.0.0.27]
[16308] [0309.0.40.40] In one embodiment, an reference to a
"protein (=polypeptide) of the invention" or as indicated in Table
XII, application no. 40, columns 5 or 7, refers to a polypeptide
having an amino acid sequence corresponding to the polypeptide of
the invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table XII, application no. 40, columns 5 or 7, refers
to a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous a polypeptide of the
invention, preferably which is not substantially homologous to a
polypeptide as indicated in Table XII, application no. 40, columns
5 or 7, e.g., a protein which does not confer the activity
described herein or annotated or known for as indicated in Table
XII, application no.
[16309] 40, column 3, and which is derived from the same or a
different organism. In one embodiment, a "non-polypeptide of the
invention" or "other polypeptide" not being indicated in Table XII,
application no. 40, columns 5 or 7, does not confer an increase of
the respective fine chemical in an organism or part thereof.
[16310] [0310.0.0.40] to [0334.0.0.40] see [0310.0.0.27] to
[0334.0.0.27]
[16311] [0335.0.40.40] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of the nucleic acid sequences of the table XI, application no. 40,
columns 5 or 7 and/or homologs thereof. As described inter alia in
WO 99/32619, dsRNAi approaches are clearly superior to traditional
antisense approaches. The invention therefore furthermore relates
to double-stranded RNA molecules (dsRNA molecules) which, when
introduced into an organism, advantageously into a plant (or a
cell, tissue, organ or seed derived there from), bring about
altered metabolic activity by the reduction in the expression of a
nucleic acid sequences of the table XI, application no. 40, columns
5 or 7 and/or homologs thereof. In a double-stranded RNA molecule
for reducing the expression of an protein encoded by a nucleic acid
sequence of one of the table XI, application no. 40, columns 5 or 7
and/or homologs thereof, one of the two RNA strands is essentially
identical to at least part of a nucleic acid sequence, and the
respective other RNA strand is essentially identical to at least
part of the complementary strand of a nucleic acid sequence.
[16312] [0336.0.0.40] to [0342.0.0.40] see [0336.0.0.27] to
[0342.0.0.27]
[16313] [0343.0.40.40] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence of one of the sequences shown in table XI, application no.
40, columns 5 or 7 or its homolog is not necessarily required in
order to bring about effective reduction in the expression. The
advantage is, accordingly, that the method is tolerant with regard
to sequence deviations as may be present as a consequence of
genetic mutations, polymorphisms or evolutionary divergences. Thus,
for example, using the dsRNA, which has been generated starting
from a sequence of one of sequences shown in table XI, application
no. 40, columns 5 or 7 or homologs thereof of the one organism, may
be used to suppress the corresponding expression in another
organism.
[16314] [0344.0.0.40] to [0361.0.0.40] see [0344.0.0.27] to
[0361.0.0.27]
[16315] [0362.0.40.40] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention or used in the method of the
invention, the nucleic acid construct of the invention, the
antisense molecule of the invention, the vector of the invention or
a nucleic acid molecule encoding the polypeptide of the invention,
e.g. encoding a polypeptide having an protein activity a protein as
shown in table XI, application no. 40, column 3 or of a protein as
indicated in Table XII, application no. 40, column 5, or being
encoded by a nucleic acid molecule indicated in Table XI,
application no. 40, column 5, or of its homologs, e.g. as indicated
in Table XII, application no. 40, column 7. Due to the above
mentioned activity the respective fine chemical content in a cell
or an organism is increased. For example, due to modulation or
manipulation, the cellular activity is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an protein activity a protein
e.g. for a protein as indicated in Table XII, application no. 40,
column 5, or being encoded by a nucleic acid molecule indicated in
Table XI, application no. 40, column 5, or of its homologs, e.g. as
indicated in Table XII, application no. 40, column 7, means herein
that due to modulation or manipulation of the genome, the activity
of a protein as shown in table XI, application no. 40, column 3 is
increased in the cell or organism or part thereof, e.g. of a
protein as indicated in Table XII, application no. 40, column 5, or
being encoded by a nucleic acid molecule indicated in Table XI,
application no. 40, column 5, or of its homologs, e.g. as indicated
in Table XII, application no. 40, column 7. Examples are described
above in context with the process of the invention.
[16316] [0363.0.0.40] to [0376.0.0.40] see [0363.0.0.27] to
[0376.0.0.27]
[16317] [0377.0.40.40] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced coenzymes are isolated to high purity, preferably, to
purity of more than 70% w/w or more, more preferred are 80% or
more, in particular preferred are 90% w/w or more, even more
preferred are 95% w/w or more, e.g. 97% w/w or more, e.g. 98% w/w,
99% w/w, or 99.9% w/w.
[16318] [0378.0.40.40] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the Coenzyme Q9
or Coenzyme Q10 produced in the process can be recovered, purified
or isolated. The resulting coenzymes, e.g. compositions comprising
the former, can, if appropriate, subsequently be further purified,
if desired mixed with other active ingredients such as vitamins,
amino acids, carbohydrates, antibiotics and the like, and, if
appropriate, formulated.
[16319] [0379.0.40.40] In one embodiment, said coenzyme is the
respective fine chemical, preferably reduced Coenzyme Q9 and/or
Coenzyme Q10.
[16320] [0380.0.40.40] The Coenzyme Q9 or Coenzyme Q10 comprising
fraction or the respective fine chemical obtained in the process
are suitable as starting material for the synthesis of further
products of value. For example, they can be used in combination
with each other or alone for the production of pharmaceuticals,
foodstuffs, animal feeds or cosmetics. Accordingly, the present
invention relates a method for the production of a pharmaceuticals,
food stuff, animal feeds, nutrients or cosmetics comprising the
steps of the process according to the invention, including the
isolation of Coenzyme Q9 or Coenzyme Q10 composition produced or
the respective fine chemical produced if desired and formulating
the product with a pharmaceutical acceptable carrier or formulating
the product in a form acceptable for an application in agriculture.
A further embodiment according to the invention is the use of the
respective fine chemical produced in the process or of the
transgenic organisms in animal feeds, foodstuffs, medicines, food
supplements, cosmetics or pharmaceuticals.
[16321] [0381.0.0.40] to [0382.0.0.40] see [0381.0.0.27] to
[0382.0.0.27]
[16322] [0383.0.40.40] For preparing antioxidant, coenzyme and/or
vitamin-containing fine chemicals, in particular the respective
fine chemical-comprising fine chemicals, it is possible to use as
source organic compounds such as, for example, waxes, oils, fats
and/or lipids or a membrane comprising fraction, in particular
compositions comprising membranes of mitochondria or of
plastids.
[16323] [0384.0.0.40] see [0384.0.0.27]
[16324] [0385.0.40.40] The fermentation broths obtained in this
way, containing in particular Coenzyme Q10 and/or Coenzyme Q9 in
mixtures with other lipids, fats and/or oils and/or with other
vitamins or coenzymes or antioxidants normally have a dry matter
content of from, 1% to 50% by weight. Depending on requirements,
the biomass can be removed entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation or a combination of these methods, from the
fermentation broth or left completely in it. The fermentation broth
can then be thickened or concentrated by known methods, such as,
for example, with the aid of a rotary evaporator, thin-film
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. This concentrated fermentation broth can then be
worked up by freeze-drying, spray drying, spray granulation or by
other processes.
[16325] [0386.0.40.40] However, it is also possible to further
purify the fraction or composition comprising the respective fine
chemical, e.g. Coenzyme Q9 and/or Coenzyme Q10, as produced. For
this purpose, the product-containing composition is subjected for
example to a thin layer chromatography on silica gel plates or to a
chromatography such as a Florisil column (Bouhours J. F., J.
Chromatrogr. 1979, 169, 462), in which case the desired product or
the impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use. An alternative method to
purify the fatty acids is for example crystallization in the
presence of urea. These methods can be combined with each
other.
[16326] [0387.0.0.40] to [0392.0.0.40] see [0387.0.0.27] to
[0392.0.0.27]
[16327] [0393.0.40.40] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the respective fine chemical production
in a cell, comprising the following steps:
(a) contacting e.g. hybridising, the nucleic acid molecules of a
sample, e.g. cells, tissues, plants or microorganisms or a nucleic
acid library, which can contain a candidate gene encoding a gene
product conferring an increase in the respective fine chemical
after expression, with the nucleic acid molecule of the present
invention; (b) identifying the nucleic acid molecules, which
hybridize under relaxed stringent conditions with the nucleic acid
molecule of the present invention in particular to the nucleic acid
molecule sequence shown in table XI, application no. 40, columns 5
or 7 and, optionally, isolating the full length cDNA clone or
complete genomic clone; (c) introducing the candidate nucleic acid
molecules in host cells, preferably in a plant cell or a
microorganism, appropriate for producing the respective fine
chemical; (d) expressing the identified nucleic acid molecules in
the host cells; (e) assaying the respective fine chemical level in
the host cells; and (f) identifying the nucleic acid molecule and
its gene product which expression confers an increase in the
respective fine chemical level in the host cell after expression
compared to the wild type.
[16328] [0394.0.32.40] to [0552.0.32.40] see [0394.0.0.32] to
[0552.0.0.32]
[16329] [0553.0.40.40] [16330] 1. A process for the production of
Coenzyme Q9, which comprises [16331] (a) increasing or generating
the activity of a protein as indicated in Table XII, application
no. 40, columns 5 or 7, or a functional equivalent thereof in a
non-human organism, or in one or more parts thereof; and [16332]
(b) growing the organism under conditions which permit the
production of Coenzyme Q9 in said organism. [16333] 2. A process
for the production of Coenzyme Q9, comprising the increasing or
generating in an organism or a part thereof the expression of at
least one nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: [16334] a) nucleic acid
molecule encoding of a polypeptide as indicated in Table XII,
application no. 40, columns 5 or 7, or a fragment thereof, which
confers an increase in the amount of Coenzyme Q9 in an organism or
a part thereof; [16335] b) nucleic acid molecule comprising of a
nucleic acid molecule as indicated in Table XI, application no. 40,
columns 5 or 7, [16336] c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of Coenzyme Q9 in an
organism or a part thereof; [16337] d) nucleic acid molecule which
encodes a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
Coenzyme Q9 in an organism or a part thereof; [16338] e) nucleic
acid molecule which hybridizes with a nucleic acid molecule of (a)
to (c) under stringent hybridisation conditions and conferring an
increase in the amount of Coenzyme Q9 in an organism or a part
thereof; [16339] f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table XIII, application no.
40, column 7, and conferring an increase in the amount of Coenzyme
Q9 in an organism or a part thereof; [16340] g) nucleic acid
molecule encoding a polypeptide which is isolated with the aid of
monoclonal antibodies against a polypeptide encoded by one of the
nucleic acid molecules of (a) to (f) and conferring an increase in
the amount of Coenzyme Q9 in an organism or a part thereof; [16341]
h) nucleic acid molecule encoding a polypeptide comprising a
consensus as indicated in Table XIV, application no. 40, column 7,
and conferring an increase in the amount of Coenzyme Q9 in an
organism or a part thereof; and [16342] i) nucleic acid molecule
which is obtainable by screening a suitable nucleic acid library
under stringent hybridization conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k) or
with a fragment thereof having at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of Coenzyme Q9 in an organism or a part thereof [16343] or
comprising a sequence which is complementary thereto. [16344] 3.
The process of claim 1 or 2, comprising recovering of the free or
bound Coenzyme Q9. [16345] 4. The process of any one of claims 1 to
3, comprising the following steps: [16346] (a) selecting an
organism or a part thereof expressing a polypeptide encoded by the
nucleic acid molecule characterized in claim 2; [16347] (b)
mutagenizing the selected organism or the part thereof; [16348] (c)
comparing the activity or the expression level of said polypeptide
in the mutagenized organism or the part thereof with the activity
or the expression of said polypeptide of the selected organisms or
the part thereof; [16349] (d) selecting the mutated organisms or
parts thereof, which comprise an increased activity or expression
level of said polypeptide compared to the selected organism or the
part thereof; [16350] (e) optionally, growing and cultivating the
organisms or the parts thereof; and [16351] (f) recovering, and
optionally isolating, the free or bound Coenzyme Q9 produced by the
selected mutated organisms or parts thereof. [16352] 5. The process
of any one of claims 1 to 4, wherein the activity of said protein
or the expression of said nucleic acid molecule is increased or
generated transiently or stably. [16353] 6. An isolated nucleic
acid molecule comprising a nucleic acid molecule selected from the
group consisting of: [16354] a) nucleic acid molecule encoding of a
polypeptide as indicated in Table XII, application no. 40, columns
5 or 7, or a fragment thereof, which confers an increase in the
amount of Coenzyme Q9 in an organism or a part thereof; [16355] b)
nucleic acid molecule comprising of a nucleic acid molecule as
indicated in
[16356] Table XI, application no. 40, columns 5 or 7; [16357] c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of Coenzyme Q9 in an organism
or a part thereof; [16358] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of Coenzyme Q9
in an organism or a part thereof; [16359] e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of Coenzyme Q9 in an organism or a part thereof; [16360]
f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table XIII, application no. 40, column 7, and
conferring an increase in the amount of Coenzyme Q9 in an organism
or a part thereof; [16361] g) nucleic acid molecule encoding a
polypeptide which is isolated with the aid of monoclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (f) and conferring an increase in the amount of Coenzyme
Q9 in an organism or a part thereof; [16362] h) nucleic acid
molecule encoding a polypeptide comprising a consensus as indicated
in Table XIV, application no. 40, column 7, and conferring an
increase in the amount of Coenzyme Q9 in an organism or a part
thereof; and [16363] i) nucleic acid molecule which is obtainable
by screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of Coenzyme Q9 in an organism or a part thereof [16364]
whereby the nucleic acid molecule distinguishes over the sequence
as indicated in Table XI, application no. 40, columns 5 or 7, by
one or more nucleotides. [16365] 7. A nucleic acid construct which
confers the expression of the nucleic acid molecule of claim 6,
comprising one or more regulatory elements. [16366] 8. A vector
comprising the nucleic acid molecule as claimed in claim 6 or the
nucleic acid construct of claim 7. [16367] 9. The vector as claimed
in claim 8, wherein the nucleic acid molecule is in operable
linkage with regulatory sequences for the expression in a
prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. [16368] 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. [16369] 11. The host cell of claim 10, which is a
transgenic host cell. [16370] 12. The host cell of claim 10 or 11,
which is a plant cell, an animal cell, a microorganism, or a yeast
cell, a fungus cell, a prokaryotic cell, an eukaryotic cell or an
archaebacterium. [16371] 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. [16372] 14. A polypeptide produced by
the process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table XII,
application no. 40, columns 5 or 7, by one or more amino acids
[16373] 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. [16374] 16. A plant tissue,
propagation material, harvested material or a plant comprising the
host cell as claimed in claim 12 which is plant cell or an
Agrobacterium. [16375] 17. A method for screening for agonists and
antagonists of the activity of a polypeptide encoded by the nucleic
acid molecule of claim 6 conferring an increase in the amount of
Coenzyme Q9 in an organism or a part thereof comprising: [16376]
(a) contacting cells, tissues, plants or microorganisms which
express the a polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of Coenzyme Q9 in an
organism or a part thereof with a candidate compound or a sample
comprising a plurality of compounds under conditions which permit
the expression the polypeptide; [16377] (b) assaying the Coenzyme
Q9 level or the polypeptide expression level in the cell, tissue,
plant or microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and [16378] (c)
identifying a agonist or antagonist by comparing the measured
Coenzyme Q9 level or polypeptide expression level with a standard
Coenzyme Q9 or polypeptide expression level measured in the absence
of said candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. [16379] 18. A process for the
identification of a compound conferring increased Coenzyme Q9
production in a plant or microorganism, comprising the steps:
[16380] (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of Coenzyme Q9 CoQ 9 in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with said readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
Coenzyme Q9 in an organism or a part thereof; [16381] (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. [16382] 19. A method for the identification of a
gene product conferring an increase in Coenzyme Q9 production in a
cell, comprising the following steps: [16383] (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in Coenzyme Q9
after expression with the nucleic acid molecule of claim 6; [16384]
(b) identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; [16385] (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing Coenzyme Q9;
[16386] (d) expressing the identified nucleic acid molecules in the
host cells; [16387] (e) assaying the Coenzyme Q9 level in the host
cells; and [16388] (f) identifying nucleic acid molecule and its
gene product which expression confers an increase in the Coenzyme
Q9 level in the host cell in the host cell after expression
compared to the wild type. [16389] 20. A method for the
identification of a gene product conferring an increase in Coenzyme
Q9 production in a cell, comprising the following steps: [16390]
(a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the Coenzyme Q9 amount or level
in an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; [16391]
(b) introducing the candidate nucleic acid molecules in host cells
appropriate for producing Coenzyme Q9; [16392] (c) expressing the
identified nucleic acid molecules in the host cells; [16393] (d)
assaying the Coenzyme Q9 level in the host cells; and [16394] (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the Coenzyme Q9 level in the host
cell after expression compared to the wild type. [16395] 21. A
method for the production of an agricultural composition comprising
the steps of the method of any one of claims 17 to 20 and
formulating the compound identified in any one of claims 17 to 20
in a form acceptable for an application in agriculture. [16396] 22.
A composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. [16397] 23. Use of
the nucleic acid molecule as claimed in claim 6 for the
identification of a nucleic acid molecule conferring an increase of
Coenzyme Q9 after expression. [16398] 24. Use of the polypeptide of
claim 14 or the nucleic acid construct claim 7 or the gene product
identified according to the method of claim 19 or 20 for
identifying compounds capable of conferring a modulation of
Coenzyme Q9 levels in an organism. [16399] 25. Cosmetic,
pharmaceutical, nutrition composition, food or feed composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20. [16400] 26. Use of the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of claim 8 or 9, the antagonist or
agonist identified according to claim 17, the antibody of claim 15,
the plant or plant tissue of claim 16, the harvested material of
claim 16, the host cell of claim 10 to 12 or the gene product
identified according to the method of claim 19 or 20 for the
protection of vegetable fats, oils, lipids or waxes comprising the
respective fine chemical.
[16401] 27. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the production of oils, lipids,
fats or waxes comprising the respective fine chemical derived from
microorganisms.
[16402] 28. Use of the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the production of of a
pharmaceutical agent for the treatment of congestive heart failure,
heart muscle dysfunction, reduced platelet size, limited of
platelet activity, idiopathic dilated cardiomyopathy, atigue, chest
pain, dyspnea and palpitations, hypertension and other
manifestations of cardiovascular disease, lowering levels of
Coenzyme Q10 due to treatments with HMG-CoA reductase inhibitors,
diseased gingival, gingival inflammation, cancer, AIDS, other
immune dysfunctions, Muscular dystrophy associated with cardiac
disease, exercise-related muscle exhaustion and damage, obesity,
neurodegenerative diseases, male infertility, chronic stable
angina, significant reduction of plasma levels of lipid
peroxidation, skin photoaging, lipid peroxidation or for reducing
the size of tumors, for remission in metastatic breast cancer, for
improving sperm motility, for protecting against oxidative stress,
for stabilizing cell membranes or protecting seminal fluid or a
cosmetic agent or a nutrition supplement for food or feed.
[16403] [0554.0.0.40] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[16404] [0000.0.41.41] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[16405] [0001.0.0.41] for the disclosure of this paragraph see
[0001.0.0.27].
[16406] [0002.0.41.41] Plants produce several important secondary
metabolites from phenylalanine through the phenylpropanoid pathway.
Such substances include flavonoids, lignins, tannins, salicylic
acid and hydroxycinnamic acid esters. Recent work on the
phenylpropanoid pathway has shown that the traditional view of
lignin biosynthesis is incorrect. Although the hydroxylation and
methylation reactions of the pathway were long thought to occur at
the level of the free hydroxycinnamic aicds, it turns now out, that
the enzymes catalyzing phenylpropanoid 3-hydroxylation and
3-O-methylation reactions uses shikimate and CoA conjugates as
substrates. The recent cloning of a aldehyde dehydrogenase involved
in ferulic acid and sinapic acid biosynthesis suggest that both
substances are derived at least in part through oxidation of
coniferaldehyde and sinapaldehyde (see Nair et al., 2004, Plant
Cell, 16, 544-554 and citations therein).
[16407] [0003.0.41.41] %
[16408] [0004.0.41.41] %
[16409] [0005.0.41.41] %
[16410] [0006.0.41.41] %
[16411] [0007.0.41.41] Cinnamic acids, which include caffeic and
ferulic acids, are also powerful antioxidants. Experiments have
found that these compounds can stop the growth of cancer cells.
[16412] In addition sinapic acid is an intermediate in syringyl
lignin biosynthesis in angiosperms, and in some taxa serves as a
precursor for soluble secondary metabolites. The biosynthesis and
accumulation of the sinapate esters sinapoylglucose,
sinapoylmalate, and sinapoylcholine are developmentally regulated
in at least Arabidopsis and other members of the Brassicaceae
(Ruegger et al., 1999, 119(1): 101-10, 1999).
[16413] Due to these interesting physiological roles and
agrobiotechnological potential of sinapic acid there is a need to
identify the genes of enzymes and other proteins involved in
sinapic acid metabolism, and to generate mutants or transgenic
plant lines with which to modify the sinapic acid content in
plants.
[16414] [0008.0.41.41] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce sinapic acid in plants. That type of
production permits control over quality, quantity and selection of
the most suitable and efficient producer organisms. The latter is
especially important for commercial production economics and
therefore availability to consumers. In addition it is desirable to
produce sinapic acid in plants in order to increase plant
productivity and resistance against biotic and abiotic stress as
discussed before.
[16415] Methods of recombinant DNA technology have been used for
some years to improve the production of fine chemicals in
microorganisms and plants by amplifying individual biosynthesis
genes and investigating the effect on production of fine chemicals.
It is for example reported, that the xanthophyll astaxanthin could
be produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. Those results indicated
that about 75 percent of the carotenoids found in the flower of the
transformed plant contained a keto group.
[16416] [0009.0.41.41] Thus, it would be advantageous if an algae,
plant or other microorganism were available which produce large
amounts sinapic acid. The invention discussed hereinafter relates
in some embodiments to such transformed prokaryotic or eukaryotic
microorganisms.
[16417] It would also be advantageous if plants were available
whose roots, leaves, stem, fruits or flowers produced large amounts
of sinapic acid. The invention discussed hereinafter relates in
some embodiments to such transformed plants.
[16418] [0010.0.41.41] Therefore improving the quality of
foodstuffs and animal feeds is an important task of the
food-and-feed industry. This is necessary since, for example
sinapic acid, as mentioned above, which occur in plants and some
microorganisms are limited with regard to the supply of mammals.
Especially advantageous for the quality of foodstuffs and animal
feeds is as balanced as possible a specific sinapic acid profile in
the diet since an excess of sinapic acid above a specific
concentration in the food has a positive effect. A further increase
in quality is only possible via addition of further sinapic acid,
which are limiting.
[16419] [0011.0.41.41] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add sinapic acid in a balanced
manner to suit the organism.
[16420] [0012.0.41.41] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
proteins which participate in the biosynthesis of sinapic acid and
make it possible to produce them specifically on an industrial
scale without unwanted byproducts forming. In the selection of
genes for biosynthesis two characteristics above all are
particularly important. On the one hand, there is as ever a need
for improved processes for obtaining the highest possible contents
of sinapic acid; on the other hand as less as possible byproducts
should be produced in the production process.
[16421] [0013.0.0.41] for the disclosure of this paragraph see
[0013.0.0.27].
[16422] [0014.0.41.41] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a sinapic acid. Accordingly,
in the present invention, the term "the fine chemical" as used
herein relates to a sinapic acid. Further, the term "the fine
chemicals" as used herein also relates to fine chemicals comprising
sinapic acid.
[16423] [0015.0.41.41] In one embodiment, the term "the fine
chemical" or "the respective fine chemical" means at least one
chemical compound with sinapic acid activity.
[16424] In one embodiment, the term "the fine chemical" means
sinapic acid depending on the context in which the term is used.
Throughout the specification the term "the fine chemical" means
sinapic acid, its salts, ester, thioester or in free form or bound
to other compounds such sugars or sugarpolymers, like glucoside,
e.g. diglucoside.
[16425] [0016.0.41.41] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [16426] (a) increasing or generating the activity of one
or more of a protein as shown in table XII, application no. 41,
column 3 encoded by the nucleic acid sequences as shown in table
XI, application no. 41, column 5, in a non-human organism or in one
or more parts thereof or [16427] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
sinapic acid of the invention or fine chemicals comprising sinapic
acid of the invention, in said organism or in the culture medium
surrounding the organism.
[16428] [0016.1.41.41] Accordingly, the term "the fine chemical"
means in one embodiment "sinapic acid" in relation to all sequences
listed in Tables XI to XIV, line 42 or homologs thereof.
[16429] [0017.0.0.41] to [0019.0.0.41] for the disclosure of the
paragraphs [0017.0.0.41] to [0019.0.0.41] see paragraphs
[0017.0.0.27] to [0019.0.0.27] above.
[16430] [0020.0.41.41] Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, column 5, line 42 in a plant conferred an increase in
sinapic acid content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of sinapic acid.
[16431] [0021.0.0.41] for the disclosure of this paragraph see
[0021.0.0.27] above.
[16432] [0022.0.41.41]
[16433] The sequence of YDR513W from Saccharomyces cerevisiae has
been published in Jacq, C. et al., Nature 387 (6632 Suppl), 75-78
(1997) and its activity is being defined as a protein having
glutaredoxin (thioltransferase) (glutathione reductase) activity.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein YDR513W having said
activity, for the production of the respective fine chemical, in
particular for increasing the amount of sinapic acid, preferably in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
activity of the protein YDR513W is increased.
[16434] [0022.1.0.41] to [0023.0.0.41] see [0022.1.0.27] to
[0023.0.0.27]
[16435] [0023.1.41.41] Homologs of the polypeptide disclosed in
table XII, application no. 41, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 41, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 41, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 41,
column 7, resp.
[16436] [0024.0.0.41] for the disclosure of this paragraph see
[0024.0.0.27] above.
[16437] [0025.0.41.41] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 41, column 3" if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 41, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[16438] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 41,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 41,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[16439] [0025.1.0.41] and [0025.2.0.41] for the disclosure of the
paragraphs [0025.1.0.41] and [0025.2.0.41] see [0025.1.0.27] and
[0025.2.0.27] above.
[16440] [0026.0.0.41] to [0033.0.0.41] for the disclosure of the
paragraphs [0026.0.0.41] to [0033.0.0.41] see [0026.0.0.27] to
[0033.0.0.27] above.
[16441] [0034.0.41.41] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 41, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[16442] [0035.0.0.41] to [0044.0.0.41] for the disclosure of the
paragraphs [0035.0.0.41] to [0044.0.0.41] see paragraphs
[0035.0.0.27] to [0044.0.0.27] above.
[16443] [0045.0.41.41] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
41, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line,
between 10% and 50%, 10% and 100%, 10% and 200%, 10% and 300%, 10%
and 400%, 10% and 500%, 20% and 50%, 20% and 250%, 20% and 500%,
50% and 100%, 50% and 250%, 50% and 500%, 500% and 1000% or
more.
[16444] [0046.0.41.41] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
41, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line
confers an increase of the respective fine chemical and of further
sinapic acid or their precursors.
[16445] [0047.0.0.41] and [0048.0.0.41] for the disclosure of the
paragraphs [0047.0.0.41] and [0048.0.0.41] see paragraphs
[0047.0.0.27] and [0048.0.0.27] above.
[16446] [0049.0.41.41] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 41, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 41, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 41, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[16447] [0050.0.41.41] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of sinapic acid, resp., or their ester, or glucoside thereof,
e.g the diglucoside thereof.
[16448] [0051.0.41.41] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g compositions comprising sinapic acid.
Depending on the choice of the organism used for the process
according to the present invention, for example a microorganism or
a plant, compositions or mixtures of sinapic acid can be
produced.
[16449] [0052.0.0.41] for the disclosure of this paragraph see
paragraph [0052.0.0.27] above.
[16450] [0053.0.41.41] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[16451] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 41, columns 5 and 7 or its homologs activity
having herein-mentioned sinapic acid of the invention increasing
activity; and/or [16452] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 41,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 41, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned sinapic acid of the invention increasing
activity; and/or [16453] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned sinapic acid
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 41, columns 5
and 7 or its homologs activity, or decreasing the inhibiitory
regulation of the polypeptide of the invention; and/or [16454] d)
generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned sinapic acid of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 41, columns 5
and 7 or its homologs activity; and/or [16455] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned sinapic acid of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 41, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [16456] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned sinapic acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 41, columns 5 and 7 or its
homologs activity, and/or [16457] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned sinapic acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 41, columns 5 and 7 or its
homologs activity; and/or [16458] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 41, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[16459] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [16460] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[16461] [0054.0.41.41] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 41 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 41, column 3 or its homologs.
[16462] [0055.0.0.41] to [0067.0.0.41] for the disclosure of the
paragraphs [0055.0.0.41] to [0067.0.0.41] see paragraphs
[0055.0.0.27] to [0067.0.0.27] above.
[16463] [0068.0.41.41] The mutation is introduced in such a way
that the production of sinapic acid is not adversely affected.
[16464] [0069.0.0.41] for the disclosure of this paragraph see
paragraph [0069.0.0.27] above.
[16465] [0070.0.41.41] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolite composition in the organism, e.g. an advantageous
composition of sinapic acid or their biochemical derivatives, e.g.
comprising a higher content of (from a viewpoint of nutritional
physiology limited) sinapic acid or their derivatives.
[16466] [0071.0.0.41] for the disclosure of this paragraph see
paragraph [0071.0.0.27] above.
[16467] [0072.0.41.41] %
[16468] [0073.0.41.41] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
(a) providing a non-human organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant; (b) increasing an activity of a polypeptide of the
invention or a homolog thereof, e.g. as indicated in Table XII,
application no. 41, columns 5 or 7, or of a polypeptide being
encoded by the nucleic acid molecule of the present invention and
described below, e.g. conferring an increase of the respective fine
chemical in an organism, preferably in a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a
plant, (c) growing an organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant under conditions which permit the production of the
respective fine chemical in the organism, preferably the
microorganism, the plant cell, the plant tissue or the plant; and
(d) if desired, recovering, optionally isolating, the free and/or
bound the respective fine chemical synthesized by the organism, the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue or the plant.
[16469] [0074.0.41.41] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound respective fine chemical.
[16470] [0075.0.0.41] to [0077.0.0.41] for the disclosure of the
paragraphs [0075.0.0.41] to [0077.0.0.41] see paragraphs
[0075.0.0.27] to [0077.0.0.27] above.
[16471] [0078.0.41.41] The organism such as microorganisms or
plants or the recovered, and if desired isolated, the respective
fine chemical can then be processed further directly into
foodstuffs or animal feeds or for other applications. The
fermentation broth, fermentation products, plants or plant products
can be purified with methods known to the person skilled in the
art. Products of these different work-up procedures are sinapic
acid or comprising compositions of sinapic acid still comprising
fermentation broth, plant particles and cell components in
different amounts, advantageously in the range of from 0 to 99% by
weight, preferably below 80% by weight, especially preferably below
50% by weight.
[16472] [0079.0.0.41] to [0084.0.0.41] for the disclosure of the
paragraphs [0079.0.0.41] to [0084.0.0.41] see paragraphs
[0079.0.0.27] to [0084.0.0.27] above.
[16473] [0085.0.41.41] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [16474] a) a nucleic acid sequence as
indicated in Table XI, application no. 41, columns 5 or 7, or a
derivative thereof, or [16475] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 41, columns
5 or 7, or a derivative thereof, or [16476] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[16477] [0086.0.0.41] and [0087.0.0.41] for the disclosure of the
paragraphs [0086.0.0.41] and [0087.0.0.41] see paragraphs
[0086.0.0.27] and [0087.0.0.27] above.
[16478] [0088.0.41.41] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for animals such as poultry is dependent on the
abovementioned fine chemicals or the plants are more resistant to
biotic and abiotic stress and the yield is increased.
[16479] [0088.1.0.41] for the disclosure of this paragraph see
paragraph [0088.1.0.27] above.
[16480] [0089.0.0.41] to [0094.0.0.41] for the disclosure of the
paragraphs [0089.0.0.41] to [0094.0.0.41] see paragraphs
[0089.0.0.27] to [0094.0.0.27] above.
[16481] [0095.0.41.41] It may be advantageous to increase the pool
of sinapic acid in the transgenic organisms by the process
according to the invention in order to isolate high amounts of the
pure respective fine chemical and/or to obtain increased resistance
against biotic and abiotic stresses and to obtain higher yield.
[16482] [0096.0.41.41] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example in
the organism, is useful to increase the production of the
respective fine chemical.
[16483] [0097.0.0.41] for the disclosure of this paragraph see
paragraph [0097.0.0.27] above.
[16484] [0098.0.41.41] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated.
[16485] [0099.0.41.41] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[16486] [0100.0.41.41] Transgenic plants which comprise the fine
chemicals such as sinapic acid synthesized in the process according
to the invention can advantageously be marketed directly without
there being any need for the fine chemicals synthesized to be
isolated. Plants for the process according to the invention are
listed as meaning intact plants and all plant parts, plant organs
or plant parts such as leaf, stem, seeds, root, tubers, anthers,
fibers, root hairs, stalks, embryos, calli, cotelydons, petioles,
harvested material, plant tissue, reproductive tissue and cell
cultures which are derived from the actual transgenic plant and/or
can be used for bringing about the transgenic plant. In this
context, the seed comprises all parts of the seed such as the seed
coats, epidermal cells, seed cells, endosperm or embryonic
tissue.
[16487] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, as extracts, e.g. ether, alcohol,
or other organic solvents or water containing extract and/or free
fine chemicals. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pretreated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[16488] Because sinapic acid in microorganisms are localized
intracellular, their recovery essentially comes down to the
isolation of the biomass. Well-established approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Of the residual
hydrocarbon, adsorbed on the cells, has to be removed. Solvent
extraction or treatment with surfactants have been suggested for
this purpose.
[16489] [0101.0.41.41] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[16490] [0102.0.41.41] Sinapic acid can for example be detected
advantageously via HPLC, LC or GC separation methods. The
unambiguous detection for the presence of sinapic acid containing
products can be obtained by analyzing recombinant organisms using
analytical standard methods: LC, LC-MS, MS or TLC). The material to
be analyzed can be disrupted by sonication, grinding in a glass
mill, liquid nitrogen and grinding, cooking, or via other
applicable methods.
[16491] [0103.0.41.41] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [16492] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide having a
sequence as indicated in Table XII, application no. 41, columns 5
or 7, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [16493] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table XI, application no. 41, columns 5 or 7,
[16494] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [16495] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[16496] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [16497]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [16498] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [16499] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers pairs
having a sequence as indicated in Table XIII, application no. 41,
columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [16500]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [16501] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequences as indicated in Table XIV, application
no. 41, column 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [16502]
k) nucleic acid molecule comprising one or more of the nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domain of the polypeptide indicated in Table XII, application no.
41, columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[16503] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[16504] [00103.1.41.41] In one embodiment, the nucleic acid
molecule used in the process of the invention distinguishes over
the sequence indicated in Table XI, application no. 41, columns 5
or 7 by one or more nucleotides. In one embodiment, the nucleic
acid molecule used in the process of the invention does not consist
of the sequence shown in indicated in Table XI, application no. 41,
columns 5 or 7. In one embodiment, the nucleic acid molecule used
in the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI,
application no. 41, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table XII, application no. 41, columns 5 or 7.
[16505] [0104.0.41.41] In one embodiment, the nucleic acid molecule
used in the process of the present invention distinguishes over the
sequence indicated in Table XI, application no. 41, columns 5 or 7,
by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence indicated in Table XI, application no. 41, columns 5
or 7. In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence indicated in Table XI, application no.
41, columns 5 or 7. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table XII, application no. 41, columns 5 or 7.
[16506] [0105.0.0.41] to [0107.0.0.41] for the disclosure of the
paragraphs [0105.0.0.41] to [0107.0.0.41] see paragraphs
[0105.0.0.27] and [0107.0.0.27] above.
[16507] [0108.0.41.41] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 41, columns 5 or 7, nucleic
acid molecules which are derived from an amino acid sequences as
indicated in Table XII, application no. 41, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 41, column 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of an activity of a polypeptide as indicated in Table XII,
application no. 41, column 3, 5 or 7, e.g. conferring the increase
of the respective fine chemical, meaning sinapic acid, resp., after
increasing its expression or activity, are advantageously increased
in the process according to the invention.
[16508] [0109.0.41.41] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular sinapic
acid, produced in the process according to the invention.
[16509] [0110.0.41.41] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide used in the
method of the invention or used in the process of the invention,
e.g. of a protein as shown in Table XII, application no. 41,
columns 5 or 7, or being encoded by a nucleic acid molecule
indicated in Table XI, application no. 41, columns 5 or 7, or of
its homologs, e.g. as indicated in Table XII, application no. 41,
columns 5 or 7, can be determined from generally accessible
databases.
[16510] [0111.0.0.41] for the disclosure of this paragraph see
[0111.0.0.27] above.
[16511] [0112.0.41.41] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XII, application no. 41,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 41, columns 5 and 7, and conferring an
increase in the sinapic acid level.
[16512] [0113.0.0.41] to [0120.0.0.41] for the disclosure of the
paragraphs [0113.0.0.41] to [0120.0.0.41] see paragraphs
[0113.0.0.27] and [0120.0.0.27] above.
[16513] [0121.0.41.41] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 41, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a sinapic acid level increase after
increasing the activity of the polypeptide sequences indicated in
Table XII, application no. 41, columns 5 or 7.
[16514] [0122.0.0.41] to [0127.0.0.41] for the disclosure of the
paragraphs [0122.0.0.41] to [0127.0.0.41] see paragraphs
[0122.0.0.27] and [0127.0.0.27] above.
[16515] [0128.0.41.41] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 41, column 7, by means of polymerase chain reaction
can be generated on the basis of a sequence shown herein, for
example the sequence as indicated in Table XI, application no. 41,
columns 5 or 7, or the sequences derived from a sequences as
indicated in Table XII, application no. 41, columns 5 or 7.
[16516] [0129.0.41.41] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence shown in Table XIV,
application no. 41, column 7, is derived from said alignments.
[16517] [0130.0.0.41] for the disclosure of this paragraph see
[0130.0.0.27].
[16518] [0131.0.0.41] to [0138.0.0.41] for the disclosure of the
paragraphs [0131.0.0.41] to [0138.0.0.41] see paragraphs
[0131.0.0.27] to [0138.0.0.27] above.
[16519] [0139.0.41.41] Polypeptides having above-mentioned
activity, i.e. conferring the respective fine chemical level
increase, derived from other organisms, can be encoded by other DNA
sequences which hybridise to a sequence indicated in Table XI,
application no. 41, columns 5 or 7, for sinapic acid under relaxed
hybridization conditions and which code on expression for peptides
having the respective fine chemical, i.e. sinapic acid, resp.,
increasing-activity.
[16520] [0140.0.0.41] to [0146.0.0.41] for the disclosure of the
paragraphs [0140.0.0.41] to [0146.0.0.41] see paragraphs
[0140.0.0.27] and [0146.0.0.27] above.
[16521] [0147.0.41.41] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 41, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences s
such that it can hybridize to one of said nucleotide sequences
thereby forming a stable duplex. Preferably, the hybridisation is
performed under stringent hybridization conditions. However, a
complement of one of the herein disclosed sequences is preferably a
sequence complement thereto according to the base pairing of
nucleic acid molecules well known to the skilled person. For
example, the bases A and G undergo base pairing with the bases T
and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[16522] [0148.0.41.41] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 41,
columns 5 or 7 or a portion thereof and preferably has above
mentioned activity, in particular, of sinapic acid increasing
activity after increasing the activity or an activity of a product
of a gene encoding said sequences or their homologs.
[16523] [0149.0.41.41] The nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 41,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity and as indicated in indicated in Table
XII.
[16524] [00149.1.41.41] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table XI, columns 5 or 7, lines 243 to 250
and 603 has further one or more of the activities annotated or
known for a protein as indicated in Table XII, column 3, lines 243
to 250 and 603.
[16525] [0150.0.41.41] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table XI, application no. 41, columns
5 or 7, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of sinapic acid, resp., if
its activity is increased. The nucleotide sequences determined from
the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table XI,
application no. 41, columns 5 or 7, an anti-sense sequence of one
of the sequences, e.g., as indicated in Table XI, application no.
41, columns 5 or 7, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table XIII, application no. 41, column 7, will result in a fragment
of a polynucleotide sequence as indicated in Table XI, application
no. 41, columns 5 or 7 or its gene product.
[16526] [0151.0.0.41]: for the disclosure of this paragraph see
[0151.0.0.27] above.
[16527] [0152.0.41.41] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 41, columns 5
or 7, such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular a sinapic acid increasing activity as mentioned above
or as described in the examples in plants or microorganisms is
comprised.
[16528] [0153.0.41.41] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 41,
columns 5 or 7, such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 41, columns 5 or 7, has for
example an activity of a polypeptide indicated in Table XII,
application no. 41, column 3.
[16529] [0154.0.41.41] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 41, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[16530] [0155.0.0.41] and [0156.0.0.41] for the disclosure of the
paragraphs [0155.0.0.41] and [0156.0.0.41] see paragraphs
[0155.0.0.27] and [0156.0.0.27] above.
[16531] [0157.0.41.41] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences as
indicated in Table XI, application no. 41, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
polypeptides comprising the sequence as indicated in Table XIV,
application no. 41, column 7, or as polypeptides depicted in Table
XII, application no. 41, columns 5 or 7, or the functional
homologues. Advantageously, the nucleic acid molecule of the
invention comprises, or in an other embodiment has, a nucleotide
sequence encoding a protein comprising, or in an other embodiment
having, an amino acid sequence of a consensus sequences as
indicated in Table XIV, application no. 41, column 7, or of the
polypeptide as indicated in Table XII, application no. 41, columns
5 or 7, or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
XIV, application no. 41, column 7, or of a polypeptide as indicated
in Table XII, application no. 41, columns 5 or 7, or the functional
homologues. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 41, columns 5 or 7.
Preferably the nucleic acid molecule of the invention is a
functional homologue or identical to a nucleic acid molecule
indicated in Table XI, application no. 41, columns 5 or 7.
[16532] [0158.0.0.41] to [0160.0.0.41] for the disclosure of the
paragraphs [0158.0.0.41] to [0160.0.0.41] see paragraphs
[0158.0.0.27] to [0160.0.0.27] above.
[16533] [0161.0.41.41] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 41, columns 5 or
7. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[16534] [0162.0.0.41] for the disclosure of this paragraph see
paragraph [0162.0.0.27] above.
[16535] [0163.0.41.41] Preferably, a nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 41, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the increase of
the amount of the respective fine chemical in a organism or a part
thereof, e.g. a tissue, a cell, or a compartment of a cell, after
increasing the expression or activity thereof or the activity of a
protein of the invention or used in the process of the
invention.
[16536] [0164.0.0.41] for the disclosure of this paragraph see
paragraph [0164.0.0.27] above.
[16537] [0165.0.41.41] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 41, columns 5 or 7.
[16538] [0166.0.0.41] and [0167.0.0.41] for the disclosure of the
paragraphs [0166.0.0.41] and [0167.0.0.41] see paragraphs
[0166.0.0.27] and [0167.0.0.27] above.
[16539] [0168.0.41.41] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 41, columns 5 or 7, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 41,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table XII, application no. 41, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 41, columns 5
or 7, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table XII, application no.
41, columns 5 or 7, and most preferably at least about 96%, 97%,
98%, or 99% identical to the sequence as indicated in Table XII,
application no. 41, columns 5 or 7.
[16540] [0169.0.0.41] to [0172.0.0.41] for the disclosure of the
paragraphs [0169.0.0.41] to [0172.0.0.41] see paragraphs
[0169.0.0.27] to [0172.0.0.27] above.
[16541] [0173.0.41.41] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108401 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108401 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[16542] [0174.0.0.41]: for the disclosure of this paragraph see
paragraph [0174.0.0.27] above.
[16543] [0175.0.41.41] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108402 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108402 by the above program algorithm with the
above parameter set, has a 80% homology.
[16544] [0176.0.41.41] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 41,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 41, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 41, columns
5 or 7.
[16545] [0177.0.41.41] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 41,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptide as indicated in Table XII,
application no. 41, columns 5 or according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 41, columns
5 or 7.
[16546] [0178.0.0.41] for the disclosure of this paragraph see
[0178.0.0.27] above.
[16547] [0179.0.41.41] A nucleic acid molecule encoding a
homologous to a protein sequence as indicated in Table XII,
application no. 41, columns 5 or 7, can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table XI, application no.
41, columns 5 or 7, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences as
indicated in Table XI, application no. 41, columns 5 or 7, by
standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[16548] [0180.0.0.41] to [0183.0.0.41] for the disclosure of the
paragraphs [0180.0.0.41] to [0183.0.0.41] see paragraphs
[0180.0.0.27] to [0183.0.0.27] above.
[16549] [0184.0.41.41] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 41,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 41, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 41,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[16550] [0185.0.41.41] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 41, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table XI, application no. 41, columns 5 or 7. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequences as indicated in Table XI, application no. 41, columns 5
or 7.
[16551] [0186.0.41.41] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table XII,
application no. 41, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 41, columns 5 or 7.
[16552] [0187.0.41.41] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table XII, application no.
41, columns 5 or 7, comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence
encoding a sequences as indicated in Table XII, application no. 41,
columns 5 or 7.
[16553] [0188.0.41.41] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 41, columns 5 or 7 and is expressed under
identical conditions.
[16554] [0189.0.41.41] Homologues of a sequences as indicated in
Table XI, application no. 41, columns 5 or 7, or of a derived
sequences as indicated in Table XII, application no. 41, columns 5
or 7 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[16555] [0190.0.0.41], [0191.0.0.41], [00191.1.0.41] and
[0192.0.0.41] to [0203.0.0.41] for the disclosure of the paragraphs
[0190.0.0.41], [0191.0.0.41], [0191.1.0.41] and [0192.0.0.41] to
[0203.0.0.41] see paragraphs [0190.0.0.27], [0191.0.0.27],
[0191.1.0.27] and [0192.0.0.27] to [0203.0.0.27] above.
[16556] [0204.0.41.41] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule which comprises a
nucleic acid molecule selected from the group consisting of:
[16557] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 41, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, in particular according to Table XII, application no. 41,
column 6 in an organism or a part thereof [16558] b) nucleic acid
molecule comprising, preferably at least the mature form, of a
nucleic acid molecule as indicated in Table XI, application no. 41,
columns 5 or 7, or a fragment thereof conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [16559] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [16560] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[16561] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [16562] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [16563] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [16564] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table XIII, application no. 41, column 7, and
conferring an increase in the amount of the respective fine
chemical, in particular according to Table XII, application no. 41,
column 6 in an organism or a part thereof; [16565] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[16566] j) nucleic acid molecule which encodes a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 41, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical, in particular according
to table XII, application no. 41, column 6 in an organism or a part
thereof; [16567] k) nucleic acid molecule encoding the amino acid
sequence of a polypeptide encoding a domaine of a polypeptide as
indicated in Table XII, application no. 41, columns 5 or 7, and
conferring an increase in the amount of the respective fine
chemical, in particular accccording to table XII, application no.
41, column 6 in an organism or a part thereof; and [16568] l)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (h) or of a nucleic
acid molecule as indicated in Table XI, application no. 41, columns
5 or 7, or a nucleic acid molecule encoding, preferably at least
the mature form of, a polypeptide as indicated in Table XII,
application no. 41, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical according to Table XII,
application no. 41, column 6 in an organism or a part thereof;
[16569] or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over the sequence indicated in Table
XI, application no. 41, columns 5 or 7, by one or more nucleotides.
In one embodiment, the nucleic acid molecule does not consist of
the sequence shown and indicated in Table XI, application no. 41,
columns 5 or 7,
[16570] In one embodiment, the nucleic acid molecule is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table XI, application no. 41, columns 5 or 7.
[16571] In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table XII,
application no. 41, columns 5 or 7.
[16572] In an other embodiment, the nucleic acid molecule of the
present invention is at least 30%, 40%, 50%, or 60% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 41, columns 5 or
7.
[16573] In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table XII,
application no. 41, columns 5 or 7.
[16574] Accordingly, in one embodiment, the nucleic acid molecule
of the differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 41, columns 5 or
7.
[16575] Accordingly, in one embodiment, the nucleic acid molecule
of the present invention encodes a polypeptide, which differs at
least in one or more amino acids from a polypeptide indicated in
Table XII, application no. 41, columns 5 or 7.
[16576] In another embodiment, a nucleic acid molecule indicated in
Table XI, application no. 41, columns 5 or 7.
[16577] Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table XII, application no.
41, columns 5 or 7.
[16578] In a further embodiment, the protein of the present
invention is at least 30%, 40%, 50%, or 60% identical to a protein
sequence indicated in Table XII, application no. 41, columns 5 or
7, and less than 100%, preferably less than 99.999%, 99.99% or
99.9%, more preferably less than 99%, 985, 97%, 96% or 95%
identical to a sequence as indicated in Table XI, columns 5 or
7.
[16579] [0205.0.0.41] and [0206.0.0.41] for the disclosure of the
paragraphs [0205.0.0.41] and [0206.0.0.41] see paragraphs
[0205.0.0.27] and [0206.0.0.27] above.
[16580] [0207.0.41.41] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the glutamic acid metabolism, the
phosphoenolpyruvate metabolism, the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[16581] [0208.0.0.41] to [0226.0.0.41] for the disclosure of the
paragraphs [0208.0.0.41] to [0226.0.0.41] see paragraphs
[0208.0.0.27] to [0226.0.0.27] above.
[16582] [0227.0.41.41] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[16583] In addition to a sequence indicated in Table XI,
application no. 41, columns 5 or 7, or its derivatives, it is
advantageous to express and/or mutate further genes in the
organisms. Especially advantageously, additionally at least one
further gene of the glutamic acid or phosphoenolpyruvate metabolic
pathway, is expressed in the organisms such as plants or
microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the fine chemicals desired since, for example,
feedback regulations no longer exist to the same extent or not at
all. In addition it might be advantageously to combine one or more
of the sequences indicated in Table XI, application no. 41, columns
5 or 7, with genes which generally support or enhances to growth or
yield of the target organisms, for example genes which lead to
faster growth rate of microorganisms or genes which produces
stress-, pathogen, or herbicide resistant plants.
[16584] [0228.0.41.41] %
[16585] [0229.0.41.41] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences indicated
in Table XI, application no. 41, columns 5 or 7, used in the
process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the aromatic amino acid
pathway, such as tryptophan, phenylalanine or tyrosine. These genes
can lead to an increased synthesis of the essential amino acids
tryptophan, phenylalanine or tyrosine.
[16586] [0230.0.0.41] for the disclosure of this paragraph see
paragraph [0230.0.0.27] above.
[16587] [0231.0.41.41] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a sinapic acid degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene. A person skilled in the art knows for example,
that the inhibition or repression of a sinapic acid degrading
enzyme will result in an increased sinapic acidaccumulation in the
plant.
[16588] [0232.0.0.41] to [0276.0.0.41] for the disclosure of the
paragraphs [0232.0.0.41] to [0276.0.0.41] see paragraphs
[0232.0.0.27] to [0276.0.0.27] above.
[16589] [0277.0.41.41] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously.
[16590] [0278.0.0.41] to [0282.0.0.41] for the disclosure of the
paragraphs [0278.0.0.41] to [0282.0.0.41] see paragraphs
[0278.0.0.27] to [0282.0.0.27] above.
[16591] [0283.0.41.41] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells, for example using the antibody
of the present invention as described below, e.g. an antibody
against a protein as indicated in Table XII, application no. 41,
column 3, or an antibody against a polypeptide as indicated in
Table XII, application no. 41, columns 5 or 7, which can be
produced by standard techniques utilizing the polypeptid of the
present invention or fragment thereof. Preferred are monoclonal
antibodies.
[16592] [0284.0.0.41] for the disclosure of this paragraph see
[0284.0.0.27] above.
[16593] [0285.0.41.41] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 41, columns 5 or 7, or as coded by a nucleic
acid molecule as indicated in Table XI, application no. 41, columns
5 or 7, or functional homologues thereof.
[16594] [0286.0.41.41] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 41, column 7, and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 41, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid.
[16595] [0287.0.0.41] to [0290.0.0.41] for the disclosure of the
paragraphs [0287.0.0.41] to [0290.0.0.41] see paragraphs
[0287.0.0.27] to [0290.0.0.27] above.
[16596] [0291.0.41.41] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. In one embodiment, said polypeptide of the
invention distinguishes over a sequence as indicated in Table XII,
application no. 41, columns 5 or 7, by one or more amino acids.
[16597] In one embodiment, polypeptide distinguishes form a
sequence as indicated in Table XII, application no. 41, columns 5
or 7, by more than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, even more
preferred are more than 40, 50, or 60 amino acids and, preferably,
the sequence of the polypeptide of the invention distinguishes from
a sequence as indicated in Table XII, application no. 41, columns 5
or 7, by not more than 80% or 70% of the amino acids, preferably
not more than 60% or 50%, more preferred not more than 40% or 30%,
even more preferred not more than 20% or 10%. In an other
embodiment, said polypeptide of the invention does not consist of a
sequence as indicated in Table XII, application no. 41, columns 5
or 7.
[16598] [0292.0.0.41] for the disclosure of this paragraph see
[0292.0.0.27] above.
[16599] [0293.0.41.41] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, the polypeptide of
the invention has a sequence which distinguishes from a sequence as
indicated in Table XII, application no. 41, columns 5 or 7, by one
or more amino acids. In an other embodiment, said polypeptide of
the invention does not consist of the sequence as indicated in
Table XII, application no. 41, columns 5 or 7. In a further
embodiment, said polypeptide of the present invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical. In one embodiment,
said polypeptide does not consist of the sequence encoded by a
nucleic acid molecules as indicated in Table XI, application no.
41, columns 5 or 7.
[16600] [0294.0.41.41] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 41, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 41, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[16601] [0295.0.0.41] to [0297.0.0.41] for the disclosure of the
paragraphs [0295.0.0.41] to [0297.0.0.41] see paragraphs
[0295.0.0.27] to [0297.0.0.27] above.
[16602] [00297.1.41.41] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity and/or the amino acid
sequence of a polypeptide indicated in Table XII, application no.
41, columns 3, 5 or 7.
[16603] [0298.0.41.41] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 41, columns 5 or 7. The
portion of the protein is preferably a biologically active portion
as described herein. Preferably, the polypeptide used in the
process of the invention has an amino acid sequence identical to a
sequence as indicated in Table XII, application no. 41, columns 5
or 7.
[16604] [0299.0.41.41] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 41,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 41, columns 5 or 7, or which is
homologous thereto, as defined above.
[16605] [0300.0.41.41] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 41, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 41, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[16606] [0301.0.0.41] for the disclosure of this paragraph see
[0301.0.0.27] above.
[16607] [0302.0.41.41] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 41, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[16608] [0303.0.0.41] for the disclosure of this paragraph see
[0303.0.0.27] above.
[16609] [0304.0.41.41] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 41, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[16610] [0305.0.0.41] to [0308.0.0.41] for the disclosure of the
paragraphs [0305.0.0.41] to [0308.0.0.41] see paragraphs
[0305.0.0.27 to [0308.0.0.27] above.
[16611] [0309.0.41.41] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table XII,
application no. 41, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas an
"other polypeptide" not being indicated in Table XII, application
no. 41, columns 5 or 7, refers to a polypeptide having an amino
acid sequence corresponding to a protein which is not substantially
homologous to a polypeptide of the invention, preferably which is
not substantially homologous to a polypeptide as indicated in Table
XII, application no. 41, columns 5 or 7, e.g., a protein which does
not confer the activity described herein or annotated or known for
as indicated in Table XII, application no. 41, column 3, and which
is derived from the same or a different organism. In one
embodiment, an "other polypeptide" not being indicated in Table
XII, application no. 41, columns 5 or 7, does not confer an
increase of the respective fine chemical in an organism or part
thereof.
[16612] [0310.0.0.41] to [0334.0.0.41] for the disclosure of the
paragraphs [0310.0.0.41] to [0334.0.0.41] see paragraphs
[0310.0.0.27] to [0334.0.0.27] above.
[16613] [0335.0.41.41] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XI, application
no. 41, columns 5 or 7, and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived there from),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
application no. 41, columns 5 or 7, and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of
aprotein encoded by a nucleic acid sequence as indicated in Table
XI, application no. 41, columns 5 or 7, and/or homologs thereof,
one of the two RNA strands is essentially identical to at least
part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[16614] [0336.0.0.41] to [0342.0.0.41] for the disclosure of the
paragraphs [0336.0.0.41] to [0342.0.0.41] see paragraphs
[0336.0.0.27] to [0342.0.0.27] above.
[16615] [0343.0.41.41] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 41, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table XI, application no. 41, columns 5 or 7, or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[16616] [0344.0.0.41] to [0361.0.0.41] for the disclosure of the
paragraphs [0344.0.0.41] to [0361.0.0.41] see paragraphs
[0344.0.0.27] to [0361.0.0.27] above.
[16617] [0362.0.41.41] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 41, columns 5 or 7, e.g. encoding a
polypeptide having protein activity, as indicated in Table XII,
application no. 41, columns 3. Due to the abovementioned activity
the respective fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity of the polypeptide of the invention or nucleic
acid molecule of the invention is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a polypeptide as
indicated in Table XII, application no. 41, columns 5 or 7, means
herein that due to modulation or manipulation of the genome, an
activity as annotated for a polypeptide as indicated in Table XII,
application no. 41, column 3, e.g. having a sequence as indicated
in Table XII, application no. 41, columns 5 or 7, is increased in a
cell or an organism or a part thereof. Examples are described above
in context with the process of the invention.
[16618] [0363.0.0.41] for the disclosure of this paragraph see
[0363.0.0.27] above.
[16619] [0364.0.41.41] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table XII, application no. 41, column 3, with the
corresponding protein-encoding sequence as indicated in Table XI,
application no. 41, column 5, becomes a transgenic expression
cassette when it is modified by non-natural, synthetic "artificial"
methods such as, for example, mutagenization. Such methods have
been described (U.S. Pat. No. 5,565,350; WO 00/15815; also see
above).
[16620] [0365.0.0.41] to [0373.0.0.41] for the disclosure of the
paragraphs [0365.0.0.41], to [0373.0.0.41] see paragraphs
[0365.0.0.27] to [0373.0.0.27] above.
[16621] [0374.0.41.41] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. sinapic acid, in particular
the respective fine chemical, produced in the process according to
the invention may, however, also be isolated from the plant in the
form of their free sinapic acid, in particular the free respective
fine chemical, or bound in or to compounds or moieties, like
glucosides, e.g. diglucosides. The respective fine chemical
produced by this process can be harvested by harvesting the
organisms either from the culture in which they grow or from the
field. This can be done via expressing, grinding and/or extraction,
salt precipitation and/or ion-exchange chromatography or other
chromatographic methods of the plant parts, preferably the plant
seeds, plant fruits, plant tubers and the like.
[16622] [0375.0.0.41] and [0376.0.0.41] for the disclosure of the
paragraphs [0375.0.0.41] and [0376.0.0.41] see paragraphs
[0375.0.0.27] and [0376.0.0.27] above.
[16623] [0377.0.41.41] Accordingly, the present invention relates
also to a process whereby the produced sinapic acid is
isolated.
[16624] [0378.0.41.41] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the sinapic acid
produced in the process can be isolated. The resulting sinapic acid
can, if appropriate, subsequently be further purified, if desired
mixed with other active ingredients such as vitamins, amino acids,
carbohydrates, antibiotics and the like, and, if appropriate,
formulated.
[16625] [0379.0.41.41] In one embodiment, sinapic acid is a mixture
of the respective fine chemicals.
[16626] [0380.0.41.41] The sinapic acid obtained in the process are
suitable as starting material for the synthesis of further products
of value. For example, they can be used in combination with each
other or alone for the production of pharmaceuticals, foodstuffs,
animal feeds or cosmetics. Accordingly, the present invention
relates a method for the production of pharmaceuticals, food stuff,
animal feeds, nutrients or cosmetics comprising the steps of the
process according to the invention, including the isolation of the
sinapic acid composition produced or the respective fine chemical
produced if desired and formulating the product with a
pharmaceutical acceptable carrier or formulating the product in a
form acceptable for an application in agriculture. A further
embodiment according to the invention is the use of the sinapic
acid produced in the process or of the transgenic organisms in
animal feeds, foodstuffs, medicines, food supplements, cosmetics or
pharmaceuticals or for the production of sinapic acid e.g. after
isolation of the respective fine chemical or without, e.g. in situ,
e.g in the organism used for the process for the production of the
respective fine chemical.
[16627] [0381.0.0.41] to [0384.0.0.41] for the disclosure of the
paragraphs [0381.0.0.41], to [0384.0.0.41] see paragraphs
[0381.0.0.27] to [0384.0.0.27] above.
[16628] [0385.0.41.41] The fermentation broths obtained in this
way, containing in particular sinapic acid in mixtures with other
organic acids, aminoacids, polypeptides or polysaccarides, normally
have a dry matter content of from 1 to 70% by weight, preferably
7.5 to 25% by weight. Sugar-limited fermentation is additionally
advantageous, e.g. at the end, for example over at least 30% of the
fermentation time. This means that the concentration of utilizable
sugar in the fermentation medium is kept at, or reduced to, 0 to 10
g/l, preferably to 0 to 3 g/I during this time. The fermentation
broth is then processed further. Depending on requirements, the
biomass can be removed or isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth or left completely in it.
[16629] The fermentation broth can then be thickened or
concentrated by known methods, such as, for example, with the aid
of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[16630] [0386.0.41.41] Accordingly, it is possible to purify the
sinapic acid produced according to the invention further. For this
purpose, the product-containing composition is subjected for
example to separation via e.g. an open column chromatography or
HPLC in which case the desired product or the impurities are
retained wholly or partly on the chromatography resin. These
chromatography steps can be repeated if necessary, using the same
or different chromatography resins. The skilled worker is familiar
with the choice of suitable chromatography resins and their most
effective use.
[16631] [0387.0.0.41] to [0392.0.0.41] for the disclosure of the
paragraphs [0387.0.0.41] to [0392.0.0.41] see paragraphs
[0387.0.0.27] to [0392.0.0.27] above.
[16632] [0393.0.41.41] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [16633] a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [16634] b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table XI, application no. 41, columns 5 or 7, and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[16635] c) introducing the candidate nucleic acid molecules in host
cells, preferably in a plant cell or a microorganism, appropriate
for producing the fine chemical; [16636] d) expressing the
identified nucleic acid molecules in the host cells; [16637] e)
assaying the the fine chemical level in the host cells; and [16638]
f) identifying the nucleic acid molecule and its gene product which
expression confers an increase in the the fine chemical level in
the host cell after expression compared to the wild type.
[16639] [0394.0.32.41] to [0552.0.32.41] for the disclosure of the
paragraphs [0394.0.32.41] to [0552.0.32.41] see paragraphs
[0394.0.0.32] to [0552.0.0.32] above.
[16640] [0553.0.41.41]
1. A process for the production of sinapic acid resp., which
comprises (a) increasing or generating the activity of a protein as
indicated in Table XII, application no. 41, columns 5 or 7, or a
functional equivalent thereof in a non-human organism, or in one or
more parts thereof; and (b) growing the organism under conditions
which permit the production of sinapic acid resp. in said organism.
2. A process for the production of sinapic acid resp., comprising
the increasing or generating in an organism or a part thereof the
expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of: a)
nucleic acid molecule encoding of a polypeptide as indicated in
Table XII, application no. 41, columns 5 or 7, or a fragment
thereof, which confers an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of sinapic acid
resp., in an organism or a part thereof; b) nucleic acid molecule
comprising of a nucleic acid molecule as indicated in Table XI,
application no. 41, columns 5 or 7, c) nucleic acid molecule whose
sequence can be deduced from a polypeptide sequence encoded by a
nucleic acid molecule of (a) or (b) as a result of the degeneracy
of the genetic code and conferring an increase in the amount of the
fine chemical as indicated in table XII, column 6, e.g of sinapic
acid resp., in an organism or a part thereof; d) nucleic acid
molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the fine chemical as indicated in table XII,
column 6, e.g of sinapic acid resp., in an organism or a part
thereof; e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of sinapic acid
resp., in an organism or a part thereof; f) nucleic acid molecule
which encompasses a nucleic acid molecule which is obtained by
amplifying nucleic acid molecules from a cDNA library or a genomic
library using the primers or primer pairs as indicated in Table
XIII, application no. 41, column 7, and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of sinapic acid resp., in an organism or a part thereof; g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of sinapic acid resp., in an organism or a part
thereof; h) nucleic acid molecule encoding a polypeptide comprising
a consensus sequence as indicated in Table XIV, application no. 41,
column 7, and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of sinapic acid
resp., in an organism or a part thereof; and i) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment thereof having at least 15 nt, preferably
20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid
molecule characterized in (a) to (k) and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of sinapic acid resp., in an organism or a part thereof. or
comprising a sequence which is complementary thereto. 3. The
process of claim 1 or 2, comprising recovering of the free or bound
sinapic acid resp. 4. The process of any one of claims 1 to 3,
comprising the following steps: (a) selecting an organism or a part
thereof expressing a polypeptide encoded by the nucleic acid
molecule characterized in claim 2; (b) mutagenizing the selected
organism or the part thereof; (c) comparing the activity or the
expression level of said polypeptide in the mutagenized organism or
the part thereof with the activity or the expression of said
polypeptide of the selected organisms or the part thereof; (d)
selecting the mutated organisms or parts thereof, which comprise an
increased activity or expression level of said polypeptide compared
to the selected organism or the part thereof; (e) optionally,
growing and cultivating the organisms or the parts thereof; and (f)
recovering, and optionally isolating, the free or bound sinapic
acid resp., produced by the selected mutated organisms or parts
thereof. 5. The process of any one of claims 1 to 4, wherein the
activity of said protein or the expression of said nucleic acid
molecule is increased or generated transiently or stably. 6. An
isolated nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: a) nucleic acid molecule
encoding of a polypeptide as indicated in Table XII, application
no. 41, columns 5 or 7, or a fragment thereof, which confers an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of sinapic acid resp., in an organism or a part
thereof; b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table XI, application no. 41, columns 5 or
7, c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of sinapic acid resp., in an
organism or a part thereof; d) nucleic acid molecule which encodes
a polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of sinapic acid
resp., in an organism or a part thereof; e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
under stringent hybridisation conditions and conferring an increase
in the amount of the fine chemical as indicated in table XII,
column 6, e.g of sinapic acid resp., in an organism or a part
thereof; f) nucleic acid molecule which encompasses a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers or
primer pairs as indicated in Table XIII, application no. 41, column
7, and conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of sinapic acid resp., in an
organism or a part thereof; g) nucleic acid molecule encoding a
polypeptide which is isolated with the aid of monoclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (f) and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of sinapic acid
resp., in an organism or a part thereof; h) nucleic acid molecule
encoding a polypeptide comprising a consensus sequence as indicated
in Table XIV, application no. 41, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of sinapic acid resp., in an organism or a part
thereof; and i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of sinapic acid resp., in an organism or a part thereof.
whereby the nucleic acid molecule distinguishes over the sequence
as indicated in Table XI, application no. 41, columns 5 or 7, by
one or more nucleotides. 7. A nucleic acid construct which confers
the expression of the nucleic acid molecule of claim 6, comprising
one or more regulatory elements. 8. A vector comprising the nucleic
acid molecule as claimed in claim 6 or the nucleic acid construct
of claim 7. 9. The vector as claimed in claim 8, wherein the
nucleic acid molecule is in operable linkage with regulatory
sequences for the expression in a prokaryotic or eukaryotic, or in
a prokaryotic and eukaryotic, host. 10. A host cell, which has been
transformed stably or transiently with the vector as claimed in
claim 8 or 9 or the nucleic acid molecule as claimed in claim 6 or
the nucleic acid construct of claim 7 or produced as described in
claim any one of claims 2 to 5. 11. The host cell of claim 10,
which is a transgenic host cell. 12. The host cell of claim 10 or
11, which is a plant cell, an animal cell, a microorganism, or a
yeast cell, a fungus cell, a prokaryotic cell, an eukaryotic cell
or an archaebacterium. 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. 14. A polypeptide produced by the
process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table XII,
application no. 41, columns 5 or 7, by one or more amino acids 15.
An antibody, which binds specifically to the polypeptide as claimed
in claim 14. 16. A plant tissue, propagation material, harvested
material or a plant comprising the host cell as claimed in claim 12
which is plant cell or an Agrobacterium. 17. A method for screening
for agonists and antagonists of the activity of a polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of sinapic acid resp., in an organism or a
part thereof comprising: (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 5 conferring an increase in the
amount of sinapic acid resp., in an organism or a part thereof with
a candidate compound or a sample comprising a plurality of
compounds under conditions which permit the expression the
polypeptide; (b) assaying the sinapic acid resp., level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and (c) identifying a
agonist or antagonist by comparing the measured sinapic acid resp.,
level or polypeptide expression level with a standard sinapic acid
resp., or polypeptide expression level measured in the absence of
said candidate compound or a sample comprising said plurality of
compounds, whereby an increased level over the standard indicates
that the compound or the sample comprising said plurality of
compounds is an agonist and a decreased level over the standard
indicates that the compound or the sample comprising said plurality
of compounds is an antagonist. 18. A process for the identification
of a compound conferring increased sinapic acid resp., production
in a plant or microorganism, comprising the steps: (a) culturing a
plant cell or tissue or microorganism or maintaining a plant
expressing the polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of sinapic acid resp.,
in an organism or a part thereof and a readout system capable of
interacting with the polypeptide under suitable conditions which
permit the interaction of the polypeptide with dais readout system
in the presence of a compound or a sample comprising a plurality of
compounds and capable of providing a detectable signal in response
to the binding of a compound to said polypeptide under conditions
which permit the expression of said readout system and of the
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of sinapic acid resp., in an
organism or a part thereof; (b) identifying if the compound is an
effective agonist by detecting the presence or absence or increase
of a signal produced by said readout system. 19. A method for the
identification of a gene product conferring an increase in sinapic
acid resp., production in a cell, comprising the following steps:
(a) contacting the nucleic acid molecules of a sample, which can
contain a candidate gene encoding a gene product conferring an
increase in sinapic acid resp., after expression with the nucleic
acid molecule of claim 6; (b) identifying the nucleic acid
molecules, which hybridise under relaxed stringent conditions with
the nucleic acid molecule of claim 6; (c) introducing the candidate
nucleic acid molecules in host cells appropriate for producing
sinapic acid resp.; (d) expressing the identified nucleic acid
molecules in the host cells; (e) assaying the sinapic acid resp.,
level in the host cells; and (f) identifying nucleic acid molecule
and its gene product which expression confers an increase in the
sinapic acid resp., level in the host cell in the host cell after
expression compared to the wild type. 20. A method for the
identification of a gene product conferring an increase in sinapic
acid resp., production in a cell, comprising the following steps:
(a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the sinapic acid resp., amount or
level in an organism or a part thereof after expression, and which
are at least 20% homolog to the nucleic acid molecule of claim 6;
(b) introducing the candidate nucleic acid molecules in host cells
appropriate for producing sinapic acid resp.; (c) expressing the
identified nucleic acid molecules in the host cells; (d) assaying
the sinapic acid resp., level in the host cells; and (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the sinapic acid resp., level in
the host cell after expression compared to the wild type. 21. A
method for the production of an agricultural composition comprising
the steps of the method of any one of claims 17 to 20 and
formulating the compound identified in any one of claims 17 to 20
in a form acceptable for an application in agriculture. 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. 23. Use of the
nucleic acid molecule as claimed in claim 6 for the identification
of a nucleic acid molecule conferring an increase of sinapic acid
resp., after expression. 24. Use of the polypeptide of claim 14 or
the nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of sinapic acid resp., levels in
an organism. 25. Agrochemical, pharmaceutical, food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. 26. The method of any one of claims 1
to 5, the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20, wherein the fine chemical is sinapic
acid.
[16641] [0554.0.0.41] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[16642] [0000.0.42.42] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[16643] [0001.0.0.42] see [0001.0.0.27] [0002.0.42.42]
[16644] Plants produce very long chain fatty acids such as melissic
acid (C30:0).
[16645] Very long-chain fatty acids (VLCFAs) are synthesized by a
membrane-bound fatty acid elongation complex (elongase, FAE) using
acyl-CoA substrates. The first reaction of elongation involves
condensation of malonyl-CoA with a long chain substrate producing a
.beta.-ketoacyl-CoA. Subsequent reactions are reduction of
.beta.-hydroxyacyl-CoA, dehydration to an enoyl-CoA, followed by a
second reduction to form the elongated acyl-CoA. The
.beta.-ketoacyl-CoA synthase (KCS) catalyzing the condensation
reaction plays a key role in determining the chain length of fatty
acid products found in seed oils and is the rate-limiting enzyme
for seed VLCFA production (Lassner et al., Plant Cell, 8(1996),
281-292).
[16646] The elongation process can be repeated to yield members
that are 20, 22, and 24 carbons long. Although such very long chain
fatty acids are minor components of the lipid membranes of the
body, they undoubtedly perform valuable functions, apparently
helping to stabilize membranes, especially those in peripheral
nerve cells.
[16647] Melissic acid (C30:0) (triacontanoic acid) is a component
of beeswax.
[16648] Beeswax (cera alba) is obtained from the product excreted
by certain glands of the honeybee from which the honeycomb is made.
It is freed of solid impurities by melting and centrifugation (cera
flava). Finally, it is bleached completely white (cera alba).
Beeswax consists of 10-15 percent paraffin carbohydrates, 35-37
percent esters of C16 to C36 fatty acids and about 15 percent
cerotic acid, melissic acid and their homologues. Beeswax is used
as a thickener and a humectant in the manufacture of ointments,
creams, lipsticks and other cosmetics and skincare products as an
emulsifier, emollient, moisturizer and film former.
[16649] Beeswax is also used for the production of candles.
[16650] Wax is a general term used to refer to the mixture of
long-chain apolar lipids forming a protective coating (cutin in the
cuticle) on plant leaves and fruits but also in animals (wax of
honeybee, cuticular lipids of insects, spermaceti of the sperm
whale, skin lipids, uropygial glands of birds, depot fat of
planktonic crustacea), algae, fungi and bacteria.
[16651] Many of the waxes found in nature have commercial uses in
the lubricant, food and cosmetic industry. Jojoba oil has long been
suggested as a putative resource of wax, since this desert shrub is
unusual in its capacity to produce waxes rather than
triacylglycerols (TAG) as seed storage lipids. These waxes are
esters of very-long-chain-fatty acids and fatty alcohols (Miwa,
1971, J Am Oil Chem Soc 48, 259-264). As the production cost for
jojoba wax, which is primarily used for cosmetic applications, is
high, there is a need to engineer crop plants to produce high level
of wax esters in its seed oil.
[16652] Plant aerial surfaces are covered by epicuticular waxes,
complex mixtures of very long (C.sub.20-C.sub.34) fatty acids,
alkanes, aldehydes, ketones and esters. In addition to repelling
atmospheric water they prevent dessication and are therefore an
important determinant of drought resistance (Riederer and
Schreiber, 2001, J. Exp. Bot 52, 2023-2032). Beside abiotic stress
resistance the wax layer is part of the plant defense against
biotic stressor, especially insects as for example described by
Marcell and Beattie, 2002, Mol Plant Microbe Interact. 15(12),
1236-44. Furthermore they provide stability to pollen grains, thus
influencing fertility and productivity.
[16653] Very-long-chain fatty acids (VLCFAs), consisting of more
than 18 carbon atoms like melissic acid, are essential components
for the vitality of higher plants. The key enzyme of VLCFA
biosynthesis, the extraplastidary fatty acid elongase, is shown for
to be the primary target site of chloroacetamide herbicides. With
an analysis of the fatty acid composition and the metabolism of
14C-labelled precursors (sterate, malonate, acetate), the reduction
of VLCFAs was determined in vivo. The inhibition of the recombinant
protein substantiates the first and rate-limiting step of VLCFA
biosynthesis, the condensation of acyl-CoA with malonyl-CoA to
.beta.-ketoacyl-CoA, to be the primary target site of
chloroacetamides (150=10-100 nM). The concentration of VLCFAs
within the untreated cell is low, the very-long-chain compounds are
found mainly in plasma membrane lipids and epicuticular waxes. A
shift of fatty acids towards shorter chain length or even the
complete depletion of very-long-chain components is the consequence
of the inhibition of VLCFA biosynthesis. Especially the loss of
plasma membrane VLCFAs is involved in phytotoxic effects of
chloroacetamides such as the inhibition of membrane biogenesis and
mitosis (Matthes, B.,
http://www.ub.uni-konstanz.de/kops/volltexte/2001/661/).
[16654] Increased wax production in transgenic plants has for
example been reported by Broun et al., 2004, Proc Natl. Acad. Sci,
101, 4706-4711. The authors overexpressed the transcriptional
activator WIN1 in Arabidopsis, leading to increased wax load on
anal organs. As this resulted in a complex change in the wax
profile and the transgenic overexpressors had characteristic
alterations in growth and development (Broun et al., 2004, Proc
Natl. Acad. Sci, 101, 4706-4711) there is still a need for a more
controlled increased production of defined VLCFAs.
[16655] Very long chain fatty alcohols obtained from plant waxes
and beeswax have also been reported to lower plasma cholesterol in
humans and existing data support the hypothesis that VLCFA exert
regulatory roles in cholesterol metabolism in the peroxisome and
also alter LDL uptake and metabolism (discussed in Hargrove et al.,
2004, Exp Biol Med (Maywood), 229(3): 215-26).
[16656] Due to these interesting physiological roles and the
nutritional, cosmetic and agrobiotechnological potential of
melissic acid (C30:0) there is a need to identify the genes of
enzymes and other proteins involved in melissic acid metabolism,
and to generate mutants or transgenic plant lines with which to
modify the melissic acid content in plants.
[16657] [0003.0.42.42] %
[16658] [0004.0.42.42] %
[16659] [0005.0.42.42] %
[16660] [0006.0.42.42] %
[16661] [0007.0.42.42] %
[16662] [0008.0.42.42] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce melissic acid in plants. That type of
production permits control over quality, quantity and selection of
the most suitable and efficient producer organisms. The latter is
especially important for commercial production economics and
therefore availability to consumers. In addition it is desirable to
produce melissic acid in plants in order to increase plant
productivity and resistance against biotic and abiotic stress as
discussed before.
[16663] Methods of recombinant DNA technology have been used for
some years to improve the production of fine chemicals in
microorganisms and plants by amplifying individual biosynthesis
genes and investigating the effect on production of fine chemicals.
It is for example reported, that the xanthophyll astaxanthin could
be produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. Those results indicated
that about 75 percent of the carotenoids found in the flower of the
transformed plant contained a keto group.
[16664] [0009.0.42.42] Thus, it would be advantageous if an algae,
plant or other microorganism were available which produce large
amounts melissic acid. The invention discussed hereinafter relates
in some embodiments to such transformed prokaryotic or eukaryotic
microorganisms.
[16665] It would also be advantageous if plants were available
whose roots, leaves, stem, fruits or flowers produced large amounts
of melissic acid. The invention discussed hereinafter relates in
some embodiments to such transformed plants.
[16666] [0010.0.42.42] Therefore improving the quality of
foodstuffs and animal feeds is an important task of the
food-and-feed industry. This is necessary since, for example
melissic acid, as mentioned above, which occur in plants and some
microorganisms are limited with regard to the supply of mammals.
Especially advantageous for the quality of foodstuffs and animal
feeds is as balanced as possible a specific melissic acid profile
in the diet since an excess of melissic acid above a specific
concentration in the food has a positive effect. A further increase
in quality is only possible via addition of further melissic acid,
which are limiting.
[16667] [0011.0.42.42] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add melissic acid in a balanced
manner to suit the organism.
[16668] [0012.0.42.42] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
proteins which participate in the biosynthesis of melissic acid and
make it possible to produce them specifically on an industrial
scale without unwanted byproducts forming. In the selection of
genes for biosynthesis two characteristics above all are
particularly important. On the one hand, there is as ever a need
for improved processes for obtaining the highest possible contents
of melissic acid; on the other hand as less as possible byproducts
should be produced in the production process.
[16669] [0013.0.0.42] see [0013.0.0.27]
[16670] [0014.0.42.42] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a melissic acid.
Accordingly, in the present invention, the term "the fine chemical"
as used herein relates to a melissic acid. Further, the term "the
fine chemicals" as used herein also relates to fine chemicals
comprising melissic acid.
[16671] In one embodiment, the term "the fine chemical" means
melissic acid. Throughout the specification the term "the fine
chemical" means melissic acid, its salts, ester, thioester or in
free form or bound to other compounds such sugars or sugarpolymers,
like glucoside, e.g. diglucoside.
[16672] [0016.0.42.42] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [16673] (a) increasing or generating the activity of one
or more [16674] of a protein as shown in table XII, application no.
42, column 3 encoded by the nucleic acid sequences as shown in
table XI, application no. 42, column 5, in a non-human organism or
in one or more parts thereof or [16675] (b) growing the organism
under conditions which permit the production of the fine chemical,
thus melissic acid of the invention or fine chemicals comprising
melissic acid of the invention, in said organism or in the culture
medium surrounding the organism.
[16676] [0016.1.42.42] Accordingly, the term "the fine chemical"
means in one embodiment "melissic acid" in relation to all
sequences listed in Tables XI to XIV, line 43 or homologs
thereof.
[16677] [0017.0.0.42] to [0019.0.0.42]: see [0017.0.0.27] to
[0019.0.0.27]
[16678] [0020.0.42.42] Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, column 5, line 43 in a plant conferred an increase in
melissic acid content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of melissic acid.
[16679] [0021.0.0.42] see [0021.0.0.27]
[16680] [0022.0.42.42]
[16681] The sequence of YDR513W from Saccharomyces cerevisiae has
been published in Jacq, C. et al., Nature 387 (6632 Suppl), 75-78
(1997) and its activity is being defined as a protein having
glutathione reductase activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
YDR513W or its homolog, e.g. as shown herein, for the production of
the respective fine chemical, in particular for increasing the
amount of melissic acid, preferably in free or bound form in an
organism or a part thereof, as mentioned. In one embodiment, in the
process of the present invention the activity of the protein
YDR513W is increased.
[16682] [0022.1.0.42] to [0023.0.42.42] see [0022.1.0.27] to
[0023.0.0.27]
[16683] [0023.1.42.42] Homologs of the polypeptide disclosed in
table XII, application no. 42, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 42, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 42, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 42,
column 7, resp.
[16684] [0024.0.0.42] see [0024.0.0.27]
[16685] [0025.0.42.42] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 42, column 3" if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 42, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[16686] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 42,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 42,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[16687] [0025.1.0.42] see [0025.1.0.27]
[16688] [0026.0.0.42] to [0033.0.0.42]: see [0026.0.0.27] to
[0033.0.0.27]
[16689] [0034.0.42.42] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 42, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[16690] [0035.0.0.42] to [0038.0.0.42]: see [0035.0.0.27] to
[0038.0.0.27]
[16691] [0039.0.0.42]: see [0039.0.0.27]
[16692] [0040.0.0.42] to [0044.0.0.42]: see [0040.0.0.27] to
[0044.0.0.27]
[16693] [0045.0.42.42] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
42, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line,
between 10% and 50%, 10% and 100%, 10% and 200%, 10% and 300%, 10%
and 400%, 10% and 500%, 20% and 50%, 20% and 250%, 20% and 500%,
50% and 100%, 50% and 250%, 50% and 500%, 500% and 1000% or
more.
[16694] [0046.0.42.42] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
42, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line
confers an increase of the respective fine chemical and of further
melissic acid or their precursors.
[16695] [0047.0.0.42] to [0048.0.0.42]: see [0047.0.0.27] to
[0048.0.0.27]
[16696] [0049.0.42.42] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 42, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 42, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 42, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[16697] [0050.0.42.42] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of melissic acid, resp., or their ester, or glucoside
thereof, e.g the diglucoside thereof.
[16698] [0051.0.42.42] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g compositions comprising melissic acid.
Depending on the choice of the organism used for the process
according to the present invention, for example a microorganism or
a plant, compositions or mixtures of melissic acid can be
produced.
[16699] [0052.0.0.42] see [0052.0.0.27]
[16700] [0053.0.42.42] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[16701] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 42, columns 5 and 7 or its homologs activity
having herein-mentioned melissic acid of the invention increasing
activity; and/or [16702] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 42,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 42, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned melissic acid of the invention increasing
activity; and/or [16703] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned wax component
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 42, columns 5
and 7 or its homologs activity, or decreasing the inhibiitory
regulation of the polypeptide of the invention; and/or [16704] d)
generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned melissic acid of the
invention increasing activity, e.g. of a polypeptide having the
activity of a protein as indicated in table XII, application no.
42, columns 5 and 7 or its homologs activity; and/or [16705] e)
stimulating activity of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
present invention or a polypeptide of the present invention having
herein-mentioned melissic acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 42, columns 5 and 7 or its
homologs activity, by adding one or more exogenous inducing factors
to the organisms or parts thereof; and/or [16706] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned melissic acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 42, columns 5 and 7 or its
homologs activity, and/or [16707] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned melissic acid of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 42, columns 5 and 7 or its
homologs activity; and/or [16708] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 42, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[16709] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [16710] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[16711] [0054.0.42.42] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 42 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 42, column 3 or its homologs.
[16712] [0055.0.0.42] to [0067.0.0.42]: see [0055.0.0.27] to
[0067.0.0.27]
[16713] [0068.0.42.42] The mutation is introduced in such a way
that the production of melissic acid is not adversely affected.
[16714] [0069.0.0.42] see [0069.0.0.27]
[16715] [0070.0.42.42] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolite composition in the organism, e.g. an advantageous
composition of melissic acid or their biochemical derivatives, e.g.
comprising a higher content of (from a viewpoint of nutritional
physiology limited) melissic acid or their derivatives.
[16716] [0071.0.0.42] see [0071.0.0.27]
[16717] [0072.0.42.42] %
[16718] [0073.0.42.42] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[16719] a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [16720] b) increasing an activity of a
polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table XII, application no. 42, columns 5 or 7, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the respective fine chemical in an organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [16721] c) growing an organism,
preferably a microorganism, a non-human animal, a plant or animal
cell, a plant or animal tissue or a plant under conditions which
permit the production of the respective fine chemical in the
organism, preferably the microorganism, the plant cell, the plant
tissue or the plant; and [16722] d) if desired, recovering,
optionally isolating, the free and/or bound respective fine
chemical synthesized by the organism, the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[16723] [0074.0.42.42] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound respective fine chemical.
[16724] [0075.0.0.42] to [0077.0.0.42]: see [0075.0.0.27] to
[0077.0.0.27]
[16725] [0078.0.42.42] The organism such as microorganisms or
plants or the recovered, and if desired isolated, respective fine
chemical can then be processed further directly into foodstuffs or
animal feeds or for other applications. The fermentation broth,
fermentation products, plants or plant products can be purified
with methods known to the person skilled in the art. Products of
these different work-up procedures are melissic acid or comprising
compositions of melissic acid still comprising fermentation broth,
plant particles and cell components in different amounts,
advantageously in the range of from 0 to 99% by weight, preferably
below 80% by weight, especially preferably below 50% by weight.
[16726] [0079.0.0.42] to [0084.0.0.42]: see [0079.0.0.27] to
[0084.0.0.27]
[16727] [0084.0.42.42] %
[16728] [0085.0.42.42] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [16729] a) a nucleic acid sequence as
indicated in Table XI, application no. 42, columns 5 or 7, or a
derivative thereof, or [16730] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 42, columns
5 or 7, or a derivative thereof, or [16731] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[16732] [0086.0.0.42] to [0087.0.0.42]: see [0086.0.0.27] to
[0087.0.0.27]
[16733] [0088.0.42.42] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned fine
chemicals or the plants are more resistant to biotic and abiotic
stress and the yield is increased.
[16734] [0088.1.0.42] see [0088.1.0.27]
[16735] [0089.0.0.42] to [0090.0.0.42]: see [0089.0.0.27] to
[0090.0.0.27]
[16736] [0091.0.0.42] see [0091.0.0.27]
[16737] [0092.0.0.42] to [0094.0.0.42]: see [0092.0.0.27] to
[0094.0.0.27]
[16738] [0095.0.42.42] It may be advantageous to increase the pool
of melissic acid in the transgenic organisms by the process
according to the invention in order to isolate high amounts of the
pure respective fine chemical and/or to obtain increased resistance
against biotic and abiotic stresses and to obtain higher yield.
[16739] [0096.0.42.42] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example in
the organism, is useful to increase the production of the
respective fine chemical.
[16740] [0097.0.42.42] %
[16741] [0098.0.42.42] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated.
[16742] [0099.0.42.42] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[16743] [0100.0.42.42] Transgenic plants which comprise the fine
chemicals such as melissic acid synthesized in the process
according to the invention can advantageously be marketed directly
without there being any need for the fine chemicals synthesized to
be isolated. Plants for the process according to the invention are
listed as meaning intact plants and all plant parts, plant organs
or plant parts such as leaf, stem, seeds, root, tubers, anthers,
fibers, root hairs, stalks, embryos, calli, cotelydons, petioles,
harvested material, plant tissue, reproductive tissue and cell
cultures which are derived from the actual transgenic plant and/or
can be used for bringing about the transgenic plant. In this
context, the seed comprises all parts of the seed such as the seed
coats, epidermal cells, seed cells, endosperm or embryonic
tissue.
[16744] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, as extracts, e.g. ether, alcohol,
or other organic solvents or water containing extract and/or free
fine chemicals. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pretreated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[16745] Because melissic acid in microorganisms are localized
intracellular, their recovery essentially comes down to the
isolation of the biomass. Well-established approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Of the residual
hydrocarbon, adsorbed on the cells, has to be removed. Solvent
extraction or treatment with surfactants have been suggested for
this purpose.
[16746] [0101.0.42.42] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michel, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[16747] [0102.0.42.42] Melissic acid can for example be analyzed
advantageously via HPLC, LC or GC separation and MS
(masspectrometry) detection methods. The unambiguous detection for
the presence of melissic acid containing products can be obtained
by analyzing recombinant organisms using analytical standard
methods: LC, LC-MS, MS or TLC). The material to be analyzed can be
disrupted by sonication, grinding in a glass mill, liquid nitrogen
and grinding, cooking, or via other applicable methods.
[16748] [0103.0.42.42] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [16749] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide having a
sequence as indicated in Table XII, application no. 42, columns 5
or 7, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [16750] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table XI, application no. 42, columns 5 or 7,
[16751] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [16752] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[16753] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [16754]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [16755] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [16756] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primer pairs
having a sequence as indicated in Table XIII, application no. 42,
columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [16757]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [16758] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence having
sequences as indicated in Table XIV, application no. 42, column 7,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [16759] k) nucleic acid
molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of the polypeptide indicated in Table XII, application no. 42,
columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[16760] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[16761] [0103.1.42.42] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table XI, application no. 42, columns 5 or 7
by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence shown in indicated in Table XI, application no. 42,
columns 5 or 7. In one embodiment, the nucleic acid molecule used
in the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI,
application no. 42, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table XII, application no. 42, columns 5 or 7.
[16762] [0104.0.42.42] In one embodiment, the nucleic acid molecule
used in the process of the present invention distinguishes over the
sequence indicated in Table XI, application no. 42, columns 5 or 7,
by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence indicated in Table XI, application no. 42, columns 5
or 7. In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence indicated in Table XI, application no.
42, columns 5 or 7. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table XII, application no. 42, columns 5 or 7.
[16763] [0105.0.0.42] to [0107.0.0.42]: see [0105.0.0.27] to
[0107.0.0.27]
[16764] [0108.0.42.42] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 42, columns 5 or 7, nucleic
acid molecules which are derived from an amino acid sequences as
indicated in Table XII, application no. 42, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 42, column 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of an activity of a polypeptide as indicated in Table XII,
application no. 42, column 3, 5 or 7, e.g. conferring the increase
of the respective fine chemical, meaning melissic acid, resp.,
after increasing its expression or activity, are advantageously
increased in the process according to the invention.
[16765] [0109.0.42.42] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular melissic
acid, produced in the process according to the invention.
[16766] [0110.0.0.42] see [0110.0.0.27]
[16767] [0111.0.0.42] see [0111.0.0.27]
[16768] [0112.0.42.42] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XII, application no. 42,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 42, columns 5 and 7, and conferring an
increase in the melissic acid level.
[16769] [0113.0.0.42] to [0114.0.0.42]: see [0113.0.0.27] to
[0114.0.0.27]
[16770] [0115.0.0.42] see [0115.0.0.27]
[16771] [0116.0.0.42] to [0120.0.0.42] see [0116.0.0.27] to
[0120.0.0.27]
[16772] [0120.1.42.42]: %
[16773] [0121.0.42.42] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 42, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a melissic acid level increase after
increasing the activity of the polypeptide sequences indicated in
Table XII, application no. 42, columns 5 or 7.
[16774] [0122.0.0.42] to [0127.0.0.42]: see [0122.0.0.27] to
[0127.0.0.27]
[16775] [0128.0.42.42] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 42, columns 7, by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table XI, application no.
42, columns 5 or 7, or the sequences derived from a sequences as
indicated in Table XII, application no. 42, columns 5 or 7.
[16776] [0129.0.42.42] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved regions are those, which show a
very little variation in the amino acid in one particular position
of several homologs from different origin. The consensus sequence
indicated in Table XIV, application no. 42, columns 7, is derived
from such alignments.
[16777] [0130.0.42.42] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of melissic
acid after increasing the expression or activity the protein
comprising said fragment.
[16778] [0131.0.0.42] to [0138.0.0.42]: see [0131.0.0.27] to
[0138.0.0.27]
[16779] [0139.0.42.42] Polypeptides having above-mentioned
activity, i.e. conferring an increase of the respective fine
chemical level, derived from other organisms, can be encoded by
other DNA sequences which hybridize to a sequences indicated in
Table XI, application no. 42, columns 5 or 7, under relaxed
hybridization conditions and which code on expression for peptides
having the respective fine chemical, in particular, of melissic
acid resp., increasing activity.
[16780] [0140.0.0.42] to [0146.0.0.42]: see [0140.0.0.27] to
[0146.0.0.27]
[16781] [0147.0.42.42] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 42, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences s
such that it can hybridize to one of said nucleotide sequences
thereby forming a stable duplex. Preferably, the hybridisation is
performed under stringent hybridization conditions. However, a
complement of one of the herein disclosed sequences is preferably a
sequence complement thereto according to the base pairing of
nucleic acid molecules well known to the skilled person. For
example, the bases A and G undergo base pairing with the bases T
and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[16782] [0148.0.42.42] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 42,
columns 5 or 7 or a portion thereof and preferably has above
mentioned activity, in particular, of melissic acid increasing
activity after increasing the activity or an activity of a product
of a gene encoding said sequences or their homologs.
[16783] [0149.0.42.42] The nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 42,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity and as indicated in indicated in Table
XII.
[16784] [00149.1.42.42] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table XI, application no. 42, columns 5 or
7, preferably the nucleic acid molecule of the invention is a
functional homologue or identical to a nucleic acid molecule
indicated in Table XI, application no. 42, columns 5 or 7, has
further one or more of the activities annotated or known for a
protein as indicated in Table XII, application no. 42, column
3.
[16785] [0150.0.42.42] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table XI, application no. 42, columns
5 or 7, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of melissic acid, resp., if
its activity is increased. The nucleotide sequences determined from
the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table XI,
application no. 42, columns 5 or 7, an anti-sense sequence of one
of the sequences, e.g., as indicated in Table XI, application no.
42, columns 5 or 7, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table XIII, application no. 42, column 7, will result in a fragment
of a polynucleotide sequence as indicated in Table XI, application
no. 42, columns 5 or 7 or its gene product.
[16786] [0151.0.0.42]: see [0151.0.0.27]
[16787] [0152.0.42.42] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 42, columns 5
or 7, such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular a melissic acid increasing activity as mentioned
above or as described in the examples in plants or microorganisms
is comprised.
[16788] [0153.0.42.42] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 42,
columns 5 or 7, such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 42, columns 5 or 7, has for
example an activity of a polypeptide indicated in Table XII,
application no. 42, column 3.
[16789] [0154.0.42.42] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 42, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[16790] [0155.0.0.42] to [0156.0.0.42]: see [0155.0.0.27] to
[0156.0.0.27]
[16791] [0157.0.42.42] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences as
indicated in Table XI, application no. 42, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
polypeptides comprising the sequence as indicated in Table XIV,
application no. 42, column 7, or as polypeptides depicted in Table
XII, application no. 42, columns 5 or 7, or the functional
homologues. Advantageously, the nucleic acid molecule of the
invention comprises, or in an other embodiment has, a nucleotide
sequence encoding a protein comprising, or in an other embodiment
having, an amino acid sequence of a consensus sequences as
indicated in Table XIV, application no. 42, column 7, or of the
polypeptide as indicated in Table XII, application no. 42, columns
5 or 7, or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
XIV, application no. 42, column 7, or of a polypeptide as indicated
in Table XII, application no. 42, columns 5 or 7, or the functional
homologues. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 42, columns 5 or 7.
[16792] [0158.0.0.42] to [0160.0.0.42]: see [0158.0.0.27] to
[0160.0.0.27]
[16793] [0161.0.42.42] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 42, columns 5 or
7. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[16794] [0162.0.0.42] see [0162.0.0.27]
[16795] [0163.0.42.42] Preferably, a nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 42, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the increase of
the amount of the respective fine chemical in an organism or a part
thereof, e.g. a tissue, a cell, or a compartment of a cell, after
increasing the expression or activity thereof or the activity of a
protein of the invention or used in the process of the
invention.
[16796] [0164.0.0.42] see [0164.0.0.27]
[16797] [0165.0.42.42] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 42, columns 5 or 7.
[16798] [0166.0.0.42] to [0167.0.0.42]: see [0166.0.0.27] to
[0167.0.0.27]
[16799] [0168.0.42.42] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 42, columns 5 or 7, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 42,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table XII, application no. 42, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 42, columns 5
or 7, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table XII, application no.
42, columns 5 or 7, and most preferably at least about 96%, 97%,
98%, or 99% identical to the sequence as indicated in Table XII,
application no. 42, columns 5 or 7.
[16800] [0169.0.0.42] to [0172.0.0.42]: see [0169.0.0.27] to
[0172.0.0.27]
[16801] [0173.0.42.42] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108401 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108401 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[16802] [0174.0.0.42]: see [0174.0.0.27]
[16803] [0175.0.42.42] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108402 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108402 by the above program algorithm with the
above parameter set, has a 80% homology.
[16804] [0176.0.42.42] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 42,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 42, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 42, columns
5 or 7.
[16805] [0177.0.42.42] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 42,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptide as indicated in Table XII,
application no. 42, columns 5 or according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 42, columns
5 or 7.
[16806] [0178.0.0.42] see [0178.0.0.27]
[16807] [0179.0.42.42] A nucleic acid molecule encoding a homologue
to a protein sequence as indicated in Table XII, application no.
42, columns 5 or 7, can be created by introducing one or more
nucleotide substitutions, additions or deletions into a nucleotide
sequence of the nucleic acid molecule of the present invention, in
particular as indicated in Table XI, application no. 42, columns 5
or 7, such that one or more amino acid substitutions, additions or
deletions are introduced into the encoded protein. Mutations can be
introduced into the encoding sequences as indicated in Table XI,
application no. 42, columns 5 or 7, by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
[16808] [0180.0.0.42] to [0183.0.0.42]: see [0180.0.0.27] to
[0183.0.0.27]
[16809] [0184.0.42.42] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 42,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 42, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 42,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[16810] [0185.0.42.42] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 42, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table XI, application no. 42, columns 5 or 7. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequences as indicated in Table XI, application no. 42, columns 5
or 7.
[16811] [0186.0.42.42] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table XII,
application no. 42, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 42, columns 5 or 7.
[16812] [0187.0.42.42] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table XII, application no.
42, columns 5 or 7, comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence
encoding a sequences as indicated in Table XII, application no. 42,
columns 5 or 7.
[16813] [0188.0.42.42] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 42, columns 5 or 7 and is expressed under
identical conditions.
[16814] [0189.0.42.42] Homologues of a sequences as indicated in
Table XI, application no. 42, columns 5 or 7, or of a derived
sequences as indicated in Table XII, application no. 42, columns 5
or 7 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[16815] [0190.0.0.42]: see [0190.0.0.27]
[16816] [0191.0.42.42] The organisms or part thereof produce
according to the herein mentioned process of the invention an
increased level of free and/or bound the respective fine chemical
compared to said control or selected organisms or parts
thereof.
[16817] [0192.0.0.42] to [0203.0.0.42]: see [0192.0.0.27] to
[0203.0.0.27]
[16818] [0204.0.42.42] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule which comprises a
nucleic acid molecule selected from the group consisting of:
[16819] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 42, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, in particular according to Table XII, application no. 42,
column 6 in an organism or a part thereof [16820] b) nucleic acid
molecule comprising, preferably at least the mature form, of a
nucleic acid molecule as indicated in Table XI, application no. 42,
columns 5 or 7, or a fragment thereof conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [16821] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [16822] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[16823] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [16824] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [16825] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [16826] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table XIII, application no. 42, column 7, and
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII, application no. 42,
column 6 in an organism or a part thereof; [16827] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[16828] j) nucleic acid molecule which encodes a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 42, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical, in particular according
to table XII, application no. 42, column 6 in an organism or a part
thereof; [16829] k) nucleic acid molecule encoding the amino acid
sequence of a polypeptide encoding a domaine of a polypeptide as
indicated in Table XII, application no. 42, columns 5 or 7, and
conferring an increase in the amount of the respective fine
chemical, in particular accccording to table XII, application no.
42, column 6 in an organism or a part thereof; and [16830] l)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (h) or of a nucleic
acid molecule as indicated in Table XI, application no. 42, columns
5 or 7, or a nucleic acid molecule encoding, preferably at least
the mature form of, a polypeptide as indicated in Table XII,
application no. 42, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical according to Table XII,
application no. 42, column 6 in an organism or a part thereof;
[16831] or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over the sequence indicated in Table
XI, application no. 42, columns 5 or 7, by one or more nucleotides.
In one embodiment, the nucleic acid molecule does not consist of
the sequence shown and indicated in Table XI, application no. 42,
columns 5 or 7,
[16832] In one embodiment, the nucleic acid molecule is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table XI, application no. 42, columns 5 or 7.
[16833] In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table XII,
application no. 42, columns 5 or 7.
[16834] In an other embodiment, the nucleic acid molecule of the
present invention is at least 30%, 40%, 50%, or 60% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 42, columns 5 or 7.
In a further embodiment the nucleic acid molecule does not encode a
polypeptide sequence as indicated in Table XII, application no. 42,
columns 5 or 7.
[16835] Accordingly, in one embodiment, the nucleic acid molecule
of the differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 42, columns 5 or
7.
[16836] Accordingly, in one embodiment, the nucleic acid molecule
of the present invention encodes a polypeptide, which differs at
least in one or more amino acids from a polypeptide indicated in
Table XII, application no. 42, columns 5 or 7.
[16837] In another embodiment, a nucleic acid molecule indicated in
Table XI, application no. 42, columns 5 or 7.
[16838] Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table XII, application no.
42, columns 5 or 7.
[16839] In a further embodiment, the protein of the present
invention is at least 30%, 40%, 50%, or 60% identical to a protein
sequence indicated in Table XII, application no. 42, columns 5 or
7, and less than 100%, preferably less than 99.999%, 99.99% or
99.9%, more preferably less than 99%, 985, 97%, 96% or 95%
identical to a sequence as indicated in Table XI, columns 5 or
7.
[16840] [0205.0.0.42] to [0206.0.0.42]: see [0205.0.0.27] to
[0206.0.0.27]
[16841] [0207.0.42.42] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the glutamic acid metabolism, the
phosphoenolpyruvate metabolism, the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[16842] [0208.0.0.42] to [0226.0.0.42]: see [0208.0.0.27] to
[0226.0.0.27]
[16843] [0227.0.42.42] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[16844] In addition to a sequence indicated in Table XI,
application no. 42, columns 5 or 7, or its derivatives, it is
advantageous to express and/or mutate further genes in the
organisms. Especially advantageously, additionally at least one
further gene of the acetyl-CoA or malonyl-CoA metabolic pathway or
a polypeptide having a very long chain fatty acid acyl (VLCFA) CoA
synthase activity, is expressed in the organisms such as plants or
microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the fine chemicals desired since, for example,
feedback regulations no longer exist to the same extent or not at
all. In addition it might be advantageously to combine one or more
of the sequences indicated in Table XI, application no. 42, columns
5 or 7, with genes which generally support or enhance to growth or
yield of the target organisms, for example genes which lead to
faster growth rate of microorganisms or genes which produces
stress-, pathogen, or herbicide resistant plants.
[16845] [0228.0.42.42] %
[16846] [0229.0.42.42] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences indicated
in Table XI, application no. 42, columns 5 or 7, used in the
process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the fatty acid pathway, such as
acetyl-CoA or malonyl-CoA or a polypeptide having a very long chain
fatty acid acyl (VLCFA) CoA synthase activity. These genes can lead
to an increased synthesis of the VLCFAs.
[16847] [0230.0.0.42] see [230.0.0.27]
[16848] [0231.0.42.42] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a melissic acid degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene. A person skilled in the art knows for example,
that the inhibition or repression of a melissic acid degrading
enzyme will result in an increased accumulation of melissic acid in
plants.
[16849] [0232.0.0.42] to [0276.0.0.42]: see [0232.0.0.27] to
[0276.0.0.27]
[16850] [0277.0.42.42] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously.
[16851] [0278.0.0.42] to [0282.0.0.42]: see [0278.0.0.27] to
[0282.0.0.27]
[16852] [0283.0.42.42] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells, for example using the antibody
of the present invention as described below, e.g. an antibody
against a protein as indicated in Table XII, application no. 42,
column 3, or an antibody against a polypeptide as indicated in
Table XII, application no. 42, columns 5 or 7, which can be
produced by standard techniques utilizing the polypeptid of the
present invention or fragment thereof. Preferred are monoclonal
antibodies.
[16853] [0284.0.0.42] see [0284.0.0.27]
[16854] [0285.0.42.42] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 42, columns 5 or 7, lines 251 to 261, 627,
resp., or as coded by a nucleic acid molecule as indicated in Table
XI, application no. 42, columns 5 or 7, or functional homologues
thereof.
[16855] [0286.0.42.42] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 42, column 7, and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 42, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table XIV, application no. 42, column 7.
[16856] [0287.0.0.42] to [0289.0.0.42]: see [0287.0.0.27] to
[0289.0.0.27]
[16857] [00290.0.42.42] The alignment was performed with the
Software AlignX (Sep. 25, 2002) a component of Vector NTI Suite
8.0, InforMax.TM., Invitrogen.TM. life science software, U.S. Main
Office, 7305 Executive Way, Frederick, Md. 21704, USA with the
following settings: For pairwise alignments: gap opening penalty:
10.0; gap extension penalty 0,1. For multiple alignments: Gap
opening penalty: 10.0; Gap extension penalty: 0.1; Gap separation
penalty range: 8; Residue substitution matrix: blosum62;
Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0,5;
Consensus calculation options: Residue fraction for consensus: 0.9.
Presettings were selected to allow also for the alignment of
conserved amino acids
[16858] [0291.0.42.42] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. In one embodiment, said polypeptide of the
invention distinguishes over a sequence as indicated in Table XII,
application no. 42, columns 5 or 7, by one or more amino acids. In
one embodiment, polypeptide distinguishes form a sequence as
indicated in Table XII, application no. 42, columns 5 or 7, by more
than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, even more preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table XII, application no. 42, columns 5 or 7, by not
more than 80% or 70% of the amino acids, preferably not more than
60% or 50%, more preferred not more than 40% or 30%, even more
preferred not more than 20% or 10%. In an other embodiment, said
polypeptide of the invention does not consist of a sequence as
indicated in Table XII, application no. 42, columns 5 or 7.
[16859] [0292.0.0.42] see [0292.0.0.27]
[16860] [0293.0.42.42] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, the polypeptide of
the invention has a sequence which distinguishes from a sequence as
indicated in Table XII, application no. 42, columns 5 or 7, by one
or more amino acids. In an other embodiment, said polypeptide of
the invention does not consist of the sequence as indicated in
Table XII, application no. 42, columns 5 or 7.
[16861] In a further embodiment, said polypeptide of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by a nucleic acid molecules as indicated in
Table XI, application no. 42, columns 5 or 7.
[16862] [0294.0.42.42] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 42, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 42, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[16863] [0295.0.0.42] to [0296.0.0.42]: see [0295.0.0.27] to
[0296.0.0.27]
[16864] [0297.0.0.42] see [0297.0.0.27]
[16865] [00297.1.42.42] %
[16866] [0298.0.42.42] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 42, columns 5 or 7. The
portion of the protein is preferably a biologically active portion
as described herein. Preferably, the polypeptide used in the
process of the invention has an amino acid sequence identical to a
sequence as indicated in Table XII, application no. 42, columns 5
or 7.
[16867] [0299.0.42.42] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 42,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 42, columns 5 or 7, or which is
homologous thereto, as defined above.
[16868] [0300.0.42.42] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 42, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 42, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[16869] [0301.0.0.42] see [0301.0.0.27]
[16870] [0302.0.42.42] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 42, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[16871] [0303.0.0.42] see [0303.0.0.27]
[16872] [0304.0.42.42] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 42, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[16873] [0305.0.0.42] to [0308.0.0.42]: see [0305.0.0.27] to
[0308.0.0.27]
[16874] [0309.0.42.42] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table XII,
application no. 42, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas an
"other polypeptide" not being indicated in Table XII, application
no. 42, columns 5 or 7, refers to a polypeptide having an amino
acid sequence corresponding to a protein which is not substantially
homologous to a polypeptide of the invention, preferably which is
not substantially homologous to a polypeptide as indicated in Table
XII, application no. 42, columns 5 or 7, e.g., a protein which does
not confer the activity described herein or annotated or known for
as indicated in Table XII, application no. 42, column 3, and which
is derived from the same or a different organism. In one
embodiment, an "other polypeptide" not being indicated in Table
XII, application no. 42, columns 5 or 7, does not confer an
increase of the respective fine chemical in an organism or part
thereof.
[16875] [0310.0.0.42] to [0334.0.0.42]: see [0310.0.0.27] to
[0334.0.0.27]
[16876] [0335.0.42.42] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XI, application
no. 42, columns 5 or 7, and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived there from),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
columns 5 or 7, and/or homologs thereof. In a double-stranded RNA
molecule for reducing the expression of a protein encoded by a
nucleic acid sequence as indicated in Table XI, application no. 42,
columns 5 or 7, and/or homologs thereof, one of the two RNA strands
is essentially identical to at least part of a nucleic acid
sequence, and the respective other RNA strand is essentially
identical to at least part of the complementary strand of a nucleic
acid sequence.
[16877] [0336.0.0.42] to [0342.0.0.42]: see [0336.0.0.27] to
[0342.0.0.27]
[16878] [0343.0.42.42] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 42, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table XI, application no. 42, columns 5 or 7, or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[16879] [0344.0.0.42] to [0350.0.0.42]: see [0344.0.0.27] to
[0350.0.0.27]
[16880] [0351.0.0.42] to [0361.0.0.42]: see [0351.0.0.27] to
[0361.0.0.27]
[16881] [0362.0.42.42] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 42, columns 5 or 7, e.g. encoding a
polypeptide having protein activity, as indicated in Table XII,
application no. 42, columns 3. Due to the above-mentioned activity
the respective fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity of the polypeptide of the invention or nucleic
acid molecule of the invention is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a polypeptide as
indicated in Table XII, application no. 42, columns 5 or 7, means
herein that due to modulation or manipulation of the genome, an
activity as annotated for a polypeptide as indicated in Table XII,
application no. 42, column 3, e.g. having a sequence as indicated
in Table XII, application no. 42, columns 5 or 7, is increased in a
cell or an organism or a part thereof. Examples are described above
in context with the process of the invention.
[16882] [0363.0.0.42] see [0363.0.0.27]
[16883] [0364.0.42.42] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table XII, application no. 42, column 3, with the
corresponding protein-encoding sequence as indicated in Table XI,
application no. 42, column 5, becomes a transgenic expression
cassette when it is modified by non-natural, synthetic "artificial"
methods such as, for example, mutagenization. Such methods have
been described (U.S. Pat. No. 5,565,350; WO 00/15815; also see
above).
[16884] [0365.0.0.42] to [0373.0.0.42]: see [0365.0.0.27] to
[0373.0.0.27]
[16885] [0374.0.42.42] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. melissic acid, in particular
the respective fine chemical, produced in the process according to
the invention may, however, also be isolated from the plant in the
form of their free melissic acid, in particular the free respective
fine chemical, or bound in or to compounds or moieties, like
glucosides, e.g. diglucosides. The respective fine chemical
produced by this process can be harvested by harvesting the
organisms either from the culture in which they grow or from the
field. This can be done via expressing, grinding and/or extraction,
salt precipitation and/or ion-exchange chromatography or other
chromatographic methods of the plant parts, preferably the plant
seeds, plant fruits, plant tubers and the like.
[16886] [0375.0.0.42] to [0376.0.0.42]: see [0375.0.0.27] to
[0376.0.0.27]
[16887] [0377.0.42.42] Accordingly, the present invention relates
also to a process whereby the produced melissic acid is
isolated.
[16888] [0378.0.42.42] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the melissic acid
produced in the process can be isolated. The resulting melissic
acid can, if appropriate, subsequently be further purified, if
desired mixed with other active ingredients such as vitamins, amino
acids, carbohydrates, antibiotics and the like, and, if
appropriate, formulated.
[16889] [0379.0.42.42] In one embodiment the product produced by
the present invention is a mixture of the respective fine chemicals
melissic acid.
[16890] [0380.0.42.42] The melissic acid obtained in the process by
carrying out the invention is suitable as starting material for the
synthesis of further products of value. For example, they can be
used in combination with each other or alone for the production of
pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates to a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the melissic acid composition
produced or the respective fine chemical produced if desired and
formulating the product with a pharmaceutical acceptable carrier or
formulating the product in a form acceptable for an application in
agriculture. A further embodiment according to the invention is the
use of the melissic acid produced in the process or of the
transgenic organism in animal feeds, foodstuffs, medicines, food
supplements, cosmetics or pharmaceuticals or for the production of
melissic acid e.g. after isolation of the respective fine chemical
or without, e.g. in situ, e.g in the organism used for the process
for the production of the respective fine chemical.
[16891] [0381.0.0.42] to [0382.0.0.42]: see [0381.0.0.27] to
[0382.0.0.27]
[16892] [0383.0.42.42]
[16893] [0384.0.0.42] see [0384.0.0.27]
[16894] [0385.0.42.42] The fermentation broths obtained in this
way, containing in particular melissic acid in mixtures with other
organic acids, amino acids, polypeptides or polysaccarides,
normally have a dry matter content of from 1 to 70% by weight,
preferably 7.5 to 25% by weight. Sugar-limited fermentation is
additionally advantageous, e.g. at the end, for example over at
least 30% of the fermentation time. This means that the
concentration of utilizable sugar in the fermentation medium is
kept at, or reduced to, 0 to 10 g/l, preferably to 0 to 3 g/l
during this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed or isolated
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation, coagulation/flocculation
or a combination of these methods, from the fermentation broth or
left completely in it.
[16895] The fermentation broth can then be thickened or
concentrated by known methods, such as, for example, with the aid
of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[16896] [0386.0.42.42] Accordingly, it is possible to purify the
melissic acid produced according to the invention further. For this
purpose, the product-containing composition is subjected for
example to separation via e.g. an open column chromatography or
HPLC in which case the desired product or the impurities are
retained wholly or partly on the chromatography resin. These
chromatography steps can be repeated if necessary, using the same
or different chromatography resins. The skilled worker is familiar
with the choice of suitable chromatography resins and their most
effective use.
[16897] [0387.0.0.42] to [0392.0.0.42]: see [0387.0.0.27] to
[0392.0.0.27]
[16898] [0393.0.42.42] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [16899] a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [16900] b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table XI, application no. 42, columns 5 or 7, and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[16901] c) introducing the candidate nucleic acid molecules in host
cells, preferably in a plant cell or a microorganism, appropriate
for producing the fine chemical; [16902] d) expressing the
identified nucleic acid molecules in the host cells; [16903] e)
assaying the the fine chemical level in the host cells; and [16904]
f) identifying the nucleic acid molecule and its gene product which
expression confers an increase in the the fine chemical level in
the host cell after expression compared to the wild type.
[16905] [0394.0.32.42] to [0552.0.32.42]: see [0394.0.0.32] to
[0552.0.0.32]
[16906] [0553.0.42.42]
1. A process for the production of melissic acid resp., which
comprises (a) increasing or generating the activity of a protein as
indicated in Table XII, application no. 42, columns 5 or 7, or a
functional equivalent thereof in a non-human organism, or in one or
more parts thereof; and (b) growing the organism under conditions
which permit the production of melissic acid resp. in said
organism. 2. A process for the production of melissic acid resp.,
comprising the increasing or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: a) nucleic acid molecule encoding of a polypeptide
as indicated in Table XII, application no. 42, columns 5 or 7, or a
fragment thereof, which confers an increase in the amount of the
fine chemical as indicated in table XII, column 6, e.g of melissic
acid resp., in an organism or a part thereof; b) nucleic acid
molecule comprising of a nucleic acid molecule as indicated in
Table XI, application no. 42, columns 5 or 7, c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of melissic acid resp., in an organism or a part thereof; d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical as indicated in
table XII, column 6, e.g of melissic acid resp., in an organism or
a part thereof; e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the fine chemical as indicated in table XII, column 6, e.g of
melissic acid resp., in an organism or a part thereof; f) nucleic
acid molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table XIII, application no. 42, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of melissic acid resp., in an organism or a part
thereof; g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of melissic acid resp., in
an organism or a part thereof; h) nucleic acid molecule encoding a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 42, column 7, and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of melissic acid resp., in an organism or a part thereof;
and i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of melissic acid resp., in
an organism or a part thereof. or comprising a sequence which is
complementary thereto. 3. The process of claim 1 or 2, comprising
recovering of the free or bound melissic acid resp. 4. The process
of any one of claims 1 to 3, comprising the following steps: (a)
selecting an organism or a part thereof expressing a polypeptide
encoded by the nucleic acid molecule characterized in claim 2; (b)
mutagenizing the selected organism or the part thereof; (c)
comparing the activity or the expression level of said polypeptide
in the mutagenized organism or the part thereof with the activity
or the expression of said polypeptide of the selected organisms or
the part thereof; (d) selecting the mutated organisms or parts
thereof, which comprise an increased activity or expression level
of said polypeptide compared to the selected organism or the part
thereof; (e) optionally, growing and cultivating the organisms or
the parts thereof; and (f) recovering, and optionally isolating,
the free or bound melissic acid resp., produced by the selected
mutated organisms or parts thereof. 5. The process of any one of
claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: a) nucleic acid molecule encoding of a polypeptide
as indicated in Table XII, application no. 42, columns 5 or 7, or a
fragment thereof, which confers an increase in the amount of the
fine chemical as indicated in table XII, column 6, e.g of melissic
acid resp., in an organism or a part thereof; b) nucleic acid
molecule comprising of a nucleic acid molecule as indicated in
Table XI, application no. 42, columns 5 or 7, c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of melissic acid resp., in an organism or a part thereof; d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical as indicated in
table XII, column 6, e.g of melissic acid resp., in an organism or
a part thereof; e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the fine chemical as indicated in table XII, column 6, e.g of
melissic acid resp., in an organism or a part thereof; f) nucleic
acid molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table XIII, application no. 42, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of melissic acid resp., in an organism or a part
thereof; g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of melissic acid resp., in
an organism or a part thereof; h) nucleic acid molecule encoding a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 42, column 7, and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of melissic acid resp., in an organism or a part thereof;
and i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of melissic acid resp., in
an organism or a part thereof. whereby the nucleic acid molecule
distinguishes over the sequence as indicated in Table XI,
application no. 42, columns 5 or 7, by one or more nucleotides. 7.
A nucleic acid construct which confers the expression of the
nucleic acid molecule of claim 6, comprising one or more regulatory
elements. 8. A vector comprising the nucleic acid molecule as
claimed in claim 6 or the nucleic acid construct of claim 7. 9. The
vector as claimed in claim 8, wherein the nucleic acid molecule is
in operable linkage with regulatory sequences for the expression in
a prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. 11. The host cell of claim 10, which is a transgenic
host cell. 12. The host cell of claim 10 or 11, which is a plant
cell, an animal cell, a microorganism, or a yeast cell, a fungus
cell, a prokaryotic cell, an eukaryotic cell or an archaebacterium.
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. 14. A polypeptide produced by the process as claimed in claim
13 or encoded by the nucleic acid molecule as claimed in claim 6
whereby the polypeptide distinguishes over a sequence as indicated
in Table XII, application no. 42, columns 5 or 7, by one or more
amino acids 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. 16. A plant tissue, propagation
material, harvested material or a plant comprising the host cell as
claimed in claim 12 which is plant cell or an Agrobacterium. 17. A
method for screening for agonists and antagonists of the activity
of a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of melissic acid resp., in an
organism or a part thereof comprising: (a) contacting cells,
tissues, plants or microorganisms which express the a polypeptide
encoded by the nucleic acid molecule of claim 5 conferring an
increase in the amount of melissic acid resp., in an organism or a
part thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; (b) assaying the melissic acid resp., level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and (c) identifying a
agonist or antagonist by comparing the measured melissic acid
resp., level or polypeptide expression level with a standard
melissic acid resp., or polypeptide expression level measured in
the absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. 18. A process for the
identification of a compound conferring increased melissic acid
resp., production in a plant or microorganism, comprising the
steps: (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of melissic acid resp., in an organism or a part thereof and
a readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with dais readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
melissic acid resp., in an organism or a part thereof; (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. 19. A method for the identification of a gene
product conferring an increase in melissic acid resp., production
in a cell, comprising the following steps: (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in melissic
acid resp., after expression with the nucleic acid molecule of
claim 6; (b) identifying the nucleic acid molecules, which
hybridise under relaxed stringent conditions with the nucleic acid
molecule of claim 6; (c) introducing the candidate nucleic acid
molecules in host cells appropriate for producing melissic acid
resp.; (d) expressing the identified nucleic acid molecules in the
host cells; (e) assaying the melissic acid resp., level in the host
cells; and (f) identifying nucleic acid molecule and its gene
product which expression confers an increase in the melissic acid
resp., level in the host cell in the host cell after expression
compared to the wild type. 20. A method for the identification of a
gene product conferring an increase in melissic acid resp.,
production in a cell, comprising the following steps: (a)
identifying in a data bank nucleic acid molecules of an organism;
which can contain a candidate gene encoding a gene product
conferring an increase in the melissic acid resp., amount or level
in an organism or a part thereof after expression, and which are at
least 20% homolog to the nucleic acid molecule of claim 6; (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing melissic acid resp.; (c) expressing the
identified nucleic acid molecules in the host cells; (d) assaying
the melissic acid resp., level in the host cells; and (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the melissic acid resp., level in
the host cell after expression compared to the wild type. 21. A
method for the production of an agricultural composition comprising
the steps of the method of any one of claims 17 to 20 and
formulating the compound identified in any one of claims 17 to 20
in a form acceptable for an application in agriculture. 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. 23. Use of the
nucleic acid molecule as claimed in claim 6 for the identification
of a nucleic acid molecule conferring an increase of melissic acid
resp., after expression. 24. Use of the polypeptide of claim 14 or
the nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of melissic acid resp., levels
in an organism. 25. Agrochemical, pharmaceutical, food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. 26. The method of any one of claims 1
to 5, the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20, wherein the fine chemical is melissic
acid. 27. A host cell or plant according to any of the claims 10 to
12 which is resistant to a herbicide inhibiting the biosynthesis of
melissic acid.
[16907] [0554.0.0.42] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
Description
[16908] [0000.0.43.43] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[16909] [0001.0.0.43] for the disclosure of this paragraph see
[0001.0.0.27].
[16910] [0002.0.43.43] Plants produce glycerol. and
glycerol-3-phosphate. Importantly lipids derived from glycerol are
the major components of eukaryotic cells. In terms of dry weight
they account for anywhere between 10% and 90% of the total mass of
the cell. Triglycerol are the major source of store energy in
eucaryotic organisms.
[16911] Glycerol-3-phosphate can be synthesized via two different
routes in plants. In one route, it is formed from dihydroxyacetone
phosphate (DHAP), an intermediate of glycolysis, by the sequential
action of triosephosphate isomerase, glyceraldehyd-phosphate
phosphatase, glyeraldehyde reductase and glycerol kinase. The last
enzyme of this pathway has been suggested as the rate-limiting step
of this route for glycerol-3-phosphate synthesis. In an second
pathway glycerol-3-phosphate dehydrogenase (NAD(+)-G-3-P
oxidoreductase, EC 1.1.1.8) (GPDH) catalyses the reduction of
dihydroxyacetone phosphate (DHAP) to form glycerol-3-phosphate
(G-3-P). Based on enzymatic studies is has been suggested that this
enzyme activity is probably the primary source of
glycerol-3-phosphate at least in Brassica campestris seeds (Sharma
et al., 2001, Plant Sci 160, 603-610).
[16912] [0003.0.43.43] In plants, at least two types of GPDH, a
cytoplasmatic and a plastidial exist, which also differ in their
reducing cosubstrate. The cytsolic GPDH uses NADH as the
cosubstrate. The mitochondrial FAD-dependent glycerol-3-phosphate
dehydrogenase (FAD-GPDH) of Arabidopsis forms a G-3-P shuttle, as
previously established in other eukaryotic organisms, and links
cytosolic G-3-P metabolism to carbon source utilization and energy
metabolism in plants--also see Shen, W. et al., FEBS Lett. 2003
Feb. 11; 536(1-3): 92-6.
[16913] Glycerol-insensitive Arabidopsis mutants: glil seedlings
lack glycerol kinase, accumulate glycerol and are more resistant to
abiotic stress, see Eastmond P. J., HYPERLINK
"http://www.ingentaconnect.com/content/bsc/tpj" \o "The Plant
Journal" The Plant Journal, 2004, 37(4), 617-625. These data show
that glycerol kinase is required for glycerol catabolism in
Arabidopsis and that the accumulation of glycerol can enhance
resistance to a variety of abiotic stresses associated with
dehydration.
[16914] [0004.0.43.43] The major storage lipids (or oils) of seeds
occur in the form of triacylglycerols (TAG), or three fatty acids
linked to glycerol by ester bonds. Triacylglycerol synthesis
involves diverse cellular compartments, including the cytoplasm,
the mitochondria, the plastids, and the endoplasmic reticulum (ER).
Glycerol-3-phosphate enters the ER for the final step in
triacylglycerol synthesis. The newly formed triacylglycerols
accumulate between the two layers of the double membrane of the ER,
forming an oil body surrounded by a single (or half) unit
membrane.
[16915] [0005.0.43.43] Glycerol-3-phosphate acyltransferase (GPAT)
is one of the most important enzymes in TAG biosynthesis, since it
initiates TAG synthesis by catalyzing the acylation of the Sn-1
position of Sn-glycerol-3-phosphate, producing
Sn-1-acyl-glycerol-3-phosphate. Lyso-phosphatidic acid (LPA) is
then acetylated by LPA acyltransferases to produce phosphatidic
acid (PA). Then diacylglycerol (DAG) is released through the
dephosphorylation of PA by PA phosphohydrolase. Finally DAG becomes
acylated by the activity of the DAG acyltransferase. In a second
pathway phosphatidylcholine (PC) is formed and its acyl residues
are desaturated further. The choline phosphate residue is then
liberated by hydrolysis and the correspondinbg DAG acylated. This
second pathway operates frequently in the synthesis of highly
unsaturated TAG (Heldt 1997, Plant biochemistry and molecular
biology. Oxford University Press, New York).
[16916] Additionally at present, many researches have proved that
the GPAT is related to plant chilling-resistance, see Liu, Ji-Mei
et al., Plant Physiol. 120(1999): 934.
[16917] Glycerol-3-phosphate is a primary substrate for
triacylglycerol synthesis. Vigeolas and Geigenberger (Planta
219(2004): 827-835) have shown that injection of developing seeds
with glycerol leads to increased glycerol-3-phosphate levels. These
increased levels of glycerol-3-phosphate were accompanied by an
increase in the flux of sucrose into total lipids and
triacylglycerol providing evidence that the prevailing levels of
glycerol-3-phosphate co-limit triacylglycerol production in
developing seeds.
[16918] The direct acylation of glycerol by a glycerol: acyl-CoA
acyltransferase to form mono-acyl-glycerol and, subsequently,
diacylglycerol and triacylglycerol has been shown in myoblast and
hepatocytes (Lee, D. P. et al. J. Lipid res. 42 (2001): 1979-1986).
This direct acylation became more prominent when the
glycerol-3-phosphate pathway was attenuated or when glycerol levels
become elevated.
[16919] [0006.0.43.43] Glycerol is used together with water and
alcohol (ethyl alcohol) in glycerinated water/alcohol plant
extracts and phytoaromatic compounds. These products are used as
food supplements, providing concentrates of the minerals, trace
elements, active ingredients (alkaloids, polyphenols, pigments,
etc.) and aromatic substances to be found in plants. Glycerin acts
as a carrier for plant extracts. It is found in the end product
(the fresh plant extract) in concentrations of up to 24% or
25%.
[16920] Raw glycerol is a by-product of the transesterification
process of rape oil to rape methyl ester (RME) and used edible oil
to used edible methyl ester (AME), both better known as
Biodiesel.
[16921] Glycerol world production is estimated to be around 750.000
t/year. Around 90% is manufactured on the basis of natural oils and
fats.
[16922] [0007.0.43.43] The green alga Dunaliella, for example,
recently has been established in mass culture as a commercial
source for glycerol. Dunaliella withstands extreme salinities while
maintaing a low intracellular salt concentration. Osmotic
adjustment is achieved by intracellular accumulation of glycerol to
a level counterbalancing the external osmoticum.
[16923] The osmoregulatory isoform of dihydroxyacetone phosphate
(DHAP) reductase (Osm-DHAPR) is an enzyme unique to Dunaliella
tertiolecta and is the osmoregulatory isoform involved in the
synthesis of free glycerol for osmoregulation in extreme
environments, such as high salinity, see Ghoshal, D., et al.,
HYPERLINK
"http://www.ingentaconnect.com/content/ap/pt;sessionid=708pg3rt74t81.vict-
oria" \o "Protein Expression and Purification" Protein Expression
and Purification, 2002, 24, (3), 404-411.
[16924] A unsolved problem in plant biochemistry is the
understanding of metabolic regulation of glycerol-3-phosphate
synthesis and its use in modifying glyceride metabolism or glycerol
production. Practically it will have significance for rationally
genetically engineering of plants for increased synthesis of
triacylglycerols or for other value added products, and for
introducing the glycerol synthesis capability into plants of
economic importance for an elevated environmental stress
tolerance--see: Durba, G. et al., J. Plant Biochemistry &
Biotechnology 10(2001), 113-120.
[16925] [0008.0.43.43] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce glycerol and/or glycerol-3-phosphate
in plants. That type of production permits control over quality,
quantity and selection of the most suitable and efficient producer
organisms. The latter is especially important for commercial
production economics and therefore availability to consumers. In
addition it is desirable to produce glycerol and/or
glycerol-3-phosphate in plants in order to increase plant
productivity and resistance against biotic and abiotic stress as
discussed before.
[16926] Methods of recombinant DNA technology have been used for
some years to improve the production of fine chemicals in
microorganisms and plants by amplifying individual biosynthesis
genes and investigating the effect on production of fine chemicals.
It is for example reported, that the xanthophyll astaxanthin could
be produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. Those results indicated
that about 75 percent of the carotenoids found in the flower of the
transformed plant contained a keto group.
[16927] [0009.0.43.43] Thus, it would be advantageous if an algae,
plant or other microorganism were available which produce large
amounts of glycerol and/or glycerol-3-phosphate. The invention
discussed hereinafter relates in some embodiments to such
transformed prokaryotic or eukaryotic microorganisms.
[16928] It would also be advantageous if plants were available
whose roots, leaves, stem, fruits or flowers produced large amounts
of glycerol and/or glycerol-3-phosphate. The invention discussed
hereinafter relates in some embodiments to such transformed
plants.
[16929] Furthermore it would be advantageous if plants were
available whose seed produced larger amounts of total lipids. The
invention discussed hereinafter relates in some embodiments to such
transformed plants.
[16930] [0010.0.43.43] Therefore improving the quality of
foodstuffs and animal feeds is an important task of the
food-and-feed industry. This is necessary since, for example
glycerol and/or glycerol-3-phosphate, as mentioned above, which
occur in plants and some microorganisms are limited with regard to
the supply of mammals. Especially advantageous for the quality of
foodstuffs and animal feeds is as balanced as possible a specific
glycerol and/or glycerol-3-phosphate profile in the diet since an
excess of glycerol and/or glycerol-3-phosphate above a specific
concentration in the food has a positive effect. A further increase
in quality is only possible via addition of further glycerol and/or
glycerol-3-phosphate, which are limiting.
[16931] [0011.0.43.43] To ensure a high quality of foods and animal
feeds, it is therefore necessary to add glycerol and/or
glycerol-3-phosphate in a balanced manner to suit the organism.
[16932] [0012.0.43.43] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes or other
proteins which participate in the biosynthesis of glycerol and/or
glycerol-3-phosphate and make it possible to produce them
specifically on an industrial scale without unwanted byproducts
forming. In the selection of genes for biosynthesis two
characteristics above all are particularly important. On the one
hand, there is as ever a need for improved processes for obtaining
the highest possible contents of glycerol and/or
glycerol-3-phosphate; on the other hand as less as possible
byproducts should be produced in the production process.
[16933] Furthermore there is still a great demand for new and more
suitable genes, which encode enzymes or other proteins, which
participate in the biosynthesis of total lipids and make it
possible to produce them specifically on an industrial scale
without unwanted byproducts forming. In the selection of genes for
biosynthesis two characteristics above all are particularly
important. On the one hand, there is as ever a need for improved
processes for obtaining the highest possible contents of total
lipids; on the other hand as less as possible byproducts should be
produced in the production process.
[16934] Glycerol or glycerol-3-phosphate is biosynthetic precusor
for the biosynthesis of monoacylglycerols, diacylglycerols,
triacylglycerols, phosphatidylglycerols and other glycerolipids
(e.g. glycosylglycerides, diphosphatidylglycerols, phosphonolipids,
phosphatidylcholines, phosphatidylethanolamines,
phosphatidylinositols, phytoglycolipids). Therefore the analysis of
the glycerol content in cells, tissues or plant parts like seeds
and leaves after total lipid extraction and lipid hydrolysis
directly correlates with the analysis of the total lipid content.
For example if the overexpression of a gene participating in the
biosynthesis of triacylglycerols in the seed results in an increase
in total lipid content in the seed or leaf this seed will also show
an increased glycerol content after total lipid extraction and
hydrolysis of the lipids.
[16935] Therefore the method as described below which leads to an
increase in glycerol in the lipid fraction after cleavage of the
ester functions for example with a mixture of methanol and
hydrochloric acid clearly represents a method for an increased
production of triacylglycerol or total lipids.
[16936] [0013.0.0.43] for the disclosure of this paragraph see
[0013.0.0.27].
[16937] [0014.0.43.43] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is glycerol and/or
glycerol-3-phosphate. Accordingly, in the present invention, the
term "the fine chemical" as used herein relates to glycerol and/or
glycerol-3-phosphate. Further, the term "the fine chemicals" as
used herein also relates to fine chemicals comprising glycerol
and/or glycerol-3-phosphate.
[16938] [0015.0.43.43] In one embodiment, the term "the fine
chemical" means glycerol. In one embodiment, the term "the fine
chemical" means glycerol-3-phosphate depending on the context in
which the term is used. Throughout the specification the term "the
fine chemical" means glycerol and/or glycerol-3-phosphate, its
salts, ester, thioester or in free form or bound to other compounds
such as sugars or sugarpolymers, like glucoside or polyols like
myo-inositol.
[16939] In one embodiment, the term "the fine chemical" means
monoacylglycerols, diacylglycerols, triacylglycerols,
phosphatidylglycerols and/or other glycerolipids (e.g. but not
limited to glycosylglycerides, diphosphatidylglycerols,
phosphonolipids, phosphatidylcholines, phosphatidylethanolamines,
phosphatidylinositols or phytoglycolipids) and is hereinafter
referred to as "total lipids".
[16940] [0016.0.43.43] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [16941] (a) increasing or generating the activity of one
or more [16942] of a protein as shown in table XII, application no.
43, column 3 encoded by the nucleic acid sequences as shown in
table XI, application no. 43, column 5, in a non-human organism or
in one or more parts thereof or [16943] (b) growing the organism
under conditions which permit the production of the fine chemical,
thus glycerol of the invention or fine chemicals comprising
glycerol of the invention, in said organism or in the culture
medium surrounding the organism.
[16944] [0016.1.43.43] Accordingly, the term "the fine chemical"
means in one embodiment "glycerol" in relation to all sequences
listed in Tables XI to XIV, line 44 and/or 45 and/or 46 or homologs
thereof.
[16945] [0017.0.0.43] and [0019.0.0.43] for the disclosure of the
paragraphs [0017.0.0.43] and [0019.0.0.43] see paragraphs
[0017.0.0.27] and [0019.0.0.27] above.
[16946] [0020.0.43.43] Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, column 5, line 44 in a plant conferred an increase in
glycerol content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of glycerol. Surprisingly it was found, that the
transgenic expression of the Zea mays protein as indicated in Table
XII, column 5, line 45 in a plant conferred an increase in glycerol
content of the transformed plants. Thus, in one embodiment, said
protein or its homologs are used for the production of glycerol.
Surprisingly it was found, that the transgenic expression of the
Glycine max protein as indicated in Table XII, column 5, line 46 in
a plant conferred an increase in glycerol content of the
transformed plants. Thus, in one embodiment, said protein or its
homologs are used for the production of glycerol.
[16947] [0021.0.0.43] for the disclosure of this paragraph see
[0021.0.0.27] above.
[16948] [0022.0.43.43] The sequence of YDR513W from Saccharomyces
cerevisiae has been published in Jacq, C. et al., Nature 387 (6632
Suppl), 75-78 (1997) and its activity is being defined as a protein
having gluthatione reductase activity. Accordingly, in one
embodiment, the process of the present invention comprises the use
of a protein YDR513W from Saccharomyces cerevisiae or a plant or
its homolog, e.g. as shown herein, for the production of the
respective fine chemical, in particular for increasing the amount
of glycerol and/or total lipid, preferably in free or bound form in
an organism or a part thereof, as mentioned. In one embodiment, in
the process of the present invention the YDR513W-protein-activity
is increased.
[16949] The sequence of YGL237C from Saccharomyces cerevisiae has
been published in Tettelin, H. et al. Nature 387 (6632 Suppl),
81-84 (1997) and its activity is being defined as transcriptional
activator and global regulator of respiratory gene expression.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein YGL237C from Saccharomyces
cerevisiae or a plant or its homolog, e.g. as shown herein, for the
production of the respective fine chemical, in particular for
increasing the amount of glycerol and/or total lipid, preferably in
free or bound form in an organism or a part thereof, as mentioned.
In one embodiment, in the process of the present invention the
YGL237C-protein-activity a is increased.
[16950] The sequence of YMR015C from Saccharomyces cerevisiae has
been published Bowman, S. et al., Nature 387 (6632 Suppl), 90-93
(1997) and its activity. is being defined as a protein having C-22
sterol desaturase activity. Accordingly, in one embodiment, the
process of the present invention comprises the use of a protein
YMR015C from Saccharomyces cerevisiae or a plant or its homolog,
e.g. as shown herein, for the production of the respective fine
chemical, in particular for increasing the amount of glycerol
and/or total lipid, preferably in free or bound form in an organism
or a part thereof, as mentioned. In one embodiment, in the process
of the present invention the YMR015C-protein-activity is
increased.
[16951] [0022.1.0.43] and [0023.0.0.43] for the disclosure of the
paragraphs [0022.1.0.43] and [0023.0.0.43] see paragraphs
[0022.1.0.27] and [0023.0.0.27] above.
[16952] [0023.1.0.43] Homologs of the polypeptide disclosed in
table XII, application no. 43, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 43, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 43, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 43,
column 7, resp.
[16953] [0024.0.0.43] for the disclosure of this paragraph see
[0024.0.0.27] above.
[16954] [0025.0.43.43] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 43, column 3" if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 43, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[16955] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 43,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 43,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[16956] [0025.1.0.43] and [0025.2.0.43] for the disclosure of the
paragraphs [0025.1.0.43] and [0025.2.0.43] see [0025.1.0.27] and
[0025.2.0.27] above.
[16957] [0026.0.0.17] to [0033.0.0.17] for the disclosure of the
paragraphs [0026.0.0.17] to [0033.0.0.17] see [0026.0.0.27] to
[0033.0.0.27] above.
[16958] [0034.0.43.43] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 43, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[16959] [0035.0.0.43] to [0044.0.0.43] for the disclosure of the
paragraphs [0035.0.0.43] to [0044.0.0.43] see paragraphs
[0035.0.0.27] to [0044.0.0.27] above.
[16960] [0045.0.43.43] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
43, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line,
between 10% and 50%, 10% and 100%, 10% and 200%, 10% and 300%, 10%
and 400%, 10% and 500%, 20% and 50%, 20% and 250%, 20% and 500%,
50% and 100%, 50% and 250%, 50% and 500%, 500% and 1000% or
more.
[16961] [0046.0.43.43] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
43, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line
confers an increase of the respective fine chemical and of further
glycerol or their precursors.
[16962] [0047.0.0.43] and [0048.0.0.43] for the disclosure of the
paragraphs [0047.0.0.43] and [0048.0.0.43] see paragraphs
[0047.0.0.27] and [0048.0.0.27] above.
[16963] [0049.0.43.43] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 43, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 43, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 43, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[16964] [0050.0.43.43] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of glycerol-3-phosphate, resp., or their esters, e.g.
monoacyl or diacyl fatty acids thereof.
[16965] %
[16966] [0051.0.43.43] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free or
bound, e.g compositions comprising glycerol and/or total lipid.
Depending on the choice of the organism used for the process
according to the present invention, for example a microorganism or
a plant, compositions or mixtures of glycerol and/or total lipid
can be produced.
[16967] [0052.0.0.43] for the disclosure of this paragraph see
paragraph [0052.0.0.27] above.
[16968] [0053.0.43.43] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[16969] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 43, columns 5 and 7 or its homologs activity
having herein-mentioned glycerol of the invention increasing
activity; and/or [16970] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 43,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 43, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned glycerol of the invention increasing
activity; and/or [16971] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned gylcerol increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 43, columns 5 and 7 or its
homologs activity, or decreasing the inhibiitory regulation of the
polypeptide of the invention; and/or [16972] d) generating or
increasing the expression of an endogenous or artificial
transcription factor mediating the expression of a protein
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention or of the polypeptide of the
invention having herein-mentioned glycerol of the invention
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 43, columns 5
and 7 or its homologs activity; and/or [16973] e) stimulating
activity of a protein conferring the increased expression of a
protein encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention having
herein-mentioned glycerol of the invention increasing activity,
e.g. of a polypeptide having the activity of a protein as indicated
in table XII, application no. 43, columns 5 and 7 or its homologs
activity, by adding one or more exogenous inducing factors to the
organisms or parts thereof; and/or [16974] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned glycerol of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 43, columns 5 and 7 or its
homologs activity, and/or [16975] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned glycerol of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 43, columns 5 and 7 or its
homologs activity; and/or [16976] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 43, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[16977] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [16978] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[16979] [0054.0.43.43] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 43 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 43, column 3 or its homologs.
[16980] [0055.0.0.43] to [0067.0.0.43] for the disclosure of the
paragraphs [0055.0.0.43] to [0067.0.0.43] see paragraphs
[0055.0.0.27] to [0067.0.0.27] above.
[16981] [0068.0.43.43] The mutation is introduced in such a way
that the production of the finde chemical, meaning glycerol and/or
total lipid is not adversely affected.
[16982] [0069.0.0.43] for the disclosure of this paragraph see
paragraph [0069.0.0.27] above.
[16983] [0070.0.43.43] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolite composition in the organism, e.g. an advantageous
composition of glycerol or their biochemical derivatives, e.g.
comprising a higher content of (from a viewpoint of nutritional
physiology limited) glycerol or their derivatives including total
lipids.
[16984] [0071.0.0.43] for the disclosure of this paragraph see
paragraph [0071.0.0.27] above.
[16985] [0072.0.43.43] %
[16986] [0073.0.43.43] Accordingly, in one embodiment, the process
according to the invention relates to a process, which comprises:
[16987] (a) providing a non-human organism, preferably a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant; [16988] (b) increasing an activity of
a polypeptide of the invention or a homolog thereof, e.g. as
indicated in Table XII, application no. 43, columns 5 or 7, or of a
polypeptide being encoded by the nucleic acid molecule of the
present invention and described below, e.g. conferring an increase
of the respective fine chemical in an organism, preferably in a
microorganism, a non-human animal, a plant or animal cell, a plant
or animal tissue or a plant, [16989] (c) growing an organism,
preferably a microorganism, a non-human animal, a plant or animal
cell, a plant or animal tissue or a plant under conditions which
permit the production of the respective fine chemical in the
organism, preferably the microorganism, the plant cell, the plant
tissue or the plant; and [16990] (d) if desired, recovering,
optionally isolating, the free and/or bound respective fine
chemical synthesized by the organism, the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[16991] [0074.0.43.43] for the disclosure of this paragraph see
paragraph [0071.0.0.17] above.
[16992] [0075.0.0.43] to [0077.0.0.43] for the disclosure of the
paragraphs [0075.0.0.43] to [0077.0.0.43] see paragraphs
[0075.0.0.27] to [0077.0.0.27] above.
[16993] [0078.0.43.43] The organism such as microorganisms or
plants or the recovered, and if desired isolated, respective fine
chemical can then be processed further directly into foodstuffs or
animal feeds or for other applications. The fermentation broth,
fermentation products, plants or plant products can be purified
with methods known to the person skilled in the art. Products of
these different work-up procedures are glycerol and/or glycerol as
a component of lipids or comprising compositions of glycerol and/or
glycerol as a component of lipids still comprising fermentation
broth, plant particles and cell components in different amounts,
advantageously in the range of from 0 to 99% by weight, preferably
below 80% by weight, especially preferably below 50% by weight.
[16994] [0079.0.0.43] to [0084.0.0.43] for the disclosure of the
paragraphs [0079.0.0.43] to [0084.0.0.43] see paragraphs
[0079.0.0.27] to [0084.0.0.27] above.
[16995] [0085.0.43.43] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [16996] a) a nucleic acid sequence as
indicated in Table XI, application no. 43, columns 5 or 7, or a
derivative thereof, or [16997] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 43, columns
5 or 7, or a derivative thereof, or [16998] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[16999] [0086.0.0.43] and [0087.0.0.43] for the disclosure of the
paragraphs [0086.0.0.43] and [0087.0.0.43] see paragraphs
[0086.0.0.27] and [0087.0.0.27] above.
[17000] [0088.0.43.43] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned fine
chemicals or the plants are more resistant to biotic and abiotic
stress and the yield is increased.
[17001] [0088.1.0.43] for the disclosure of this paragraph see
paragraph [0088.1.0.27] above.
[17002] [0089.0.0.43] to [0094.0.0.43] for the disclosure of the
paragraphs [0089.0.0.43] to [0094.0.0.43] see paragraphs
[0089.0.0.27] to [0094.0.0.27] above.
[17003] [0095.0.43.43] It may be advantageous to increase the pool
of glycerol and/or total lipid in the transgenic organisms by the
process according to the invention in order to isolate high amounts
of the pure respective fine chemical and/or to obtain increased
resistance against biotic and abiotic stresses and to obtain higher
yield.
[17004] [0096.0.43.43] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptid or a compound, which
functions as a sink for the desired fine chemical, for example in
the organism, is useful to increase the production of the
respective fine chemical.
[17005] [0097.0.0.43] for the disclosure of this paragraph see
paragraph [0097.0.0.27] above.
[17006] [0098.0.43.43] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated.
[17007] [0099.0.43.43] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[17008] [0100.0.43.43] Transgenic plants which comprise the fine
chemicals such as glycerol and/or total lipids synthesized in the
process according to the invention can advantageously be marketed
directly without there being any need for the fine chemicals
synthesized to be isolated. Plants for the process according to the
invention are listed as meaning intact plants and all plant parts,
plant organs or plant parts such as leaf, stem, seeds, root,
tubers, anthers, fibers, root hairs, stalks, embryos, calli,
cotelydons, petioles, harvested material, plant tissue,
reproductive tissue and cell cultures which are derived from the
actual transgenic plant and/or can be used for bringing about the
transgenic plant. In this context, the seed comprises all parts of
the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue.
[17009] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, as extracts, e.g. ether, alcohol,
or other organic solvents or water containing extract and/or free
fine chemicals. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. To allow for
greater ease of disruption of the plant parts, specifically the
seeds, they can previously be comminuted, steamed or roasted.
Seeds, which have been pretreated in this manner can subsequently
be pressed or extracted with solvents such as warm hexane. The
solvent is subsequently removed. In the case of microorganisms, the
latter are, after harvesting, for example extracted directly
without further processing steps or else, after disruption,
extracted via various methods with which the skilled worker is
familiar. Thereafter, the resulting products can be processed
further, i.e. degummed and/or refined. In this process, substances
such as the plant mucilages and suspended matter can be first
removed. What is known as desliming can be affected enzymatically
or, for example, chemico-physically by addition of acid such as
phosphoric acid.
[17010] Because glycerol and/or total lipids in microorganisms are
localized intracellular, their recovery essentially comes down to
the isolation of the biomass. Well-established approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Of the residual
hydrocarbon, adsorbed on the cells, has to be removed. Solvent
extraction or treatment with surfactants have been suggested for
this purpose.
[17011] [0101.0.43.43] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[17012] [0102.0.43.43] Glycerol can for example be analyzed
advantageously via HPLC, LC or GC separation and MS
(masspectrometry) detection methods. The unambiguous detection for
the presence of glycerol containing products can be obtained by
analyzing recombinant organisms using analytical standard methods:
LC, LC-MS, MS or TLC). The material to be analyzed can be disrupted
by sonication, grinding in a glass mill, liquid nitrogen and
grinding, cooking, or via other applicable methods.
[17013] Total lipids in the seed of plants can for example be
analyzed advantageously by lipid extraction as described by Bligh
and Dyer (Can J Biochem Phys 1959, 37, 911-917) followed by the
determination of the lipid content by gaschromatography as
described by Benning and Sommerville (J Bacteriol 1992, 174,
6479-6487).
[17014] [0103.0.43.43] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [17015] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide having a
sequence as indicated in Table XII, application no. 43, columns 5
or 7, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [17016] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table XI, application no. 43, columns 5 or 7;
[17017] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [17018] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[17019] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [17020]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [17021] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [17022] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primer pairs
having a sequence as indicated in Table XIII, application no. 43,
column 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [17023]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [17024] j) nucleic acid molecule which
encodes a polypeptide comprising the consensus sequence having
sequences as indicated in Table XIV, application no. 43, column 7,
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [17025] k) nucleic acid
molecule comprising one or more of the nucleic acid molecule
encoding the amino acid sequence of a polypeptide encoding a domain
of the polypeptide indicated in Table XII, application no. 43,
columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[17026] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[17027] [00103.1.43.43] In one embodiment, the nucleic acid
molecule used in the process of the invention distinguishes over
the sequence indicated in Table XI, application no. 43, columns 5
or 7 by one or more nucleotides. In one embodiment, the nucleic
acid molecule used in the process of the invention does not consist
of the sequence shown in indicated in Table XI, application no. 43,
columns 5 or 7. In one embodiment, the nucleic acid molecule used
in the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI,
application no.
[17028] 43, columns 5 or 7. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table XII, application no. 43, columns 5 or 7.
[17029] [0104.0.43.43] In one embodiment, the nucleic acid molecule
used in the process of the present invention distinguishes over the
sequence indicated in Table XI, application no. 43, columns 5 or 7,
by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence indicated in Table XI, application no. 43, columns 5
or 7. In one embodiment, the nucleic acid molecule of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical to the sequence indicated in Table XI, application no.
43, columns 5 or 7. In another embodiment, the nucleic acid
molecule does not encode a polypeptide of a sequence indicated in
Table XII, application no. 43, columns 5 or 7.
[17030] [0105.0.0.43] to [0107.0.0.43] for the disclosure of the
paragraphs [0105.0.0.43] to [0107.0.0.43] see paragraphs
[0105.0.0.27] to [0107.0.0.27] above.
[17031] [0108.0.43.43] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 43, columns 5 or 7, nucleic
acid molecules which are derived from an amino acid sequences as
indicated in Table XII, application no. 43, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 43, column 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of an activity of a polypeptide as indicated in Table XII,
application no. 43, column 3, 5 or 7, e.g. conferring the increase
of the respective fine chemical, meaning glycerol and/or total
lipid, resp., after increasing its expression or activity, are
advantageously increased in the process according to the
invention.
[17032] [0109.0.43.43] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular glycerol
and/or total lipids, produced in the process according to the
invention.
[17033] [0110.0.43.43] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide used in the
method of the invention or used in the process of the invention,
e.g. of a protein as shown in Table XII, application no. 43,
columns 5 or 7 or being encoded by a nucleic acid molecule
indicated in Table XI, application no. 43, columns 5 or 7, or of
its homologs, e.g. as indicated in Table XII, application no. 43,
columns 5 or 7, can be determined from generally accessible
databases.
[17034] [0111.0.0.43] for the disclosure of this paragraph see
[0111.0.0.27] above.
[17035] [0112.0.43.43] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XII, application no. 43,
column 3, lines 173, 262 to 274 and 628 to 632 or having the
sequence of a polypeptide as indicated in Table XII, application
no. 43, columns 5 and 7, and conferring an increase in the glycerol
and/or total lipid level.
[17036] [0113.0.0.43] to [0120.0.0.43] for the disclosure of the
paragraphs [0113.0.0.43] to [0120.0.0.43] see paragraphs
[0113.0.0.27] and [0120.0.0.27] above.
[17037] [0121.0.43.43] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 43, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring a glycerol and/or total lipid level
increase after increasing the activity of the polypeptide sequences
indicated in Table XII, application no. 43, columns 5 or 7.
[17038] [0122.0.0.43] to [0127.0.0.43] for the disclosure of the
paragraphs [0122.0.0.43] to [0127.0.0.43] see paragraphs
[0122.0.0.27] and [0127.0.0.27] above.
[17039] [0128.0.43.43] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 43, column 7, by means of polymerase chain reaction
can be generated on the basis of a sequence shown herein, for
example the sequence as indicated in Table XI, application no. 43,
columns 5 or 7, or the sequences derived from a sequences as
indicated in Table XII, application no. 43, columns 5 or 7.
[17040] [0129.0.43.43] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved regions are those, which show a
very little variation in the amino acid in one particular position
of several homologs from different origin. The consensus sequence
indicated in Table XIV, application no. 43, columns 7, is derived
from such alignments.
[17041] [0130.0.43.43] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of glycerol
and/or total lipid after increasing the expression or activity the
protein comprising said fragment.
[17042] [0131.0.0.43] to [0138.0.0.43] for the disclosure of the
paragraphs [0131.0.0.43] to [0138.0.0.43] see paragraphs
[0131.0.0.27] to [0138.0.0.27] above.
[17043] [0139.0.43.43] Polypeptides having above-mentioned
activity, i.e. conferring the respective fine chemical level
increase, derived from other organisms, can be encoded by other DNA
sequences which hybridise to a sequence indicated in Table XI,
application no. 43, columns 5 or 7, for glycerol, and/or total
lipid under relaxed hybridization conditions and which code on
expression for peptides having the respective fine chemical, i.e.
glycerol and/or total lipids, resp., increasing-activity.
[17044] [0140.0.0.43] to [0146.0.0.43] for the disclosure of the
paragraphs [0140.0.0.43] to [0146.0.0.43] see paragraphs
[0140.0.0.27] to [0146.0.0.27] above.
[17045] [0147.0.43.43] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 43, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences s
such that it can hybridize to one of said nucleotide sequences
thereby forming a stable duplex. Preferably, the hybridisation is
performed under stringent hybridization conditions. However, a
complement of one of the herein disclosed sequences is preferably a
sequence complement thereto according to the base pairing of
nucleic acid molecules well known to the skilled person. For
example, the bases A and G undergo base pairing with the bases T
and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[17046] [0148.0.43.43] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 43,
columns 5 or 7 or a portion thereof and preferably has above
mentioned activity, in particular, of glycerol increasing activity
after increasing the activity or an activity of a product of a gene
encoding said sequences or their homologs.
[17047] [0149.0.43.43] The nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 43,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity and as indicated in indicated in Table
XII.
[17048] [00149.1.43.43] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table XI, application no. 43, columns 5 or
7, has further one or more of the activities annotated or known for
a protein as indicated in Table XII, application no. 43, column
3.
[17049] [0150.0.43.43] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table XI, application no. 43, columns
5 or 7, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of glycerol, resp., if its
activity is increased. The nucleotide sequences determined from the
cloning of the present protein-according-to-the-invention-encoding
gene allows for the generation of probes and primers designed for
use in identifying and/or cloning its homologues in other cell
types and organisms. The probe/primer typically comprises
substantially purified oligonucleotide. The oligonucleotide
typically comprises a region of nucleotide sequence that hybridizes
under stringent conditions to at least about 12, 15 preferably
about 20 or 25, more preferably about 40, 50 or 75 consecutive
nucleotides of a sense strand of one of the sequences set forth,
e.g., as indicated in Table XI, application no. 43, columns 5 or 7,
an anti-sense sequence of one of the sequences, e.g., as indicated
in Table XI, application no. 43, columns 5 or 7, or naturally
occurring mutants thereof. Primers based on a nucleotide of
invention can be used in PCR reactions to clone homologues of the
polypeptide of the invention or of the polypeptide used in the
process of the invention, e.g. as the primers described in the
examples of the present invention, e.g. as shown in the examples. A
PCR with the primer pairs indicated in Table XIII, application no.
43, column 7, will result in a fragment of a polynucleotide
sequence as indicated in Table XI, application no. 43, columns 5 or
7 or its gene product.
[17050] [0151.0.0.43]: for the disclosure of this paragraph see
paragraph [0151.0.0.27] above.
[17051] [0152.0.43.43] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 43, columns 5
or 7, such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular a glycerol and total lipid increasing activity as
mentioned above or as described in the examples in plants or
microorganisms is comprised.
[17052] [0153.0.43.43] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 43,
columns 5 or 7, such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 43, columns 5 or 7, has for
example an activity of a polypeptide indicated in Table XII,
application no. 43, column 3.
[17053] [0154.0.43.43] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 43, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[17054] [0155.0.0.43] and [0156.0.0.43] for the disclosure of the
paragraphs [0155.0.0.43] and [0156.0.0.43] see paragraphs
[0155.0.0.27] and [0156.0.0.27] above.
[17055] [0157.0.43.43] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences as
indicated in Table XI, application no. 43, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
polypeptides comprising the sequence as indicated in Table XIV,
application no. 43, column 7, or as polypeptides depicted in Table
XII, application no. 43, columns 5 or 7, or the functional
homologues. Advantageously, the nucleic acid molecule of the
invention comprises, or in an other embodiment has, a nucleotide
sequence encoding a protein comprising, or in an other embodiment
having, an amino acid sequence of a consensus sequences as
indicated in Table XIV, application no. 43, column 7, or of the
polypeptide as indicated in Table XII, application no. 43, columns
5 or 7, or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
XIV, application no. 43, column 7, or of a polypeptide as indicated
in Table XII, application no. 43, columns 5 or 7, or the functional
homologues. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 43, columns 5 or 7.
[17056] [0158.0.0.43] to [0160.0.0.43] for the disclosure of the
paragraphs [0158.0.0.43] to [0160.0.0.43] see paragraphs
[0158.0.0.27] to [0160.0.0.27] above.
[17057] [0161.0.43.43] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no.
[17058] 43, columns 5 or 7. The nucleic acid molecule is preferably
at least 20, 30, 50, 100, 250 or more nucleotides in length.
[17059] [0162.0.0.43] for the disclosure of this paragraph see
paragraph [0162.0.0.27] above.
[17060] [0163.0.43.43] Preferably, a nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 43, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the increase of
the amount of the respective fine chemical in an organism or a part
thereof, e.g. a tissue, a cell, or a compartment of a cell, after
increasing the expression or activity thereof or the activity of a
protein of the invention or used in the process of the
invention.
[17061] [0164.0.0.43] for the disclosure of this paragraph see
paragraph [0164.0.0.27] above.
[17062] [0165.0.43.43] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 43, columns 5 or 7.
[17063] [0166.0.0.43] and [0167.0.0.43] for the disclosure of the
paragraphs [0166.0.0.43] and [0167.0.0.43] see paragraphs
[0166.0.0.27] and [0167.0.0.27] above.
[17064] [0168.0.43.43] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 43, columns 5 or 7, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 43,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table XII, application no. 43, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 43, columns 5
or 7, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table XII, application no.
43, columns 5 or 7, and most preferably at least about 96%, 97%,
98%, or 99% identical to the sequence as indicated in Table XII,
application no. 43, columns 5 or 7.
[17065] [0169.0.0.43] to [0172.0.0.43] for the disclosure of the
paragraphs [0169.0.0.43] to [0172.0.0.43] see paragraphs
[0169.0.0.27] to [0172.0.0.27] above.
[17066] [0173.0.43.43] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108401 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108401 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[17067] [0174.0.0.43]: for the disclosure of this paragraph see
paragraph [0174.0.0.27] above.
[17068] [0175.0.43.43] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108402 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108402 by the above program algorithm with the
above parameter set, has a 80% homology.
[17069] [0176.0.43.43] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 43,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 43, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 43, columns
5 or 7.
[17070] [0177.0.43.43] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 43,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptide as indicated in Table XII,
application no. 43, columns 5 or according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 43, columns
5 or 7.
[17071] [0178.0.0.43] for the disclosure of this paragraph see
[0178.0.0.27] above.
[17072] [0179.0.43.43] A nucleic acid molecule encoding a homologue
to a protein sequence as indicated in Table XII, application no.
43, columns 5 or 7, can be created by introducing one or more
nucleotide substitutions, additions or deletions into a nucleotide
sequence of the nucleic acid molecule of the present invention, in
particular as indicated in Table XI, application no. 43, columns 5
or 7, such that one or more amino acid substitutions, additions or
deletions are introduced into the encoded protein. Mutations can be
introduced into the encoding sequences as indicated in Table XI,
application no. 43, columns 5 or 7, by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis.
[17073] [0180.0.0.43] to [0183.0.0.43] for the disclosure of the
paragraphs [0180.0.0.43] to [0183.0.0.43] see paragraphs
[0180.0.0.27] to [0183.0.0.27] above.
[17074] [0184.0.43.43] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 43,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 43, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 43,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[17075] [0185.0.43.43] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 43, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table XI, application no. 43, columns 5 or 7. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequences as indicated in Table XI, application no. 43, columns 5
or 7.
[17076] [0186.0.43.43] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table XII,
application no. 43, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 43, columns 5 or 7.
[17077] [0187.0.43.43] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table XII, application no.
43, columns 5 or 7, comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence
encoding a sequences as indicated in Table XII, application no. 43,
columns 5 or 7.
[17078] [0188.0.43.43] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 43, columns 5 or 7 and is expressed under
identical conditions.
[17079] [0189.0.43.43] Homologues of a sequences as indicated in
Table XI, application no.
[17080] 43, columns 5 or 7, or of a derived sequences as indicated
in Table XII, application no. 43, columns 5 or 7 also mean
truncated sequences, cDNA, single-stranded DNA or RNA of the coding
and noncoding DNA sequence. Homologues of said sequences are also
understood as meaning derivatives, which comprise noncoding regions
such as, for example, UTRs, terminators, enhancers or promoter
variants. The promoters upstream of the nucleotide sequences stated
can be modified by one or more nucleotide substitution(s),
insertion(s) and/or deletion(s) without, however, interfering with
the functionality or activity either of the promoters, the open
reading frame (=ORF) or with the 3'-regulatory region such as
terminators or other 3' regulatory regions, which are far away from
the ORF. It is furthermore possible that the activity of the
promoters is increased by modification of their sequence, or that
they are replaced completely by more active promoters, even
promoters from heterologous organisms. Appropriate promoters are
known to the person skilled in the art and are mentioned herein
below.
[17081] [0190.0.0.43]: for the disclosure of this paragraph see
[0190.0.0.27] above.
[17082] [0191.0.43.43] In one embodiment, the organisms or part
thereof produce according to the herein mentioned process of the
invention an increased level of free and/or bound the respective
fine chemical compared to said control or selected organisms or
parts thereof.
[17083] [0191.1.0.43]: for the disclosure of this paragraph see
[0191.1.0.27] above.
[17084] [0192.0.0.43] to [0203.0.0.43] for the disclosure of the
paragraphs [0192.0.0.43] to [0203.0.0.43] see paragraphs
[0192.0.0.27] to [0203.0.0.27] above.
[17085] [0204.0.43.43] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule which comprises a
nucleic acid molecule selected from the group consisting of:
[17086] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 43, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII, application no. 43,
column 6 in an organism or a part thereof [17087] b) nucleic acid
molecule comprising, preferably at least the mature form, of a
nucleic acid molecule as indicated in Table XI, application no. 43,
columns 5 or 7, or a fragment thereof conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [17088] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [17089] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[17090] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [17091] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [17092] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [17093] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table XIII, application no. 43, column 7, and
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII, application no. 43,
column 6 in an organism or a part thereof; [17094] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[17095] j) nucleic acid molecule which encodes a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 43, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical, in particular according
to table XII, application no. 43, column 6 in an organism or a part
thereof; [17096] k) nucleic acid molecule encoding the amino acid
sequence of a polypeptide encoding a domaine of a polypeptide as
indicated in Table XII, application no. 43, columns 5 or 7, and
conferring an increase in the amount of the respective fine
chemical, in particular accccording to table XII, application no.
43, column 6 in an organism or a part thereof; and [17097] l)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (h) or of a nucleic
acid molecule as indicated in Table XI, application no. 43, columns
5 or 7, or a nucleic acid molecule encoding, preferably at least
the mature form of, a polypeptide as indicated in Table XII,
application no. 43, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical according to table XII,
application no. 43, column 6 in an organism or a part thereof;
[17098] or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over the sequence indicated in Table
XI, application no. 43, columns 5 or 7, by one or more nucleotides.
In one embodiment, the nucleic acid molecule does not consist of
the sequence shown and indicated in Table XI, application no. 43,
columns 5 or 7, In one embodiment, the nucleic acid molecule is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 43, columns 5 or
7.
[17099] In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table XII,
application no. 43, columns 5 or 7.
[17100] In an other embodiment, the nucleic acid molecule of the
present invention is at least 30%, 40%, 50%, or 60% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 43, columns 5 or
7.
[17101] In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table XII,
application no. 43, columns 5 or 7.
[17102] Accordingly, in one embodiment, the nucleic acid molecule
of the differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 43, columns 5 or
7.
[17103] Accordingly, in one embodiment, the nucleic acid molecule
of the present invention encodes a polypeptide, which differs at
least in one or more amino acids from a polypeptide indicated in
Table XII, application no. 43, columns 5 or 7.
[17104] In another embodiment, a nucleic acid molecule indicated in
Table XI, application no. 43, columns 5 or 7.
[17105] Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table XII, application no.
43, columns 5 or 7.
[17106] In a further embodiment, the protein of the present
invention is at least 30%, 40%, 50%, or 60% identical to a protein
sequence indicated in Table XII, application no. 43, columns 5 or
7, and less than 100%, preferably less than 99.999%, 99.99% or
99.9%, more preferably less than 99%, 985, 97%, 96% or 95%
identical to a sequence as indicated in Table XI, columns 5 or
7.
[17107] [0205.0.0.43] to [0206.0.0.43]: see [0205.0.0.27] to
[0206.0.0.27]
[17108] [0207.0.43.43] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the glutamic acid metabolism, the
phosphoenolpyruvate metabolism, the amino acid metabolism, of
glycolysis, of the tricarboxylic acid metabolism or their
combinations. As described herein, regulator sequences or factors
can have a positive effect on preferably the gene expression of the
genes introduced, thus increasing it. Thus, an enhancement of the
regulator elements may advantageously take place at the
transcriptional level by using strong transcription signals such as
promoters and/or enhancers. In addition, however, an enhancement of
translation is also possible, for example by increasing mRNA
stability or by inserting a translation enhancer sequence.
[17109] [0208.0.0.43] to [0226.0.0.43] for the disclosure of the
paragraphs [0208.0.0.43] to [0226.0.0.43] see paragraphs
[0208.0.0.27] to [0226.0.0.27] above.
[17110] [0227.0.43.43] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[17111] In addition to a sequence indicated in Table XI,
application no. 43, columns 5 or 7, or its derivatives, it is
advantageous to express and/or mutate further genes in the
organisms. Especially advantageously, additionally at least one
further gene of the acetyl-CoA or malonyl-CoA metabolic pathway or
a polypeptide having a very long chain fatty acid acyl (VLCFA) CoA
synthase activity, is expressed in the organisms such as plants or
microorganisms. It is also possible that the regulation of the
natural genes has been modified advantageously so that the gene
and/or its gene product is no longer subject to the regulatory
mechanisms which exist in the organisms. This leads to an increased
synthesis of the fine chemicals desired since, for example,
feedback regulations no longer exist to the same extent or not at
all. In addition it might be advantageously to combine one or more
of the sequences indicated in Table XI, application no. 43, columns
5 or 7, with genes which generally support or enhance to growth or
yield of the target organisms, for example genes which lead to
faster growth rate of microorganisms or genes which produces
stress-, pathogen, or herbicide resistant plants.
[17112] [0228.0.43.43] In a further embodiment of the process of
the invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the glycerol
metabolism, in particular in synthesis of glycerol.
[17113] [0229.0.43.43] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences indicated
in Table XI, application no. 43, columns 5 or 7, used in the
process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the fatty acid pathway, such as
acetyl-CoA or malonyl-CoA or a polypeptide having a very long chain
fatty acid acyl (VLCFA) CoA synthase activity. These genes can lead
to an increased synthesis of the VLCFAs.
[17114] [0230.0.0.43] for the disclosure of this paragraph see
paragraph [0230.0.0.27] above.
[17115] [0231.0.43.43] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a glycerol and/or a lipid degrading
protein is attenuated, in particular by reducing the rate of
expression of the corresponding gene. A person skilled in the art
knows for example, that the inhibition or repression of a glycerol
or lipid degrading enzyme will result in an increased accumulation
of glycerol and/or total lipids in plants.
[17116] [0232.0.0.43] to [0276.0.0.43] for the disclosure of the
paragraphs [0232.0.0.43] to [0276.0.0.43] see paragraphs
[0232.0.0.27] to [0276.0.0.27] above.
[17117] [0277.0.43.43] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously.
[17118] [0278.0.0.43] to [0282.0.0.43] for the disclosure of the
paragraphs [0278.0.0.43] to [0282.0.0.43] see paragraphs
[0278.0.0.27] to [0282.0.0.27] above.
[17119] [0283.0.43.43] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells, for example using the antibody
of the present invention as described below, e.g. an antibody
against a protein as indicated in Table XII, application no. 43,
column 3, or an antibody against a polypeptide as indicated in
Table XII, application no. 43, columns 5 or 7, which can be
produced by standard techniques utilizing the polypeptid of the
present invention or fragment thereof. Preferred are monoclonal
antibodies specifically binding to polypeptides as indicated in
Table XII, application no. 43, columns 5 or 7, more preferred
specifically binding to polypeptides as indicated in Table XII,
application no. 43, column 5.
[17120] [0284.0.0.43] for the disclosure of this paragraph see
[0284.0.0.27] above.
[17121] [0285.0.43.43] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 43, columns 5 or 7, or as coded by a nucleic
acid molecule as indicated in Table XI, application no. 43, columns
5 or 7, or functional homologues thereof.
[17122] [0286.0.43.43] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 43, column 7, and in one
another embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 43, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid or, in an further
embodiment, can be replaced and/or absent. In one embodiment, the
present invention relates to the method of the present invention
comprising a polypeptide or to a polypeptide comprising more than
one consensus sequences (of an individual line) as indicated in
Table XIV, application no. 43, column 7.
[17123] [0287.0.0.43] to [0290.0.0.43] for the disclosure of the
paragraphs [0287.0.0.43] to [0290.0.0.43] see paragraphs
[0287.0.0.27] to [0290.0.0.27] above.
[17124] [0291.0.43.43] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[17125] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table XII,
application no. 43, columns 5 or 7, by one or more amino acids.
[17126] In one embodiment, polypeptide distinguishes form a
sequence as indicated in Table XII, application no. 43, columns 5
or 7, by more than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids,
preferably by more than 10, 15, 20, 25 or 30 amino acids, even more
preferred are more than 40, 50, or 60 amino acids and, preferably,
the sequence of the polypeptide of the invention distinguishes from
a sequence as indicated in Table XII, application no. 43, columns 5
or 7, by not more than 80% or 70% of the amino acids, preferably
not more than 60% or 50%, more preferred not more than 40% or 30%,
even more preferred not more than 20% or 10%. In an other
embodiment, said polypeptide of the invention does not consist of a
sequence as indicated in Table XII, application no. 43, columns 5
or 7.
[17127] [0292.0.0.43] for the disclosure of this paragraph see
[0292.0.0.27] above.
[17128] [0293.0.43.43] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, the polypeptide of
the invention has a sequence which distinguishes from a sequence as
indicated in Table XII, application no. 43, columns 5 or 7, by one
or more amino acids. In an other embodiment, said polypeptide of
the invention does not consist of the sequence as indicated in
Table XII, application no. 43, columns 5 or 7. In a further
embodiment, said polypeptide of the present invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical. In one embodiment,
said polypeptide does not consist of the sequence encoded by a
nucleic acid molecules as indicated in Table XI, application no.
43, columns 5 or 7.
[17129] [0294.0.43.43] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 43, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 43, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[17130] [0295.0.0.43] to [0297.0.0.43] for the disclosure of the
paragraphs [0295.0.0.43] to [0297.0.0.43] see paragraphs
[0295.0.0.27] to [0297.0.0.27] above.
[17131] [00297.1.43.43] Non polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table XII, application no. 43, columns 3, 5 or 7.
[17132] [0298.0.43.43] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 43, columns 5 or 7. The
portion of the protein is preferably a biologically active portion
as described herein. Preferably, the polypeptide used in the
process of the invention has an amino acid sequence identical to a
sequence as indicated in Table XII, application no. 43, columns 5
or 7.
[17133] [0299.0.43.43] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 43,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 43, columns 5 or 7, or which is
homologous thereto, as defined above.
[17134] [0300.0.43.43] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 43, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 43, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[17135] [0301.0.0.43] for the disclosure of this paragraph see
[0301.0.0.27] above.
[17136] [0302.0.43.43] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 43, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[17137] [0303.0.0.43] for the disclosure of this paragraph see
[0303.0.0.27] above.
[17138] [0304.0.43.43] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 43, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[17139] [0305.0.0.43] to [0308.0.0.43] for the disclosure of the
paragraphs [0305.0.0.43] to [0308.0.0.43] see paragraphs
[0305.0.0.27] to [0308.0.0.27] above.
[17140] [0306.1.43.43]%
[17141] [0309.0.43.43] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table XII,
application no. 43, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas an
"other polypeptide" not being indicated in Table XII, application
no. 43, columns 5 or 7, refers to a polypeptide having an amino
acid sequence corresponding to a protein which is not substantially
homologous to a polypeptide of the invention, preferably which is
not substantially homologous to a polypeptide as indicated in Table
XII, application no. 43, columns 5 or 7, e.g., a protein which does
not confer the activity described herein or annotated or known for
as indicated in Table XII, application no. 43, column 3, and which
is derived from the same or a different organism. In one
embodiment, an "other polypeptide" not being indicated in Table
XII, application no. 43, columns 5 or 7, does not confer an
increase of the respective fine chemical in an organism or part
thereof.
[17142] [0310.0.0.43] to [0334.0.0.43] for the disclosure of the
paragraphs [0310.0.0.43] to [0334.0.0.43] see paragraphs
[0310.0.0.27] to [0334.0.0.27] above.
[17143] [0335.0.43.43] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XI, application
no. 43, columns 5 or 7, and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived there from),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
application no. 43, columns 5 or 7, and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of a
protein encoded by a nucleic acid sequence as indicated in Table
XI, application no. 43, columns 5 or 7, and/or homologs thereof,
one of the two RNA strands is essentially identical to at least
part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[17144] [0336.0.0.43] to [0342.0.0.43] for the disclosure of the
paragraphs [0336.0.0.43] to [0342.0.0.43] see paragraphs
[0336.0.0.27] to [0342.0.0.27] above.
[17145] [0343.0.43.43] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 43, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table XI, application no. 43, columns 5 or 7, or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[17146] [0344.0.0.43] to [0361.0.0.43] for the disclosure of the
paragraphs [0344.0.0.43] to [0361.0.0.43] see paragraphs
[0344.0.0.27] to [0361.0.0.27] above.
[17147] [0362.0.43.43] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 43, columns 5 or 7, e.g. encoding a
polypeptide having protein activity, as indicated in Table XII,
application no. 43, columns 3. Due to the above-mentioned activity
the respective fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity of the polypeptide of the invention or nucleic
acid molecule of the invention is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a polypeptide as
indicated in Table XII, application no. 43, columns 5 or 7, means
herein that due to modulation or manipulation of the genome, an
activity as annotated for a polypeptide as indicated in Table XII,
application no. 43, column 3, e.g. having a sequence as indicated
in Table XII, application no. 43, columns 5 or 7, is increased in a
cell or an organism or a part thereof. Examples are described above
in context with the process of the invention.
[17148] [0363.0.0.43] for the disclosure of this paragraph see
paragraph [0363.0.0.27] above.
[17149] [0364.0.43.43] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table XII, application no. 43, column 3, with the
corresponding protein-encoding sequence as indicated in Table XI,
application no. 43, column 5, becomes a transgenic expression
cassette when it is modified by non-natural, synthetic "artificial"
methods such as, for example, mutagenization. Such methods have
been described (U.S. Pat. No. 5,565,350; WO 00/15815; also see
above).
[17150] [0365.0.0.43] to [0373.0.0.43] for the disclosure of the
paragraphs [0365.0.0.43], to [0373.0.0.43] see paragraphs
[0365.0.0.27] to [0373.0.0.27] above.
[17151] [0374.0.43.43] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. Glycerol, in particular the
respective fine chemical, produced in the process according to the
invention may, however, also be isolated from the plant in the form
of their free glycerol, in particular the free respective fine
chemical, or bound in or to compounds or moieties, as for example
but not limited to in mono-, di- or triacylglyceroles,
phosphoglycerides, monoacylglycerol phosphate or diacylglycerol
phosphate. The respective fine chemical produced by this process
can be harvested by harvesting the organisms either from the
culture in which they grow or from the field. This can be done via
expressing, grinding and/or extraction, salt precipitation and/or
ion-exchange chromatography or other chromatographic methods of the
plant parts, preferably the plant seeds, plant fruits, plant tubers
and the like.
[17152] [0375.0.0.43] and [0376.0.0.43] for the disclosure of the
paragraphs [0375.0.0.43] and [0376.0.0.43] see paragraphs
[0375.0.0.27] and [0376.0.0.27] above.
[17153] [0377.0.43.43] Accordingly, the present invention relates
also to a process whereby the produced glycerol and/or total lipid
is isolated.
[17154] [0378.0.43.43] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the glycerol
and/or total lipid produced in the process can be isolated. The
resulting glycerol and/or total lipid can, if appropriate,
subsequently be further purified, if desired mixed with other
active ingredients such as vitamins, amino acids, carbohydrates,
antibiotics and the like, and, if appropriate, formulated.
[17155] [0379.0.43.43] In one embodiment the product produced by
the present invention is a mixture of the respective fine chemicals
glycerol and total lipids.
[17156] [0380.0.43.43] The glycerol or total lipids obtained in the
process by carrying out the invention is suitable as starting
material for the synthesis of further products of value. For
example, they can be used in combination with each other or alone
for the production of pharmaceuticals, foodstuffs, animal feeds or
cosmetics. Accordingly, the present invention relates to a method
for the production of pharmaceuticals, food stuff, animal feeds,
nutrients or cosmetics comprising the steps of the process
according to the invention, including the isolation of the glycerol
composition produced or the respective fine chemical produced if
desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the glycerol and/or total lipid
produced in the process or of the transgenic organism in animal
feeds, foodstuffs, medicines, food supplements, cosmetics or
pharmaceuticals or for the production of glycerol e.g. after
isolation of the respective fine chemical or without, e.g. in situ,
e.g in the organism used for the process for the production of the
respective fine chemical.
[17157] [0381.0.0.43] and [0382.0.0.43] for the disclosure of the
paragraphs [0381.0.0.43] and [0382.0.0.43] see paragraphs
[0381.0.0.27] and [0382.0.0.27] above.
[17158] [0383.0.43.43] %
[17159] [0384.0.0.43] for the disclosure of this paragraph see
[0384.0.0.27] above.
[17160] [0385.0.43.43] The fermentation broths obtained in this
way, containing in particular glycerol and/or total lipids in
mixtures with other organic acids, amino acids, polypeptides or
polysaccarides, normally have a dry matter content of from 1 to 70%
by weight, preferably 7.5 to 25% by weight. Sugar-limited
fermentation is additionally advantageous, e.g. at the end, for
example over at least 30% of the fermentation time. This means that
the concentration of utilizable sugar in the fermentation medium is
kept at, or reduced to, 0 to 10 g/l, preferably to 0 to 3 g/l
during this time. The fermentation broth is then processed further.
Depending on requirements, the biomass can be removed or isolated
entirely or partly by separation methods, such as, for example,
centrifugation, filtration, decantation, coagulation/flocculation
or a combination of these methods, from the fermentation broth or
left completely in it.
[17161] The fermentation broth can then be thickened or
concentrated by known methods, such as, for example, with the aid
of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[17162] [0386.0.43.43] Accordingly, it is possible to purify the
glycerol produced according to the invention further. For this
purpose, the product-containing composition is subjected for
example to separation via e.g. an open column chromatography or
HPLC in which case the desired product or the impurities are
retained wholly or partly on the chromatography resin. These
chromatography steps can be repeated if necessary, using the same
or different chromatography resins. The skilled worker is familiar
with the choice of suitable chromatography resins and their most
effective use.
[17163] [0387.0.0.43] to [0392.0.0.43] for the disclosure of the
paragraphs [0387.0.0.43] to [0392.0.0.43] see paragraphs
[0387.0.0.27] to [0392.0.0.27] above.
[17164] [0393.0.43.43] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [17165] a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [17166] b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table XI, application no. 43, columns 5 or 7, and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[17167] c) introducing the candidate nucleic acid molecules in host
cells, preferably in a plant cell or a microorganism, appropriate
for producing the fine chemical; [17168] d) expressing the
identified nucleic acid molecules in the host cells; [17169] e)
assaying the the fine chemical level in the host cells; and [17170]
f) identifying the nucleic acid molecule and its gene product which
expression confers an increase in the the fine chemical level in
the host cell after expression compared to the wild type.
[17171] [0394.0.32.43] to [0552.0.32.43] for the disclosure of the
paragraphs [0394.0.32.43] to [0552.0.32.43] see paragraphs
[0394.0.0.32] to [0552.0.0.32] above.
[17172] [0553.0.43.43]
1. A process for the production of glycerol resp., which comprises
(a) increasing or generating the activity of a protein as indicated
in Table XII, application no. 43, columns 5 or 7, or a functional
equivalent thereof in a non-human organism, or in one or more parts
thereof; and (b) growing the organism under conditions which permit
the production of glycerol resp. in said organism. 2. A process for
the production of glycerol resp., comprising the increasing or
generating in an organism or a part thereof the expression of at
least one nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: a) nucleic acid molecule
encoding of a polypeptide as indicated in Table XII, application
no. 43, columns 5 or 7, or a fragment thereof, which confers an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of glycerol resp., in an organism or a part
thereof; b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table XI, application no. 43, columns 5 or
7, c) nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of glycerol resp., in an
organism or a part thereof; d) nucleic acid molecule which encodes
a polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of glycerol
resp., in an organism or a part thereof; e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
under stringent hybridisation conditions and conferring an increase
in the amount of the fine chemical as indicated in table XII,
column 6, e.g of glycerol resp., in an organism or a part thereof;
f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table XIII, application no. 43, column 7, and
conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of glycerol resp., in an
organism or a part thereof; g) nucleic acid molecule encoding a
polypeptide which is isolated with the aid of monoclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (f) and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of glycerol
resp., in an organism or a part thereof; h) nucleic acid molecule
encoding a polypeptide comprising a consensus sequence as indicated
in Table XIV, application no. 43, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of glycerol resp., in an organism or a part
thereof; and i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of glycerol resp., in an organism or a part thereof. or
comprising a sequence which is complementary thereto. 3. The
process of claim 1 or 2, comprising recovering of the free or bound
glycerol resp. 4. The process of any one of claims 1 to 3,
comprising the following steps: (a) selecting an organism or a part
thereof expressing a polypeptide encoded by the nucleic acid
molecule characterized in claim 2; (b) mutagenizing the selected
organism or the part thereof; (c) comparing the activity or the
expression level of said polypeptide in the mutagenized organism or
the part thereof with the activity or the expression of said
polypeptide of the selected organisms or the part thereof; (d)
selecting the mutated organisms or parts thereof, which comprise an
increased activity or expression level of said polypeptide compared
to the selected organism or the part thereof; (e) optionally,
growing and cultivating the organisms or the parts thereof; and (f)
recovering, and optionally isolating, the free or bound glycerol
resp., produced by the selected mutated organisms or parts thereof.
5. The process of any one of claims 1 to 4, wherein the activity of
said protein or the expression of said nucleic acid molecule is
increased or generated transiently or stably. 6. An isolated
nucleic acid molecule comprising a nucleic acid molecule selected
from the group consisting of: a) nucleic acid molecule encoding of
a polypeptide as indicated in Table XII, application no. 43,
columns 5 or 7, or a fragment thereof, which confers an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of glycerol resp., in an organism or a part thereof; b)
nucleic acid molecule comprising of a nucleic acid molecule as
indicated in Table XI, application no. 43, columns 5 or 7, c)
nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of glycerol resp., in an
organism or a part thereof; d) nucleic acid molecule which encodes
a polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of glycerol
resp., in an organism or a part thereof; e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
under stringent hybridisation conditions and conferring an increase
in the amount of the fine chemical as indicated in table XII,
column 6, e.g of glycerol resp., in an organism or a part thereof;
f) nucleic acid molecule which encompasses a nucleic acid molecule
which is obtained by amplifying nucleic acid molecules from a cDNA
library or a genomic library using the primers or primer pairs as
indicated in Table XIII, application no. 43, column 7, and
conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of glycerol resp., in an
organism or a part thereof; g) nucleic acid molecule encoding a
polypeptide which is isolated with the aid of monoclonal antibodies
against a polypeptide encoded by one of the nucleic acid molecules
of (a) to (f) and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of glycerol
resp., in an organism or a part thereof; h) nucleic acid molecule
encoding a polypeptide comprising a consensus sequence as indicated
in Table XIV, application no. 43, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of glycerol resp., in an organism or a part
thereof; and i) nucleic acid molecule which is obtainable by
screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of glycerol resp., in an organism or a part thereof. whereby
the nucleic acid molecule distinguishes over the sequence as
indicated in Table XI, application no. 43, columns 5 or 7, by one
or more nucleotides. 7. A nucleic acid construct which confers the
expression of the nucleic acid molecule of claim 6, comprising one
or more regulatory elements. 8. A vector comprising the nucleic
acid molecule as claimed in claim 6 or the nucleic acid construct
of claim 7. 9. The vector as claimed in claim 8, wherein the
nucleic acid molecule is in operable linkage with regulatory
sequences for the expression in a prokaryotic or eukaryotic, or in
a prokaryotic and eukaryotic, host. 10. A host cell, which has been
transformed stably or transiently with the vector as claimed in
claim 8 or 9 or the nucleic acid molecule as claimed in claim 6 or
the nucleic acid construct of claim 7 or produced as described in
claim any one of claims 2 to 5. 11. The host cell of claim 10,
which is a transgenic host cell. 12. The host cell of claim 10 or
11, which is a plant cell, an animal cell, a microorganism, or a
yeast cell, a fungus cell, a prokaryotic cell, an eukaryotic cell
or an archaebacterium. 13. A process for producing a polypeptide,
wherein the polypeptide is expressed in a host cell as claimed in
any one of claims 10 to 12. 14. A polypeptide produced by the
process as claimed in claim 13 or encoded by the nucleic acid
molecule as claimed in claim 6 whereby the polypeptide
distinguishes over a sequence as indicated in Table XII,
application no. 43, columns 5 or 7, by one or more amino acids 15.
An antibody, which binds specifically to the polypeptide as claimed
in claim 14. 16. A plant tissue, propagation material, harvested
material or a plant comprising the host cell as claimed in claim 12
which is plant cell or an Agrobacterium. 17. A method for screening
for agonists and antagonists of the activity of a polypeptide
encoded by the nucleic acid molecule of claim 6 conferring an
increase in the amount of glycerol resp., in an organism or a part
thereof comprising: (a) contacting cells, tissues, plants or
microorganisms which express the a polypeptide encoded by the
nucleic acid molecule of claim 5 conferring an increase in the
amount of glycerol resp., in an organism or a part thereof with a
candidate compound or a sample comprising a plurality of compounds
under conditions which permit the expression the polypeptide; (b)
assaying the glycerol resp., level or the polypeptide expression
level in the cell, tissue, plant or microorganism or the media the
cell, tissue, plant or microorganisms is cultured or maintained in;
and (c) identifying a agonist or antagonist by comparing the
measured glycerol resp., level or polypeptide expression level with
a standard glycerol resp., or polypeptide expression level measured
in the absence of said candidate compound or a sample comprising
said plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. 18. A process for the
identification of a compound conferring increased glycerol resp.,
production in a plant or microorganism, comprising the steps: (a)
culturing a plant cell or tissue or microorganism or maintaining a
plant expressing the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
glycerol resp., in an organism or a part thereof and a readout
system capable of interacting with the polypeptide under suitable
conditions which permit the interaction of the polypeptide with
dais readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
glycerol resp., in an organism or a part thereof; (b) identifying
if the compound is an effective agonist by detecting the presence
or absence or increase of a signal produced by said readout system.
19. A method for the identification of a gene product conferring an
increase in glycerol resp., production in a cell, comprising the
following steps: (a) contacting the nucleic acid molecules of a
sample, which can contain a candidate gene encoding a gene product
conferring an increase in glycerol resp., after expression with the
nucleic acid molecule of claim 6; (b) identifying the nucleic acid
molecules, which hybridise under relaxed stringent conditions with
the nucleic acid molecule of claim 6; (c) introducing the candidate
nucleic acid molecules in host cells appropriate for producing
glycerol resp.; (d) expressing the identified nucleic acid
molecules in the host cells; (e) assaying the glycerol resp., level
in the host cells; and (f) identifying nucleic acid molecule and
its gene product which expression confers an increase in the
glycerol resp., level in the host cell in the host cell after
expression compared to the wild type. 20. A method for the
identification of a gene product conferring an increase in glycerol
resp., production in a cell, comprising the following steps: (a)
identifying in a data bank nucleic acid molecules of an organism;
which can contain a candidate gene encoding a gene product
conferring an increase in the glycerol resp., amount or level in an
organism or a part thereof after expression, and which are at least
20% homolog to the nucleic acid molecule of claim 6; (b)
introducing the candidate nucleic acid molecules in host cells
appropriate for producing glycerol resp.; (c) expressing the
identified nucleic acid molecules in the host cells; (d) assaying
the glycerol resp., level in the host cells; and (e) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the glycerol resp., level in the host cell after
expression compared to the wild type. 21. A method for the
production of an agricultural composition comprising the steps of
the method of any one of claims 17 to 20 and formulating the
compound identified in any one of claims 17 to 20 in a form
acceptable for an application in agriculture. 22. A composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of any
one of claim 8 or 9, an antagonist or agonist identified according
to claim 17, the compound of claim 18, the gene product of claim 19
or 20, the antibody of claim 15, and optionally an agricultural
acceptable carrier. 23. Use of the nucleic acid molecule as claimed
in claim 6 for the identification of a nucleic acid molecule
conferring an increase of glycerol resp., after expression. 24. Use
of the polypeptide of claim 14 or the nucleic acid construct claim
7 or the gene product identified according to the method of claim
19 or 20 for identifying compounds capable of conferring a
modulation of glycerol resp., levels in an organism.
25. Agrochemical, pharmaceutical, food or feed composition
comprising the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20. 26. The method of any one of claims 1 to
5, the nucleic acid molecule of claim 6, the polypeptide of claim
14, the nucleic acid construct of claim 7, the vector of claim 8 or
9, the antagonist or agonist identified according to claim 17, the
antibody of claim 15, the plant or plant tissue of claim 16, the
harvested material of claim 16, the host cell of claim 10 to 12 or
the gene product identified according to the method of claim 19 or
20, wherein the fine chemical is glycerol. 27. A host cell or plant
according to any of the claims 10 to 12 which is resistant to a
herbicide inhibiting the biosynthesis of glycerol. 28. A process
for the increased production of total lipids, comprising the
increasing or generating in an organism or a part thereof the
expression of at least one nucleic acid molecule comprising a
nucleic acid molecule selected from the group consisting of:
[17173] a) nucleic acid molecule encoding a polypeptide as
indicated in Table XII, application no. 43, columns 5 or 7, or a
fragment thereof, which confers an increase in the amount of total
lipids in an organism or a part thereof; [17174] b) nucleic acid
molecule comprising of a nucleic acid molecule as indicated in
Table XI, application no. 43, columns 5 or 7; [17175] c) nucleic
acid molecule whose sequence can be deduced from a polypeptide
sequence encoded by a nucleic acid molecule of (a) or (b) as a
result of the degeneracy of the genetic code and conferring an
increase in the amount of total lipids in an organism or a part
thereof; [17176] d) nucleic acid molecule which encodes a
polypeptide which has at least 50% identity with the amino acid
sequence of the polypeptide encoded by the nucleic acid molecule of
(a) to (c) and conferring an increase in the amount of total lipids
in an organism or a part thereof; [17177] e) nucleic acid molecule
which hybridizes with a nucleic acid molecule of (a) to (c) under
stringent hybridisation conditions and conferring an increase in
the amount of total lipids in an organism or a part thereof;
[17178] f) nucleic acid molecule which encompasses a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers or
primer pairs as indicated in Table XIII, application no. 43, column
7, and conferring an increase in the amount of total lipids in an
organism or a part thereof; [17179] g) nucleic acid molecule
encoding a polypeptide which is isolated with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (f) and conferring an increase in the amount of
total lipids in an organism or a part thereof; [17180] h) nucleic
acid molecule encoding a polypeptide comprising a consensus as
indicated in Table XIV, application no. 43, column 7, and
conferring an increase in the amount of total lipids in an organism
or a part thereof; and [17181] i) nucleic acid molecule which is
obtainable by screening a suitable nucleic acid library under
stringent hybridization conditions with a probe comprising one of
the sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of total lipids in an organism or a part thereof. [17182] or
comprising a sequence which is complementary thereto. 29. The use
of the nucleic acid molecule encoding a polypeptide as indicated in
Table XII, application no. 43, columns 5 or 7, or a fragment
thereof, which confers an increase in the amount of total lipids in
an organism or a part thereof for the identification of a nucleic
acid molecule conferring an increase in the amount of total lipids
after expression.
[17183] [0554.0.0.43] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[17184] [0000.0.44.44] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[17185] [0001.0.0.44] see [0001.0.0.27]
[17186] [0002.0.44.44] Carotenoids are red, yellow and orange
pigments that are widely distributed in nature. Although specific
carotenoids have been identified in photosynthetic centers in
plants, in bird feathers, in crustaceans and in marigold petals,
they are especially abundant in yellow-orange fruits and vegetables
and dark green, leafy vegetables. Of the more than 700 naturally
occurring carotenoids identified thus far, as many as 50 may be
absorbed and metabolized by the human body. To date, only 14
carotenoids have been identified in human serum.
[17187] In animals some carotenoids (particularly beta-carotene)
serve as dietary precursors to Vitamin A, and many of them may
function as fat-soluble antioxidants. In plants carotenes serve for
example as antioxidants to protect the highly reactive photosystems
and act as accessory photopigments. In vitro experiments have shown
that lycopene, alpha-carotene, zeaxanthin, lutein and cryptoxanthin
quench singlet oxygen and inhibit lipid peroxidation. The isolation
and identification of oxidized metabolites of lutein, zeaxanthin
and lycopene provide direct evidence of the antioxidant action of
these carotenoids.
[17188] Carotenoids are 40-carbon (C.sub.40) terpenoids generally
comprising eight isoprene (C.sub.5) units joined together. Linking
of the units is reversed at the center of the molecule.
"Ketocarotenoid" is a general term for carotenoid pigments that
contain a keto group in the ionene ring portion of the molecule,
whereas "hydroxycarotenoid" refers to carotenoid pigments that
contain a hydroxyl group in the ionene ring. Trivial names and
abbreviations will be used throughout this disclosure, with
IUPAC-recommended semi-systematic names usually being given in
parentheses after first mention of a trivial name.
[17189] Carotenoids are synthesized from a five carbon atom
metabolic precursor, isopentenyl pyrophosphate (IPP). There are at
least two known biosynthetic pathways in the formation of IPP, the
universal isoprene unit. One pathway begins with mevalonic acid,
the first specific precursor of terpenoids, formed from acetyl-CoA
via HMG-CoA (3-hydroxy-3-methylglutaryl-CoA), that is itself
converted to isopentenyl pyrophosphate (IPP). Later, condensation
of two geranylgeranyl pyrophosphate (GGPP) molecules with each
other produces colorless phytoene, which is the initial carotenoid.
Studies have also shown the existence of an alternative,
mevalonate-independent pathway for IPP formation that was
characterized initially in several species of eubacteria, a green
alga, and in the plastids of higher plants. The first reaction in
this alternative pathway is the transketolase-type condensation
reaction of pyruvate and D-glyceraldehylde-3-phosphate to yield
1-deoxy-D-xylulose-5-phosphate (DXP) as an intermediate.
[17190] Through a series of desaturation reactions, phytoene is
converted to phytofluenell, -tcarotene, neurosporene and finally to
lycopene. Subsequently, lycopene is converted by a cyclization
reaction to .beta.-carotene that contains two .beta.-ionene rings.
A keto-group and/or a hydroxyl group are introduced into each ring
of .beta.-carotene to thereby synthesize canthaxanthin, zeaxanthin,
astaxanthin. A hydroxylase enzyme has been shown to convert
canthaxanthin to astaxanthin. Similarly, a ketolase enzyme has been
shown to convert zeaxanthin to astaxanthin. The ketolase also
converts .beta.-carotene to canthaxanthin and the hydroxylase
converts .beta.-carotene to zeaxanthin.
[17191] Carotenoids absorb light in the 400-500 nm region of the
visible spectrum. This physical property imparts the characteristic
red/yellow color of the pigments. A conjugated backbone composed of
isoprene units is usually inverted at the center of the molecule,
imparting symmetry. Changes in geometrical configuration about the
double bonds result in the existence of many cis- and
trans-isomers. Hydroxylated, oxidized, hydrogenated or
ring-containing derivatives also exist. Hydrocarbon carotenoids are
classified as carotenes while those containing oxygen are known as
xanthophylls.
[17192] In animals, carotenoids are absorbed from the intestine
with the aid of dietary fat and incorporated into chylomicrons for
transport in the serum. The different structural features possessed
by carotenoids account for selective distribution in organ tissue,
biological activity and pro-vitamin A potency, or in vivo
conversion to vitamin A. Due to the hydrophobic character,
carotenoids are associated with lipid portions of human tissues,
cells, and membranes. In general, 80-85% of carotenoids are
distributed in adipose tissue, with smaller amounts found in the
liver, muscle, adrenal glands, and reproductive organs.
Approximately 1% circulate in the serum on high and low density
lipoproteins. Serum concentrations are fairly constant and slow to
change during periods of low intake. The estimated half-life was
estimated to be 11-14 days for lycopene, alpha-carotene,
beta-carotene, lutein and zeaxanthin. Evidence for the existence of
more than one body pool has been published. The major serum
carotenoids are alpha-carotene, beta-carotene, lutein, zeaxanthin,
lycopene and cryptoxanthin. Smaller amounts of polyenes such as
phytoene and phytofluene are also present.
[17193] Human serum levels reflect lifestyle choices and dietary
habits within and between cultures. Approximately only 15 circulate
in the blood, on HDL and LDL. Variations can be attributed to
different intakes, unequal abilities to absorb certain carotenoids,
and different rates of metabolism and tissue uptake. Decreased
serum levels occur with alcohol consumption, the use of oral
contraceptives, smoking and prolonged exposure to UV light.
[17194] Alpha-Carotene, beta-carotene and beta-cryptoxanthin can be
converted to retinol or vitamin A in the intestine and liver by the
enzyme 15-15'-b-carotenoid dioxygenase. Such in vivo formation of
retinol appears to be homeostatically controlled, such that
conversion to retinol is limited in persons having adequate vitamin
A status.
[17195] [0003.0.44.44] The established efficacy of beta-carotene in
quenching singlet oxygen and intercepting deleterious free radicals
and reactive oxygen species makes it part of the diverse
antioxidant defense system in humans. Reactive oxygen species have
been implicated in the development of many diseases, including
ischemic heart disease, various cancers, cataracts and macular
degeneration. Because the conjugated polyene portion of
beta-carotene confers its antioxidant capability and all
carotenoids possess this structural feature, research efforts have
been directed at evaluating the efficacy of other carotenoids in
the prevention of free radical-mediated diseases. Indeed, in vitro
experiments have demonstrated that lycopene, alpha-carotene,
zeaxanthin, lutein and cryptoxanthin quench singlet oxygen and
inhibit lipid peroxidation. The isolation and identification of
oxidized metabolites of lutein, zeaxanthin and lycopene may provide
direct evidence of the antioxidant action of these carotenoids.
[17196] In addition to antioxidant capability, other biological
actions of carotenoids include the ability to enhance
immunocompetence and in vitro gap junction communication, reduce or
inhibited mutagenesis and inhibit cell transformations in
vitro.
[17197] Many epidemiological studies have established an inverse
correlation between dietary intake of yellow-orange fruit and dark
green, leafy vegetables and the incidence of various cancers,
especially those of the mouth, pharynx, larynx, esophagus, lung,
stomach, cervix and bladder. While a number of protective compounds
may be responsible for this observation, the co-incidence of
carotenoids in these foods has been noted. Because nutritionists
and medical professionals currently recognize the occurrence of a
large number of distinct carotenoids in food, interest in their
functions and biological impact on health is burgeoning.
[17198] Lutein exists in the retina. It functions to protect
photoreceptor cells from light-generated oxygen radicals, and thus
plays a key role in preventing advanced macular degeneration.
Lutein possesses chemopreventive activity, induces gap junction
communication between cells and inhibits lipid peroxidation in
vitro more effectively than beta-carotene, alpha-carotene and
lycopene. High levels of lutein in serum have been inversely
correlated with lung cancer.
[17199] In addition to lutein, zeaxanthin exists in the retina and
confers protection against macular degeneration. Zeaxanthin is also
prevalent in ovaries and adipocyte tissue. This xanthophyll does
not possess provitamin A activity.
[17200] Alcohol consumption has been shown to influence lipid
peroxidation. Anhydrolutein, an oxidative by-product of lutein and
zeaxanthin, was higher in plasma after alcohol ingestion, while
concentrations of these xanthophylls were reduced. Lutein and
zeaxanthin may therefore have protective effects against LDL
oxidation.
[17201] The all-trans isomer of Lycopene is typically quantified in
serum, although signals for 9-, 13- and 15-cis isomers are
detectable and account for as much as 50% of the total lycopene. In
experiments performed in vitro, lycopene quenched singlet oxygen
more efficiently than alpha-carotene, beta-carotene, zeaxanthin,
lutein and cryptoxanthin.
[17202] Lycopene induces gap junction communication, inhibits lipid
peroxidation and has displays chemopreventive activity. Serum
levels of lycopene have been inversely related to the risk of
cancer in the pancreas and cervix. This carotenoid has been
identified in tissues of the thyroid, kidneys, adrenals, spleen,
liver, heart, testes and pancreas. Lycopene is not converted to
retinol in vivo.
[17203] beta-Cryptoxanthin is capable of quenching singlet oxygen.
beta-Cryptoxanthin is used to color butter. beta-Cryptoxanthin
exhibits provitamin A activity.
[17204] The all-trans isomer of this carotenoid is the major source
of dietary retinoids, due to its high provitamin A activity. One
molecule of trans-beta-carotene can theoretically provide two
molecules of trans retinaldehyde in vivo. Signals for 13- and
15-cis isomers of beta-carotene are also observed in the carotenoid
profile and account for 10% or less of the total beta-carotene in
serum. beta-Carotene quenches singlet oxygen, induces gap junction
communication and inhibits lipid peroxidation. High serum levels of
beta-carotene are correlated with low incidences of cancer in the
mouth, lung, breast, cervix, skin and stomach. beta-Carotene has
been identified in tissues of the thyroid, kidney, spleen, liver,
heart, pancreas, fat, ovaries and adrenal glands.
[17205] alpha-Carotene is similar to beta-carotene in its
biological activity, but quenches singlet oxygen more effectively.
alpha-Carotene improves gap junction communication, prevents lipid
peroxidation and inhibits the formation and uptake of carcinogens
in the body. High serum levels have been associated with lower
risks of lung cancer. With one half the provitamin A potency of
beta-carotene, alpha-carotene also restores normal cell growth and
differentiation. Serum levels are usually between 10 and 20% of the
values for total beta-carotene.
[17206] Alpha-Carotene, beta-carotene and beta-cryptoxanthin can be
converted to Vitamin A in the intestine and liver. Vitamin A is
essential for the immune response and is also involved in other
defenses against infectious agents. Nevertheless, in many
individuals, this conversion is slow and ineffectual, particularly
for older. Some individuals are known as non or low-responders
because they do not convert beta-carotene to Vitamin
[17207] A at the rate as expected. A number of factors can inhibit
this conversion of beta-carotene to Vitamin A. The major reason why
so many Americans have a poor vitamin A status is the regular use
of excessive alcohol. Intestinal parasites can be a factor. And,
any prescription drug that requires liver metabolism will decrease
the liver conversion of beta-carotene to retinol in the liver.
Diabetics and individuals with hypothyroidism or even borderline
hypothyroidism are likely to be low-responders.
[17208] [0004.0.44.44] In plants, approximately 80-90% of the
carotenoids present in green, leafy vegetables such as broccoli,
kale, spinach and brussel sprouts are xanthophylls, whereas 10-20%
are carotenes. Conversely, yellow and orange vegetables including
carrots, sweet potatoes and squash contain predominantly carotenes.
Up to 60% of the xanthophylls and 15% of the carotenes in these
foods are destroyed during microwave cooking. Of the xanthophylls,
lutein appears to be the most stable.
[17209] Lutein occurs in mango, papaya, oranges, kiwi, peaches,
squash, peas, lima beans, green beans, broccoli, brussel sprouts,
cabbage, kale, lettuce, prunes, pumpkin, sweet potatoes and
honeydew melon. Commercial sources are obtained from the extraction
of marigold petals. Lutein does not possess provitamin A
activity.
[17210] Dietary sources of Zeaxanthin include peaches, squash,
apricots, oranges, papaya, prunes, pumpkin, mango, kale, kiwi,
lettuce, honeydew melon and yellow corn.
[17211] The red color of fruits and vegetables such as tomatoes,
pink grapefruit, the skin of red grapes, watermelon and red guavas
is due to lycopene. Other dietary sources include papaya and
apricots.
[17212] beta-Cryptoxanthin occurs in oranges, mango, papaya,
cantaloupe, peaches, prunes, squash.
[17213] Dietary sources of beta-Carotene include mango, cantaloupe,
carrots, pumpkin, papaya, peaches, prunes, squash, sweet potato,
apricots, cabbage, lima beans, green beans, broccoli, brussel
sprouts, kale, kiwi, lettuce, peas, spinach, tomatoes, pink
grapefruit, honeydew melon and oranges.
[17214] Dietary sources of alpha-Carotene include sweet potatoes,
apricots, pumpkin, cantaloupe, green beans, lima beans, broccoli,
brussel sprouts, cabbage, kale, kiwi, lettuce, peas, spinach,
prunes, peaches, mango, papaya, squash and carrots.
[17215] [0005.0.44.44] Some carotenoids occur particularly in a
wide variety of marine animals including fish such as salmonids and
sea bream, and crustaceans such as crab, lobster, and shrimp.
Because animals generally cannot biosynthesize carotenoids, they
obtain those carotenoids present in microorganisms or plants upon
which they feed.
[17216] Carotenoids e.g. xanthophylls, e.g. as astaxanthin,
supplied from biological sources, such as crustaceans, yeast, and
green alga is limited by low yield and costly extraction methods
when compared with that obtained by organic synthetic methods.
Usual synthetic methods, however, produce by-products that can be
considered unacceptable. It is therefore desirable to find a
relatively inexpensive source of carotenoids, in particular
xantophylls, to be used as a feed supplement in aquaculture and as
a valuable chemical for other industrial uses and for diets.
Sources of Xanthophylls include crustaceans such as a krill in the
Antarctic Ocean, cultured products of the yeast Phaffia, cultured
products of a green alga Haematococcus pluvialis, and products
obtained by organic synthetic methods. However, when crustaceans
such as a krill or the like are used, a great deal of work and
expense are required for the isolation of xanthophylls from
contaminants such as lipids and the like during the harvesting and
extraction. Moreover, in the case of the cultured product of the
yeast Phaffia, a great deal of expense is required for the
gathering and extraction of astaxanthin because the yeast has rigid
cell walls and produces xantophylls only in a low yield. One
approach to increase the productivity of some xantophylls'
production in a biological system is to use genetic engineering
technology.
[17217] [0006.0.44.44] In many plants, lycopene is a branch point
in carotenoid biosynthesis. Thus, some of the plant's lycopene is
made into beta-carotene and zeaxanthin, and sometimes zeaxanthin
diglucoside, whereas remaining portions of lycopene are formed into
alpha-carotene and lutein (3,3'-dihydroxy-.alpha.-carotene),
another hydroxylated compound. Carotenoids in higher plants; i.e.,
angiosperms, are found in plastids; i.e., chloroplasts and
chromoplasts. Plastids are intracellular storage bodies that differ
from vacuoles in being surrounded by a double membrane rather than
a single membrane. Plastids such as chloroplasts can also contain
their own DNA and ribosomes, can reproduce independently and
synthesize some of their own proteins. Plastids thus share several
characteristics of mitochondria. In leaves, carotenoids are usually
present in the grana of chloroplasts where they provide a
photoprotective function. Beta-carotene and lutein are the
predominant carotenoids, with the epoxidized carotenoids
violaxanthin and neoxanthin being present in smaller amounts.
Carotenoids accumulate in developing chromoplasts of flower petals,
usually with the disappearance of chlorophyll. As in flower petals,
carotenoids appear in fruit chromoplasts as they develop from
chloroplasts. Most enzymes that take part in conversion of phytoene
to carotenes and xanthophylls are labile, membrane-associated
proteins that lose activity upon solubilization. In maize,
cartonoids were present in horny endosperm (74% to 86%), floury
endosperm (9%-23%) and in the germ and bran of the kernel.
[17218] [0007.0.44.44] At the present time only a few plants are
widely used for commercial colored carotenoid production. However,
the productivity of colored carotenoid synthesis in most of these
plants is relatively low and the resulting carotenoids are
expensively produced.
[17219] Dried marigold petals and marigold petal concentrates
obtained from so-called xanthophyll marigolds are used as feed
additives in the poultry industry to intensify the yellow color of
egg yolks and broiler skin. The pigmenting ability of marigold
petal meal resides largely in the carotenoid fraction known as the
xanthophylls, primarily lutein esters. The xanthophyll zeaxanthin,
also found in marigold petals, has been shown to be effective as a
broiler pigmenter, producing a highly acceptable yellow to
yellow-orange color. Of the xanthophylls, the pigments lutein and
zeaxanthin are the most abundant in commercially available hybrids.
Structural formulas for lutein and zeaxanthin are shown below.
[17220] Carotenoids have been found in various higher plants in
storage organs and in flower petals. For example, marigold flower
petals accumulate large quantities of esterified lutein as their
predominant xanthophyll carotenoid (about 75 to more than 90
percent), with smaller amounts of esterified zeaxanthin. Besides
lutein and zeaxanthin, marigold flower petals also typically
exhibit a small accumulation of .beta.-carotene and epoxidized
xanthophylls, but do not produce or accumulate canthaxanthin or
astaxanthin because a 4-keto-.beta.-ionene ring-forming enzyme is
absent in naturally-occurring marigolds or their hybrids.
[17221] [0008.0.44.44] One way to increase the productive capacity
of biosynthesis is to apply recombinant DNA technology. Thus, it
would be desirable to produce colored carotenoids generally and,
with the use of recent advances in determining carotenoid
biosynthesis from .beta.-carotene to xanthophylls to control the
production of carotenoids. That type of production permits control
over quality, quantity and selection of the most suitable and
efficient producer organisms. The latter is especially important
for commercial production economics and therefore availability to
consumers. Methods of recombinant DNA technology have been used for
some years to improve the production of Xanthophylls in
microorganisms, in particular algae or in plants by amplifying
individual xanthophyll biosynthesis genes and investigating the
effect on xanthophyll production. It is for example reported, that
the five ketocarotenoids, e.g. the xanthophyll astaxanthin could be
produced in the nectaries of transgenic tobacco plants. Those
transgenic plants were prepared by Argobacterium
tumifaciens-mediated transformation of tobacco plants using a
vector that contained a ketolase-encoding gene from H. pluvialis
denominated crtO along with the Pds gene from tomato as the
promoter and to encode a leader sequence. The Pds gene was said by
those workers to direct transcription and expression in
chloroplasts and/or chromoplast-containing tissues of plants. Those
results indicated that about 75 percent of the carotenoids found in
the flower of the transformed plant contained a keto group.
Further, in maize the phytonene synthase (Psy), Phytone desaturase
(Pds), and the -carotene desaturase were identified and it was
shown, that PSY activity is an important control point for the
regulation of the flux.
[17222] Genes suitable for conversion of microorganisms have also
been reported (U.S. Pat. No. 6,150,130 WO 99/61652). Two different
genes that can convert a carotenoid .beta.-ionene ring compound
into astaxanthin have been isolated from the green alga
Haematococcus pluvialis. Zeaxanthin or -carotene were also found in
the marine bacteria Agrobacterium aurantiacum, Alcaligenes PC-1,
Erwinia uredovora. An A. aurantiacum crtZ gene was introduced to an
E. coli transformant that accumulated all-trans-.beta.-carotene.
The transformant so formed produced zeaxanthin. A gene cluster
encoding the enzymes for a carotenoid biosynthesis pathway has been
also cloned from the purple photosynthetic bacterium Rhodobacter
capsulatus. A similar cluster for carotenoid biosynthesis from
ubiquitous precursors such as farnesyl pyrophosphate and geranyl
pyrophosphate has been cloned from the non-photosynthetic bacteria
Erwinia herbicola. Yet another carotenoid biosynthesis gene cluster
has been cloned from Erwinia uredovora. It is yet unknown and
unpredictable as to whether enzymes encoded by other organisms
behave similarly to that of A. aurantiacum in vitro or in vivo
after transformation into the cells of a higher plant.
[17223] [0009.0.44.44] In addition to the above said about the
biological importance of carotenoids, e.g. in vision, bone growth,
reproduction, immune function, gene expression, emboryonic
expression, cell division and cell differation, and respiration, it
should be mentioned that in the world, the prevalence of vitamin A
deficiency ranges from 100 to 250 million children and an estimated
250.000 to 500.000 children go blind each year from vitamin A
deficiency.
[17224] Thus, it would be advantageous if an algae or other
microorganism were available who produce large amounts of
.beta.-carotene, beta-cryptoxanthin, lutein, zeaxanthin, or other
carotenoids. It might be advantageous that only small amounts or no
lutein is produced so that such organisms could be transformed with
e.g. one or more of an appropriate hydroxylase gene and/or an
appropriate ketolase gene to produce cryptoxanthin, zeaxanthin or
astaxanthin. The invention discussed hereinafter relates in some
embodiments to such transformed prokaryotic or eukaryotic
microorganisms. It would also be advantageous if a marigold or
other plants were available whose flowers produced large amounts of
.beta.-carotene, beta-cryptoxanthin, lutein, zeaxanthin, or other
carotenoids. It might be advantageous that only small amounts or no
lutein is produced so that such plants could be transformed with
one or more of an appropriate hydroxylase gene and an appropriate
ketolase gene to produce cryptoxanthin, zeaxanthin or astaxanthin
from e.g the flowers of the resulting transformants. The invention
discussed hereinafter relates in some embodiments to such
transformed plants.
[17225] [0010.0.44.44] Therefore improving the quality of
foodstuffs and animal feeds is an important task of the
food-and-feed industry. This is necessary since, for example, as
mentioned above beta-carotene, which occur in plants and some
microorganisms are limited with regard to the supply of mammals.
Especially advantageous for the quality of foodstuffs and animal
feeds is as balanced as possible a carotenoids profile in the diet
since a great excess of some carotenoids above a specific
concentration in the food has only some positive effect. A further
increase in quality is only possible via addition of further
carotenoids, which are limiting.
[17226] [0011.0.44.44]
[17227] To ensure a high quality of foods and animal feeds, it is
therefore necessary to add one or a plurality of carotenoids in a
balanced manner to suit the organism.
[17228] [0012.0.44.44] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes which
participate in the biosynthesis of carotenoids, e.g. beta-carotene
or its/their precursor, e.g. isopentyl pyrophosphate (IPP), and
make it possible to produce them specifically on an industrial
scale without unwanted byproducts forming.
[17229] In the selection of genes for biosynthesis two
characteristics above all are particularly important. On the one
hand, there is as ever a need for improved processes for obtaining
the highest possible contents of carotenoids like beta-carotene; on
the other hand as less as possible byproducts should be produced in
the production process.
[17230] [0013.0.0.44] see [0013.0.0.27]
[17231] [0014.0.44.44] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is a carotene or a precursor
thereof. In a preferred embodiment, the fine chemical is
beta-carotene, resp. Accordingly, in the present invention, the
term "the fine chemical" as used herein relates to a "carotene, in
particular beta-carotene. Further, the term "the fine chemicals" as
used herein also relates to fine chemicals comprising carotene, in
particular beta-carotene.
[17232] [0015.0.44.44] In one embodiment, the term "carotene, in
particular beta-carotene," or "the fine chemical" or "the
respective fine chemical" means at least one chemical compound with
a carotene-, in particular beta-carotene-like activity.
[17233] In one embodiment, the term "the fine chemical" means a
"carotene, in particular beta-carotene". In one embodiment, the
term "the fine chemical" means beta-carotene depending on the
context in which the term is used. Throughout the specification the
term "the fine chemical" includes the free fine chemicals, its
salts, ester, thioester or bound to other compounds such sugars or
sugarpolymers, like glucoside, e.g. diglucoside. In one embodiment,
the term "the fine chemical" means beta-carotene, resp., in free
form or their salts or their ester or bound to a glucoside, e.g a
diglucoside, resp.
[17234] [0016.0.44.44] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [17235] (a) increasing or generating the activity of one
or more [17236] of a protein as shown in table XII, application no.
44, column 3 encoded by the nucleic acid sequences as shown in
table XI, application no. 44, column 5, in a non-human organism or
in one or more parts thereof or [17237] (b) growing the organism
under conditions which permit the production of the fine chemical,
thus beta-Carotene of the invention or fine chemicals comprising
beta-Carotene of the invention, in said organism or in the culture
medium surrounding the organism.
[17238] [0016.1.44.44] Accordingly, the term "the fine chemical"
means in one embodiment "beta-Carotene" in relation to all
sequences listed in Tables XI to XIV, line 47 or homologs
thereof.
[17239] [0017.0.0.44] to [0019.0.0.44]: see [0017.0.0.27] to
[0019.0.0.27]
[17240] [0020.0.44.44] Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, column 5, line 47 in a plant conferred an increase in
beta-Carotene content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of beta-Carotene.
[17241] [0021.0.0.44] see [0021.0.0.27]
[17242] [0022.0.44.44]
[17243] The sequence of YDR513W from Saccharomyces cerevisiae has
been published in Jacq et al., Nature 387 (6632 Suppl), 75-78,
1997, and Goffeau et al., Science 274 (5287), 546-547, 1996, and
its activity is being defined as glutathione reductase.
Accordingly, in one embodiment, the process of the present
invention comprises the use of a protein of the superfamily of
glutaredoxin, in particular being involved in deoxyribonucleotide
metabolism, cytoplasm, stress response, detoxification, electron
transport and membrane-associated energy conservation, preferably a
glutathione reductase, or its homolog, e.g. as shown herein, for
the production of the respective fine chemical, meaning of
carotene, in particular beta-carotene, in particular for increasing
the amount in free or bound form in an organism or a part thereof,
as mentioned. In one embodiment, in the process of the present
invention said activity, e.g. the activity of a glutathione
reductase is increased or generated, e.g. from Saccharomyces
cerevisiae or a plant or a homolog thereof.
[17244] [0022.1.0.44] to [0023.0.0.44] see [0022.1.0.27] to
[0023.0.0.27]
[17245] [0023.1.44.44] Homologs of the polypeptide disclosed in
table XII, application no. 44, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 44, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 44, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 44,
column 7, resp.
[17246] [0024.0.0.44] see [0024.0.0.27]
[17247] [0025.0.44.44] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 44, column 3" if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 44, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[17248] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 44,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 44,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[17249] [0025.1.0.44] see [0025.1.0.27]
[17250] [0026.0.0.44] to [0033.0.0.44]: see [0026.0.0.27] to
[0033.0.0.27]
[17251] [0034.0.44.44] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 44, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[17252] [0035.0.0.44] to [0038.0.0.44]: see [0035.0.0.27] to
[0038.0.0.27]
[17253] [0039.0.0.44] see [0039.0.0.27]
[17254] [0040.0.0.44] to [0044.0.0.44]: see [0040.0.0.27] to
[0044.0.0.27]
[17255] [0045.0.44.44] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
44, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line,
between 10% and 50%, 10% and 100%, 10% and 200%, 10% and 300%, 10%
and 400%, 10% and 500%, 20% and 50%, 20% and 250%, 20% and 500%,
50% and 100%, 50% and 250%, 50% and 500%, 500% and 1000% or
more.
[17256] [0046.0.44.44] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
44, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line
confers an increase of the respective fine chemical and of further
beta-carotene or their precursors.
[17257] [0047.0.0.44] to [0048.0.0.44]: see [0047.0.0.27] to
[0048.0.0.27]
[17258] [0049.0.44.44] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 44, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 44, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 44, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[17259] [0050.0.44.44] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, such as, for example, the potassium or sodium
salts of beta-carotene, resp., or their ester, or glucoside
thereof, e.g the diglucoside thereof.
[17260] [0051.0.44.44] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g compositions comprising beta-carotene.
Depending on the choice of the organism used for the process
according to the present invention, for example a microorganism or
a plant, compositions or mixtures of various beta-carotene can be
produced.
[17261] [0052.0.0.44] see [0052.0.0.27]
[17262] [0053.0.44.44] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[17263] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 44, columns 5 and 7 or its homologs activity
having herein-mentioned beta-Carotenes of the invention increasing
activity; and/or [17264] b) stabilizing a mRNA conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention as shown in table XI, application no. 44,
columns 5 and 7, e.g. a nucleic acid sequence encoding a
polypeptide having the activity of a protein as indicated in table
XII, application no. 44, columns 5 and 7 or its homologs activity
or of a mRNA encoding the polypeptide of the present invention
having herein-mentioned beta-Carotenes of the invention increasing
activity; and/or [17265] c) increasing the specific activity of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the present invention having herein-mentioned beta-Carotene
increasing activity, e.g. of a polypeptide having the activity of a
protein as indicated in table XII, application no. 44, columns 5
and 7 or its homologs activity, or decreasing the inhibiitory
regulation of the polypeptide of the invention; and/or [17266] d)
generating or increasing the expression of an endogenous or
artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned beta-Carotenes of the
invention increasing activity, e.g. of a polypeptide having the
activity of a protein as indicated in table XII, application no.
44, columns 5 and 7 or its homologs activity; and/or [17267] e)
stimulating activity of a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
present invention or a polypeptide of the present invention having
herein-mentioned beta-Carotenes of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 44, columns 5 and 7 or its
homologs activity, by adding one or more exogenous inducing factors
to the organisms or parts thereof; and/or [17268] f) expressing a
transgenic gene encoding a protein conferring the increased
expression of a polypeptide encoded by the nucleic acid molecule of
the present invention or a polypeptide of the present invention,
having herein-mentioned beta-Carotenes of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 44, columns 5 and 7 or its
homologs activity, and/or [17269] g) increasing the copy number of
a gene conferring the increased expression of a nucleic acid
molecule encoding a polypeptide encoded by the nucleic acid
molecule of the invention or the polypeptide of the invention
having herein-mentioned beta-Carotenes of the invention increasing
activity, e.g. of a polypeptide having the activity of a protein as
indicated in table XII, application no. 44, columns 5 and 7 or its
homologs activity; and/or [17270] h) increasing the expression of
the endogenous gene encoding the polypeptide of the invention, e.g.
a polypeptide having the activity of a protein as indicated in
table XII, application no. 44, columns 5 and 7 or its homologs
activity, by adding positive expression or removing negative
expression elements, e.g. homologous recombination can be used to
either introduce positive regulatory elements like for plants the
35S enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[17271] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [17272] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[17273] [0054.0.44.44] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 44 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 44, column 3 or its homologs.
[17274] [0055.0.0.44] to [0064.0.0.44]: see [0055.0.0.27] to
[0064.0.0.27]
[17275] [0065.0.0.44]: see [0065.0.0.27]
[17276] [0066.0.0.44] to [0067.0.0.44]: see [0066.0.0.27] to
[0067.0.0.27]
[17277] [0068.0.44.44] The mutation is introduced in such a way
that the production of the respective fine chemical, in particular
beta-carotene is not adversely affected.
[17278] [0069.0.0.44] see [0069.0.0.27]
[17279] [0070.0.44.44] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolites composition in the organism, e.g. an advantageous
composition of carotenoids, in particular carotenes, in particular
beta-carotene, e.g. comprising a higher content of (from a
viewpoint of nutritional physiology limited) carotenoids, in
particular carotene, in particular beta-carotene.
[17280] [0071.0.0.44] see [0071.0.0.27]
[17281] [0072.0.44.44] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are
further carotenoids, e.g. carotenes or carotene, e.g.
ketocarentoids, or hydrocarotenoids, e.g. lutein, lycopene,
alpha-carotene, or beta-carentene, or other compounds for which IPP
is a precursor well known for the skilled.
[17282] [0073.0.44.44] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
(a) providing a non-human organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant; (b) increasing an activity of a polypeptide of the
invention or a homolog thereof, e.g. as indicated in Table XII,
application no. 44, columns 5 or 7, or of a polypeptide being
encoded by the nucleic acid molecule of the present invention and
described below, e.g. conferring an increase of the respective fine
chemical in an organism, preferably in a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a
plant, (c) growing an organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant under conditions which permit the production of the
respective fine chemical in the organism, preferably the
microorganism, the plant cell, the plant tissue or the plant; and
(d) if desired, recovering, optionally isolating, the free and/or
bound the respective fine chemical and, optionally further free
and/or bound carotenoids, in particular carotene, in particular
beta-carotene synthesized by the organism, the microorganism, the
non-human animal, the plant or animal cell, the plant or animal
tissue or the plant.
[17283] [0074.0.44.44] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the respective fine chemical or the free and
bound the respective fine chemical but as option it is also
possible to produce, recover and, if desired isolate, other free
or/and bound carotenoids, in particular carotene, in particular
beta-carotene.
[17284] [0075.0.0.44] to [0077.0.0.44]: see [0075.0.0.27] to
[0077.0.0.27]
[17285] [0078.0.44.44] The organism such as microorganisms or
plants or the recovered, and if desired isolated, the respective
fine chemical can then be processed further directly into
foodstuffs or animal feeds or for other applications, for example
according to the disclosures made in U.S. Pat. No. 6,399,115:
Method and composition for the treatment of benign prostate
hypertrophy (BPH) and prevention of prostate cancer
U.S. Pat. No. 6,399,114: Nutritional system for nervous system
disorders U.S. Pat. No. 6,399,060: Composition having nematicidal
activity U.S. Pat. No. 6,399,046: Use of a content of catechins or
a content of green tea extract in cosmetic preparations for tanning
the skin U.S. Pat. No. 6,395,782: Method of increasing longevity
and preventing body weight wasting in autoimmune disease by using
conjugated linoleic acid U.S. Pat. No. 6,395,508: Peptide mixture
and products thereof U.S. Pat. No. 6,395,315: Fermentation
composition, process for preparing the same, and use thereof U.S.
Pat. No. 6,395,311: Multicomponent biological vehicle U.S. Pat. No.
6,394,230: Sterol esters as food additives U.S. Pat. No. 6,391,640:
Methods and compositions for cellular and metabolic engineering
U.S. Pat. No. 6,391,332: Therapeutic micronutrient composition for
severe trauma, burns and critical illness U.S. Pat. No. 6,391,321:
Emulsifier-free finely disperse systems of the oil-in-water and
water-in-oil type U.S. Pat. No. 6,391,319: Cosmetic and
dermatological emulsions comprising alkyl glucosides and increased
electrolyte concentrations U.S. Pat. No. 6,391,289: Use of
sunscreen combinations comprising, as essential constituent,
4,4'-diarylbutadienes as photostable UV filters in cosmetic and
pharmaceutical preparations U.S. Pat. No. 6,387,961: Alkyl
2-acetamido-2-deoxyglucopyranoside and methods of emulsifying U.S.
Pat. No. 6,387,927: Epothilone derivatives and their use as
antitumor agents U.S. Pat. No. 6,387,883: Method for the prevention
and treatment of cachexia and anorexia U.S. Pat. No. 6,387,878:
Methods of treating intestinal ischemia using heparin-binding
epidermal growth factor U.S. Pat. No. 6,387,862: Bleach
compositions U.S. Pat. No. 6,387,418: Pomegranate extracts and
methods of using thereof U.S. Pat. No. 6,387,370: Compositions
containing extracts of Morinda citrifolia, red wine, prune,
blueberry, pomegranate, apple and enzyme mixture U.S. Pat. No.
6,387,355: Use of sunscreen combinations comprising, as essential
constituent, amino-substituted hydroxybenzophenones as photostable
UV filters in cosmetic and pharmaceutical preparations U.S. Pat.
No. 6,384,090: Preparation of active ingredient dispersions and
apparatus therefor U.S. Pat. No. 6,384,085: Material separated from
Ecklonia cava, method for extracting and purifying the same, and
use thereof as antioxidants U.S. Pat. No. 6,383,751: Assessing
lipid metabolism U.S. Pat. No. 6,383,543: Process for the
extraction of an organic salt from plants, the salt, and other
similar salts U.S. Pat. No. 6,383,524: Compositions and methods for
enhancing therapeutic effects U.S. Pat. No. 6,383,523:
Pharmaceutical compositions and methods for managing skin
conditions U.S. Pat. No. 6,383,503: PREPARATIONS OF THE W/O
EMULSION TYPE WITH AN INCREASED WATER CONTENT, ADDITIONALLY
COMPRISING ONE OR MORE ALKYLMETHICONE COPOLYOLS AND/OR
ALKYLDIMETHICONE COPOLYOLS, AND, IF DESIRED, CATIONIC POLYMERS U.S.
Pat. No. 6,383,474: Carotenoid preparation U.S. Pat. No. 6,383,473:
Solid composition for reducing tooth erosion U.S. Pat. No.
6,380,442: Process for the isolation of mixed carotenoids from
plants U.S. Pat. No. 6,380,232: Benzimidazole urea derivatives, and
pharmaceutical compositions and unit dosages thereof U.S. Pat. No.
6,380,227: Fermentative preparation process for and crystal forms
of cytostatics U.S. Pat. No. 6,379,697: Stabilization of
photosensitive materials U.S. Pat. No. 6,379,683: Nanocapsules
based on dendritic polymers U.S. Pat. No. 6,376,722: Lutein to
zeaxanthin isomerization process and product U.S. Pat. No.
6,376,717: Preparation of astaxanthin U.S. Pat. No. 6,376,544:
Nutritional product for a person having renal failure U.S. Pat. No.
6,376,498: Pharmaceutical, cosmetic and/or food composition with
antioxidant properties U.S. Pat. No. 6,376,455: Quaternary ammonium
compounds, compositions containing them, and uses thereof U.S. Pat.
No. 6,376,005: Antimicrobial composition for food and beverage
products U.S. Pat. No. 6,375,993: Pomegranate extracts and methods
of using thereof U.S. Pat. No. 6,375,992: Methods of hydrating
mammalian skin comprising oral administration of a defined
composition U.S. Pat. No. 6,375,963: Bioadhesive hot-melt extruded
film for topical and mucosal adhesion applications and drug
delivery and process for preparation thereof U.S. Pat. No.
6,375,956: Strip pack U.S. Pat. No. 6,375,873: Process and
apparatus for producing stably fine-particle powders U.S. Pat. No.
6,372,964: For higher basidiomycetes mushrooms grown as biomass in
submerged culture U.S. Pat. No. 6,372,946: Preparation of
4,4'-diketo-.beta.-carotene derivatives
[17286] The cited literature describe some preferred embodiments.
Said applications describe some advantageous embodiments without
meant to be limiting.
[17287] The fermentation broth, fermentation products, plants or
plant products can be purified as described in above mentioned
applications and other methods known to the person skilled in the
art, e.g. as described in Methods in Enzymology: Carotenoids, Part
A: Chemistry, Separation, Quantitation and Antioxidation, by John N
Abelson or Part B, Metabolism, Genetics, and Biochemistry, or
described herein below. Products of these different work-up
procedures are IPP and/or beta-carotenes and optional other
carotenoids, comprising compositions which still comprise
fermentation broth, plant particles and cell components in
different amounts, advantageously in the range of from 0 to 99% by
weight, preferably below 80% by weight, especially preferably
between below 50% by weight.
[17288] [0079.0.0.44] to [0084.0.0.44]: see [0079.0.0.27] to
[0084.0.0.27]
[17289] [0084.2.44.44] The invention also contemplates embodiments
in which the .beta.-carotene is present in the flowers of the
flowering plant chosen as the host (for example, marigolds). The
invention also contemplates embodiments in which a host plant's
flowers lack .beta.-carotene. In a plant of the latter type, the
inserted DNA includes genes that code for carotenoid precursors
(compounds that can be converted biologically into .beta.-carotene)
and a ketolase, as well as a hydroxylase, if otherwise absent.
[17290] In one embodiment, preferred flowering plants include, but
are not limited to: Amaryllidaceae (Allium, Narcissus); Apocynaceae
(Catharanthus); Asteraceae, alternatively Compositae (Aster,
Calendula, Callistephus, Cichorium, Coreopsis, Dahlia,
Dendranthema, Gazania, Gerbera, Helianthus, Helichrysum, Lactuca,
Rudbeckia, Tagetes, Zinnia); Balsaminaceae (Impatiens); Begoniaceae
(Begonia); Caryophyllaceae (Dianthus); Chenopodiaceae (Beta,
Spinacia); Cucurbitaceae (Citrullus, Curcurbita, Cucumis);
Cruciferae (Alyssum, Brassica, Erysimum, Matthiola, Raphanus);
Gentinaceae (Eustoma); Geraniaceae (Pelargonium); Graminae,
alternatively Poaceae, (Avena, Horedum, Oryza, Panicum, Pennisetum,
Poa, Saccharum, Secale, Sorghum, Triticum, Zea); Euphorbiaceae
(Poinsettia); Labiatae (Salvia); Leguminosae (Glycine, Lathyrus,
Medicago, Phaseolus, Pisum); Liliaceae (Cilium); Lobeliaceae
(Lobelia); Malvaceae (Abelmoschus, Gossypium, MaIva);
Plumbaginaceae (Limonium); Polemoniaceae (Phlox); Primulaceae
(Cyclamen); Ranunculaceae (Aconitum, Anemone, Aquilegia, Caltha,
Delphinium, Ranunculus); Rosaceae (Rosa); Rubiaceae (Pentas);
Scrophulariaceae (Angelonia, Antirrhinum, Torenia); Solanaceae
(Capsicum, Lycopersicon, Nicotiana, Petunia, Solanum); Umbelliferae
(Apium, Daucus, Pastinaca); Verbenaceae (Verbena, Lantana);
Violaceae (Viola).
[17291] [0085.0.44.44] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [17292] a) a nucleic acid sequence as
indicated in Table XI, application no. 44, columns 5 or 7, or a
derivative thereof, or [17293] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 44, columns
5 or 7, or a derivative thereof, or [17294] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[17295] [0086.0.0.44] to [0087.0.0.44]: see [0086.0.0.27] to
[0087.0.0.27]
[17296] [0088.0.44.44] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content of
the respective fine chemical is modified advantageously owing to
the nucleic acid molecule of the present invention expressed. This
is important for plant breeders since, for example, the nutritional
value of plants for poultry is dependent on the abovementioned
carotene, in particular beta-carotene and the general amount of
xanthohylls as energy source in feed. Further, this is also
important for the production of cosmetic compostions since, for
example, the antioxidant level of plant extracts is dependent on
the amount of th abovementioned carotene, in particular
beta-carotene and/or the amount of carotenoids as antioxidants.
[17297] [0088.1.0.44] see [0088.1.0.27]
[17298] [0089.0.0.44] to [0090.0.0.44]: see [0089.0.0.27] to
[0090.0.0.27]
[17299] [0091.0.44.44] Thus, the content of plant components and
preferably also further impurities is as low as possible, and the
abovementioned carotene, in particular beta-carotene, in particular
the fine chemical, is/are obtained in as pure form as possible. In
these applications, the content of plant components advantageously
amounts to less than 10%, preferably 1%, more preferably 0.1%, very
especially preferably 0.01% or less.
[17300] [0092.0.0.44] to [0094.0.0.44]: see [0092.0.0.27] to
[0094.0.0.27]
[17301] [0095.0.44.44] It may be advantageous to increase the pool
of said carotenoids, in particular carotene, in particular
beta-carotene in the transgenic organisms by the process according
to the invention in order to isolate high amounts of the pure
respective fine chemical.
[17302] [0096.0.44.44] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptide or a compound, which
functions as a sink for the desired fine chemical, for example
zeaxanthin or cryptoxanthin in the organism, is useful to increase
the production of the respective fine chemical. It has been
reported, that the inhibition of Lyopene production increases the
amount of other carotene, in particular beta-carotene in the cell.
Further it may be, that the inhibition of enzymes using zeaxanthin
or cryptoxanthin as substrate increases the amount of said
chemicals in a cell. For example, in one embodiment, it can be
advantageous to inhibit the production of astaxanthin, if a high
amount of cryptoxanthin or zeaxanthin is desired.
[17303] [0097.0.44.44] Glucosides, in particular, dicglucosides of
the zeaxanthin and beta-cryptoxanthin as well as other modification
of zeaxanthin and cryptoxanthin are known to a person skilled in
the art. In may also be advantageous to increase the content of the
bound respective fine chemical, e.g. of modification of zeaxanthin
and cryptoxanthin, in particular its glucosides, e.g.
diglucosides.
[17304] [0098.0.44.44] In a preferred embodiment, the respective
fine chemical is produced in accordance with the invention and, if
desired, is isolated. The production of further carotenoids, e.g.
carotenes or carotene, in particular beta-carotene, in particular
ketocarentoids or hydrocarotenoids, e.g. lutein, lycopene,
alpha-carotene, or beta-carentene, or compounds for which the
respective fine chemical is a biosynthesis precursor compounds,
e.g. astaxanthin, or mixtures thereof or mixtures of other
carotenoids, in particular of carotene, in particular
beta-carotene, by the process according to the invention is
advantageous.
[17305] [0099.0.44.44] In the case of the fermentation of
microorganisms, the abovementioned desired fine chemical may
accumulate in the medium and/or the cells. If microorganisms are
used in the process according to the invention, the fermentation
broth can be processed after the cultivation. Depending on the
requirement, all or some of the biomass can be removed from the
fermentation broth by separation methods such as, for example,
centrifugation, filtration, decanting or a combination of these
methods, or else the biomass can be left in the fermentation broth.
The fermentation broth can subsequently be reduced, or
concentrated, with the aid of known methods such as, for example,
rotary evaporator, thin-layer evaporator, falling film evaporator,
by reverse osmosis or by nanofiltration. Afterwards advantageously
further compounds for formulation can be added such as corn starch
or silicates. This concentrated fermentation broth advantageously
together with compounds for the formulation can subsequently be
processed by lyophilization, spray drying, and spray granulation or
by other methods. Preferably the respective fine chemical
comprising compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[17306] [0100.0.44.44] Transgenic plants which comprise the
carotenoids such as said carotene, in particular beta-carotene,
e.g. cryptoxanthin or zeaxanthin (or astaxanthin as it is
synthesized from cryptoxanthin or zeaxanthin) synthesized in the
process according to the invention can advantageously be marketed
directly without there being any need for the carotenoids
synthesized to be isolated. Plants for the process according to the
invention are listed as meaning intact plants and all plant parts,
plant organs or plant parts such as leaf, stem, seeds, root,
tubers, anthers, fibers, root hairs, stalks, embryos, calli,
cotelydons, petioles, harvested material, plant tissue,
reproductive tissue and cell cultures which are derived from the
actual transgenic plant and/or can be used for bringing about the
transgenic plant. In this context, the seed comprises all parts of
the seed such as the seed coats, epidermal cells, seed cells,
endosperm or embryonic tissue.
[17307] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, (in the form of their oils, fats,
lipids, as extracts, e.g. ether, alcohol, or other organic solvents
or water containing extract and/or free carotene, in particular
beta-carotene. The respective fine chemical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts. To increase the
efficiency of extraction it is beneficial to clean, to temper and
if necessary to hull and to flake the plant material. E.g., oils,
fats, and/or lipids comprising carotene, in particular
beta-carotene can be obtained by what is known as cold beating or
cold pressing without applying heat. To allow for greater ease of
disruption of the plant parts, specifically the seeds, they can
previously be comminuted, steamed or roasted. Seeds, which have
been pretreated in this manner can subsequently be pressed or
extracted with solvents such as warm hexane. The solvent is
subsequently removed. In the case of microorganisms, the latter
are, after harvesting, for example extracted directly without
further processing steps or else, after disruption, extracted via
various methods with which the skilled worker is familiar.
Thereafter, the resulting products can be processed further, i.e.
degummed and/or refined. In this process, substances such as the
plant mucilages and suspended matter can be first removed. What is
known as desliming can be affected enzymatically or, for example,
chemico-physically by addition of acid such as phosphoric acid.
[17308] Because carotenoids in microorganisms are localized
intracellular, their recovery essentials comes down to the
isolation of the biomass. Well-established approaches for the
harvesting of cells include filtration, centrifugation and
coagulation/flocculation as described herein. Of the residual
hydrocarbon, adsorbed on the cells, has to be removed. Solvent
extraction or treatment with surfactants have been suggested for
this purpose. However, it can be advantageous to avoid this
treatment as it can result in cells devoid of most carotenoids.
[17309] [0101.0.44.44] The identity and purity of the compound(s)
isolated can be determined by prior-art techniques. They encompass
high-performance liquid chromatography (HPLC), gas chromatography
(GC), spectroscopic methods, mass spectrometry (MS), staining
methods, thin-layer chromatography, NIRS, enzyme assays or
microbiological assays. These analytical methods are compiled in:
Patek et al. (1994) Appl. Environ. Microbiol. 60:133-140; Malakhova
et al. (1996) Biotekhnologiya 11 27-32; and Schmidt et al. (1998)
Bioprocess Engineer. 19:67-70. Ulmann's Encyclopedia of Industrial
Chemistry (1996) Bd. A27, VCH Weinheim, pp. 89-90, pp. 521-540, pp.
540-547, pp. 559-566, 575-581 and pp. 581-587; Michal, G (1999)
Biochemical Pathways: An Atlas of Biochemistry and Molecular
Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications
of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry
and Molecular Biology, vol. 17.
[17310] [0102.0.44.44] Carotene, in particular beta-carotene, in
particular beta-cryptoxanthin or zeaxanthin can for example be
detected advantageously via HPLC, LC or GC separation methods. The
unambiguous detection for the presence of carotene, in particular
beta-carotene, in particular beta-cryptoxanthin or zeaxanthin
containing products can be obtained by analyzing recombinant
organisms using analytical standard methods: LC, LC-MS, MS or TLC).
The material to be analyzed can be disrupted by sonication,
grinding in a glass mill, liquid nitrogen and grinding, cooking, or
via other applicable methods.
[17311] [0103.0.44.44] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [17312] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide having a
sequence as indicated in Table XII, application no. 44, columns 5
or 7, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [17313] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table XI, application no. 44, columns 5 or 7,
[17314] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [17315] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[17316] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridization
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [17317]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [17318] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [17319] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers pairs
having a sequence as indicated in Table XIII, application no. 44,
columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [17320]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [17321] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequences as indicated in Table XIV, application
no. 44, columns 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [17322]
k) nucleic acid molecule comprising one or more of the nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domain of the polypeptide indicated in Table XII, application no.
44, columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[17323] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[17324] [0103.1.44.44] In one embodiment, the nucleic acid molecule
used in the process of the invention distinguishes over the
sequence indicated in Table XI, application no. 44, columns 5 or 7
by one or more nucleotides. In one embodiment, the nucleic acid
molecule used in the process of the invention does not consist of
the sequence shown in indicated in Table XI, application no. 44,
columns 5 or 7. In one embodiment, the nucleic acid molecule used
in the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI,
application no. 44, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table XII, application no. 44, columns 5 or 7.
[17325] [0104.0.44.44] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence indicated in
Table XI, application no. 44, columns 5 or 7, by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of the sequence
indicated in Table XI, application no. 44, columns 5 or 7. In one
embodiment, the nucleic acid molecule of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to the
sequence indicated in Table XI, application no. 44, columns 5 or 7.
In another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table XII, application no.
44, columns 5 or 7.
[17326] [0105.0.0.44] to [0107.0.0.44]: see [0105.0.0.27] to
[0107.0.0.27]
[17327] [0108.0.44.44] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 44, columns 5 or 7, nucleic
acid molecules which are derived from an amino acid sequences as
indicated in Table XII, application no. 44, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 44, columns 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of an activity of a polypeptide as indicated in Table XII,
application no. 44, column 3, 5 or 7, e.g. conferring the increase
of the respective fine chemical, meaning carotenoids, in particular
carotene, in particular beta-carotene, resp., after increasing its
expression or activity, are advantageously increased in the process
according to the invention.
[17328] [0109.0.44.44] In one embodiment, said sequences are cloned
into nucleic acid constructs, either individually or in
combination. These nucleic acid constructs enable an optimal
synthesis of the respective fine chemicals, in particular
beta-carotene, produced in the process according to the
invention.
[17329] [0110.0.0.44] see. [0110.0.0.27]
[17330] [0111.0.0.44] see [0111.0.0.27]
[17331] [0112.0.44.44] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XII, application no. 44,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 44, columns 5 and 7, and conferring an
increase in the beta-carotene level, respectively.
[17332] [0113.0.0.44] to [0114.0.0.44]: see [0113.0.0.27] to
[0114.0.0.27]
[17333] [0115.0.0.44] see [0115.0.0.27]
[17334] [0116.0.0.44] to [0120.0.0.44] see [0116.0.0.27] to
[0120.0.0.27]
[17335] [0120.1.44.44] Production strains which are also
advantageously selected in the process according to the invention
are microorganisms selected from the group green algae, like
Spongioccoccum exentricum, Chlorella sorokiniana (pyrenoidosa,
7-11-05), or form the group of fungi like fungi belonging to the
Daccrymycetaceae family, or non-photosynthetic bacteria, like
methylotrophs, flavobacteria, actinomycetes, like streptomyces
chrestomyceticus, Mycobacteria like Mycobacterim phlei, or
Rhodobacter capsulatus.
[17336] [0121.0.44.44] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 44, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity I.e. they confer a beta-carotene increase after increasing
the activity of the polypeptide sequences indicated in Table XII,
application no. 44, columns 5 or 7.
[17337] [0122.0.0.44] to [0127.0.0.44]: see [0122.0.0.27] to
[0127.0.0.27]
[17338] [0128.0.44.44] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 44, columns 7, by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table XI, application no.
44, columns 5 or 7, or the sequences derived from a sequences as
indicated in Table XII, application no. 44, columns 5 or 7.
[17339] [0129.0.44.44] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved region for the polypeptide of the
invention are indicated in the alignments shown in the figures.
Conserved regions are those, which show a very little variation in
the amino acid in one particular position of several homologs from
different origin. The consensus sequence indicated in Table XIV,
application no. 44, columns 7, is derived from said alignments.
[17340] [0130.0.44.44] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of the
respective fine chemical after increasing the expression or
activity the protein comprising said fragment.
[17341] [0131.0.0.44] to [0138.0.0.44]: see [0131.0.0.27] to
[0138.0.0.27]
[17342] [0139.0.44.44] Polypeptides having above-mentioned
activity, i.e. conferring the respective fine chemical level
increase, derived from other organisms, can be encoded by other DNA
sequences which hybridise to a sequence indicated in Table XI,
application no. 44, columns 5 or 7, for beta-carotene under relaxed
hybridization conditions and which code on expression for peptides
having the respective fine chemical, i.e. beta-carotene, resp.,
--increasing activity.
[17343] [0140.0.0.44] to [0146.0.0.44]: see [0140.0.0.27] to
[0146.0.0.27]
[17344] [0147.0.44.44] Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 44, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences s
such that it can hybridize to one of said nucleotide sequences
thereby forming a stable duplex. Preferably, the hybridisation is
performed under stringent hybridization conditions. However, a
complement of one of the herein disclosed sequences is preferably a
sequence complement thereto according to the base pairing of
nucleic acid molecules well known to the skilled person. For
example, the bases A and G undergo base pairing with the bases T
and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[17345] [0148.0.44.44] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 44,
columns 5 or 7 or a portion thereof and preferably has above
mentioned activity, in particular, of beta-Carotene increasing
activity after increasing the activity or an activity of a product
of a gene encoding said sequences or their homologs.
[17346] [0149.0.44.44] The nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 44,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity and as indicated in indicated in Table
XII.
[17347] [00149.1.44.44] Optionally, in one embodiment, the
nucleotide sequence, which hybridises to one of the nucleotide
sequences indicated in Table XI, application no. 44, columns 5 or
7, has further one or more of the activities annotated or known for
the a protein as indicated in Table XII, application no. 44, column
3.
[17348] [0150.0.44.44] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table XI, application no. 44, columns
5 or 7, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of beta-Carotene, resp., if
its activity is increased. The nucleotide sequences determined from
the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table XI,
application no. 44, columns 5 or 7, an anti-sense sequence of one
of the sequences, e.g., as indicated in Table XI, application no.
44, columns 5 or 7, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table XIII, application no. 44, column 7, will result in a fragment
of a polynucleotide sequence as indicated in Table XI, application
no. 44, columns 5 or 7 or its gene product.
[17349] [0151.0.0.44]: see [0151.0.0.27]
[17350] [0152.0.44.44] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 44, columns 5
or 7, such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular a beta-carotene increasing activity as mentioned
above or as described in the examples in plants or microorganisms
is comprised.
[17351] [0153.0.44.44] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 44,
columns 5 or 7, such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 44, columns 5 or 7, has for
example an activity of a polypeptide indicated in Table XII,
application no. 44, column 3.
[17352] [0154.0.44.44] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 44, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[17353] [0155.0.0.44] to [0156.0.0.44]: see [0155.0.0.27] to
[0156.0.0.27]
[17354] [0157.0.44.44] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences as
indicated in Table XI, application no. 44, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
polypeptides comprising the sequence as indicated in Table XIV,
application no. 44, columns 7, or as polypeptides depicted in Table
XII, application no. 44, columns 5 or 7, or the functional
homologues. Advantageously, the nucleic acid molecule of the
invention comprises, or in an other embodiment has, a nucleotide
sequence encoding a protein comprising, or in an other embodiment
having, an amino acid sequence of a consensus sequences as
indicated in Table XIV, application no. 44, columns 7, or of the
polypeptide as indicated in Table XII, application no. 44, columns
5 or 7, or the functional homologues. In a still further
embodiment, the nucleic acid molecule of the invention encodes a
full length protein which is substantially homologous to an amino
acid sequence comprising a consensus sequence as indicated in Table
XIV, application no. 44, columns 5 or 7, or of a polypeptide as
indicated in Table XII, application no. 44, columns 5 or 7, or the
functional homologues. However, in a preferred embodiment, the
nucleic acid molecule of the present invention does not consist of
a sequence as indicated in Table XI, application no. 44, columns 5
or 7.
[17355] [0158.0.0.44] to [0160.0.0.44]: see [0158.0.0.27] to
[0160.0.0.27]
[17356] [0161.0.44.44] Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 44, columns 5 or
7. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[17357] [0162.0.0.44] see [0162.0.0.27]
[17358] [0163.0.44.44] Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 44, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the increase of
the amount of the respective fine chemical in a organism or a part
thereof, e.g. a tissue, a cell, or a compartment of a cell, after
increasing the expression or activity thereof or the activity of a
protein of the invention or used in the process of the
invention.
[17359] [0164.0.0.44] see [0164.0.0.27]
[17360] [0165.0.44.44] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 44, columns 5 or 7.
[17361] [0166.0.0.44] to [0167.0.0.44]: see [0166.0.0.27] to
[0167.0.0.27]
[17362] [0168.0.44.44] Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 44, columns 5 or 7, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 44,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table XII, application no. 44, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 44, columns 5
or 7, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table XII, application no.
44, columns 5 or 7, and most preferably at least about 96%, 97%,
98%, or 99% identical to the sequence as indicated in Table XII,
application no. 44, columns 5 or 7.
[17363] [0169.0.0.44] to [0172.0.0.44]: see [0169.0.0.27] to
[0172.0.0.27]
[17364] [0173.0.44.44] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108401 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108401 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[17365] [0174.0.0.44]: see [0174.0.0.27]
[17366] [0175.0.44.44] For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108402 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108402 by the above program algorithm with the
above parameter set, has a 80% homology.
[17367] [0176.0.44.44] Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 44,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 44, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 44, columns
5 or 7.
[17368] [0177.0.44.44] Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 44,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptide as indicated in Table XII,
application no. 44, columns 5 or according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 44, columns
5 or 7.
[17369] [0178.0.0.44] see [0178.0.0.27]
[17370] [0179.0.44.44] A nucleic acid molecule encoding an
homologous to a protein sequence as indicated in Table XII,
application no. 44, columns 5 or 7, can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table XI, application no.
44, columns 5 or 7, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences of a
sequences as indicated in Table XI, application no. 44, columns 5
or 7, by standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[17371] [0180.0.0.44] to [0183.0.0.44]: see [0180.0.0.27] to
[0183.0.0.27]
[17372] [0184.0.44.44] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, application no. 44,
columns 5 or 7, or of the nucleic acid sequences derived from a
sequences as indicated in Table XII, application no. 44, columns 5
or 7, comprise also allelic variants with at least approximately
30%, 35%, 40% or 45% homology, by preference at least approximately
50%, 60% or 70%, more preferably at least approximately 90%, 91%,
92%, 93%, 94% or 95% and even more preferably at least
approximately 96%, 97%, 98%, 99% or more homology with one of the
nucleotide sequences shown or the abovementioned derived nucleic
acid sequences or their homologues, derivatives or analogues or
parts of these. Allelic variants encompass in particular functional
variants which can be obtained by deletion, insertion or
substitution of nucleotides from the sequences shown, preferably
from a sequence as indicated in Table XI, application no. 44,
columns 5 or 7, or from the derived nucleic acid sequences, the
intention being, however, that the enzyme activity or the
biological activity of the resulting proteins synthesized is
advantageously retained or increased.
[17373] [0185.0.44.44] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 44, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table XI, application no. 44, columns 5 or 7. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequences as indicated in Table XI, application no. 44, columns 5
or 7.
[17374] [0186.0.44.44] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table XII,
application no. 44, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 44, columns 5 or 7.
[17375] [0187.0.44.44] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table XII, application no.
44, columns 5 or 7, comprises less than 100 further nucleotides. In
a further embodiment, said nucleic acid molecule comprises less
than 30 further nucleotides. In one embodiment, the nucleic acid
molecule used in the process is identical to a coding sequence
encoding a sequences as indicated in Table XII, application no. 44,
columns 5 or 7.
[17376] [0188.0.44.44] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 44, columns 5 or 7 and is expressed under
identical conditions.
[17377] [0189.0.44.44] Homologues of a sequences as indicated in
Table XI, application no. 44, columns 5 or 7, or of a derived
sequences as indicated in Table XII, application no. 44, columns 5
or 7 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[17378] [0190.0.0.44]: see [0190.0.0.27]
[17379] [0191.0.44.44] The organisms or part thereof produce
according to the herein mentioned process of the invention an
increased level of free and/or bound the respective fine chemical
compared to said control or selected organisms or parts
thereof.
[17380] [0192.0.0.44] to [0203.0.0.44]: see [0192.0.0.27] to
[0203.0.0.27]
[17381] [0204.0.44.44] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule which comprises a
nucleic acid molecule selected from the group consisting of:
[17382] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 44, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, in particular according to tableTable XII, application
no. 44, column 6 in an organism or a part thereof [17383] b)
nucleic acid molecule comprising, preferably at least the mature
form, of a nucleic acid molecule as indicated in Table XI,
application no. 44, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [17384] c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as result of the
degeneracy of the genetic code and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[17385] d) nucleic acid molecule encoding a polypeptide whose
sequence has at least 50% identity with the amino acid sequence of
the polypeptide encoded by the nucleic acid molecule of (a) to (c)
and conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [17386] e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[17387] f) nucleic acid molecule encoding a polypeptide, the
polypeptide being derived by substituting, deleting and/or adding
one or more amino acids of the amino acid sequence of the
polypeptide encoded by the nucleic acid molecules (a) to (d),
preferably to (a) to (c), and conferring an increase in the amount
of the fine chemical in an organism or a part thereof; [17388] g)
nucleic acid molecule encoding a fragment or an epitope of a
polypeptide which is encoded by one of the nucleic acid molecules
of (a) to (e), preferably to (a) to (c) and conferring an increase
in the amount of the fine chemical in an organism or a part
thereof; [17389] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying a cDNA library or a
genomic library using primers or primer pairs as indicated in Table
XIII, application no. 44, column 7, and conferring an increase in
the amount of the respective fine chemical, in particular according
to tableTable XII, application no. 44, column 6 in an organism or a
part thereof; [17390] i) nucleic acid molecule encoding a
polypeptide which is isolated, e.g. from a expression library, with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (g), preferably to (a)
to (c) and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [17391] j) nucleic acid
molecule which encodes a polypeptide comprising a consensus
sequence as indicated in Table XIV, application no. 44, columns 5
or 7, and conferring an increase in the amount of the respective
fine chemical, in particular according to table XII, application
no. 44, column 6 in an organism or a part thereof; [17392] k)
nucleic acid molecule encoding the amino acid sequence of a
polypeptide encoding a domaine of a polypeptide as indicated in
Table XII, application no. 44, columns 5 or 7, and conferring an
increase in the amount of the respective fine chemical, in
particular accccording to table XII, application no. 44, column 6
in an organism or a part thereof; and [17393] l) nucleic acid
molecule which is obtainable by screening a suitable nucleic acid
library under stringent hybridization conditions with a probe
comprising one of the sequences of the nucleic acid molecule of (a)
to (k) or with a fragment of at least 15 nt, preferably 20 nt, 30
nt, 50 nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (h) or of a nucleic acid molecule as
indicated in Table XI, application no. 44, columns 5 or 7, or a
nucleic acid molecule encoding, preferably at least the mature form
of, a polypeptide as indicated in Table XII, application no. 44,
columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical according to tableTable XII, application
no. 44, column 6 in an organism or a part thereof; [17394] or which
encompasses a sequence which is complementary thereto; whereby,
preferably, the nucleic acid molecule according to (a) to (l)
distinguishes over the sequence indicated in Table XI, application
no. 44, columns 5 or 7, by one or more nucleotides. In one
embodiment, the nucleic acid molecule does not consist of the
sequence shown and indicated in Table XI, application no. 44,
columns 5 or 7,
[17395] In one embodiment, the nucleic acid molecule is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical to a sequence
indicated in Table XI, application no. 44, columns 5 or 7.
[17396] In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table XII,
application no. 44, columns 5 or 7.
[17397] In an other embodiment, the nucleic acid molecule of the
present invention is at least 30%, 40%, 50%, or 60% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 44, columns 5 or
7.
[17398] In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table XII,
application no. 44, columns 5 or 7.
[17399] Accordingly, in one embodiment, the nucleic acid molecule
of the differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 44, columns 5 or
7.
[17400] Accordingly, in one embodiment, the nucleic acid molecule
of the present invention encodes a polypeptide, which differs at
least in one or more amino acids from a polypeptide indicated in
Table XII, application no. 44, columns 5 or 7.
[17401] In another embodiment, a nucleic acid molecule indicated in
Table XI, application no. 44, columns 5 or 7.
[17402] Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table XII, application no.
44, columns 5 or 7.
[17403] In a further embodiment, the protein of the present
invention is at least 30%, 40%, 50%, or 60% identical to a protein
sequence indicated in Table XII, application no. 44, columns 5 or
7, and less than 100%, preferably less than 99.999%, 99.99% or
99.9%, more preferably less than 99%, 985, 97%, 96% or 95%
identical to a sequence as indicated in Table XI, columns 5 or
7.
[17404] [0205.0.0.44] to [0206.0.0.44]: see [0205.0.0.27] to
[0206.0.0.27]
[17405] [0207.0.44.44] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the carotenoid metabolism, the
carotene, in particular beta-carotene metabolism, the astaxanthin
metabolism, the amino acid metabolism, of glycolysis, of the
tricarboxylic acid metabolism or their combinations. As described
herein, regulator sequences or factors can have a positive effect
on preferably the gene expression of the genes introduced, thus
increasing it. Thus, an enhancement of the regulator elements may
advantageously take place at the transcriptional level by using
strong transcription signals such as promoters and/or enhancers. In
addition, however, an enhancement of translation is also possible,
for example by increasing mRNA stability or by inserting a
translation enhancer sequence.
[17406] [0208.0.0.44] to [0226.0.0.44]: see [0208.0.0.27] to
[0226.0.0.27]
[17407] [0227.0.44.44] The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[17408] In addition to a sequence indicated in Table XI,
application no. 44, columns 5 or 7, or its derivatives, it is
advantageous to express and/or mutate further genes in the
organisms. Especially advantageously, additionally at least one
further gene of the carotenoid, in particular carotene biosynthetic
pathway, e.g. one of the above mentioned genes of this pathway, or
e.g. for the synthesis of astaxanthin or for another provitamin A
or for another carotenoids or carotene, is expressed in the
organisms such as plants or microorganisms. It is also possible
that the regulation of the natural genes has been modified
advantageously so that the gene and/or its gene product is no
longer subject to the regulatory mechanisms which exist in the
organisms. This leads to an increased synthesis of the amino acids
desired since, for example, feedback regulations no longer exist to
the same extent or not at all. In addition it might be
advantageously to combine one or more of the sequences indicated in
Table XI, application no. 44, columns 5 or 7, with genes which
generally support or enhances to growth or yield of the target
organisms, for example genes which lead to faster growth rate of
microorganisms or genes which produces stress-, pathogen, or
herbicide resistant plants.
[17409] [0228.0.44.44] In a further embodiment of the process of
the invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the carotenoid, in
particular carotenes metabolism, in particular in synthesis of
beta-cryptoxanthin, zeaxanthin, astaxanthin or lutein.
[17410] [0229.0.44.44] Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the carotenoids biosynthetic
pathway, such as phytoene synthase (Psy), which is an important
control point for the regulation of the flux (Fraser et al., 2002),
phytoene desaturase (Pds), z-carotene desaturase, above mentioned
enzymes (s. introduction of the application), e.g. hydroxylases
such as beta-carotene hydroxylase (U.S. Pat. No. 6,214,575),
ketolases, or cyclases such as the beta-cyclase (U.S. Pat. No.
6,232,530) or oxygenases such as the beta-C4-oxygenase described in
U.S. Pat. No. 6,218,599 or homologs thereof, astaxanthin synthase
(U.S. Pat. No. 6,365,386), or other genes as described in U.S. Pat.
No. 6,150,130. These genes can lead to an increased synthesis of
the essential carotenoids, in particular carotene, in particular
beta-carotenes.
[17411] [0230.0.0.44] see [230.0.0.27].
[17412] [0231.0.44.44] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a beta-carotene degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[17413] [0232.0.0.44] to [0276.0.0.44]: see [0232.0.0.27] to
[0276.0.0.27]
[17414] [0277.0.44.44] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
are familiar, for example via extraction, salt precipitation,
and/or different chromatography methods. The process according to
the invention can be conducted batchwise, semibatchwise or
continuously. The fine respective chemical, i.e. e.g.
beta-carotene, and other carotenoids, in particular carotenes
produced by this process can be obtained by harvesting the
organisms, either from the crop in which they grow, or from the
field. This can be done via pressing or extraction of the plant
parts
[17415] [0278.0.0.44] to [0282.0.0.44]: see [0278.0.0.27] to
[0282.0.0.27]
[17416] [0283.0.44.44] Moreover, native polypeptide conferring the
increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table
XII, application no. 44, column 3, or an antibody against a
polypeptide as indicated in Table XII, application no. 44, columns
5 or 7, which can be produced by standard techniques utilizing the
polypeptid of the present invention or fragment thereof, i.e., the
polypeptide of this invention. Preferred are monoclonal
antibodies.
[17417] [0284.0.0.44] see [0284.0.0.27]
[17418] [0285.0.44.44] In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 44, columns 5 or 7, or as coded by a nucleic
acid molecule as indicated in Table XI, application no. 44, columns
5 or 7, or functional homologues thereof.
[17419] [0286.0.44.44] In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 44, columns 5 or 7, and in
one another embodiment, the present invention relates to a
polypeptide comprising or consisting of a consensus sequence as
indicated in Table XIV, application no. 44, columns 5 or 7, whereby
20 or less, preferably 15 or 10, preferably 9, 8, 7, or 6, more
preferred 5 or 4, even more preferred 3, even more preferred 2,
even more preferred 1, most preferred 0 of the amino acids
positions indicated can be replaced by any amino acid or, in an
further embodiment, can be replaced and/or absent. In one
embodiment, the present invention relates to the method of the
present invention comprising a polypeptide or to a polypeptide
comprising more than one consensus sequences (of an individual
line) as indicated in Table XIV, application no. 44, column 7.
[17420] [0287.0.0.44] to [0289.0.0.44]: see [0287.0.0.27] to
[0289.0.0.27]
[17421] [00290.0.44.44] The alignment was performed with the
Software AlignX (Sep. 25, 2002) a component of Vector NTI Suite
8.0, InforMax.TM., Invitrogen.TM. life science software, U.S. Main
Office, 7305 Executive Way, Frederick, Md. 21704, USA with the
following settings: For pairwise alignments: gap opening penalty:
10.0; gap extension penalty 0,1. For multiple alignments: Gap
opening penalty: 10.0; Gap extension penalty: 0.1; Gap separation
penalty range: 8; Residue substitution matrix: blosum62;
Hydrophilic residues: G P S N D Q E K R; Transition weighting: 0,5;
Consensus calculation options: Residue fraction for consensus: 0.9.
Presettings were selected to allow also for the alignment of
conserved amino acids
[17422] [0291.0.44.44] In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences. In one embodiment, said polypeptide of the
invention distinguishes over a sequence as indicated in Table XII,
application no. 44, columns 5 or 7, by one or more amino acids. In
one embodiment, polypeptide distinguishes form a sequence as
indicated in Table XII, application no. 44, columns 5 or 7, by more
than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, even more preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table XII, application no. 44, columns 5 or 7, by not
more than 80% or 70% of the amino acids, preferably not more than
60% or 50%, more preferred not more than 40% or 30%, even more
preferred not more than 20% or 10%. In an other embodiment, said
polypeptide of the invention does not consist of a sequence as
indicated in Table XII, application no. 44, columns 5 or 7.
[17423] [0292.0.0.44] see [0292.0.0.27]
[17424] [0293.0.44.44] In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, the polypeptide of
the invention has a sequence which distinguishes from a sequence as
indicated in Table XII, application no. 44, columns 5 or 7, by one
or more amino acids. In an other embodiment, said polypeptide of
the invention does not consist of the sequence as indicated in
Table XII, application no. 44, columns 5 or 7. In a further
embodiment, said polypeptide of the present invention is less than
100%, 99.999%, 99.99%, 99.9% or 99% identical. In one embodiment,
said polypeptide does not consist of the sequence encoded by a
nucleic acid molecules as indicated in Table XI, application no.
44, columns 5 or 7.
[17425] [0294.0.44.44] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 44, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 44, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[17426] [0295.0.0.44] to [0296.0.0.44]: see [0295.0.0.27] to
[0296.0.0.27]
[17427] [0297.0.0.44] see [0297.0.0.27]
[17428] [00297.1.44.44] Non-polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table XII, application no. 44, columns 3, 5 or 7.
[17429] [0298.0.44.44] A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 44, columns 5 or 7. The
portion of the protein is preferably a biologically active portion
as described herein. Preferably, the polypeptide used in the
process of the invention has an amino acid sequence identical to a
sequence as indicated in Table XII, application no. 44, columns 5
or 7.
[17430] [0299.0.44.44] Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 44,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 44, columns 5 or 7, or which is
homologous thereto, as defined above.
[17431] [0300.0.44.44] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 44, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 44, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[17432] [0301.0.0.44] see [0301.0.0.27]
[17433] [0302.0.44.44] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 44, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[17434] [0303.0.0.44] see [0303.0.0.27]
[17435] [0304.0.44.44] Manipulation of the nucleic acid molecule of
the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 44, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[17436] [0305.0.0.44] to [0308.0.0.44]: see [0305.0.0.27] to
[0308.0.0.27]
[17437] [0309.0.44.44] In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table XII,
application no. 44, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table XII, application no. 44, columns 5 or 7, refers
to a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous a polypeptide of the
invention, preferably which is not substantially homologous to a
polypeptide as indicated in Table XII, application no. 44, columns
5 or 7, e.g., a protein which does not confer the activity
described herein or annotated or known for as indicated in Table
XII, application no. 44, column 3, and which is derived from the
same or a different organism. In one embodiment, a "non-polypeptide
of the invention" or "other polypeptide" not being indicated in
Table XII, application no. 44, columns 5 or 7, does not confer an
increase of the respective fine chemical in an organism or part
thereof.
[17438] [0310.0.0.44] to [0334.0.0.44]: see [0310.0.0.27] to
[0334.0.0.27]
[17439] [0335.0.44.44] The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XI, application
no. 44, columns 5 or 7, and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived there from),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
application no. 44, columns 5 or 7, and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of an
protein encoded by a nucleic acid sequence sequences as indicated
in Table XI, application no. 44, columns 5 or 7, and/or homologs
thereof, one of the two RNA strands is essentially identical to at
least part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[17440] [0336.0.0.44] to [0342.0.0.44]: see [0336.0.0.27] to
[0342.0.0.27]
[17441] [0343.0.44.44] As has already been described, 100% sequence
identity between the dsRNA and a gene transcript of a nucleic acid
sequence as indicated in Table XI, application no. 44, columns 5 or
7, or its homolog is not necessarily required in order to bring
about effective reduction in the expression. The advantage is,
accordingly, that the method is tolerant with regard to sequence
deviations as may be present as a consequence of genetic mutations,
polymorphisms or evolutionary divergences. Thus, for example, using
the dsRNA, which has been generated starting from a sequence as
indicated in Table XI, application no. 44, columns 5 or 7, or
homologs thereof of the one organism, may be used to suppress the
corresponding expression in another organism.
[17442] [0344.0.0.44] to [0350.0.0.44]: see [0344.0.0.27] to
[0350.0.0.27]
[17443] [0351.0.0.44] to [0361.0.0.44]: see [0351.0.0.27] to
[0361.0.0.27]
[17444] [0362.0.44.44] Accordingly the present invention relates to
any cell transgenic for any nucleic acid characterized as part of
the invention, e.g. conferring the increase of the respective fine
chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 44, columns 5 or 7, e.g. encoding a
polypeptide having protein activity, as indicated in Table XII,
application no. 44, columns 3. Due to the above mentioned activity
the respective fine chemical content in a cell or an organism is
increased. For example, due to modulation or manipulation, the
cellular activity of the polypeptide of the invention or nucleic
acid molecule of the invention is increased, e.g. due to an
increased expression or specific activity of the subject matters of
the invention in a cell or an organism or a part thereof.
Transgenic for a polypeptide having an activity of a polypeptide as
indicated in Table XII, application no. 44, columns 5 or 7, means
herein that due to modulation or manipulation of the genome, an
activity as annotated for a polypeptide as indicated in Table XII,
application no. 44, column 3, e.g. having a sequence as indicated
in Table XII, application no. 44, columns 5 or 7, is increased in a
cell or an organism or a part thereof. Examples are described above
in context with the process of the invention.
[17445] [0363.0.0.44] see [0363.0.0.27]
[17446] [0364.0.44.44] A naturally occurring expression
cassette--for example the naturally occurring combination of a
promoter of a gene encoding a polypeptide of the invention as
indicated in Table XII, application no. 44, column 3, 5 or 7, with
the corresponding protein-encoding sequence as indicated in Table
XI, application no. 44, column 5 or 7, --becomes a transgenic
expression cassette when it is modified by non-natural, synthetic
"artificial" methods such as, for example, mutagenization. Such
methods have been described (U.S. Pat. No. 5,565,350; WO 00/15815;
also see above).
[17447] [0365.0.0.44] to [0373.0.0.44]: see [0365.0.0.27] to
[0373.0.0.27]
[17448] [0374.0.44.44] Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. The respective fine chemical
produced in the process according to the invention may, however,
also be isolated from the plant in the form of their free
beta-carotene or bound in or to compounds or moieties, like
glucosides, e.g. diglucosides. The respective fine chemical
produced by this process can be harvested by harvesting the
organisms either from the culture in which they grow or from the
field. This can be done via expressing, grinding and/or extraction,
salt precipitation and/or ion-exchange chromatography or other
chromatographic methods of the plant parts, preferably the plant
seeds, plant fruits, plant tubers and the like.
[17449] [0375.0.0.44] to [0376.0.0.44]: see [0375.0.0.27] to
[0376.0.0.27]
[17450] [0377.0.44.44] Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced carotenoids, e.g the fine chemical(s) comprising
composition is isolated. In one embodiment, the produced respective
fine chemical is isolated.
[17451] [0378.0.44.44] In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the respective
fine chemical produced in the process can be isolated. The
resulting respective fine chemical can, if appropriate,
subsequently be further purified, if desired mixed with other
active ingredients such as vitamins, amino acids, carbohydrates,
antibiotics and the like, and, if appropriate, formulated.
[17452] [0379.0.44.44] In one embodiment, the product of the
process is a mixture of the fine chemicals. In one embodiment, the
product is a mixture of the respective fine chemicals with other
carotenoids.
[17453] [0380.0.44.44] The respective fine chemicals obtained in
the process are suitable as starting material for the synthesis of
further products of value. For example, they can be used in
combination with each other or alone for the production of
pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates to a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the respective fine chemical
containing composition produced by the process of the invention or
the respective fine chemical produced by the process of the
invention--if desired--and formulating the product with a
pharmaceutical or cosmetic acceptable carrier or formulating the
product in a form acceptable for an application in agriculture. A
further embodiment according to the invention is the use of the the
respective fine chemical produced in the process or of the
transgenic organisms in animal feeds, foodstuffs, medicines, food
supplements, cosmetics or pharmaceuticals or for the production of
other carotenoids, e.g. in after isolation of the respective fine
chemical or without, e.g. in situ, e.g within the organism used for
the process for the production of the respective fine chemical.
[17454] [0381.0.0.44] to [0382.0.0.44]: see [0381.0.0.27] to
[0382.0.0.27] [0383.0.44.44] ./.
[17455] [0384.0.0.44] see [0384.0.0.27]
[17456] [0385.0.44.44] The fermentation broths obtained in this
way, containing in particular Beta-carotene in mixtures with other
carotenoids, in particular with other carotenoids, in particular
carotenes, or containing microorganisms or parts of microorganisms,
like plastids, containing the respective fine chemical or the
carotenoids produced in mixtures with other carotenoids, in
particular with other carotene, normally have a dry matter content
of from 1 to 70% by weight, preferably 7.5 to 25% by weight.
Sugar-limited fermentation is additionally advantageous, e.g. at
the end, for example over at least 30% of the fermentation time.
This means that the concentration of utilizable sugar in the
fermentation medium is kept at, or reduced to, 0 to 10 g/l,
preferably to 0 to 3 g/l during this time. The fermentation broth
is then processed further. Depending on requirements, the biomass
can be removed or isolated entirely or partly by separation
methods, such as, for example, centrifugation, filtration,
decantation, coagulation/flocculation or a combination of these
methods, from the fermentation broth or left completely in it.
[17457] The fermentation broth can then be thickened or
concentrated by known methods, such as, for example, with the aid
of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[17458] As carotenoids are often localized in membranes or
plastids, in one embodiment it is advantageous to avoid a leaching
of the cells when the biomass is isolated entirely or partly by
separation methods, such as, for example, centrifugation,
filtration, decantation, coagulation/flocculation or a combination
of these methods, from the fermentation broth. The dry biomass can
directly be added to animal feed, provided the carotenoids
concentration is sufficiently high and no toxic compounds are
present. In view of the instability of carentoids, conditions for
drying, e.g. spray or flash-drying, can be mild and can be avoiding
oxidation and cis/trans isomerization. For example antioxidants,
e.g. BHT, ethoxyquin or other, can be added. In case the
carotenoids concentration in the biomass is to dilute, solvent
extraction can be used for their isolation, e.g. with alcohols,
ether or other organic solvents, e.g. with methanol, ethanol,
aceton, alcoholic potassium hydroxide, glycerol-phenol, liquefied
phenol or for example with acids or bases, like trichloroacetatic
acid or potassium hydroxide. A wide range of advantageous Methods
and techniques for the isolation of carotenoids, in particular of
the respective fine chemical, in particular of beta-carotene can be
found in the state of the art. In case phenol is used it can for
example be removed with ether and water extraction and the dry
eluate comprises a mixture of the carotenoids of the biomass.
[17459] [0386.0.44.44] Accordingly, it is possible to further
purify the respective fine chemicals or other carotenoids, in
particular the carotenes produced according to the invention. For
this purpose, the product-containing composition, e.g. a total or
partial lipid extraction fraction using organic solvents, e.g. as
described above, is subjected for example (without meaning to be
limited) to a saponification to remove triglycerides, partition
between e.g. hexane/methanol (separation of non-polar epiphase from
more polar hypophasic derivates) and separation via e.g. an open
column chromatography or HPLC in which case the desired product or
the impurities are retained wholly or partly on the chromatography
resin. These chromatography steps can be repeated if necessary,
using the same or different chromatography resins. The skilled
worker is familiar with the choice of suitable chromatography
resins and their most effective use.
[17460] [0387.0.0.44] to [0392.0.0.44]: see [0387.0.0.27] to
[0392.0.0.27]
[17461] [0393.0.44.44] In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [17462] a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [17463] b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table XI, application no. 44, columns 5 or 7, and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[17464] c) introducing the candidate nucleic acid molecules in host
cells, preferably in a plant cell or a microorganism, appropriate
for producing the fine chemical; [17465] d) expressing the
identified nucleic acid molecules in the host cells; [17466] e)
assaying the the fine chemical level in the host cells; and [17467]
f) identifying the nucleic acid molecule and its gene product which
expression confers an increase in the the fine chemical level in
the host cell after expression compared to the wild type.
[17468] [0394.0.32.44] to [0552.0.32.44]: see [0394.0.0.32] to
[0552.0.0.32]
[17469] [0553.0.44.44]
1. A process for the production of beta-Carotene resp., which
comprises (a) increasing or generating the activity of a protein as
indicated in Table XII, application no. 44, columns 5 or 7, or a
functional equivalent thereof in a non-human organism, or in one or
more parts thereof; and (b) growing the organism under conditions
which permit the production of beta-Carotene resp. in said
organism. 2. A process for the production of beta-Carotene resp.,
comprising the increasing or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: a) nucleic acid molecule encoding of a polypeptide
as indicated in Table XII, application no. 44, columns 5 or 7, or a
fragment thereof, which confers an increase in the amount of the
fine chemical as indicated in table XII, column 6, e.g of
beta-Carotene resp., in an organism or a part thereof; b) nucleic
acid molecule comprising of a nucleic acid molecule as indicated in
Table XI, application no. 44, columns 5 or 7, c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of beta-Carotene resp., in an organism or a part thereof; d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical as indicated in
table XII, column 6, e.g of beta-Carotene resp., in an organism or
a part thereof; e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the fine chemical as indicated in table XII, column 6, e.g of
beta-Carotene resp., in an organism or a part thereof; f) nucleic
acid molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table XIII, application no. 44, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of beta-Carotene resp., in an organism or a part
thereof; g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of beta-Carotene resp., in
an organism or a part thereof; h) nucleic acid molecule encoding a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 44, column 7, and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of beta-Carotene resp., in an organism or a part thereof;
and i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of beta-Carotene resp., in
an organism or a part thereof. or comprising a sequence which is
complementary thereto. 3. The process of claim 1 or 2, comprising
recovering of the free or bound beta-Carotene resp. 4. The process
of any one of claims 1 to 3, comprising the following steps: (a)
selecting an organism or a part thereof expressing a polypeptide
encoded by the nucleic acid molecule characterized in claim 2; (b)
mutagenizing the selected organism or the part thereof; (c)
comparing the activity or the expression level of said polypeptide
in the mutagenized organism or the part thereof with the activity
or the expression of said polypeptide of the selected organisms or
the part thereof; (d) selecting the mutated organisms or parts
thereof, which comprise an increased activity or expression level
of said polypeptide compared to the selected organism or the part
thereof; (e) optionally, growing and cultivating the organisms or
the parts thereof; and (f) recovering, and optionally isolating,
the free or bound beta-Carotene resp., produced by the selected
mutated organisms or parts thereof. 5. The process of any one of
claims 1 to 4, wherein the activity of said protein or the
expression of said nucleic acid molecule is increased or generated
transiently or stably. 6. An isolated nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: a) nucleic acid molecule encoding of a polypeptide
as indicated in Table XII, application no. 44, columns 5 or 7, or a
fragment thereof, which confers an increase in the amount of the
fine chemical as indicated in table XII, column 6, e.g of
beta-Carotene resp., in an organism or a part thereof; b) nucleic
acid molecule comprising of a nucleic acid molecule as indicated in
Table XI, application no. 44, columns 5 or 7, c) nucleic acid
molecule whose sequence can be deduced from a polypeptide sequence
encoded by a nucleic acid molecule of (a) or (b) as a result of the
degeneracy of the genetic code and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of beta-Carotene resp., in an organism or a part thereof; d)
nucleic acid molecule which encodes a polypeptide which has at
least 50% identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid molecule of (a) to (c) and conferring
an increase in the amount of the fine chemical as indicated in
table XII, column 6, e.g of beta-Carotene resp., in an organism or
a part thereof; e) nucleic acid molecule which hybridizes with a
nucleic acid molecule of (a) to (c) under under stringent
hybridisation conditions and conferring an increase in the amount
of the fine chemical as indicated in table XII, column 6, e.g of
beta-Carotene resp., in an organism or a part thereof; f) nucleic
acid molecule which encompasses a nucleic acid molecule which is
obtained by amplifying nucleic acid molecules from a cDNA library
or a genomic library using the primers or primer pairs as indicated
in Table XIII, application no. 44, column 7, and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of beta-Carotene resp., in an organism or a part
thereof; g) nucleic acid molecule encoding a polypeptide which is
isolated with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(f) and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of beta-Carotene resp., in
an organism or a part thereof; h) nucleic acid molecule encoding a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 44, column 7, and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of beta-Carotene resp., in an organism or a part thereof;
and i) nucleic acid molecule which is obtainable by screening a
suitable nucleic acid library under stringent hybridization
conditions with a probe comprising one of the sequences of the
nucleic acid molecule of (a) to (k) or with a fragment thereof
having at least 15 nt, preferably 20 nt, 30 nt, 50 nt, 100 nt, 200
nt or 500 nt of the nucleic acid molecule characterized in (a) to
(k) and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of beta-Carotene resp., in
an organism or a part thereof. whereby the nucleic acid molecule
distinguishes over the sequence as indicated in Table XI,
application no. 44, columns 5 or 7, by one or more nucleotides. 7.
A nucleic acid construct which confers the expression of the
nucleic acid molecule of claim 6, comprising one or more regulatory
elements. 8. A vector comprising the nucleic acid molecule as
claimed in claim 6 or the nucleic acid construct of claim 7. 9. The
vector as claimed in claim 8, wherein the nucleic acid molecule is
in operable linkage with regulatory sequences for the expression in
a prokaryotic or eukaryotic, or in a prokaryotic and eukaryotic,
host. 10. A host cell, which has been transformed stably or
transiently with the vector as claimed in claim 8 or 9 or the
nucleic acid molecule as claimed in claim 6 or the nucleic acid
construct of claim 7 or produced as described in claim any one of
claims 2 to 5. 11. The host cell of claim 10, which is a transgenic
host cell. 12. The host cell of claim 10 or 11, which is a plant
cell, an animal cell, a microorganism, or a yeast cell, a fungus
cell, a prokaryotic cell, an eukaryotic cell or an archaebacterium.
13. A process for producing a polypeptide, wherein the polypeptide
is expressed in a host cell as claimed in any one of claims 10 to
12. 14. A polypeptide produced by the process as claimed in claim
13 or encoded by the nucleic acid molecule as claimed in claim 6
whereby the polypeptide distinguishes over a sequence as indicated
in Table XII, application no. 44, columns 5 or 7, by one or more
amino acids 15. An antibody, which binds specifically to the
polypeptide as claimed in claim 14. 16. A plant tissue, propagation
material, harvested material or a plant comprising the host cell as
claimed in claim 12 which is plant cell or an Agrobacterium. 17. A
method for screening for agonists and antagonists of the activity
of a polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of beta-Carotene resp., in an
organism or a part thereof comprising: (a) contacting cells,
tissues, plants or microorganisms which express the a polypeptide
encoded by the nucleic acid molecule of claim 5 conferring an
increase in the amount of beta-Carotene resp., in an organism or a
part thereof with a candidate compound or a sample comprising a
plurality of compounds under conditions which permit the expression
the polypeptide; (b) assaying the beta-Carotene resp., level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and (c) identifying a
agonist or antagonist by comparing the measured beta-Carotene
resp., level or polypeptide expression level with a standard
beta-Carotene resp., or polypeptide expression level measured in
the absence of said candidate compound or a sample comprising said
plurality of compounds, whereby an increased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an agonist and a decreased level over the
standard indicates that the compound or the sample comprising said
plurality of compounds is an antagonist. 18. A process for the
identification of a compound conferring increased beta-Carotene
resp., production in a plant or microorganism, comprising the
steps: (a) culturing a plant cell or tissue or microorganism or
maintaining a plant expressing the polypeptide encoded by the
nucleic acid molecule of claim 6 conferring an increase in the
amount of beta-Carotene resp., in an organism or a part thereof and
a readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with dais readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
beta-Carotene resp., in an organism or a part thereof; (b)
identifying if the compound is an effective agonist by detecting
the presence or absence or increase of a signal produced by said
readout system. 19. A method for the identification of a gene
product conferring an increase in beta-Carotene resp., production
in a cell, comprising the following steps: (a) contacting the
nucleic acid molecules of a sample, which can contain a candidate
gene encoding a gene product conferring an increase in
beta-Carotene resp., after expression with the nucleic acid
molecule of claim 6; (b) identifying the nucleic acid molecules,
which hybridise under relaxed stringent conditions with the nucleic
acid molecule of claim 6; (c) introducing the candidate nucleic
acid molecules in host cells appropriate for producing
beta-Carotene resp.; (d) expressing the identified nucleic acid
molecules in the host cells; (e) assaying the beta-Carotene resp.,
level in the host cells; and (f) identifying nucleic acid molecule
and its gene product which expression confers an increase in the
beta-Carotene resp., level in the host cell in the host cell after
expression compared to the wild type. 20. A method for the
identification of a gene product conferring an increase in
beta-Carotene resp., production in a cell, comprising the following
steps: (a) identifying in a data bank nucleic acid molecules of an
organism; which can contain a candidate gene encoding a gene
product conferring an increase in the beta-Carotene resp., amount
or level in an organism or a part thereof after expression, and
which are at least 20% homolog to the nucleic acid molecule of
claim 6; (b) introducing the candidate nucleic acid molecules in
host cells appropriate for producing beta-Carotene resp.; (c)
expressing the identified nucleic acid molecules in the host cells;
(d) assaying the beta-Carotene resp., level in the host cells; and
(e) identifying nucleic acid molecule and its gene product which
expression confers an increase in the beta-Carotene resp., level in
the host cell after expression compared to the wild type. 21. A
method for the production of an agricultural composition comprising
the steps of the method of any one of claims 17 to 20 and
formulating the compound identified in any one of claims 17 to 20
in a form acceptable for an application in agriculture. 22. A
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of any one of claim 8 or 9, an antagonist or agonist
identified according to claim 17, the compound of claim 18, the
gene product of claim 19 or 20, the antibody of claim 15, and
optionally an agricultural acceptable carrier. 23. Use of the
nucleic acid molecule as claimed in claim 6 for the identification
of a nucleic acid molecule conferring an increase of beta-Carotene
resp., after expression. 24. Use of the polypeptide of claim 14 or
the nucleic acid construct claim 7 or the gene product identified
according to the method of claim 19 or 20 for identifying compounds
capable of conferring a modulation of beta-Carotene resp., levels
in an organism. 25. Cosmetic, pharmaceutical, food or feed
composition comprising the nucleic acid molecule of claim 6, the
polypeptide of claim 14, the nucleic acid construct of claim 7, the
vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. 26. The method of any one of claims 1
to 5, the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20, wherein the carotene is beta-carotene 27.
Use of the nucleic acid molecule of claim 6, the polypeptide of
claim 14, the nucleic acid construct of claim 7, the vector of
claim 8 or 9, the antagonist or agonist identified according to
claim 17, the antibody of claim 15, the plant or plant tissue of
claim 16, the harvested material of claim 16, the host cell of
claim 10 to 12 or the gene product identified according to the
method of claim 19 or 20 for the protection of a plant against a
oxidative stress. 28. Use of the nucleic acid molecule of claim 6,
the polypeptide of claim 14, the nucleic acid construct of claim 7,
the vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20 for the protection of a plant against
a oxidative stress causing or a carotenoid synthesis inhibiting
herbicide. 29. Use of the agonist identified according to claim 17,
the plant or plant tissue of claim 16, the harvested material of
claim 16, or the host cell of claim 10 to 12 for the production of
a cosmetic composition.
[17470] [0554.0.0.44] Abstract: see [0554.0.0.27]
NEW GENES FOR A PROCESS FOR THE PRODUCTION OF FINE CHEMICALS
[17471] [0000.0.45.45] In a further embodiment, the present
invention relates to a further process for the production of fine
chemicals as defined below and the corresponding embodiments as
described herein as follows.
[17472] [0001.0.0.45] see [0001.0.0.27]
[17473] [0002.0.45.45] Carbohydrates are aldehyde or ketone
compounds with multiple hydroxyl groups. Many carbohydrates have
the empirical formula (CH.sub.2O)n; some also contain nitrogen,
phosphorus, or sulfur.
[17474] Carbohydrates are classsfied in monosaccharides,
oligosaccharides, and polysaccharides.
[17475] Monosaccharides, or simple sugars, consist of a single
polyhydroxy aldehyde or ketone unit. Monosaccharides of more than
four carbons tend to have cyclic structures.
[17476] Oligosaccharides consist of short chains of monosaccharide
units, or residues, usually 2 to 19 units, joined by glycosidic
bonds.
[17477] The polysaccharides are sugar polymers containing more than
20 or so monosaccharide units, and some have hundreds or thousands
of units. Some polysaccharides are linear chains; others are
branched.
[17478] Carbohydrates are called saccharides or, if they are
relatively small, sugars.
[17479] In the present invention, saccharides means all of the
aforementioned carbohydrate, e.g. monosaccharides, preferably
fructose, glucose, inositol, galactose, arabinose xylose or other
pentoses or hexoses; oligosaccharides, preferably disaccharides
like sucrose, lactose or trisaccharides like raffinose; or
polysaccharides like starch or cellulose.
[17480] [0003.0.45.45] Carbohydrates are the most abundant class of
organic compounds found in living organisms.
[17481] They are a major source of metabolic energy, both for
plants and for animals. Aside from the sugars and starches that
meet this vital nutritional role, carbohydrates function in energy
storage (for example starch or glycogen), in signaling (by
glycoproteins and glycolipids, e.g. blood group determinants), fuel
the nervous system, muscle and virtually all cells, are parts of
nucleic acids (in genes, mRNA, tRNA, ribosomes), and as cell
surface markers as recognition sites on cell surfaces and signaling
in glycolipids and glycoproteins and also serve as a structural
material for example as cell wall components (cellulose).
[17482] [0004.0.45.45] Glucose, also called dextrose, is the most
widely distributed sugar in the plant and animal kingdoms and it is
the sugar present in blood as "blood sugar. It occupies a central
position in the metabolism of plants, animals, and many
microorganisms. Glucose is rich in potential energy, and thus a
good fuel; in the body is catabolised to produce ATP. It is stored
as a high molecular weight polymer such as starch or glycogen or is
converted to fatty acids. It is also a remarkably versatile
precursor, capable of supplying a huge array of metabolic
intermediates for biosynthetic reactions.
[17483] Based on its manifold features, glucose is used in
nutrition and medicine. Fructose, also called levulose or "fruit
sugar, is the most important ketose sugar. Fructose is a hexose and
is a reducing sugar. Fructose is used a a sweetener by diabetics
because it does not rise the blood sugar level, even in large
amounts. Fructose and glucose are the main carbohydrate
constituents of honey. Those hexoses are further the main
components of many oligo- and polysaccharides, like sucrose,
raffinose, stachyose, trehalose, starch, cellulose or dextran.
[17484] The most frequent disaccharide is sucrose (saccharose,
b-D-fructofuranosyl-a-D-glucopyranosid, cane sugar, beet sugar,
sugar in a narrow sense of a name for commercially available
sucrose meaning sucrose is the sugar that is commonly called
"sugar) which consists of the six-carbon sugars D-glucose and
D-fructose. It is formed by plants but not by animals. Sucrose is a
major intermediate product of photosynthesis; in many plants it is
the principal form in which sugar is transported from the leaves to
other parts of the plant body. In mammalians sucrose is an
obligatory component of blood and its content in blood is kept at
the stable level. It is strongly necessary for brain cells as well
as for normal functioning of the central nervous system. Sugar is
widely-known as a source of glycogen--a substance, feeding liver,
heart and muscles. It is one of the most widely-used food products
and is the major disaccharide in most diets. It is present in
honey, maple sugar, fruits, berries, and vegetables. It may be
added to food products as liquid or crystalline sucrose or as
invert sugar. It is commercially prepared from sugar cane or sugar
beets. Sucrose can provide a number of desirable functional
qualities to food products including sweetness, mouth-feel, and the
ability to transform between amorphous and crystalline states.
High-concentrated sucrose is a natural preserving agent, it
determines gel-formation processes, gives necessary viscosity to
the products. Sucrose is a raw material for caramel, colour
etc.
[17485] Sucrose is further an excellent fermentation feedstock,
which is of specific interest for fermentation industry (including
a number of non-food industries--pharmaceutical industries). The
presence of eight hydroxyl groups in the sucrose molecule provides
a theoretical possibility of a very large number of sucrose
derivatives. Sucrose derivatives are used by industries in
production of detergents, emulsifiers (sucrose+fatty acids) and
adhesives (sucrose octa acetate).
[17486] Sucrose is a precursor to a group of carbohydrates in
plants known as the raffinose family of oligosaccharides found in
many plant seeds especially legumes. This family contains the
trisaccharide raffinose, the tetrasaccharide stachyose and the
pentasaccharide verbascose. Oligosaccharides of the
raffinose-series are major components in many food legumes
(Shallenberger et al., J. Agric. Food Chem., 9,1372; 1961).
Raffinose
(beta-D-fructofuranosyl-6-O-alpha-D-galactopyranosyl-alpha-D-gl-
ucopyranosid, melitriose, gossypose, melitose), which consists of
sucrose with .alpha.-galactose attached through its C-4 atom to the
1 position on the fructose residue and is thought to be second only
to sucrose among the nonstructural carbohydrates with respect to
abundance in the plant kingdom. It may be ubiquitous, at least
among higher plants. Raffinose accumulate in significant quantities
in the edible portion of many economically significant crop
species. Examples include soybean, sugar beet, cotton, canola and
all of the major edible leguminous crops including beans, peas,
lentil and lupine.
[17487] An important key intermediates in the formation of
raffinose and stachyose is myo-inositol
(cyclohexan-1,2,3,4,5,6-hexaole), the most common cyclitol.
Myo-inositol is fundamental to many different aspects of plant
growth and development. In addition to its role as the precursor
for phytic acid biosynthesis, myo-inositol is also used for uronide
and pentose biosynthesis, it is also present in phosphoinositides
of plant cell membranes, as well as other complex plant lipids
including glycophosphoceramides. Furthermore, it is also a
precursor of other naturally occurring inositol isomers, and many
of these as well as myo-inositol are distributed as methyl ethers
in a species specific pattern throughout the plant kingdom.
Myo-inositol is an important growth factor.
[17488] The most carbohydrates found in nature occur as
polysaccharides which are polymers of medium to high molecular
weight. Polysaccharides, also called glycans, differ from each
other in the identity of their recurring monosaccharide units, in
the length of their chains, in the types of bonds linking the
units, and in the degree of branching.
[17489] Starch is the most valuable polysaccharide. Normal native
starches consist of a mixture of 15-30% amylose and 70-85%
amylopectin. Amylose structurally is a linear polymer of
anhydroglucose units, of molecular weight approximately between 40
000 and 340 000, the chains containing 250 to 2000 anhydroglucose
units. Amylopectin is considered to be composed of anhydroglucose
chains with many branch points; the molecular weight may reach as
high as 80 000 000.
[17490] Starch is the most important, abundant, digestible food
polysaccharide. It occurs as the reserve polysaccharide in the
leaf, stem, root, seed, fruit and pollen of many higher plants. It
occurs as discrete, partially-crystalline granules whose size,
shape, and gelatinization temperature depend on the botanical
source of the starch. Common food starches are derived from seed
(wheat, maize, rice, barley) and root (potato, cassava/tapioca)
sources. Starches have been modified to improve desired functional
characteristics and are added in relatively small amounts to foods
as food additives. Another important polysaccharide is cellulose.
Cellulose is the most commonly seen polysaccharide and scientist
estimate that over one trillion tons of cellulose are synthesized
by plants each year. Cellulose forms the cell wall of plants. It is
yet a third polymer of the monosaccharide glucose. Cellulose
differs from starch and glycogen because the glucose units form a
two-dimensional structure, with hydrogen bonds holding together
nearby polymers, thus giving the molecule added stability. A single
"cellulose fiber" can consist of up to 10000 individual
anhydroglucose units. In cellulose, the individual fiber molecules
are arranged in bundles and thus form so called micro fibrils which
ultimately result in a "densely woven" net like structure of
cellulose molecules. The strong cohesion between the individual
cellulose fibers is due to the huge number of strong hydrogen
bonds.
[17491] Cellulose is the major polysaccharide of grass, leaves and
trees and is said to include around 50% of all biological carbon
found on our planet. It is the basic material of natural substances
such as wood, flax or cotton and consists of long, unbranched fiber
molecules. Cellulose, as plant fiber, cannot be digested by human
beings therefore cellulose passes through the digestive tract
without being absorbed into the body. Some animals, such as cows
and termites, contain bacteria in their digestive tract that help
them to digest cellulose. Nevertheless, cellulose is of importance
in human nutrition in that fiber is an essential part of the diet,
giving bulk to food and promoting intestinal motility.
[17492] [0005.0.45.45] The polysaccharides starch and cellulose are
the most important raw material in the industrial and commercial
production of glucose. In the common procedure starch or cellulose
are acidly or enzymatically hydrolysed to glucose.
[17493] Fructose is usually also produced from starch by
enzymatically transforming it into glucose syrup and subsequently
treating with isomerase, leading to a conversion of glucose to
fructose.
[17494] Succrose is obtained commercially from the expressed juice
of sugar cane or of sugar beet.
[17495] Myo-inositol exists in nature either in its free form
(found, for example, in sugarcane, beet molasses, and almond hulls)
or as a hexaphosphate called phytin (found, for example, in corn
steep liquor). Industrial purification of phytin from corn steep
liquor involves precipitation with calcium, followed by hydrolysis
with a strong acid. Separation of free form inositols from plant
extracts involves treatment with acid and separation of
myo-inositol by column (U.S. Pat. No. 5,482,631) or the use of
ion-exchange (U.S. Pat. No. 4,482,761).
[17496] Cellulose is a very important industrial product. As
disclosed above, it serves as row material for monosaccharides. It
is further used in the manufacture of paper, textiles, plastics,
explosives, packaging material (Cellophane.RTM.), feed, food and
fermentation products. Cellulose is obtained primarily by acid or
alkaline hydrolize.
[17497] Starch occurs intracellularly as large clusters or
granules. These granular starch consists of microscopic granules,
which differ in size and shape, depending on the plant source. The
granules are insoluble in water at room temperature. There is a
quite number of methods known for the extraction of starch. For
example a slurry of grinded starch containing plant material is
heated, whereby the granules swell and eventually burst, dispersing
the starch molecules into the solution. During the liquefaction
step, the long-chained starch is further degraded into smaller
branched and linear units (maltodextrins) by an alpha-amylase. A
large number of processes have been described for converting starch
to starch hydrolysates, such as maltose, glucose or specialty
syrups, either for use as sweeteners or as precursors for other
saccharides such as fructose. A process for enzymatic hydrolysis of
granular starch into a soluble starch hydrolysate is disclosed in
US 20050042737.
[17498] [0006.0.45.45] Carbohydrates play a major role in human and
animal diets, comprising some 40-75% of energy intake. Their most
important nutritional property is digestibility. Some of them are
hydrolyzed by enzymes of the human gastrointestinal system to
monosaccharides that are absorbed in the small intestine and enter
the pathways of carbohydrate metabolism. Others can be digested by
certain animals. Carbohydrates, fat and protein are the energy
yielding nutrients in animal feed. In the average diet for farm
animals, carbohydrates are included at levels of 70-80%. For
example pig diets are mainly based on cereals which contain the
main part of the energy providing nutrients that are essential for
pigs.
[17499] With view to the increasing global demand for food because
of the growing world population and at the same time the shrinking
availability of arable land, it is important to increase the food
and feed quality, particulary the availability of certain essential
nutrients, preferably carbohydrates, preferably polysaccharides
like starch or cellulose and/or monosaccharides like myo-inositol.
Nutritional improvements in foods and feeds could help to meet
these demands for improved quality. Modern agricultural
biotechnology, which involves the application of cellular and
molecular techniques to transfer DNA that encodes a desired trait
to food and feed crops, is a powerful complement to traditional
methods to meet global food and feed requirements.
[17500] [0007.0.45.45] Furthermore the physicochemical properties
such as viscosity and the capacity to bind water and ions, vary
between different cereals. Consequently, different cereal
properties affect digestion and fermentation as well as microbial
populations in the gastro-intestinal tract in various ways.
Gastro-intestinal disturbances comprise a major problem for health
of humans and animals. There is a need for suitable dietary
composition and food or feed ingredients, preferably cereals,
legumes or fruits which promotes a beneficial gut environment and
thereby preventing gastro-intestinal disorders.
[17501] Therefore improving the quality of foodstuffs and animal
feeds is an important task of the food-and-feed industry. This is
necessary since, for example, carbohydrates, which occur in plants
and some microorganisms are limited with regard to the supply of
mammals. Especially advantageous for the quality of foodstuffs and
animal feeds is as balanced as possible a carbohydrate profile in
the diet since a great excess of some sugars above a specific
concentration in the food has only some or little or no positive
effect.
[17502] [0008.0.45.45] Genetically modified plants having improved
nutritional profiles are known in the state of art. US 20030070192
discloses a DNA expression cassette which alters the sugar alcohol
of tranformed plants.
[17503] U.S. Pat. No. 5,908,975 concerns methods for synthesis and
accumulation of fructose polymers in transgenic plants by selective
expression of bacterial fructosyltransferase genes using tissue
specific promoters and a vacuole targeting sequence.
[17504] WO89/12386 describes a method for the production of glucose
and fructose polymers in transgenic tomato plants.
[17505] A stress tolerance sequences including proteins like
galactinol synthase (GOLS) and raffinose synthase (RAFS), which are
up regulated in response to stress and lead to the production of
raffinose is disclosed in US 20050055748.
[17506] U.S. Pat. No. 6,887,708 provides nucleotide sequences
encoding polypeptides having the function of GIGANTEA gene of
Arabidopsis thaliana which allows the manipulation of the starch
accumulation process in plants.
[17507] Grain having an embryo with a genotype heterozygous for two
or more wild type genes (for example, Aa/Bb) and an endosperm
having a genotype heterozygous for such genes and leading to plants
with altered the normal starch synthesis pathway is disclosed in US
20050091716.
[17508] [0009.0.45.45] Nevertheles, there is a constant need for
providing novel enzyme activities or direct or indirect regulators
and thus alternative methods with advantageous properties for
producing carbohydrates, preferably polysaccharides like starch or
cellulose and/or monosaccharides like myo-inositol or its precursor
in organisms, e.g. in transgenic organisms.
[17509] [0010.0.45.45] Another problem is the seasonal change in
carbohydrate composition of plants and optimum harvest periods for
are complicated by issues of timing.
[17510] [0011.0.45.45] To ensure constantly a high quality of foods
and animal feeds, it is necessary to add one or a plurality of
carbohydrates, preferably polysaccharides like starch or cellulose
and/or monosaccharides like myo-inositol in a balanced manner to
suit the organism.
[17511] [0012.0.45.45] Accordingly, there is still a great demand
for new and more suitable genes which encode enzymes which
participate in the biosynthesis of carbohydrates, preferably
polysaccharides like starch or cellulose and/or monosaccharides
like myo-inositol and make it possible to produce them specifically
on an industrial scale without unwanted byproducts forming. In the
selection of genes for or regulators of biosynthesis two
characteristics above all are particularly important. On the one
hand, there is as ever a need for improved processes for obtaining
the highest possible contents of carbohydrates, preferably
polysaccharides like starch or cellulose and/or monosaccharides
like myo-inositol; on the other hand as less as possible byproducts
should be produced in the production process.
[17512] The added carbohydrates further beneficially affects the
microflora by selectively stimulating the growth and/or activity of
beneficial bacteria.
[17513] Another aspect is the significant reduction of cost of
production and manufacturing not only to the nutrition, in
particular sweetener industry, but also agriculture and cosmetic
and health industry.
[17514] [0013.0.0.45] see [0013.0.0.27]
[17515] [0014.0.45.45] Accordingly, in a first embodiment, the
invention relates to a process for the production of a fine
chemical, whereby the fine chemical is carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol. Accordingly, in the
present invention, the term "the fine chemical" as used herein
relates to (a) "carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably myo-inositol. Further, the term "the fine chemicals" as
used herein also relates to compositions of fine chemicals
comprising carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably myo-inositol
[17516] [0015.0.45.45] In one embodiment, the term "carbohydrate"
or "the fine chemical" or "the respective fine chemical" means at
least one chemical compound with carbohydrate activity selected
from the group of preferably polysaccharides, more preferably
starch and/or cellulose and/or monosaccharides, more preferably
myo-inositol. In an preferred embodiment, the term "the fine
chemical" or the term "carbohydrate" or the term "the respective
fine chemical" means at least one chemical compound with
carbohydrate activity selected from the group "carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably myo-inositol. An increased
carbohydrate content normally means an increased total carbohydrate
content. However, an increased carbohydrate content also means, in
particular, a modified content of the above-described compounds
with carbohydrate activity, without the need for an inevitable
Xlncrease in the total carbohydrate content. In a preferred
embodiment, the term "the fine chemical" means carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably myo-inositol in free form
or its salts or its ester or its ether or bound to
acylglycerol.
[17517] [0015.1.45.45] A measure for the content of the
polysaccharides, preferably starch and cellulose, of the invention
can be the content of anhydroglucose. This compound is the analyte
which indicates the presence of the polysaccharides, preferably
starch and cellulose, of the invention if the samples are prepared
and measured as described in the examples.
[17518] [0016.0.45.45] Accordingly, the present invention relates
to a process for the production of the respective fine chemical
comprising [17519] (a) increasing or generating the activity of one
or more of a protein as shown in table XII, application no. 45,
column 3 encoded by the nucleic acid sequences as shown in table
XI, application no. 45, column 5, in a non-human organism or in one
or more parts thereof or [17520] (b) growing the organism under
conditions which permit the production of the fine chemical, thus
myo-inositol and/or anhydroglucose of the invention or fine
chemicals comprising myo-inositol and/or anhydroglucose of the
invention, in said organism or in the culture medium surrounding
the organism.
[17521] [0016.1.45.45] Accordingly, the term "the fine chemical"
means in one embodiment "myo-inositol" in relation to all sequences
listed in Tables XI to XIV, line 48 or homologs thereof and
means in one embodiment "anhydroglucose", preferably "starch"
and/or "cellulose" in relation to all sequences listed in Table XI
to XIV, line 49 and/or 50 or homologs thereof.
[17522] [0017.0.45.45] to [0019.0.0.45]: see [0017.0.0.27] to
[0019.0.0.27]
[17523] [0020.0.45.45] Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, column 5, line 48 in a plant conferred an increase in
myo-inositol content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of myo-inositol. Surprisingly it was found, that the
transgenic expression of the Brassica napus protein as indicated in
Table XII, column 5, line 49 in a plant conferred an increase in
anhydroglucose content of the transformed plants. Thus, in one
embodiment, said protein or its homologs are used for the
production of anhydroglucose. Surprisingly it was found, that the
transgenic expression of the Glycine max protein as indicated in
Table XII, column 5, line 50 in a plant conferred an increase in
anhydroglucose-inositol content of the transformed plants. Thus, in
one embodiment, said protein or its homologs are used for the
production of anhydroglucose.
[17524] [0021.0.45.45] see [0021.0.0.27]
[17525] [0022.0.45.45]
[17526] The sequence of b3430 from Escherichia coli K12 has been
published in Blattner et al., Science 277(5331), 1453-1474, 1997,
and its activity is being defined as a glucose-1-phosphate
adenylyltransferase. Accordingly, in one embodiment, the process of
the present invention comprises the use of a gene product with an
activity of the glucose-1-phosphate adenylyltransferase
superfamily, preferably a protein with a glucose-1-phosphate
adenylyltransferase activity or its homolog, e.g. as shown herein,
from Escherichia coli K12 or a plant or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of
carbohydrate, preferably myo-inositol in free or bound form in an
organism or a part thereof, as mentioned.
[17527] The sequence of b1539 (Accession number NP.sub.--416057)
from Escherichia coli K12 has been published in Blattner et al.,
Science 277 (5331), 1453-1474, 1997, and its activity is being
defined as a NADP-dependent L-serine/L-allo-threonine
dehydrogenase. Accordingly, in one embodiment, the process of the
present invention comprises the use of a gene product with an
activity of ribitol dehydrogenase, short-chain alcohol
dehydrogenase homology superfamily, preferably a protein with the
activity of a NADP-dependent L-serine/L-allo-threonine
dehydrogenase from E. coli or a plant or its homolog, e.g. as shown
herein, for the production of the fine chemical, meaning of starch
and/or cellulose, particular for increasing the amount of starch
and/or cellulose, preferably starch and/or cellulose in free or
bound form in an organism or a part thereof, as mentioned.
[17528] The sequence of b4232 from Escherichia coli K12 (ACCESSION
No. NP.sub.--416986) has been published in Blattner, Science
277(5331), 1453-1474,1997, and its activity is beeing defined as a
fructose-1,6-bisphosphatase. Accordingly, in one embodiment, the
process of the present invention comprises the use of a
"fructose-1,6-bisphosphatase" from E. coli or a plant or its
homolog, e.g. as shown herein, for the production of the fine
chemical, meaning of methionin, in particular for increasing the
amount of methionine in free or bound form in an organism or a part
thereof, as mentioned. In one embodiment, in the process of the
present invention the activity of a protein of the superfamily
"fructose-bisphosphatase", preferably having an activity in
C-compound and carbohydrate metabolism, C-compound and carbohydrate
utilization, energy, glycolysis and gluconeogenesis, plastid,
photosynthesis, more preferred having an
"fructose-1,6-bisphosphatase"-activity, is increased or generated,
e.g. from E. coli or a plant or a homolog thereof. Accordingly, in
one embodiment, in the process of the present invention the
activity of a "fructose-1,6-bisphosphatase" or its homolog is
increased for the production of the fine chemical, meaning of
starch and/or cellulose, in particular for increasing the amount of
starch and/or cellulose in free or bound form in an organism or a
part thereof, as mentioned.
[17529] [0022.1.0.45] to [0023.0.0.45]: see [0022.1.0.27] to
[0023.0.0.27]
[17530] [0023.1.45.45] Homologs of the polypeptide disclosed in
table XII, application no. 45, column 5, may have the activity of a
protein as disclosed in Table XI or XII, application no. 45, column
3 and may be the polypeptides encoded by the nucleic acid molecules
indicated in table XI, application no. 45, column 7, resp., or may
be the polypeptides indicated in table XII, application no. 45,
column 7, resp.
[17531] [0024.0.0.45] see [0024.0.0.27]
[17532] [0025.0.45.45] In accordance with the invention, a protein
or polypeptide has the "activity of an protein as shown in table
XII, application no. 45, column 3" if its de novo activity, or its
increased expression directly or indirectly leads to an increased
in the level of the fine chemical indicated in the respective line
of table XII, application no. 45, column 6 "metabolite" in the
organism or a part thereof, preferably in a cell of said
organism.
[17533] Throughout the specification the activity or preferably the
biological activity of such a protein or polypeptide or an nucleic
acid molecule or sequence encoding such protein or polypeptide is
identical or similar if it still has the biological or enzymatic
activity of a protein as shown in table XII, application no. 45,
column 3, or which has at least 10% of the original enzymatic or
biological activity, preferably 20%, particularly preferably 30%,
most particularly preferably 40% in comparison to a protein as
shown in the respective line of table XII, application no. 45,
column 3 of a E. coli or Saccharomyces cerevisae, respectively,
protein as mentioned in table XI to XIV, column 3 respectively and
as disclosed in paragraph [0022] of the respective invention.
[17534] [0025.1.0.45]: see [0025.1.0.27]
[17535] [0026.0.0.45] to [0033.0.0.45]: see [0026.0.0.27] to
[0033.0.0.27]
[17536] [0034.0.45.45] Preferably, the reference, control or wild
type differs form the subject of the present invention only in the
cellular activity, preferably of the activity of the polypeptide of
the invention, e.g. as result of an increase in the level of the
nucleic acid molecule of the present invention or an increase of
the specific activity of the polypeptide of the invention, e.g. by
or in the expression level or activity of an protein having the
activity of a respective protein as shown in table XII, application
no. 45, column 3 its biochemical or genetical causes and the
increased amount of the respective fine chemical.
[17537] [0035.0.0.45] to [0044.0.0.45]: see [0035.0.0.27] to
[0044.0.0.27]
[17538] [0045.0.45.45] In one embodiment, the activity of the
protein. as indicated in Table XII, columns 5 or 7, application no.
45, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line,
between 10% and 50%, 10% and 100%, 10% and 200%, 10% and 300%, 10%
and 400%, 10% and 500%, 20% and 50%, 20% and 250%, 20% and 500%,
50% and 100%, 50% and 250%, 50% and 500%, 500% and 1000% or
more.
[17539] [0046.0.45.45] In one embodiment, the activity of the
protein as indicated in Table XII, columns 5 or 7, application no.
45, is increased conferring an increase of the respective fine
chemical, indicated in Table XII, column 6 of the respective line
confers an increase of the respective fine chemical and of further
myo-inositol and/or anhydroglucose or their precursors.
[17540] [0047.0.0.45] to [0048.0.0.45]: see [0047.0.0.27] to
[0048.0.0.27]
[17541] [0049.0.45.45] A protein having an activity conferring an
increase in the amount or level of the fine chemical, has in one
embodiment the structure of the polypeptide described herein, in
particular of the polypeptides comprising the consensus sequence
shown in table XIV, application no. 45, column 7 or of the
polypeptide as shown in the amino acid sequences as disclosed in
table XII, application no. 45, columns 5 and 7 or the functional
homologues thereof as described herein, or is encoded by the
nucleic acid molecule characterized herein or the nucleic acid
molecule according to the invention, for example by the nucleic
acid molecule as shown in table XI, application no. 45, columns 5
and 7 or its herein described functional homologues and has the
herein mentioned activity.
[17542] [0050.0.45.45] For the purposes of the present invention,
the term "the respective fine chemical" also encompass the
corresponding salts, ester and ethers, preferably etherificated
with other mono-, di-oligo- or polysaccharides, with alkyl, alkenyl
or alkinyl alcohols.
[17543] [0051.0.45.45] Owing to the biological activity of the
proteins which are used in the process according to the invention
and which are encoded by nucleic acid molecules according to the
invention, it is possible to produce compositions comprising the
respective fine chemical, i.e. an increased amount of the free
chemical free or bound, e.g compositions comprising carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably myo-inositol resp.,
Depending on the choice of the organism used for the process
according to the present invention, for example a microorganism or
a plant, compositions or mixtures of various respective fine
chemicals can be produced.
[17544] [0052.0.0.45] see [0052.0.0.27]
[17545] [0053.0.45.45] In one embodiment, the process of the
present invention comprises one or more of the following steps:
[17546] a) stabilizing a protein conferring the increased
expression of a protein encoded by the nucleic acid molecule of the
invention or of the polypeptid of the invention, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 45, columns 5 and 7 or its homologs activity
having herein-mentioned myo-inositol and/or anhydroglucose of the
invention increasing activity; and/or [17547] b) stabilizing a mRNA
conferring the increased expression of a protein encoded by the
nucleic acid molecule of the invention as shown in table XI,
application no. 45, columns 5 and 7, e.g. a nucleic acid sequence
encoding a polypeptide having the activity of a protein as
indicated in table XII, application no. 45, columns 5 and 7 or its
homologs activity or of a mRNA encoding the polypeptide of the
present invention having herein-mentioned myo-inositol and/or
anhydroglucose of the invention increasing activity; and/or [17548]
c) increasing the specific activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the invention or of the polypeptide of the present
invention having herein-mentioned myo-inositol and/or
anhydroglucose increasing activity, e.g. of a polypeptide having
the activity of a protein as indicated in table XII, application
no. 45, columns 5 and 7 or its homologs activity, or decreasing the
inhibiitory regulation of the polypeptide of the invention; and/or
[17549] d) generating or increasing the expression of an endogenous
or artificial transcription factor mediating the expression of a
protein conferring the increased expression of a protein encoded by
the nucleic acid molecule of the invention or of the polypeptide of
the invention having herein-mentioned myo-inositol and/or
anhydroglucose of the invention increasing activity, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 45, columns 5 and 7 or its homologs activity;
and/or [17550] e) stimulating activity of a protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or a polypeptide of the present
invention having herein-mentioned myo-inositol and/or
anhydroglucose of the invention increasing activity, e.g. of a
polypeptide having the activity of a protein as indicated in table
XII, application no. 45, columns 5 and 7 or its homologs activity,
by adding one or more exogenous inducing factors to the organisms
or parts thereof; and/or [17551] f) expressing a transgenic gene
encoding a protein conferring the increased expression of a
polypeptide encoded by the nucleic acid molecule of the present
invention or a polypeptide of the present invention, having
herein-mentioned myo-inositol and/or anhydroglucose of the
invention increasing activity, e.g. of a polypeptide having the
activity of a protein as indicated in table XII, application no.
45, columns 5 and 7 or its homologs activity, and/or [17552] g)
increasing the copy number of a gene conferring the increased
expression of a nucleic acid molecule encoding a polypeptide
encoded by the nucleic acid molecule of the invention or the
polypeptide of the invention having herein-mentioned myo-inositol
and/or anhydroglucose of the invention increasing activity, e.g. of
a polypeptide having the activity of a protein as indicated in
table XII, application no. 45, columns 5 and 7 or its homologs
activity; and/or [17553] h) increasing the expression of the
endogenous gene encoding the polypeptide of the invention, e.g. a
polypeptide having the activity of a protein as indicated in table
XII, application no. 45, columns 5 and 7 or its homologs activity,
by adding positive expression or removing negative expression
elements, e.g. homologous recombination can be used to either
introduce positive regulatory elements like for plants the 35S
enhancer into the promoter or to remove repressor elements form
regulatory regions. Further gene conversion methods can be used to
disrupt repressor elements or to enhance to activity of positive
elements. Positive elements can be randomly introduced in plants by
T-DNA or transposon mutagenesis and lines can be identified in
which the positive elements have be integrated near to a gene of
the invention, the expression of which is thereby enhanced; and/or
[17554] i) modulating growth conditions of an organism in such a
manner, that the expression or activity of the gene encoding the
protein of the invention or the protein itself is enhanced for
example microorganisms or plants can be grown for example under a
higher temperature regime leading to an enhanced expression of heat
shock proteins, which can lead an enhanced fine chemical
production; and/or [17555] j) selecting of organisms with
especially high activity of the proteins of the invention from
natural or from mutagenized resources and breeding them into the
target organisms, eg the elite crops.
[17556] [0054.0.45.45] Preferably, said mRNA is the nucleic acid
molecule of the present invention and/or the protein conferring the
increased expression of a protein encoded by the nucleic acid
molecule of the present invention or the polypeptide having the
herein mentioned activity, e.g. conferring the increase of the
respective fine chemical as indicated in column 6 of application
no. 45 in Table XI to XIV, resp., after increasing the expression
or activity of the encoded polypeptide or having the activity of a
polypeptide having an activity as the protein as shown in table
XII, application no. 45, column 3 or its homologs.
[17557] [0055.0.0.45] to [0064.0.0.45]: see [0055.0.0.27] to
[0064.0.0.27]
[17558] [0065.0.0.45]: see [0065.0.0.27]
[17559] [0066.0.0.45] to [0067.0.0.45]: see [0066.0.0.27] to
[0067.0.0.27]
[17560] [0068.0.45.45] The mutation is introduced in such a way
that the production of the carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositolis not adversely
affected.
[17561] [0069.0.0.45] see [0069.0.0.27]
[17562] [0070.0.45.45] Owing to the introduction of a gene or a
plurality of genes conferring the expression of the nucleic acid
molecule of the invention or the polypeptide of the invention, for
example the nucleic acid construct mentioned below, or encoding a
protein of the invention into an organism alone or in combination
with other genes, it is possible not only to increase the
biosynthetic flux towards the end product, but also to increase,
modify or create de novo an advantageous, preferably novel
metabolites composition in the organism, e.g. an advantageous
composition carbohydrates comprising a higher content of (from a
viewpoint of nutritional physiology limited) preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol.
[17563] [0071.0.45.45] Preferably the composition further comprises
higher amounts of metabolites positively affecting or lower amounts
of metabolites negatively affecting the nutrition or health of
animals or humans provided with said compositions or organisms of
the invention or parts thereof. Likewise, the number or activity of
further genes which are required for the import or export of
nutrients or metabolites, including carbohydrates or its
precursors, required for the cell's biosynthesis of
carbohydratesmay be increased so that the concentration of
necessary or relevant precursors, cofactors or intermediates within
the cell(s) or within the corresponding storage compartments is
increased. Owing to the increased or novel generated activity of
the polypeptide of the invention or the polypeptide used in the
method of the invention or owing to the increased number of nucleic
acid sequences of the invention and/or to the modulation of further
genes which are involved in the biosynthesis of the carbohydrates,
e.g. by increasing the activity of enzymes synthesizing precursors
or by destroying the activity of one or more genes which are
involved in the breakdown of the carbohydrates, it is possible to
increase the yield, production and/or production efficiency of
carbohydratesin the host organism, such as the plants or the
microorganims.
[17564] [0072.0.45.45] By influencing the metabolism thus, it is
possible to produce, in the process according to the invention,
further advantageous compounds. Examples of such compounds are, in
addition to polysaccharides, more preferably starch and/or
cellulose and/or monosaccharides, more preferably myo-inositol,
further carbohydrates, preferably saccharides.
[17565] [0073.0.45.45] Accordingly, in one embodiment, the process
according to the invention relates to a process, which
comprises:
a) providing a non-human organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant; b) increasing an activity of a polypeptide of the
invention or a homolog thereof, e.g. as indicated in Table XII,
application no. 45, columns 5 or 7, or of a polypeptide being
encoded by the nucleic acid molecule of the present invention and
described below, e.g. conferring an increase of the respective fine
chemical in an organism, preferably in a microorganism, a non-human
animal, a plant or animal cell, a plant or animal tissue or a
plant, c) growing an organism, preferably a microorganism, a
non-human animal, a plant or animal cell, a plant or animal tissue
or a plant under conditions which permit the production of the
respective fine chemical in the organism, preferably the
microorganism, the plant cell, the plant tissue or the plant; and
d) if desired, revovering, optionally isolating, the free and/or
bound the respective fine chemical and, optionally further free
and/or bound carbohydrate(s) synthetized by the organism, the
microorganism, the non-human animal, the plant or animal cell, the
plant or animal tissue or the plant.
[17566] [0074.0.45.45] The organism, in particular the
microorganism, non-human animal, the plant or animal cell, the
plant or animal tissue or the plant is advantageously grown in such
a way that it is not only possible to recover, if desired isolate
the free or bound the respective fine chemical or the free and
bound the respective fine chemical but as option it is also
possible to produce, recover and, if desired isolate, other free
or/and bound carbohydrate(s).
[17567] [0075.0.0.45] to [0077.0.0.45]: see [0075.0.0.27] to
[0077.0.0.27]
[17568] [0078.0.45.45] The organism such as microorganisms or
plants or the recovered, and if desired isolated, respective fine
chemical can then be processed further directly into foodstuffs or
animal feeds or for other applications in nutrition or medicine or
cosmetics, for example according to the disclosures made in U.S.
Pat. No. 6,669,962: Starch microcapsules for delivery of active
agents; US 20050042737: Starch process; US 20050054071: Enzymes for
starch processing; US 20050091716: Novel plants and processes for
obtaining them; U.S. Pat. Nos. 5,096,594 and 5,482,631 discloses a
method of purifying cyclitols; U.S. Pat. No. 4,997,489 discloses
soaking almond hulls in water to obtain a syrup containing
fructose, glucose, inositol, and sorbitol; U.S. Pat. No. 5,296,364
discloses a microbial method for producing inositol; U.S. Pat. No.
4,734,402; U.S. Pat. No. 4,788,065; U.S. Pat. No. 6,465,037 and
U.S. Pat. No. 6,355,295: relates to soy food ingredient based on
carbohydrates, U.S. Pat. No. 6,653,451; US 20040128713: pertains to
soybean plants having in their seeds significantly lower contents
of raffinose, stachyose and phytic acid and significantly higher
contents of sucrose and inorganic phosphate; US 20050008713
discloses compositions of plant carbohydrates for dietary
supplements and nutritional support; which are expressly
incorporated herein by reference. The fermentation broth,
fermentation products, plants or plant products can be treated and
processed as described in above mentioned applications or by other
methods known to the person skilled in the art and described herein
below.
[17569] In the method for producing carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol according to the
invention, the cultivation step of the genetically modified
organisms, also referred to as transgenic organisms hereinbelow, is
preferably followed by harvesting said organisms and isolating the
respective carbohydrate(s) from said organisms.
[17570] The organisms are harvested in a manner known per se and
appropriate for the particular organism. Microorganisms such as
bacteria, mosses, yeasts and fungi or plant cells which are
cultured in liquid media by fermentation may be removed, for
example, by centrifugation, decanting or filtration. Plants are
grown on solid media in a manner known per se and harvested
accordingly.
[17571] Carbohydrates, preferably polysaccharides, more preferably
starch and/or cellulose and/or monosaccharides, more preferably
myo-inositol are isolated from the harvested biomass in a manner
known per se, for example by extraction and, where appropriate,
further chemical or physical purification processes such as, for
example, chemical and/or enzymatical degradation, precipitation
methods, crystallography, thermal separation methods such as
rectification methods or physical separation methods such as, for
example, chromatography.
[17572] Products of these different work-up procedures are
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably
myo-inositol comprising compositions, e.g. compostions comprising
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably
myo-inositol which still comprise fermentation broth, plant
particles and/or cell components in different amounts,
advantageously in the range of from 0 to 99% by weight, preferably
below 80% by weight, especially preferably between 50%, 40%, 30%,
20%, 20%, 10%, 5%, 3%, 2%, 1%, 05%, 0.1%, 0.01% and 0% by weight
resp.
[17573] In one embodiment, preferred plants include, but are not
limited to: sugar beet, sugar cane, soybeans and/or potato (Solanum
tuberosum).
[17574] [0079.0.0.45] to [0084.0.0.45]: see [0079.0.0.27] to
[0084.0.0.27]
[17575] [0085.0.45.45] With regard to the nucleic acid sequence as
depicted a nucleic acid construct which contains a nucleic acid
sequence mentioned herein or an organism (=transgenic organism)
which is transformed with said nucleic acid sequence or said
nucleic acid construct, "transgene" means all those constructs
which have been brought about by genetic manipulation methods,
preferably in which either [17576] a) a nucleic acid sequence as
indicated in Table XI, application no. 45, columns 5 or 7, or a
derivative thereof, or [17577] b) a genetic regulatory element, for
example a promoter, which is functionally linked to the nucleic
acid sequence as indicated in Table XI, application no. 45, columns
5 or 7, or a derivative thereof, or [17578] c) (a) and (b) is/are
not present in its/their natural genetic environment or has/have
been modified by means of genetic manipulation methods, it being
possible for the modification to be, by way of example, a
substitution, addition, deletion, inversion or insertion of one or
more nucleotide. "Natural genetic environment" means the natural
chromosomal locus in the organism of origin or the presence in a
genomic library. In the case of a genomic library, the natural,
genetic environment of the nucleic acid sequence is preferably at
least partially still preserved. The environment flanks the nucleic
acid sequence at least on one side and has a sequence length of at
least 50 bp, preferably at least 500 bp, particularly preferably at
least 1000 bp, very particularly preferably at least 5000 bp.
[17579] [0086.0.0.45] to [0087.0.0.45]: see [0086.0.0.27] to
[0087.0.0.27]
[17580] [0088.0.45.45] In an advantageous embodiment of the
invention, the organism takes the form of a plant whose content the
respective fine chemical is modified advantageously owing to the
nucleic acid molecule of the present invention expressed. This is
important for humans and animals since, for example, the
nutritional value of plants for nutrition is dependent on the
abovementioned carbohydrates and the general amount of saccharides
as energy source in feed.
[17581] [0088.1.0.45] to [0095.0.0.45]: see [0088.1.0.27] to
[0095.0.0.27]
[17582] [0096.0.45.45] In another preferred embodiment of the
invention a combination of the increased expression of the nucleic
acid sequence or the protein of the invention together with the
transformation of a protein or polypeptide or a compound, which
functions as a sink for the desired fine chemical, for example
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably
myo-inositol in the organism, is useful to increase the production
of the respective fine chemical.
[17583] [0097.0.0.45] see [0097.0.0.27]
[17584] [0098.0.45.45] In a preferred embodiment, the respective
fine chemical (carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably myo-inositol) is produced in accordance with the
invention and, if desired, is isolated. The production of
carbohydrates, or mixtures thereof or mixtures with other compounds
by the process according to the invention is advantageous.
[17585] [0099.0.45.45] In the case of the fermentation of
microorganisms, the abovementioned carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol may accumulate in the
medium and/or the cells. If microorganisms are used in the process
according to the invention, the fermentation broth can be processed
after the cultivation. Depending on the requirement, all or some of
the biomass can be removed from the fermentation broth by
separation methods such as, for example, centrifugation,
filtration, decanting or a combination of these methods, or else
the biomass can be left in the fermentation broth. The fermentation
broth can subsequently be reduced, or concentrated, with the aid of
known methods such as, for example, rotary evaporator, thin-layer
evaporator, falling film evaporator, by reverse osmosis or by
nanofiltration. Afterwards advantageously further compounds for
formulation can be added such as corn starch or silicates. This
concentrated fermentation broth advantageously together with
compounds for the formulation can subsequently be processed by
lyophilization, spray drying, and spray granulation or by other
methods. Preferably the respective fine chemical or the
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably
myo-inositol compositions are isolated from the organisms, such as
the microorganisms or plants or the culture medium in or on which
the organisms have been grown, or from the organism and the culture
medium, in the known manner, for example via extraction,
distillation, crystallization, chromatography or a combination of
these methods. These purification methods can be used alone or in
combination with the aforementioned methods such as the separation
and/or concentration methods.
[17586] [0100.0.45.45] Transgenic plants which comprise the
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably
myo-inositol synthesized in the process according to the invention
can advantageously be marketed directly without there being any
need for the oils, lipids or fatty acids synthesized to be
isolated. Plants for the process according to the invention are
listed as meaning intact plants and all plant parts, plant organs
or plant parts such as leaf, stem, seeds, root, tubers, anthers,
fibers, fruits, root hairs, stalks, embryos, calli, cotelydons,
petioles, harvested material, plant tissue, reproductive tissue and
cell cultures which are derived from the actual transgenic plant
and/or can be used for bringing about the transgenic plant. In this
context, the seed comprises all parts of the seed such as the seed
coats, epidermal cells, seed cells, endosperm or embryonic
tissue.
[17587] However, the respective fine chemical produced in the
process according to the invention can also be isolated from the
organisms, advantageously plants, in the form of their extracts,
e.g. water containing extract, or as fibre in case of starch and/or
cellulose, as degradation products (chemical ar enzymatical
degradation) or crystallisation product or as their ethers and/or
esters and/or free carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably myo-inositol. The respective fine chemical produced by
this process can be obtained by harvesting the organisms, either
from the crop in which they grow, or from the field. This can be
done via pressing or extraction of the plant parts, e.g. in the
plant stem, seeds, root, tubers, anthers, fibers, fruits. To
increase the efficiency of extraction it is beneficial to clean, to
temper and if necessary to hull and to flake the plant material
especially the stem, seeds, root, tubers, anthers, fibers, fruits.
extracts and/carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably myo-inositol can be obtained by what is known as cold
beating or cold pressing without applying heat. To allow for
greater ease of disruption of the plant parts, specifically the
stem, seeds, root, tubers, anthers, fibers, fruits, they can
previously be comminuted, steamed or roasted. Plant parts, which
have been pretreated in this manner can subsequently be pressed or
extracted with solvents or water. The solvent is subsequently
removed. In the case of microorganisms, the latter are, after
harvesting, for example extracted directly without further
processing steps or else, after disruption, extracted via various
methods with which the skilled worker is familiar. Thereafter, the
resulting products can be processed further, i.e. degradiated,
crystallized and/or refined.
[17588] [0101.0.45.45] see [0101.0.0.27]
[17589] [0102.0.45.45] Carbohydrates, preferably polysaccharides,
more preferably starch and/or cellulose and/or monosaccharides,
more preferably myo-inositol can for example be analyzed
advantageously via HPLC or GC separation methods and detected by MS
oder MSMS methods. The unambiguous detection for the presence of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably
myo-inositolcontaining products can be obtained by analyzing
recombinant organisms using analytical standard methods: GC, GC-MS,
LC, LC-MSMS or TLC, as described on several occasions. The
carbohydrates can be analized further in plant extracts by
anion-exchange chromatography with pulsed amperometric detection
(Cataldi et al., Anal Chem.; 72(16):3902-7, 2000), by enzymatic
"BioAnalysis" usind test kits from R-Biopharm and Roche or from
Megazyme, Ireland.
[17590] [0103.0.45.45] In a preferred embodiment, the present
invention relates to a process for the production of the respective
fine chemical comprising or generating in an organism or a part
thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: [17591] a) nucleic acid molecule encoding,
preferably at least the mature form, of the polypeptide having a
sequence as indicated in Table XII, application no. 45, columns 5
or 7, or a fragment thereof, which confers an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [17592] b) nucleic acid molecule comprising, preferably at
least the mature form, of a nucleic acid molecule having a sequence
as indicated in Table XI, application no. 45, columns 5 or 7,
[17593] c) nucleic acid molecule whose sequence can be deduced from
a polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as result of the degeneracy of the genetic code and conferring
an increase in the amount of the respective fine chemical in an
organism or a part thereof; [17594] d) nucleic acid molecule
encoding a polypeptide which has at least 50% identity with the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecule of (a) to (c) and conferring an increase in the amount of
the respective fine chemical in an organism or a part thereof;
[17595] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [17596]
f) nucleic acid molecule encoding a polypeptide, the polypeptide
being derived by substituting, deleting and/or adding one or more
amino acids of the amino acid sequence of the polypeptide encoded
by the nucleic acid molecules (a) to (d), preferably to (a) to (c)
and conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [17597] g) nucleic acid
molecule encoding a fragment or an epitope of a polypeptide which
is encoded by one of the nucleic acid molecules of (a) to (e),
preferably to (a) to (c) and and conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [17598] h) nucleic acid molecule comprising a nucleic acid
molecule which is obtained by amplifying nucleic acid molecules
from a cDNA library or a genomic library using the primers pairs
having a sequence as indicated in Table XIII, application no. 45,
column 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [17599]
i) nucleic acid molecule encoding a polypeptide which is isolated,
e.g. from an expression library, with the aid of monoclonal
antibodies against a polypeptide encoded by one of the nucleic acid
molecules of (a) to (h), preferably to (a) to (c), and and
conferring an increase in the amount of the respective fine
chemical in an organism or a part thereof; [17600] j) nucleic acid
molecule which encodes a polypeptide comprising the consensus
sequence having a sequences as indicated in Table XIV, application
no. 45, column 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; [17601]
k) nucleic acid molecule comprising one or more of the nucleic acid
molecule encoding the amino acid sequence of a polypeptide encoding
a domain of the polypeptide indicated in Table XII, application no.
45, columns 5 or 7, and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; and
[17602] l) nucleic acid molecule which is obtainable by screening a
suitable library under stringent conditions with a probe comprising
one of the sequences of the nucleic acid molecule of (a) to (k),
preferably to (a) to (c), or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (k), preferably to
(a) to (c), and conferring an increase in the amount of the
respective fine chemical in an organism or a part thereof; or which
comprises a sequence which is complementary thereto.
[17603] [00103.1.45.45] In one embodiment, the nucleic acid
molecule used in the process of the invention distinguishes over
the sequence indicated in Table XI, application no. 45, columns 5
or 7 by one or more nucleotides. In one embodiment, the nucleic
acid molecule used in the process of the invention does not consist
of the sequence shown in indicated in Table XI, application no. 45,
columns 5 or 7. In one embodiment, the nucleic acid molecule used
in the process of the invention is less than 100%, 99.999%, 99.99%,
99.9% or 99% identical to a sequence indicated in Table XI,
application no. 45, columns 5 or 7. In another embodiment, the
nucleic acid molecule does not encode a polypeptide of a sequence
indicated in Table XII, application no. 45, columns 5 or 7.
[17604] [0104.0.45.45] In one embodiment, the nucleic acid molecule
used in the process distinguishes over the sequence indicated in
Table XI, application no. 45, columns 5 or 7, by one or more
nucleotides. In one embodiment, the nucleic acid molecule used in
the process of the invention does not consist of the sequence
indicated in Table XI, application no. 45, columns 5 or 7. In one
embodiment, the nucleic acid molecule of the present invention is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to the
sequence indicated in Table XI, application no. 45, columns 5 or 7.
In another embodiment, the nucleic acid molecule does not encode a
polypeptide of a sequence indicated in Table XII, application no.
45, columns 5 or 7.
[17605] [0105.0.0.45] to [0107.0.0.45]: see [0105.0.0.27] to
[0107.0.0.27]
[17606] [0108.0.45.45] Nucleic acid molecules with the sequence as
indicated in Table XI, application no. 45, columns 5 or 7, nucleic
acid molecules which are derived from an amino acid sequences as
indicated in Table XII, application no. 45, columns 5 or 7, or from
polypeptides comprising the consensus sequence as indicated in
Table XIV, application no. 45, column 7, or their derivatives or
homologues encoding polypeptides with the enzymatic or biological
activity of an activity of a polypeptide as indicated in Table XII,
application no. 45, column 3, 5 or 7, or conferring an increase of
the respective fine chemical, meaning carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol after increasing its
expression or activity are advantageously increased in the process
according to the invention.
[17607] [0109.0.0.45] see [0109.0.0.27]
[17608] [0110.0.45.45] Nucleic acid molecules, which are
advantageous for the process according to the invention and which
encode polypeptides with an activity of a polypeptide of the
invention or the polypeptide used in the method of the invention or
used in the process of the invention, e.g. of a protein as
indicated in Table XII, application no. 45, column 5, or being
encoded by a nucleic acid molecule indicated in
[17609] Table XI, application no. 45, column 5, or of its homologs,
e.g. as indicated in Table XII, application no. 45, column 7, can
be determined from generally accessible databases.
[17610] [0111.0.0.45] see [0111.0.0.27]
[17611] [0112.0.45.45] The nucleic acid molecules used in the
process according to the invention take the form of isolated
nucleic acid sequences, which encode polypeptides with an activity
of a polypeptide as indicated in Table XII, application no. 45,
column 3, or having the sequence of a polypeptide as indicated in
Table XII, application no. 45, columns 5 and 7, and conferring an
increase in the level of carbohydrates, preferably polysaccharides,
more preferably starch and/or cellulose and/or monosaccharides,
more preferably myo-inositol.
[17612] [0113.0.0.45] to [0120.0.0.45]: see [0113.0.0.27] to
[0120.0.0.27]
[17613] [0121.0.45.45] However, it is also possible to use
artificial sequences, which differ in one or more bases from the
nucleic acid sequences found in organisms, or in one or more amino
acid molecules from polypeptide sequences found in organisms, in
particular from the polypeptide sequences indicated in Table XII,
application no. 45, columns 5 or 7, or the functional homologues
thereof as described herein, preferably conferring above-mentioned
activity, i.e. conferring an increase in the level of the fine
chemical of the invention after increasing the activity of the
polypeptide sequences indicated in Table XII, application no. 45,
columns 5 or 7.
[17614] [0122.0.0.45] to [0127.0.0.45]: see [0122.0.0.27] to
[0127.0.0.27]
[17615] [0128.0.45.45] Synthetic oligonucleotide primers for the
amplification, e.g. as the pairs indicated in Table XIII,
application no. 45, columns 7, by means of polymerase chain
reaction can be generated on the basis of a sequence shown herein,
for example the sequence as indicated in Table XI, application no.
45, columns 5 or 7, or the sequences derived from a sequence as
indicated in Table XII, application no. 45, columns 5 or 7.
[17616] [0129.0.45.45] Moreover, it is possible to identify
conserved regions from various organisms by carrying out protein
sequence alignments with the polypeptide used in the process of the
invention, in particular with sequences of the polypeptide of the
invention, from which conserved regions, and in turn, degenerate
primers can be derived. Conserved regions are those, which show a
very little variation in the amino acid in one particular position
of several homologs from different origin. The consensus sequences
indicated in Table XIV, application no. 45, column 7, are derived
from said alignments.
[17617] [0130.0.45.45] Degenerated primers can then be utilized by
PCR for the amplification of fragments of novel proteins having
above-mentioned activity, e.g. conferring the increase of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably
myo-inositol after increasing the expression or activity of the
protein comprising said fragment.
[17618] [0131.0.0.45] to [0138.0.0.45]: see [0131.0.0.27] to
[0138.0.0.27]
[17619] [0139.0.45.45] Polypeptides having above-mentioned
activity, i.e. conferring the increase of the respective fine
chemical level, derived from other organisms, can be encoded by
other DNA sequences which hybridize to a sequences indicated in
Table XI, application no. 45, columns 5 or 7, under relaxed
hybridization conditions and which code on expression for peptides
having the respective fine chemical, in particular, of starch
and/or cellulose and/or myo-inositol, resp., increasing
activity.
[17620] [0140.0.0.45] to [0146.0.0.45]: see [0140.0.0.27] to
[0146.0.0.27]
[17621] [0147.0.45.45]: Further, the nucleic acid molecule of the
invention comprises a nucleic acid molecule, which is a complement
of one of the nucleotide sequences of above mentioned nucleic acid
molecules or a portion thereof. A nucleic acid molecule which is
complementary to one of the nucleotide sequences indicated in Table
XI, application no. 45, columns 5 or 7, is one which is
sufficiently complementary to one of said nucleotide sequences s
such that it can hybridize to one of said nucleotide sequences
thereby forming a stable duplex. Preferably, the hybridisation is
performed under stringent hybridization conditions. However, a
complement of one of the herein disclosed sequences is preferably a
sequence complement thereto according to the base pairing of
nucleic acid molecules well known to the skilled person. For
example, the bases A and G undergo base pairing with the bases T
and U or C, resp. and visa versa. Modifications of the bases can
influence the base-pairing partner.
[17622] [0148.0.45.45] The nucleic acid molecule of the invention
comprises a nucleotide sequence which is at least about 30%, 35%,
40% or 45%, preferably at least about 50%, 55%, 60% or 65%, more
preferably at least about 70%, 80%, or 90%, and even more
preferably at least about 95%, 97%, 98%, 99% or more homologous to
a nucleotide sequence indicated in Table XI, application no. 45,
columns 5 or 7 or a portion thereof and preferably has above
mentioned activity, in particular, of myo-inositol and/or
anhydroglucose increasing activity after increasing the activity or
an activity of a product of a gene encoding said sequences or their
homologs.
[17623] [0149.0.45.45] The nucleic acid molecule of the invention
or the nucleic acid molecule used in the method of the invention
comprises a nucleotide sequence which hybridizes, preferably
hybridizes under stringent conditions as defined herein, to one of
the nucleotide sequences indicated in Table XI, application no. 45,
columns 5 or 7, or a portion thereof and encodes a protein having
above-mentioned activity and as indicated in indicated in Table
XII.
[17624] [00149.1.45.45] Optionally, the nucleotide sequence, which
hybridises to one of the nucleotide sequences indicated in Table
XI, application no. 45, columns 5 or 7, has further one or more of
the activities annotated or known for the a protein as indicated in
Table XII, application no. 45, column 3.
[17625] [0150.0.45.45] Moreover, the nucleic acid molecule of the
invention can comprise only a portion of the coding region of one
of the sequences indicated in Table XI, application no. 45, columns
5 or 7, for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of the
polypeptide of the present invention or of a polypeptide used in
the process of the present invention, i.e. having above-mentioned
activity, e.g. conferring an increase of myo-inositol and/or
anhydroglucose, resp., if its activity is increased. The nucleotide
sequences determined from the cloning of the present
protein-according-to-the-invention-encoding gene allows for the
generation of probes and primers designed for use in identifying
and/or cloning its homologues in other cell types and organisms.
The probe/primer typically comprises substantially purified
oligonucleotide. The oligonucleotide typically comprises a region
of nucleotide sequence that hybridizes under stringent conditions
to at least about 12, 15 preferably about 20 or 25, more preferably
about 40, 50 or 75 consecutive nucleotides of a sense strand of one
of the sequences set forth, e.g., as indicated in Table XI,
application no. 45, columns 5 or 7, anti-sense sequence of one of
the sequences, e.g., as indicated in Table XI, application no. 45,
columns 5 or 7, or naturally occurring mutants thereof. Primers
based on a nucleotide of invention can be used in PCR reactions to
clone homologues of the polypeptide of the invention or of the
polypeptide used in the process of the invention, e.g. as the
primers described in the examples of the present invention, e.g. as
shown in the examples. A PCR with the primer pairs indicated in
Table XIII, application no. 45, column 7, will result in a fragment
of a polynucleotide sequence as indicated in Table XI, application
no. 45, columns 5 or 7 or its gene product.
[17626] [0151.0.0.45]: see [0151.0.0.27]
[17627] [0152.0.45.45] The nucleic acid molecule of the invention
encodes a polypeptide or portion thereof which includes an amino
acid sequence which is sufficiently homologous to an amino acid
sequence as indicated in Table XII, application no. 45, columns 5
or 7, such that the protein or portion thereof maintains the
ability to participate in the respective fine chemical production,
in particular an activity increasing the level of carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably myo-inositol resp., as
mentioned above or as described in the examples in plants or
microorganisms is comprised.
[17628] [0153.0.45.45] As used herein, the language "sufficiently
homologous" refers to proteins or portions thereof which have amino
acid sequences which include a minimum number of identical or
equivalent amino acid residues (e.g., an amino acid residue which
has a similar side chain as an amino acid residue in one of the
sequences of the polypeptide of the present invention) to an amino
acid sequence as indicated in Table XII, application no. 45,
columns 5 or 7, such that the protein or portion thereof is able to
participate in the increase of the respective fine chemical
production. In one embodiment, a protein or portion thereof as
indicated in Table XII, application no. 45, columns 5 or 7, has for
example an activity of a polypeptide as indicated in Table XII,
application no. 45, column 3.
[17629] [0154.0.45.45] In one embodiment, the nucleic acid molecule
of the present invention comprises a nucleic acid that encodes a
portion of the protein of the present invention. The protein is at
least about 30%, 35%, 40%, 45% or 50%, preferably at least about
55%, 60%, 65% or 70%, and more preferably at least about 75%, 80%,
85%, 90%, 91%, 92%, 93% or 94% and most preferably at least about
95%, 97%, 98%, 99% or more homologous to an entire amino acid
sequence as indicated in Table XII, application no. 45, columns 5
or 7, and has above-mentioned activity, e.g. conferring preferably
the increase of the respective fine chemical.
[17630] [0155.0.0.45] to [0156.0.0.45]: see [0155.0.0.27] to
[0156.0.0.27]
[17631] [0157.0.45.45] The invention further relates to nucleic
acid molecules that differ from one of the nucleotide sequences
indicated in Table XI, application no. 45, columns 5 or 7, (and
portions thereof) due to degeneracy of the genetic code and thus
encode a polypeptide of the present invention, in particular a
polypeptide having above mentioned activity, e.g. conferring an
increase in the respective fine chemical in a organism, e.g. as
that polypeptides comprising a consensus sequence as indicated in
Table XIV, application no. 45, column 7, or of the polypeptide as
indicated in Table XII, application no. 45, columns 5 or 7, or
their functional homologues. Advantageously, the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention comprises, or in an other embodiment has, a
nucleotide sequence encoding a protein comprising, or in an other
embodiment having, a consensus sequences as indicated in Table XIV,
application no. 45, column 7, or of the polypeptide as indicated in
Table XII, application no. 45, column 7, or the functional
homologues. In a still further embodiment, the nucleic acid
molecule of the invention or the nucleic acid molecule used in the
method of the invention encodes a full length protein which is
substantially homologous to an amino acid sequence comprising a
consensus sequence as indicated in Table XIV, application no. 45,
column 7, or of a polypeptide as indicated in Table XII,
application no. 45, columns 5 or 7, or the functional homologues
thereof. However, in a preferred embodiment, the nucleic acid
molecule of the present invention does not consist of a sequence as
indicated in Table XI, application no. 45, columns 5 or 7.
[17632] [0158.0.0.45] to [0160.0.0.45]: see [0158.0.0.27] to
[0160.0.0.27]
[17633] [0161.0.45.45]: Accordingly, in another embodiment, a
nucleic acid molecule of the invention is at least 15, 20, 25 or 30
nucleotides in length. Preferably, it hybridizes under stringent
conditions to a nucleic acid molecule comprising a nucleotide
sequence of the nucleic acid molecule of the present invention or
used in the process of the present invention, e.g. comprising a
sequence as indicated in Table XI, application no. 45, columns 5 or
7. The nucleic acid molecule is preferably at least 20, 30, 50,
100, 250 or more nucleotides in length.
[17634] [0162.0.0.45]: see [0162.0.0.27]
[17635] [0163.0.45.45]: Preferably, nucleic acid molecule of the
invention that hybridizes under stringent conditions to a sequence
as indicated in Table XI, application no. 45, columns 5 or 7,
corresponds to a naturally-occurring nucleic acid molecule of the
invention. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural protein).
Preferably, the nucleic acid molecule encodes a natural protein
having above-mentioned activity, e.g. conferring the respective
fine chemical increase after increasing the expression or activity
thereof or the activity of a protein of the invention or used in
the process of the invention.
[17636] [0164.0.0.45]: see [0164.0.0.27]
[17637] [0165.0.45.45] For example, nucleotide substitutions
leading to amino acid substitutions at "non-essential" amino acid
residues can be made in a sequence of the nucleic acid molecule of
the invention or used in the process of the invention, e.g. as
indicated in Table XI, application no. 45, columns 5 or 7.
[17638] [0166.0.0.45] to [0167.0.0.45]: see [0166.0.0.27] to
[0167.0.0.27]
[17639] [0168.0.45.45]: Accordingly, the invention relates to
nucleic acid molecules encoding a polypeptide having
above-mentioned activity, e.g. conferring an increase in the the
respective fine chemical in an organisms or parts thereof that
contain changes in amino acid residues that are not essential for
said activity. Such polypeptides differ in amino acid sequence from
a sequence contained in a sequence as indicated in Table XII,
application no. 45, columns 5 or 7, yet retain said activity
described herein. The nucleic acid molecule can comprise a
nucleotide sequence encoding a polypeptide, wherein the polypeptide
comprises an amino acid sequence at least about 50% identical to an
amino acid sequence as indicated in Table XII, application no. 45,
columns 5 or 7, and is capable of participation in the increase of
production of the respective fine chemical after increasing its
activity, e.g. its expression. Preferably, the protein encoded by
the nucleic acid molecule is at least about 60% identical to a
sequence as indicated in Table XII, application no. 45, columns 5
or 7, more preferably at least about 70% identical to one of the
sequences as indicated in Table XII, application no. 45, columns 5
or 7, even more preferably at least about 80%, 90%, or 95%
homologous to a sequence as indicated in Table XII, application no.
45, columns 5 or 7, and most preferably at least about 96%, 97%,
98%, or 99% identical to the sequence as indicated in Table XII,
application no. 45, columns 5 or 7.
[17640] [0169.0.0.45] to [0172.0.0.45]: see [0169.0.0.27] to
[0172.0.0.27]
[17641] [0173.0.45.45]: For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108333 at the nucleic acid level
is understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108333 by the above Gap program algorithm with
the above parameter set, has a 80% homology.
[17642] [0174.0.0.45]: see [0174.0.0.27]
[17643] [0175.0.45.45]: For example a sequence which has a 80%
homology with sequence SEQ ID NO: 108334 at the protein level is
understood as meaning a sequence which, upon comparison with the
sequence SEQ ID NO: 108334 by the above program algorithm with the
above parameter set, has a 80% homology.
[17644] [0176.0.45.45]: Functional equivalents derived from one of
the polypeptides as indicated in Table XII, application no. 45,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of the polypeptides as indicated in Table XII,
application no. 45, columns 5 or 7, according to the invention and
are distinguished by essentially the same properties as a
polypeptide as indicated in Table XII, application no. 45, columns
5 or 7.
[17645] [0177.0.45.45]: Functional equivalents derived from a
nucleic acid sequence as indicated in Table XI, application no. 45,
columns 5 or 7, according to the invention by substitution,
insertion or deletion have at least 30%, 35%, 40%, 45% or 50%,
preferably at least 55%, 60%, 65% or 70% by preference at least
80%, especially preferably at least 85% or 90%, 91%, 92%, 93% or
94%, very especially preferably at least 95%, 97%, 98% or 99%
homology with one of a polypeptide as indicated in Table XII,
application no. 45, columns 5 or according to the invention and
encode polypeptides having essentially the same properties as a
polypeptide as indicated in Table XII, application no. 45, columns
5 or 7.
[17646] [0178.0.0.45]: see [0178.0.0.27]
[17647] [0179.0.45.45]: A nucleic acid molecule encoding an
homologous to a protein sequence as indicated in Table XII,
application no. 45, columns 5 or 7, can be created by introducing
one or more nucleotide substitutions, additions or deletions into a
nucleotide sequence of the nucleic acid molecule of the present
invention, in particular as indicated in Table XI, application no.
45, columns 5 or 7, such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations can be introduced into the encoding sequences of a
sequences as indicated in Table XI, application no. 45, columns 5
or 7, by standard techniques, such as site-directed mutagenesis and
PCR-mediated mutagenesis.
[17648] [0180.0.0.45] to [0183.0.0.45]: see [0180.0.0.27] to
[0183.0.0.27]
[17649] [0184.0.45.45] Homologues of the nucleic acid sequences
used, with a sequence as indicated in Table XI, columns 5 or 7,
application no. 45, column 7, or of the nucleic acid sequences
derived from a sequences as indicated in Table XII, application no.
45, columns 5 or 7, comprise also allelic variants with at least
approximately 30%, 35%, 40% or 45% homology, by preference at least
approximately 50%, 60% or 70%, more preferably at least
approximately 90%, 91%, 92%, 93%, 94% or 95% and even more
preferably at least approximately 96%, 97%, 98%, 99% or more
homology with one of the nucleotide sequences shown or the
abovementioned derived nucleic acid sequences or their homologues,
derivatives or analogues or parts of these. Allelic variants
encompass in particular functional variants which can be obtained
by deletion, insertion or substitution of nucleotides from the
sequences shown, preferably from a sequence as indicated in Table
XI, application no. 45, columns 5 or 7, or from the derived nucleic
acid sequences, the intention being, however, that the enzyme
activity or the biological activity of the resulting proteins
synthesized is advantageously retained or increased.
[17650] [0185.0.45.45] In one embodiment of the present invention,
the nucleic acid molecule of the invention or used in the process
of the invention comprises one or more sequences as indicated in
Table XI, application no. 45, columns 5 or 7. In one embodiment, it
is preferred that the nucleic acid molecule comprises as little as
possible other nucleotides not shown in any one of sequences as
indicated in Table XI, application no. 45, columns 5 or 7. In one
embodiment, the nucleic acid molecule comprises less than 500, 400,
300, 200, 100, 90, 80, 70, 60, 50 or 40 further nucleotides. In a
further embodiment, the nucleic acid molecule comprises less than
30, 20 or 10 further nucleotides. In one embodiment, a nucleic acid
molecule used in the process of the invention is identical to a
sequences as indicated in Table XI, application no. 45, columns 5
or 7.
[17651] [0186.0.45.45] Also preferred is that one or more nucleic
acid molecule(s) used in the process of the invention encodes a
polypeptide comprising a sequence as indicated in Table XII,
application no. 45, columns 5 or 7. In one embodiment, the nucleic
acid molecule encodes less than 150, 130, 100, 80, 60, 50, 40 or 30
further amino acids. In a further embodiment, the encoded
polypeptide comprises less than 20, 15, 10, 9, 8, 7, 6 or 5 further
amino acids. In one embodiment, the encoded polypeptide used in the
process of the invention is identical to the sequences as indicated
in Table XII, application no. 45, columns 5 or 7.
[17652] [0187.0.45.45] In one embodiment, a nucleic acid molecule
of the invention or used in the process encodes a polypeptide
comprising the sequence as indicated in Table XII, application no.
45, columns 5 or 7, and comprises less than 100 further
nucleotides. In a further embodiment, said nucleic acid molecule
comprises less than 30 further nucleotides. In one embodiment, the
nucleic acid molecule used in the process is identical to a coding
sequence encoding a polypeptide as indicated in Table XII,
application no. 45, columns 5 or 7.
[17653] [0188.0.45.45] Polypeptides (=proteins), which still have
the essential biological or enzymatic activity of the polypeptide
of the present invention conferring an increase of the respective
fine chemical i.e. whose activity is essentially not reduced, are
polypeptides with at least 10% or 20%, by preference 30% or 40%,
especially preferably 50% or 60%, very especially preferably 80% or
90 or more of the wild type biological activity or enzyme activity,
advantageously, the activity is essentially not reduced in
comparison with the activity of a polypeptide as indicated in Table
XII, application no. 45, columns 5 or 7 and is expressed under
identical conditions.
[17654] [0189.0.45.45] Homologues of a sequences as indicated in
Table XI, application no. 45, columns 5 or 7, or of a derived
sequences as indicated in Table XII, application no. 45, columns 5
or 7 also mean truncated sequences, cDNA, single-stranded DNA or
RNA of the coding and noncoding DNA sequence. Homologues of said
sequences are also understood as meaning derivatives, which
comprise noncoding regions such as, for example, UTRs, terminators,
enhancers or promoter variants. The promoters upstream of the
nucleotide sequences stated can be modified by one or more
nucleotide substitution(s), insertion(s) and/or deletion(s)
without, however, interfering with the functionality or activity
either of the promoters, the open reading frame (=ORF) or with the
3'-regulatory region such as terminators or other 3' regulatory
regions, which are far away from the ORF. It is furthermore
possible that the activity of the promoters is increased by
modification of their sequence, or that they are replaced
completely by more active promoters, even promoters from
heterologous organisms. Appropriate promoters are known to the
person skilled in the art and are mentioned herein below.
[17655] [0190.0.0.45] see [0190.0.0.27]
[17656] [0191.0.0.45] see [0191.0.0.27]:
[17657] [0192.0.0.45] to [0203.0.0.45] see [0192.0.0.27] to
[0203.0.0.27]
[17658] [0204.0.45.45] Accordingly, in one embodiment, the
invention relates to a nucleic acid molecule which comprises a
nucleic acid molecule selected from the group consisting of:
[17659] a) nucleic acid molecule encoding, preferably at least the
mature form, of a polypeptide as indicated in Table XII,
application no. 45, columns 5 or 7, or a fragment thereof
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII, application no. 45,
column 6 in an organism or a part thereof [17660] b) nucleic acid
molecule comprising, preferably at least the mature form, of a
nucleic acid molecule as indicated in Table XI, application no. 45,
columns 5 or 7, or a fragment thereof conferring an increase in the
amount of the respective fine chemical in an organism or a part
thereof; [17661] c) nucleic acid molecule whose sequence can be
deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
in an organism or a part thereof; [17662] d) nucleic acid molecule
encoding a polypeptide whose sequence has at least 50% identity
with the amino acid sequence of the polypeptide encoded by the
nucleic acid molecule of (a) to (c) and conferring an increase in
the amount of the fine chemical in an organism or a part thereof;
[17663] e) nucleic acid molecule which hybridizes with a nucleic
acid molecule of (a) to (c) under under stringent hybridisation
conditions and conferring an increase in the amount of the fine
chemical in an organism or a part thereof; [17664] f) nucleic acid
molecule encoding a polypeptide, the polypeptide being derived by
substituting, deleting and/or adding one or more amino acids of the
amino acid sequence of the polypeptide encoded by the nucleic acid
molecules (a) to (d), preferably to (a) to (c), and conferring an
increase in the amount of the fine chemical in an organism or a
part thereof; [17665] g) nucleic acid molecule encoding a fragment
or an epitope of a polypeptide which is encoded by one of the
nucleic acid molecules of (a) to (e), preferably to (a) to (c) and
conferring an increase in the amount of the fine chemical in an
organism or a part thereof; [17666] h) nucleic acid molecule
comprising a nucleic acid molecule which is obtained by amplifying
a cDNA library or a genomic library using primers or primer pairs
as indicated in Table XIII, application no. 45, column 7, and
conferring an increase in the amount of the respective fine
chemical, in particular according to table XII, application no. 45,
column 6 in an organism or a part thereof; [17667] i) nucleic acid
molecule encoding a polypeptide which is isolated, e.g. from a
expression library, with the aid of monoclonal antibodies against a
polypeptide encoded by one of the nucleic acid molecules of (a) to
(g), preferably to (a) to (c) and conferring an increase in the
amount of the fine chemical in an organism or a part thereof;
[17668] j) nucleic acid molecule which encodes a polypeptide
comprising a consensus sequence as indicated in Table XIV,
application no. 45, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical, in particular according
to table XII, application no. 45, column 6 in an organism or a part
thereof; [17669] k) nucleic acid molecule encoding the amino acid
sequence of a polypeptide encoding a domaine of a polypeptide as
indicated in Table XII, application no. 45, columns 5 or 7, and
conferring an increase in the amount of the respective fine
chemical, in particular accccording to table XII, application no.
45, column 6 in an organism or a part thereof; and [17670] l)
nucleic acid molecule which is obtainable by screening a suitable
nucleic acid library under stringent hybridization conditions with
a probe comprising one of the sequences of the nucleic acid
molecule of (a) to (k) or with a fragment of at least 15 nt,
preferably 20 nt, 30 nt, 50 nt, 100 nt, 200 nt or 500 nt of the
nucleic acid molecule characterized in (a) to (h) or of a nucleic
acid molecule as indicated in Table XI, application no. 45, columns
5 or 7, or a nucleic acid molecule encoding, preferably at least
the mature form of, a polypeptide as indicated in Table XII,
application no. 45, columns 5 or 7, and conferring an increase in
the amount of the respective fine chemical according to table XII,
application no. 45, column 6 in an organism or a part thereof;
[17671] or which encompasses a sequence which is complementary
thereto; whereby, preferably, the nucleic acid molecule according
to (a) to (l) distinguishes over the sequence indicated in Table
XI, application no. 45, columns 5 or 7, by one or more nucleotides.
In one embodiment, the nucleic acid molecule does not consist of
the sequence shown and indicated in Table XI, application no. 45,
columns 5 or 7, In one embodiment, the nucleic acid molecule is
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 45, columns 5 or
7.
[17672] In another embodiment, the nucleic acid molecule does not
encode a polypeptide of a sequence indicated in Table XII,
application no. 45, columns 5 or 7.
[17673] In an other embodiment, the nucleic acid molecule of the
present invention is at least 30%, 40%, 50%, or 60% identical and
less than 100%, 99.999%, 99.99%, 99.9% or 99% identical to a
sequence indicated in Table XI, application no. 45, columns 5 or
7.
[17674] In a further embodiment the nucleic acid molecule does not
encode a polypeptide sequence as indicated in Table XII,
application no. 45, columns 5 or 7.
[17675] Accordingly, in one embodiment, the nucleic acid molecule
of the differs at least in one or more residues from a nucleic acid
molecule indicated in Table XI, application no. 45, columns 5 or
7.
[17676] Accordingly, in one embodiment, the nucleic acid molecule
of the present invention encodes a polypeptide, which differs at
least in one or more amino acids from a polypeptide indicated in
Table XII, application no. 45, columns 5 or 7.
[17677] In another embodiment, a nucleic acid molecule indicated in
Table XI, application no. 45, columns 5 or 7.
[17678] Accordingly, in one embodiment, the protein encoded by a
sequences of a nucleic acid according to (a) to (l) does not
consist of a sequence as indicated in Table XII, application no.
45, columns 5 or 7.
[17679] In a further embodiment, the protein of the present
invention is at least 30%, 40%, 50%, or 60% identical to a protein
sequence indicated in Table XII, application no. 45, columns 5 or
7, and less than 100%, preferably less than 99.999%, 99.99% or
99.9%, more preferably less than 99%, 985, 97%, 96% or 95%
identical to a sequence as indicated in Table XI, columns 5 or
7.
[17680] [0205.0.0.45] to [0206.0.0.45] see [0205.0.0.27] to
[0206.0.0.27]
[17681] [0207.0.45.45] As described herein, the nucleic acid
construct can also comprise further genes, which are to be
introduced into the organisms or cells. It is possible and
advantageous to introduce into, and express in, the host organisms
regulatory genes such as genes for inductors, repressors or
enzymes, which, owing to their enzymatic activity, engage in the
regulation of one or more genes of a biosynthetic pathway. These
genes can be of heterologous or homologous origin. Moreover,
further biosynthesis genes may advantageously be present, or else
these genes may be located on one or more further nucleic acid
constructs. Genes, which are advantageously employed as
biosynthesis genes, are genes of the carbohydrate or starch
metabolism, the glucose metabolism, the saccharide metabolism, the
metabolism of glycolysis, or their combinations. As described
herein, regulator sequences or factors can have a positive effect
on preferably the gene expression of the genes introduced, thus
increasing it. Thus, an enhancement of the regulator elements may
advantageously take place at the transcriptional level by using
strong transcription signals such as promoters and/or enhancers. In
addition, however, an enhancement of translation is also possible,
for example by increasing mRNA stability or by inserting a
translation enhancer sequence.
[17682] [0208.0.0.45] to [0226.0.0.45]: see [0208.0.0.27] to
[0226.0.0.27]
[17683] [0227.0.45.45]: The abovementioned nucleic acid molecules
can be cloned into the nucleic acid constructs or vectors according
to the invention in combination together with further genes, or
else different genes are introduced by transforming several nucleic
acid constructs or vectors (including plasmids) into a host cell,
advantageously into a plant cell or a microorganisms.
[17684] In addition to a sequence indicated in Table XI,
application no. 45, columns 5 or 7, lines 290 to 294 and/or 604 to
607; or its derivatives, it is advantageous additionally to express
and/or mutate further genes in the organisms. Especially
advantageously, additionally at least one further gene of the
carbohydrate biosynthetic pathway is expressed in the organisms
such as plants or microorganisms. It is also possible that the
regulation of the natural genes has been modified advantageously so
that the gene and/or its gene product is no longer subject to the
regulatory mechanisms which exist in the organisms. This leads to
an increased synthesis of the carbohydrate desired since, for
example, feedback regulations no longer exist to the same extent or
not at all. In addition it might be advantageously to combine one
or more of the sequences indicated in Table XI, application no. 45,
columns 5 or 7, with genes which generally support or enhances to
growth or yield of the target organisms, for example genes which
lead to faster growth rate of microorganisms or genes which
produces stress-, pathogen, or herbicide resistant plants.
[17685] [0228.0.45.45: In a further embodiment of the process of
the invention, therefore, organisms are grown, in which there is
simultaneous overexpression of at least one nucleic acid or one of
the genes which code for proteins involved in the carbohydrate
metabolism, in particular in synthesis of polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably myo-inositol.
[17686] [0229.0.45.45]: Further advantageous nucleic acid sequences
which can be expressed in combination with the sequences used in
the process and/or the abovementioned biosynthesis genes are the
sequences encoding further genes of the carbohydrate biosynthetic
pathway. These genes can lead to an increased synthesis of the
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably
myo-inositol resp.
[17687] [0230.0.0.45] see [0230.0.0.27]
[17688] [0231.0.45.45] In a further advantageous embodiment of the
process of the invention, the organisms used in the process are
those in which simultaneously a carbohydrate degrading protein is
attenuated, in particular by reducing the rate of expression of the
corresponding gene.
[17689] [0232.0.0.45] to [0276.0.0.45]: see [0232.0.0.27] to
[0276.0.0.27]
[17690] [0277.0.45.45] The respective fine chemical produced can be
isolated from the organism by methods with which the skilled worker
is familiar. For example, via extraction, chemical or enzymatical
deg radiation, crystallization, salt precipitation, and/or
different chromatography methods. The process according to the
invention can be conducted batchwise, semibatchwise or
continuously. The respective fine chemcical produced by this
process can be obtained by harvesting the organisms, either from
the crop in which they grow, or from the field. This can be done
via pressing or extraction of the plant parts
[17691] [0278.0.0.45] to [0282.0.0.45]: see [0278.0.0.27] to
[0282.0.0.27]
[17692] [0283.0.45.45]: Moreover, a native polypeptide conferring
the increase of the respective fine chemical in an organism or part
thereof can be isolated from cells (e.g., endothelial cells), for
example using the antibody of the present invention as described
below, e.g. an antibody against a protein as indicated in Table
XII, application no. 45, column 3, or an antibody against a
polypeptide as indicated in Table XII, application no. 45, columns
5 or 7, which can be produced by standard techniques utilizing the
polypeptid of the present invention or fragment thereof, i.e., the
polypeptide of this invention. Preferred are monoclonal
antibodies.
[17693] [0284.0.0.45]: see [0284.0.0.27]
[17694] [0285.0.45.45]: In one embodiment, the present invention
relates to a polypeptide having a sequence as indicated in Table
XII, application no. 45, columns 5 or 7, or as encoded by a nucleic
acid molecule as indicated in Table XI, application no. 45, columns
5 or 7, or functional homologues thereof.
[17695] [0286.0.45.45]: In one advantageous embodiment, in the
method of the present invention the activity of a polypeptide is
increased which comprises or consists of a consensus sequence as
indicated in Table XIV, application no. 45, column 7. In another
embodiment, the present invention relates to a polypeptide
comprising or consisting of a consensus sequence as indicated in
Table XIV, application no. 45, column 7, whereby 20 or less,
preferably 15 or 10, preferably 9, 8, 7, or 6, more preferred 5 or
4, even more preferred 3, even more preferred 2, even more
preferred 1, most preferred 0 of the amino acids positions
indicated can be replaced by any amino acid and/or be absent.
[17696] In one embodiment, the present invention relates to the
method of the present invention comprising a polypeptide or to a
polypeptide comprising more than one consensus sequences (of an
individual line) as indicated in Table XIV, application no. 45,
column 7.
[17697] [0287.0.0.45] to [0290.0.0.45]: see [0287.0.0.27] to
[0290.0.0.27]
[17698] [0291.0.45.45]: In one advantageous embodiment, the method
of the present invention comprises the increasing of a polypeptide
comprising or consisting of plant or microorganism specific
consensus sequences.
[17699] In one embodiment, said polypeptide of the invention
distinguishes over a sequence as indicated in Table XII,
application no. 45, columns 5 or 7, by one or more amino acids. In
one embodiment, polypeptide distinguishes form a sequence as
indicated in Table XII, application no. 45, columns 5 or 7, by more
than 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids, preferably by more
than 10, 15, 20, 25 or 30 amino acids, even more preferred are more
than 40, 50, or 60 amino acids and, preferably, the sequence of the
polypeptide of the invention distinguishes from a sequence as
indicated in Table XII, application no. 45, columns 5 or 7, by not
more than 80% or 70% of the amino acids, preferably not more than
60% or 50%, more preferred not more than 40% or 30%, even more
preferred not more than 20% or 10%. In an other embodiment, said
polypeptide of the invention does not consist of a sequence as
indicated in Table XII, application no. 45, columns 5 or 7.
[17700] [0292.0.0.45]: see [0292.0.0.27]
[17701] [0293.0.45.45]: In one embodiment, the invention relates to
polypeptide conferring an increase in the respective fine chemical
in an organism or part thereof and being encoded by the nucleic
acid molecule of the invention or a nucleic acid molecule used in
the process of the invention. In one embodiment, the polypeptide of
the invention has a sequence which distinguishes from a sequence as
indicated in Table XII, application no. 45, columns 5 or 7, by one
or more amino acids. In an other embodiment, said polypeptide of
the invention does not consist of the sequence as indicated in
Table XII, application no. 45, columns 5 or 7.
[17702] In a further embodiment, said polypeptide of the present
invention is less than 100%, 99.999%, 99.99%, 99.9% or 99%
identical. In one embodiment, said polypeptide does not consist of
the sequence encoded by a nucleic acid molecules as indicated in
Table XI, application no. 45, columns 5 or 7.
[17703] [0294.0.45.45] In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 45, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 45, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, evenmore preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[17704] [0295.0.0.45] to [0297.0.0.45]: see [0295.0.0.27] to
[0297.0.0.27]
[17705] [0297.1.45.45] Non polypeptide of the invention-chemicals
are e.g. polypeptides having not the activity of a polypeptide
indicated in Table XII, application no. 45, columns 3, 5 or 7.
[17706] [0298.0.45.45]: A polypeptide of the invention can
participate in the process of the present invention. The
polypeptide or a portion thereof comprises preferably an amino acid
sequence which is sufficiently homologous to an amino acid sequence
as indicated in Table XII, application no. 45, columns 5 or 7. The
portion of the protein is preferably a biologically active portion
as described herein. Preferably, the polypeptide used in the
process of the invention has an amino acid sequence identical to a
sequence as indicated in Table XII, application no. 45, columns 5
or 7.
[17707] [0299.0.45.45]: Further, the polypeptide can have an amino
acid sequence which is encoded by a nucleotide sequence which
hybridizes, preferably hybridizes under stringent conditions as
described above, to a nucleotide sequence of the nucleic acid
molecule of the present invention. Accordingly, the polypeptide has
an amino acid sequence which is encoded by a nucleotide sequence
that is at least about 35%, 40%, 45%, 50%, 55%, 60%, 65% or 70%,
preferably at least about 75%, 80%, 85% or 90, and more preferably
at least about 91%, 92%, 93%, 94% or 95%, and even more preferably
at least about 96%, 97%, 98%, 99% or more homologous to one of the
nucleotide sequence as indicated in Table XI, application no. 45,
columns 5 or 7. The preferred polypeptide of the present invention
preferably possesses at least one of the activities according to
the invention and described herein. A preferred polypeptide of the
present invention includes an amino acid sequence encoded by a
nucleotide sequence which hybridizes, preferably hybridizes under
stringent conditions, to a nucleotide sequence as indicated in
Table XI, application no. 45, columns 5 or 7, or which is
homologous thereto, as defined above.
[17708] [0300.0.45.45]: In one embodiment, the present invention
relates to a polypeptide having an activity of a protein as
indicated in Table XII, application no. 45, column 3, which
distinguishes over a sequence as indicated in Table XII,
application no. 45, columns 5 or 7, by one or more amino acids,
preferably by more than 5, 6, 7, 8 or 9 amino acids, preferably by
more than 10, 15, 20, 25 or 30 amino acids, even more preferred are
more than 40, 50, or 60 amino acids but even more preferred by less
than 70% of the amino acids, more preferred by less than 50%, even
more preferred my less than 30% or 25%, more preferred are 20% or
15%, even more preferred are less than 10%.
[17709] [0301.0.0.45] see [0301.0.0.27]
[17710] [0302.0.45.45] Biologically active portions of an
polypeptide of the present invention include peptides comprising
amino acid sequences derived from the amino acid sequence of the
polypeptide of the present invention or used in the process of the
present invention, e.g., an amino acid sequence as indicated in
Table XII, application no. 45, columns 5 or 7, or the amino acid
sequence of a protein homologous thereto, which include fewer amino
acids than a full length polypeptide of the present invention or
used in the process of the present invention or the full length
protein which is homologous to an polypeptide of the present
invention or used in the process of the present invention depicted
herein, and exhibit at least one activity of polypeptide of the
present invention or used in the process of the present
invention.
[17711] [0303.0.0.45]: see [0303.0.0.27]
[17712] [0304.0.45.45]: Manipulation of the nucleic acid molecule
of the invention may result in the production of a protein having
essentially the activity of the polypeptides as indicated in Table
XII, application no. 45, column 3, but having differences in the
sequence from said wild-type protein. These proteins may be
improved in efficiency or activity, may be present in greater
numbers in the cell than is usual, or may be decreased in
efficiency or activity in relation to the wild type protein.
[17713] [0305.0.0.45] to [0308.0.0.45]: see [0305.0.0.27] to
[0308.0.0.27]
[17714] [0309.0.45.45]: In one embodiment, a reference to a protein
(=polypeptide) of the invention or as indicated in Table XII,
application no. 45, columns 5 or 7, refers to a polypeptide having
an amino acid sequence corresponding to the polypeptide of the
invention or used in the process of the invention, whereas a
"non-polypeptide of the invention" or "other polypeptide" not being
indicated in Table XII, application no. 45, columns 5 or 7, refers
to a polypeptide having an amino acid sequence corresponding to a
protein which is not substantially homologous a polypeptide of the
invention, preferably which is not substantially homologous to a
polypeptide as indicated in Table XII, application no. 45, columns
5 or 7, e.g., a protein which does not confer the activity
described herein or annotated or known for as indicated in Table
XII, application no. 45, column 3, and which is derived from the
same or a different organism. In one embodiment, a "non-polypeptide
of the invention" or "other polypeptide" not being indicated in
Table XII, application no. 45, columns 5 or 7, does not confer an
increase of the respective fine chemical in an organism or part
therof.
[17715] [0310.0.0.45] to [0334.0.0.45]: see [0310.0.0.27] to
[0334.0.0.27]
[17716] [0335.0.45.45]: The dsRNAi method has proved to be
particularly effective and advantageous for reducing the expression
of a nucleic acid sequences as indicated in Table XI, application
no. 45, columns 5 or 7, and/or homologs thereof. As described inter
alia in WO 99/32619, dsRNAi approaches are clearly superior to
traditional antisense approaches. The invention therefore
furthermore relates to double-stranded RNA molecules (dsRNA
molecules) which, when introduced into an organism, advantageously
into a plant (or a cell, tissue, organ or seed derived therefrom),
bring about altered metabolic activity by the reduction in the
expression of a nucleic acid sequences as indicated in Table XI,
application no. 45, columns 5 or 7, and/or homologs thereof. In a
double-stranded RNA molecule for reducing the expression of an
protein encoded by a nucleic acid sequence sequences as indicated
in Table XI, application no. 45, columns 5 or 7, and/or homologs
thereof, one of the two RNA strands is essentially identical to at
least part of a nucleic acid sequence, and the respective other RNA
strand is essentially identical to at least part of the
complementary strand of a nucleic acid sequence.
[17717] [0336.0.0.45] to [0342.0.0.45]: see [0336.0.0.27] to
[0342.0.0.27]
[17718] [0343.0.45.45]: As has already been described, 100%
sequence identity between the dsRNA and a gene transcript of a
nucleic acid sequence as indicated in Table XI, application no. 45,
columns 5 or 7, or its homolog is not necessarily required in order
to bring about effective reduction in the expression. The advantage
is, accordingly, that the method is tolerant with regard to
sequence deviations as may be present as a consequence of genetic
mutations, polymorphisms or evolutionary divergences. Thus, for
example, using the dsRNA, which has been generated starting from a
sequence as indicated in Table XI, application no. 45, columns 5 or
7, or homologs thereof of the one organism, may be used to suppress
the corresponding expression in another organism.
[17719] [0344.0.0.45] to [0361.0.0.45]: see [0344.0.0.27] to
[0361.0.0.27]
[17720] [0362.0.45.45]: Accordingly the present invention relates
to any cell transgenic for any nucleic acid characterized as part
of the invention, e.g. conferring the increase of the respective
fine chemical in a cell or an organism or a part thereof, e.g. the
nucleic acid molecule of the invention, the nucleic acid construct
of the invention, the antisense molecule of the invention, the
vector of the invention or a nucleic acid molecule encoding the
polypeptide of the invention, e.g. the polypeptide as indicated in
Table XII, application no. 45, e.g. encoding a polypeptide having
protein activity, as indicated in Table XII, application no. 45,
columns 3. Due to the above mentioned activity the respective fine
chemical content in a cell or an organism is increased. For
example, due to modulation or manipulation, the cellular activity
of the polypeptide of the invention or nucleic acid molecule of the
invention is increased, e.g. due to an increased expression or
specific activity of the subject matters of the invention in a cell
or an organism or a part thereof. Transgenic for a polypeptide
having an activity of a polypeptide as indicated in Table XII,
application no. 45, columns 5 or 7, means herein that due to
modulation or manipulation of the genome, an activity as annotated
for a polypeptide as indicated in Table XII, application no. 45,
column 3, e.g. having a sequence as indicated in Table XII,
application no. 45, columns 5 or 7, is increased in a cell or an
organism or a part thereof. Examples are described above in context
with the process of the invention [0363.0.0.45]: see
[0363.0.0.27]
[17721] [0364.0.0.45]: see [0364.0.0.27]
[17722] [0365.0.0.45] to [0373.0.0.45]: see [0365.0.0.27] to
[0373.0.0.27]
[17723] [0374.0.45.45]: Transgenic plants comprising the respective
fine chemical synthesized in the process according to the invention
can be marketed directly without isolation of the compounds
synthesized. In the process according to the invention, plants are
understood as meaning all plant parts, plant organs such as leaf,
stalk, root, tubers or seeds or propagation material or harvested
material or the intact plant. In this context, the seed encompasses
all parts of the seed such as the seed coats, epidermal cells, seed
cells, endosperm or embryonic tissue. Carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol resp., produced in
the process according to the invention may, however, also be
isolated from the plant in the form of their free form or bound in
or to compounds or moieties. Carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol resp., produced by
this process can be harvested by harvesting the organisms either
from the culture in which they grow or from the field. This can be
done via expressing, grinding and/or extraction, salt precipitation
and/or ion-exchange chromatography or other chromatographic methods
of the plant parts, preferably the plant seeds, plant fruits, plant
tubers and the like.
[17724] [0375.0.0.45] to [0376.0.0.45]: see [0375.0.0.27] to
[0376.0.0.27]
[17725] [0377.0.45.45]: Accordingly, the present invention relates
also to a process according to the present invention whereby the
produced carbohydrates, preferably polysaccharides, more preferably
starch and/or cellulose and/or monosaccharides, more preferably
myo-inositol comprising composition or the produced the respective
fine chemical is isolated.
[17726] [0378.0.45.45]: In this manner, more than 50% by weight,
advantageously more than 60% by weight, preferably more than 70% by
weight, especially preferably more than 80% by weight, very
especially preferably more than 90% by weight, of the
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably
myo-inositol resp., produced in the process can be isolated. The
resulting carbohydrates, preferably polysaccharides, more
preferably starch and/or cellulose and/or monosaccharides, more
preferably myo-inositol resp., can, if appropriate, subsequently be
further purified, if desired mixed with other active ingredients
such as vitamins, amino acids, carbohydrates, antibiotics and the
like, and, if appropriate, formulated.
[17727] [0379.0.45.45]: In one embodiment, the carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably myo-inositol resp., is a
mixture comprising of one or more the respective fine chemicals. In
one embodiment, the respective fine chemical means here
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably
myo-inositol. In one embodiment, carbohydrate means here a mixture
of the respective fine chemicals.
[17728] [0380.0.45.45]: The carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol resp., obtained in
the process are suitable as starting material for the synthesis of
further products of value. For example, they can be used in
combination with each other or alone for the production of
pharmaceuticals, foodstuffs, animal feeds or cosmetics.
Accordingly, the present invention relates a method for the
production of pharmaceuticals, food stuff, animal feeds, nutrients
or cosmetics comprising the steps of the process according to the
invention, including the isolation of the carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol comprising
composition produced or the respective fine chemical produced if
desired and formulating the product with a pharmaceutical
acceptable carrier or formulating the product in a form acceptable
for an application in agriculture. A further embodiment according
to the invention is the use of the carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol resp., produced in
the process or of the transgenic organisms in animal feeds,
foodstuffs, medicines, food supplements, cosmetics or
pharmaceuticals.
[17729] [0381.0.0.45] to [0382.0.0.45]: see [0381.0.0.27] to
[0382.0.0.27]
[17730] [0383.0.45.45]: ./.
[17731] [0384.0.0.45]: see [0384.0.0.27]
[17732] [0385.0.45.45]: The fermentation broths obtained in this
way, containing in particular carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol, resp., in mixtures
with other compounds, in particular with other carbohydrates, or
fatty acids building lipids or containing microorganisms or parts
of microorganisms, like plastids, containing carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably myo-inositol resp., in
mixtures with other compounds, e.g. carbohydrates, normally have a
dry matter content of from 7.5 to 25% by weight.
[17733] The fermentation broth can then be thickened or
concentrated by known methods, such as, for example, with the aid
of a rotary evaporator, thin-film evaporator, falling film
evaporator, by reverse osmosis or by nanofiltration. This
concentrated fermentation broth can then be worked up by
freeze-drying, spray drying, spray granulation or by other
processes.
[17734] [0386.0.0.45] -/-
[17735] [0387.0.0.45] to [0392.0.0.45]: see [0387.0.0.27] to
[0392.0.0.27]
[17736] [0393.0.45.45]: In one embodiment, the present invention
relates to a method for the identification of a gene product
conferring an increase in the fine chemical production in a cell,
comprising the following steps: [17737] a) contacting e.g.
hybridising, the nucleic acid molecules of a sample, e.g. cells,
tissues, plants or microorganisms or a nucleic acid library, which
can contain a candidate gene encoding a gene product conferring an
increase in the fine chemical after expression, with the nucleic
acid molecule of the present invention; [17738] b) identifying the
nucleic acid molecules, which hybridize under relaxed stringent
conditions with the nucleic acid molecule of the present invention
in particular to the nucleic acid molecule sequence as indicated in
Table XI, application no. 45, columns 5 or 7, and, optionally,
isolating the full length cDNA clone or complete genomic clone;
[17739] c) introducing the candidate nucleic acid molecules in host
cells, preferably in a plant cell or a microorganism, appropriate
for producing the fine chemical; [17740] d) expressing the
identified nucleic acid molecules in the host cells; [17741] e)
assaying the the fine chemical level in the host cells; and [17742]
f) identifying the nucleic acid molecule and its gene product which
expression confers an increase in the the fine chemical level in
the host cell after expression compared to the wild type.
[17743] [0394.0.0.45] to [0399.0.0.45]: see [0394.0.0.27] to
[0399.0.0.27]
[17744] [00399.1.45.45]: One can think to screen for increased
production of the respective fine chemical by for example searching
for a resistance to a drug blocking the synthesis of the respective
fine chemical and looking whether this effect is dependent on the
activity or expression of a polypeptide as indicated in Table XII,
application no. 45, columns 5 or 7, or a homolog thereof, e.g.
comparing the phenotyp of nearly identical organisms with low and
high activity of a protein as indicated in Table XII, application
no. 45, columns 5 or 7, after incubation with the drug.
[17745] [0400.0.0.45] to [0416.0.0.45]: see [0400.0.0.27] to
[0416.0.0.27]
[17746] [0417.0.45.45]: The nucleic acid molecule of the invention,
the vector of the invention or the nucleic acid construct of the
invention may also be useful for the production of organisms
resistant to inhibitors of the carbohydrates, preferably
polysaccharides, more preferably starch and/or cellulose and/or
monosaccharides, more preferably myo-inositol, resp., production
biosynthesis pathways. In particular, the overexpression of the
polypeptide of the present invention may protect an organism such
as a microorganism or a plant against inhibitors, which block the
carbohydrate, in particular the respective fine chemical synthesis
in said organism. Examples of inhibitors or herbicides blocking the
synthesis in organism such as microorganism or plants are for
example the cellulose synthesis inhibitors 2,6-dichlorobenzonitrile
(DCB),
N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-
-diamine, called AE F150944 or isoxaben.
[17747] [0418.0.0.45] to [0423.0.0.45]: see [0418.0.0.27] to
[0423.0.0.27]
[17748] [0424.0.45.45]: Accordingly, the nucleic acid of the
invention, the polypeptide of the invention, the nucleic acid
construct of the invention, the organisms, the host cell, the
microorganisms, the plant, plant tissue, plant cell, or the part
thereof of the invention, the vector of the invention, the agonist
identified with the method of the invention, the nucleic acid
molecule identified with the method of the present invention, can
be used for the production of the respective fine chemical or of
the respective fine chemical and one or more other
carbohydrate.
[17749] Accordingly, the nucleic acid of the invention, or the
nucleic acid molecule identified with the method of the present
invention or the complement sequences thereof, the polypeptide of
the invention, the nucleic acid construct of the invention, the
organisms, the host cell, the microorganisms, the plant, plant
tissue, plant cell, or the part thereof of the invention, the
vector of the invention, the antagonist identified with the method
of the invention, the antibody of the present invention, the
antisense molecule of the present invention, can be used for the
reduction of the respective fine chemical in a organism or part
thereof, e.g. in a cell.
[17750] [0425.0.0.45] to [0453.0.0.45]: see [0425.0.0.27] to
[0453.0.0.27]
[0454.0.45.45] Example 8
Analysis of the Effect of the Nucleic Acid Molecule on the
Production of the Fine Chemical Resp
[17751] [0455.0.45.45] The effect of the genetic modification in C.
glutamicum or other microbial especially bacterial strains for
carbohydrate production on the production of an carbohydrate can be
determined by growing the modified microorganisms under suitable
conditions (such as those described above) and analyzing the medium
and/or the cellular components for the increased production of the
amino acid. Such analytical techniques are well known to the
skilled worker and encompass spectroscopy, thin-layer
chromatography, various types of staining methods, enzymatic and
microbiological methods and analytical chromatography such as
high-performance liquid chromatography (see, for example, Ullman,
Encyclopedia of Industrial Chemistry,
[17752] Vol. A2, pp. 89-90 and pp. 443-613, VCH: Weinheim (1985);
Fallon, A., et al., (1987) "Applications of HPLC in Biochemistry"
in: Laboratory Techniques in Biochemistry and Molecular Biology,
Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3, Chapter III:
"Product recovery and purification", pp. 469-714, VCH: Weinheim;
Better, P. A. et al. (1988) Bioseparations: downstream processing
for Biotechnology, John Wiley and Sons; Kennedy, J. F. and Cabral,
J. M. S. (1992) Recovery processes for biological Materials, John
Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D. (1988)
Biochemical Separations, in Ullmann's Encyclopedia of Industrial
Chemistry, Vol. B3; chapter 11, pp. 1-27, VCH: Weinheim; and
Dechow, F. J. (1989) Separation and purification techniques in
biotechnology, Noyes Publications).
[17753] [0456.0.0.45]: see [0456.0.0.27]
[0457.0.45.45]: Example 9
Purification of the Carbohydrates, Preferably Polysaccharides, More
Preferably Starch and/or Cellulose and/or Monosaccharides, More
Preferably Myo-Inositol
[17754] [0458.0.45.45]: Abbreviations: GC-MS, gas liquid
chromatography/mass spectrometry; TLC, thin-layer
chromatography.
[17755] The unambiguous detection for the presence of
carbohydrates, preferably polysaccharides, more preferably starch
and/or cellulose and/or monosaccharides, more preferably
myo-inositolcan be obtained by analyzing recombinant organisms
using analytical standard methods: GC, GC-MS or TLC, as described
(1997, in: Advances on Lipid Methodology, Fourth Edition: Christie,
Oily Press, Dundee, 119-169; 1998,
Gaschromatographie-Massenspektrometrie-Verfahren [Gas
chromatography/mass spectrometric methods], Lipide 33:343-353). The
total carbohydrate produced in the organism for example in yeasts
used in the inventive process can be analysed for example according
to the following procedure: The material such as yeasts, E. coli or
plants to be analyzed can be disrupted by sonication, grinding in a
glass mill, liquid nitrogen and grinding or via other applicable
methods.
[17756] Plant material is initially homogenized mechanically by
comminuting in a pestle and mortar to make it more amenable to
extraction.
[17757] [0459.0.45.45]: -/-
[17758] [0460.0.0.45] see [0460.0.0.27]
[0461.0.45.45] Example 10
Cloning SEQ ID NO: 108333 for the Expression in Plants
[17759] [0462.0.0.45]: see [0462.0.0.27]
[17760] [0463.45.45] SEQ ID NO: 108333 is amplified by PCR as
described in the protocol of the Pfu Turbo or DNA Herculase
polymerase (Stratagene).
[17761] [0464.0.0.45] to [0466.0.0.45]: see [0464.0.0.27] to
[0466.0.0.27]
[17762] [0467.0.45.45] The following primer sequences were selected
for the gene SEQ ID NO: 108333:
i) forward primer: SEQ ID NO: 108339 ii) reverse primer: SEQ ID NO:
108340
[17763] [0468.0.0.45] to [0479.0.0.45]: see [0468.0.0.27] to
[0479.0.0.27]
[0480.0.45.45]: Example 11
Generation of Transgenic Plants which Express SEQ ID NO: 108333
[17764] [0481.0.0.45] to [0513.0.0.45]: see [0481.0.0.27] to
[0513.0.0.27]
[17765] [0514.0.45.45]: As an alternative, the carbohydrates,
preferably polysaccharides, more preferably starch and/or cellulose
and/or monosaccharides, more preferably myo-inositol, can be
detected advantageously as for example described by Sonnebald et
al., (Nat Biotechnol. 1997 August; 15(8):794-7), or Panikulangara
et al., Plant Physiol. 2004 October; 136(2):3148-58.
[17766] [0515.0.0.45] to [0552.0.0.45]: see [0515.0.0.27] to
[0552.0.0.27]
[17767] [0553.0.45.45]
1. A process for the production of myo-inositol and/or
anhydroglucose resp., which comprises (a) increasing or generating
the activity of a protein as indicated in Table XII, application
no. 45, columns 5 or 7, or a functional equivalent thereof in a
non-human organism, or in one or more parts thereof; and (b)
growing the organism under conditions which permit the production
of myo-inositol and/or anhydroglucose resp. in said organism. 2. A
process for the production of myo-inositol and/or anhydroglucose
resp., comprising the increasing or generating in an organism or a
part thereof the expression of at least one nucleic acid molecule
comprising a nucleic acid molecule selected from the group
consisting of: a) nucleic acid molecule encoding of a polypeptide
as indicated in Table XII, application no. 45, columns 5 or 7, or a
fragment thereof, which confers an increase in the amount of the
fine chemical as indicated in table XII, column 6, e.g of
myo-inositol and/or anhydroglucose resp., in an organism or a part
thereof; b) nucleic acid molecule comprising of a nucleic acid
molecule as indicated in Table XI, application no. 45, columns 5 or
7, nucleic acid molecule whose sequence can be deduced from a
polypeptide sequence encoded by a nucleic acid molecule of (a) or
(b) as a result of the degeneracy of the genetic code and
conferring an increase in the amount of the fine chemical as
indicated in table XII, column 6, e.g of myo-inositol and/or
anhydroglucose resp., in an organism or a part thereof; d) nucleic
acid molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the fine chemical as indicated in table XII,
column 6, e.g of myo-inositol and/or anhydroglucose resp., in an
organism or a part thereof; e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of myo-inositol and/or anhydroglucose resp., in an organism
or a part thereof; f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table XIII, application no.
45, column 7, and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of myo-inositol
and/or anhydroglucose resp., in an organism or a part thereof; g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of myo-inositol and/or anhydroglucose resp., in
an organism or a part thereof; h) nucleic acid molecule encoding a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 45, column 7, and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of myo-inositol and/or anhydroglucose resp., in an organism
or a part thereof; and i) nucleic acid molecule which is obtainable
by screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of myo-inositol and/or anhydroglucose resp., in an organism or
a part thereof. or comprising a sequence which is complementary
thereto. 3. The process of claim 1 or 2, comprising recovering of
the free or bound myo-inositol and/or anhydroglucose resp. 4. The
process of any one of claims 1 to 3, comprising the following
steps: (a) selecting an organism or a part thereof expressing a
polypeptide encoded by the nucleic acid molecule characterized in
claim 2; (b) mutagenizing the selected organism or the part
thereof; (c) comparing the activity or the expression level of said
polypeptide in the mutagenized organism or the part thereof with
the activity or the expression of said polypeptide of the selected
organisms or the part thereof; (d) selecting the mutated organisms
or parts thereof, which comprise an increased activity or
expression level of said polypeptide compared to the selected
organism or the part thereof; (e) optionally, growing and
cultivating the organisms or the parts thereof; and (f) recovering,
and optionally isolating, the free or bound myo-inositol and/or
anhydroglucose resp., produced by the selected mutated organisms or
parts thereof. 5. The process of any one of claims 1 to 4, wherein
the activity of said protein or the expression of said nucleic acid
molecule is increased or generated transiently or stably. 6. An
isolated nucleic acid molecule comprising a nucleic acid molecule
selected from the group consisting of: a) nucleic acid molecule
encoding of a polypeptide as indicated in Table XII, application
no. 45, columns 5 or 7, or a fragment thereof, which confers an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of myo-inositol and/or anhydroglucose resp., in
an organism or a part thereof; b) nucleic acid molecule comprising
of a nucleic acid molecule as indicated in Table XI, application
no. 45, columns 5 or 7, c) nucleic acid molecule whose sequence can
be deduced from a polypeptide sequence encoded by a nucleic acid
molecule of (a) or (b) as a result of the degeneracy of the genetic
code and conferring an increase in the amount of the fine chemical
as indicated in table XII, column 6, e.g of myo-inositol and/or
anhydroglucose resp., in an organism or a part thereof; d) nucleic
acid molecule which encodes a polypeptide which has at least 50%
identity with the amino acid sequence of the polypeptide encoded by
the nucleic acid molecule of (a) to (c) and conferring an increase
in the amount of the fine chemical as indicated in table XII,
column 6, e.g of myo-inositol and/or anhydroglucose resp., in an
organism or a part thereof; e) nucleic acid molecule which
hybridizes with a nucleic acid molecule of (a) to (c) under under
stringent hybridisation conditions and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of myo-inositol and/or anhydroglucose resp., in an organism
or a part thereof; f) nucleic acid molecule which encompasses a
nucleic acid molecule which is obtained by amplifying nucleic acid
molecules from a cDNA library or a genomic library using the
primers or primer pairs as indicated in Table XIII, application no.
45, column 7, and conferring an increase in the amount of the fine
chemical as indicated in table XII, column 6, e.g of myo-inositol
and/or anhydroglucose resp., in an organism or a part thereof; g)
nucleic acid molecule encoding a polypeptide which is isolated with
the aid of monoclonal antibodies against a polypeptide encoded by
one of the nucleic acid molecules of (a) to (f) and conferring an
increase in the amount of the fine chemical as indicated in table
XII, column 6, e.g of myo-inositol and/or anhydroglucose resp., in
an organism or a part thereof; h) nucleic acid molecule encoding a
polypeptide comprising a consensus sequence as indicated in Table
XIV, application no. 45, column 7, and conferring an increase in
the amount of the fine chemical as indicated in table XII, column
6, e.g of myo-inositol and/or anhydroglucose resp., in an organism
or a part thereof; and i) nucleic acid molecule which is obtainable
by screening a suitable nucleic acid library under stringent
hybridization conditions with a probe comprising one of the
sequences of the nucleic acid molecule of (a) to (k) or with a
fragment thereof having at least 15 nt, preferably 20 nt, 30 nt, 50
nt, 100 nt, 200 nt or 500 nt of the nucleic acid molecule
characterized in (a) to (k) and conferring an increase in the
amount of the fine chemical as indicated in table XII, column 6,
e.g of myo-inositol and/or anhydroglucose resp., in an organism or
a part thereof. whereby the nucleic acid molecule distinguishes
over the sequence as indicated in Table XI, application no. 45,
columns 5 or 7, by one or more nucleotides. 7. A nucleic acid
construct which confers the expression of the nucleic acid molecule
of claim 6, comprising one or more regulatory elements. 8. A vector
comprising the nucleic acid molecule as claimed in claim 6 or the
nucleic acid construct of claim 7. 9. The vector as claimed in
claim 8, wherein the nucleic acid molecule is in operable linkage
with regulatory sequences for the expression in a prokaryotic or
eukaryotic, or in a prokaryotic and eukaryotic, host. 10. A host
cell, which has been transformed stably or transiently with the
vector as claimed in claim 8 or 9 or the nucleic acid molecule as
claimed in claim 6 or the nucleic acid construct of claim 7 or
produced as described in claim any one of claims 2 to 5. 11. The
host cell of claim 10, which is a transgenic host cell. 12. The
host cell of claim 10 or 11, which is a plant cell, an animal cell,
a microorganism, or a yeast cell, a fungus cell, a prokaryotic
cell, an eukaryotic cell or an archaebacterium. 13. A process for
producing a polypeptide, wherein the polypeptide is expressed in a
host cell as claimed in any one of claims 10 to 12. 14. A
polypeptide produced by the process as claimed in claim 13 or
encoded by the nucleic acid molecule as claimed in claim 6 whereby
the polypeptide distinguishes over a sequence as indicated in Table
XII, application no. 45, columns 5 or 7, by one or more amino acids
15. An antibody, which binds specifically to the polypeptide as
claimed in claim 14. 16. A plant tissue, propagation material,
harvested material or a plant comprising the host cell as claimed
in claim 12 which is plant cell or an Agrobacterium. 17. A method
for screening for agonists and antagonists of the activity of a
polypeptide encoded by the nucleic acid molecule of claim 6
conferring an increase in the amount of myo-inositol and/or
anhydroglucose resp., in an organism or a part thereof comprising:
(a) contacting cells, tissues, plants or microorganisms which
express the a polypeptide encoded by the nucleic acid molecule of
claim 5 conferring an increase in the amount of myo-inositol and/or
anhydroglucose resp., in an organism or a part thereof with a
candidate compound or a sample comprising a plurality of compounds
under conditions which permit the expression the polypeptide; (b)
assaying the myo-inositol and/or anhydroglucose resp., level or the
polypeptide expression level in the cell, tissue, plant or
microorganism or the media the cell, tissue, plant or
microorganisms is cultured or maintained in; and (c) identifying a
agonist or antagonist by comparing the measured myo-inositol and/or
anhydroglucose resp., level or polypeptide expression level with a
standard myo-inositol and/or anhydroglucose resp., or polypeptide
expression level measured in the absence of said candidate compound
or a sample comprising said plurality of compounds, whereby an
increased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an agonist and
a decreased level over the standard indicates that the compound or
the sample comprising said plurality of compounds is an antagonist.
18. A process for the identification of a compound conferring
increased myo-inositol, and/or anhydroglucose resp., production in
a plant or microorganism, comprising the steps: (a) culturing a
plant cell or tissue or microorganism or maintaining a plant
expressing the polypeptide encoded by the nucleic acid molecule of
claim 6 conferring an increase in the amount of myo-inositol and/or
anhydroglucose resp., in an organism or a part thereof and a
readout system capable of interacting with the polypeptide under
suitable conditions which permit the interaction of the polypeptide
with dais readout system in the presence of a compound or a sample
comprising a plurality of compounds and capable of providing a
detectable signal in response to the binding of a compound to said
polypeptide under conditions which permit the expression of said
readout system and of the polypeptide encoded by the nucleic acid
molecule of claim 6 conferring an increase in the amount of
myo-inositol and/or anhydroglucose resp., in an organism or a part
thereof; (b) identifying if the compound is an effective agonist by
detecting the presence or absence or increase of a signal produced
by said readout system. 19. A method for the identification of a
gene product conferring an increase in myo-inositol and/or
anhydroglucose resp., production in a cell, comprising the
following steps: (a) contacting the nucleic acid molecules of a
sample, which can contain a candidate gene encoding a gene product
conferring an increase in myo-inositol and/or anhydroglucose resp.,
after expression with the nucleic acid molecule of claim 6; (b)
identifying the nucleic acid molecules, which hybridise under
relaxed stringent conditions with the nucleic acid molecule of
claim 6; (c) introducing the candidate nucleic acid molecules in
host cells appropriate for producing myo-inositol and/or
anhydroglucose resp.; (d) expressing the identified nucleic acid
molecules in the host cells; (e) assaying the myo-inositol and/or
anhydroglucose resp., level in the host cells; and (f) identifying
nucleic acid molecule and its gene product which expression confers
an increase in the myo-inositol and/or anhydroglucose resp., level
in the host cell in the host cell after expression compared to the
wild type. 20. A method for the identification of a gene product
conferring an increase in myo-inositol and/or anhydroglucose resp.,
production in a cell, comprising the following steps: (a)
identifying in a data bank nucleic acid molecules of an organism;
which can contain a candidate gene encoding a gene product
conferring an increase in the myo-inositol and/or anhydroglucose
resp., amount or level in an organism or a part thereof after
expression, and which are at least 20% homolog to the nucleic acid
molecule of claim 6; (b) introducing the candidate nucleic acid
molecules in host cells appropriate for producing myo-inositol
and/or anhydroglucose resp.; (c) expressing the identified nucleic
acid molecules in the host cells; (d) assaying the myo-inositol
and/or anhydroglucose resp., level in the host cells; and (e)
identifying nucleic acid molecule and its gene product which
expression confers an increase in the myo-inositol and/or
anhydroglucose resp., level in the host cell after expression
compared to the wild type. 21. A method for the production of an
agricultural composition comprising the steps of the method of any
one of claims 17 to 20 and formulating the compound identified in
any one of claims 17 to 20 in a form acceptable for an application
in agriculture. 22. A composition comprising the nucleic acid
molecule of claim 6, the polypeptide of claim 14, the nucleic acid
construct of claim 7, the vector of any one of claim 8 or 9, an
antagonist or agonist identified according to claim 17, the
compound of claim 18, the gene product of claim 19 or 20, the
antibody of claim 15, and optionally an agricultural acceptable
carrier. 23. Use of the nucleic acid molecule as claimed in claim 6
for the identification of a nucleic acid molecule conferring an
increase of myo-inositol and/or anhydroglucose resp., after
expression. 24. Use of the polypeptide of claim 14 or the nucleic
acid construct claim 7 or the gene product identified according to
the method of claim 19 or 20 for identifying compounds capable of
conferring a modulation of myo-inositol and/or anhydroglucose
resp., levels in an organism. 25. Cosmetic, pharmaceutical, food or
feed composition comprising the nucleic acid molecule of claim 6,
the polypeptide of claim 14, the nucleic acid construct of claim 7,
the vector of claim 8 or 9, the antagonist or agonist identified
according to claim 17, the antibody of claim 15, the plant or plant
tissue of claim 16, the harvested material of claim 16, the host
cell of claim 10 to 12 or the gene product identified according to
the method of claim 19 or 20. 26. Use of the nucleic acid molecule
of claim 6, the polypeptide of claim 14, the nucleic acid construct
of claim 7, the vector of claim 8 or 9, the antagonist or agonist
identified according to claim 17, the antibody of claim 15, the
plant or plant tissue of claim 16, the harvested material of claim
16, the host cell of claim 10 to 12 or the gene product identified
according to the method of claim 19 or 20 for the protection of a
plant against a myo-inositol and/or anhydroglucose synthesis
inhibiting herbicide.
TABLE-US-00149 Lengthy table referenced here
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TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140325709A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140325709A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140325709A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References