U.S. patent application number 11/274317 was filed with the patent office on 2006-08-10 for nucleotide sequences which code for the dep34 gene.
Invention is credited to Brigitte Bathe, Mike Farwick, Thomas Hermann, Klaus Huthmacher, Walter Pfefferle.
Application Number | 20060177912 11/274317 |
Document ID | / |
Family ID | 26006992 |
Filed Date | 2006-08-10 |
United States Patent
Application |
20060177912 |
Kind Code |
A1 |
Farwick; Mike ; et
al. |
August 10, 2006 |
Nucleotide sequences which code for the dep34 gene
Abstract
The invention relates to an isolated polynucleotide having a
polynucleotide sequence which codes for the dep34 gene, and a
host-vector system having a coryneform host bacterium in which the
dep34 gene is present in attenuated form and a vector which carries
at least the dep34 gene according to SEQ ID No 1, and the use of
polynucleotides which comprise the sequences according to the
invention as hybridization probes.
Inventors: |
Farwick; Mike; (Bielefeld,
DE) ; Huthmacher; Klaus; (Gelnhausen, DE) ;
Pfefferle; Walter; (Halle (Westf.), DE) ; Hermann;
Thomas; (Bielefeld, DE) ; Bathe; Brigitte;
(Salzkotten, DE) |
Correspondence
Address: |
SMITH, GAMBRELL & RUSSELL, LLP
1850 M STREET, N.W., SUITE 800
WASHINGTON
DC
20036
US
|
Family ID: |
26006992 |
Appl. No.: |
11/274317 |
Filed: |
November 16, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09946763 |
Sep 6, 2001 |
7029904 |
|
|
11274317 |
Nov 16, 2005 |
|
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Current U.S.
Class: |
435/106 ;
435/252.3; 435/471; 536/23.2 |
Current CPC
Class: |
C12P 13/08 20130101;
C07K 14/34 20130101 |
Class at
Publication: |
435/106 ;
435/471; 435/252.3; 536/023.2 |
International
Class: |
C12P 13/04 20060101
C12P013/04; C07H 21/04 20060101 C07H021/04; C12N 1/21 20060101
C12N001/21; C12N 15/74 20060101 C12N015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 9, 2000 |
DE |
100 44 708.2 |
Mar 15, 2001 |
DE |
101 12 429.5 |
Claims
1-25. (canceled)
26. A process for the fermentative preparation of L-amino acids in
Corynebacterium glutamicum bacteria, comprising: a) fermenting the
bacteria, in which at least the dep34 gene is eliminated in a
medium and for a time suitable for the formation of the L-amino
acids, wherein prior to said elimination said bacteria contain the
dep34 gene which comprises a nucleotide sequence which encodes a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
27. The method according to claim 26, further comprising: b)
isolating the L-amino acids.
28. The method as claimed in claim 26, wherein the L-amino acid
produced is L-lysine.
29. The method as claimed in claim 26, wherein the bacteria
comprise, at the same time, one or more endogenous Corynebacterium
glutamicum genes which are overexpressed, wherein the one or more
genes is/are selected from the group consisting of: the gene which
encodes dihydrodipicolinate synthase, the gene which encodes
glyceraldehyde 3-phosphate dehydrogenase, the gene which encodes
triose phosphate isomerase, the gene which encodes
3-phosphoglycerate kinase, the gene which encodes glucose
6-phosphate dehydrogenase, the gene which encodes pyruvate
carboxylase, the gene which encodes malate-quinone oxidoreductase,
the gene which encodes an aspartate kinase, the gene which encodes
a protein which exports lysine, the gene which encodes homoserine
dehydrogenase, the gene which encodes threonine dehydratase, the
gene which encodes acetohydroxy-acid synthase, the gene which
encodes dihydroxy-acid dehydratase, and the gene which encodes for
the Zwa1 protein.
30. The method as claimed in claim 26, wherein the bacteria
comprise, at the same time, one or more endogenous Corynebacterium
glutamicum genes which are eliminated, wherein said one or more
genes is/are selected from the group consisting of: the gene which
encodes phosphoenol pyruvate carboxykinase, the gene which encodes
glucose 6-phosphate isomerase, the gene which encodes pyruvate
oxidase, and the gene which encodes the Zwa2 protein.
31. The method as claimed in claim 26, wherein said polynucleotide
comprises the nucleotides 259 to 1905 of SEQ ID NO: 1.
32. The method as claimed in claim 26, wherein said polynucleotide
comprises the nucleotides sequence of SEQ ID NO: 1.
33. The method as claimed in claim 26, wherein said elimination is
achieved by one of more methods of mutagenesis selected from the
group consisting of deletion mutagenesis with deletion of two or
more codons in the dep34 gene; insertion mutagenesis due to
homologous mutagenesis recombination; and transition or
transversion mutagenesis with the incorporation of a non-sense
mutation in the dep34 gene.
Description
BACKGROUND OF THE INVENTION
[0001] The invention provides nucleotide sequences from coryneform
bacteria which code for the dep34 gene and a process for the
fermentative preparation of amino acids using bacteria in which the
dep34 gene is attenuated. All references cited herein are expressly
incorporated by reference. Incorporation by reference is also
designated by the term "I.B.R." following any citation.
[0002] L-Amino acids, in particular L-lysine, are used in human
medicine and in the pharmaceuticals industry, in the foodstuffs
industry and very particularly in animal nutrition.
[0003] It is known that amino acids are prepared by fermentation
from strains of coryneform bacteria, in particular Corynebacterium
glutamicum. Because of their great importance, work is constantly
being undertaken to improve the preparation processes. Improvements
to the process can relate to fermentation measures, such as, for
example, stirring and supply of oxygen, or the composition of the
nutrient media, such as, for example, the sugar concentration
during the fermentation, or the working up to the product form by,
for example, ion exchange chromatography, or the intrinsic output
properties of the microorganism itself.
[0004] Methods of mutagenesis, selection and mutant selection are
used to improve the output properties of these micro-organisms.
Strains which are resistant to antimetabolites or are auxotrophic
for metabolites of regulatory importance and which produce amino
acids are obtained in this manner.
[0005] Methods of the recombinant DNA technique have also been
employed for some years for improving the strain of Corynebacterium
strains which produce L-amino acid, by amplifying individual amino
acid biosynthesis genes and investigating the effect on the amino
acid production.
[0006] The invention provides new measures for improved
fermentative preparation of amino acids.
BRIEF SUMMARY OF THE INVENTION
[0007] Where L-amino acids or amino acids are mentioned in the
following, this means one or more amino acids, including their
salts, chosen from the group consisting of L-asparagine,
L-threonine, L-serine, L-glutamate, L-glycine, L-alanine,
L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine,
L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan
and L-arginine. L-Lysine is particularly preferred.
[0008] When L-lysine or lysine are mentioned in the following, not
only the bases but also the salts, such as e.g. lysine
monohydrochloride or lysine sulfate, are meant by this.
[0009] The invention provides an isolated polynucleotide from
coryneform bacteria, comprising a polynucleotide sequence which
codes for the dep34 gene, chosen from the group consisting of
[0010] a) polynucleotide which is identical to the extent of at
least 70% to a polynucleotide which codes for a polypeptide which
comprises the amino acid sequence of SEQ ID No. 2, [0011] b)
polynucleotide which codes for a polypeptide which comprises an
amino acid sequence which is identical to the extent of at least
70% to the amino acid sequence of SEQ ID No. 2, [0012] c)
polynucleotide which is complementary to the polynucleotides of a)
or b), and [0013] d) polynucleotide comprising at least 15
successive nucleotides of the polynucleotide sequence of a), b) or
c), the polypeptide preferably having the activity of the efflux
protein Dep34.
[0014] The invention also provides the above-mentioned
polynucleotide, this preferably being a DNA which is capable of
replication, comprising: [0015] (i) the nucleotide sequence, shown
in SEQ ID No.1, or [0016] (ii) at least one sequence which
corresponds to sequence (i) within the range of the degeneration of
the genetic code, or [0017] (iii) at least one sequence which
hybridizes with the sequences complementary to sequences (i) or
(ii), and optionally [0018] (iv) sense mutations of neutral
function in (i).
[0019] The invention also provides: [0020] a polynucleotide, in
particular DNA, which is capable of replication and comprises the
nucleotide sequence as shown in SEQ ID No.1; [0021] a
polynucleotide which codes for a polypeptide which comprises the
amino acid sequence as shown in SEQ ID No. 2; [0022] a vector
containing parts of the polynucleotide according to the invention,
but at least 15 successive nucleotides of the sequence claimed,
[0023] and coryneform bacteria in which the dep34 gene is
attenuated, in particular by an insertion or deletion.
[0024] The invention also provides polynucleotides, which
substantially comprise a polynucleotide sequence, which are
obtainable by screening by means of hybridization of a
corresponding gene library of a coryneform bacterium, which
comprises the complete gene or parts thereof, with a probe which
comprises the sequence of the polynucleotide according to the
invention according to SEQ ID No.1 or a fragment thereof, and
isolation of the polynucleotide sequence mentioned.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1: Map of the plasmid pCR2.1dep34int.
[0026] The abbreviations and designations used have the following
meaning. [0027] KMR: Kanamycin resistance gene [0028] KpnI:
Cleavage site of the restriction enzyme KpnI [0029] EcoRI: Cleavage
site of the restriction enzyme EcoRI [0030] PstI: Cleavage site of
the restriction enzyme PstI [0031] dep34int: Internal fragment of
the dep34 gene [0032] ColE1: Replication origin of the plasmid
ColE1
DETAILED DESCRIPTION OF THE INVENTION
[0033] Polynucleotides which comprise the sequences according to
the invention are suitable as hybridization probes for RNA, cDNA
and DNA, in order to isolate, in the full length, nucleic acids or
polynucleotides or genes which code for the efflux protein Dep34 or
to isolate those nucleic acids or polynucleotides or genes which
have a high similarity with the sequence of the dep34 gene. They
are also suitable for incorporation into so-called "arrays", "micro
arrays" or "DNA chips" in order to detect and determine the
corresponding polynucleotides.
[0034] Polynucleotides which comprise the sequences according to
the invention are furthermore suitable as primers with the aid of
which DNA of genes which code for the efflux protein Dep34 can be
prepared by the polymerase chain reaction (PCR).
[0035] Such oligonucleotides which serve as probes or primers
comprise at least 25, 26, 27, 28, 29 or 30, preferably at least 20,
21, 22, 23 or 24, very particularly preferably at least 15, 16, 17,
18 or 19 successive nucleotides. Oligonucleotides with a length of
at least 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, or at least 41,
42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides are also suitable.
Oligonucleotides with a length of at least 100, 150, 200, 250 or
300 nucleotides are optionally also suitable.
[0036] "Isolated" means separated out of its natural
environment.
[0037] "Polynucleotide" in general relates to polyribonucleotides
and polydeoxyribonucleotides, it being possible for these to be
non-modified RNA or DNA or modified RNA or DNA.
[0038] The polynucleotides according to the invention include a
polynucleotide according to SEQ ID No. 1 or a fragment prepared
therefrom and also those which are at least 70% to 80%, preferably
at least 81% to 85%, particularly preferably at least 86% to 90%,
and very particularly preferably at least 91%, 93%, 95%, 97% or 99%
identical to the polynucleotide according to SEQ ID No. 1 or a
fragment prepared therefrom.
[0039] "Polypeptides" are understood as meaning peptides or
proteins which comprise two or more amino acids bonded via peptide
bonds.
[0040] The polypeptides according to the invention include a
polypeptide according to SEQ ID No. 2, in particular those with the
biological activity of the efflux protein Dep34 and also those
which are at least 70% to 80%, preferably at least 81% to 85%,
particularly preferably at least 86% to 90% and very particularly
preferably at least 91%, 93%, 95%, 97% or 99% identical to the
polypeptide according to SEQ ID No. 2 and have the activity
mentioned.
[0041] The invention furthermore relates to a process for the
fermentative preparation of amino acids chosen from the group
consisting of L-asparagine, L-threonine, L-serine, L-glutamate,
L-glycine, L-alanine, L-cysteine, L-valine, L-methionine,
L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine,
L-lysine, L-tryptophan and L-arginine using coryneform bacteria
which in particular already produce amino acids and in which the
nucleotide sequences which code for the dep34 gene are attenuated,
in particular eliminated or expressed at a low level.
[0042] The term "attenuation" in this connection describes the
reduction or elimination of the intracellular activity of one or
more enzymes (proteins) in a microorganism which are coded by the
corresponding DNA, for example by using a weak promoter or using a
gene or allele which codes for a corresponding enzyme with a low
activity or inactivates the corresponding gene or enzyme (protein),
and optionally combining these measures.
[0043] By attenuation measures, the activity or concentration of
the corresponding protein is in general reduced to 0 to 75%, 0 to
50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration
of the wild-type protein or of the activity or concentration of the
protein in the starting microorganism.
[0044] The microorganisms provided by the present invention can
prepare amino acids from glucose, sucrose, lactose, fructose,
maltose, molasses, starch, cellulose or from glycerol and ethanol.
They can be representatives of coryneform bacteria, in particular
of the genus Corynebacterium. Of the genus Corynebacterium, there
may be mentioned in particular the species Corynebacterium
glutamicum, which is known among experts for its ability to produce
L-amino acids.
[0045] Suitable strains of the genus Corynebacterium, in particular
of the species Corynebacterium glutamicum (C. glutamicum), are in
particular the known wild-type strains [0046] Corynebacterium
glutamicum ATCC13032 [0047] Corynebacterium acetoglutamicum
ATCC15806 [0048] Corynebacterium acetoacidophilum ATCC13870 [0049]
Corynebacterium melassecola ATCC17965 [0050] Corynebacterium
thermoaminogenes FERM BP-1539 [0051] Brevibacterium flavum
ATCC14067 [0052] Brevibacterium lactofermentum ATCC13869 and [0053]
Brevibacterium divaricatum ATCC14020 and L-amino acid-producing
mutants or strains prepared therefrom.
[0054] The new dep34 gene from C. glutamicum which codes for the
efflux protein Dep34 has been isolated.
[0055] To isolate the dep34 gene or also other genes of C.
glutamicum, a gene library of this microorganism is first set up in
Escherichia coli (E. coli). The setting up of gene libraries is
described in generally known textbooks and handbooks. The textbook
by Winnacker: Gene und Klone, Eine Einfuhrung in die Gentechnologie
[Genes and Clones, An Introduction to Genetic Engineering] (Verlag
Chemie, Weinheim, Germany, 1990) I.B.R., or the handbook by
Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold
Spring Harbor Laboratory Press, 1989) I.B.R. may be mentioned as an
example. A well-known gene library is that of the E. coli K-12
strain W3110 set up in .lamda. vectors by Kohara et al. (Cell 50,
495 -508 (1987)). Bathe et al. (Molecular and General Genetics,
252:255-265, 1996) I.B.R. describe a gene library of C. glutamicum
ATCC13032, which was set up with the aid of the cosmid vector
SuperCos I (Wahl et al., 1987, Proceedings of the National Academy
of Sciences USA, 84:2160-2164) I.B.R. in the E. coli K-12 strain
NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575)
I.B.R.
[0056] Bormann et al. (Molecular Microbiology 6(3), 317-326))
(1992)) I.B.R. in turn describe a gene library of C. glutamicum
ATCC13032 using the cosmid pHC79 (Hohn and Collins, 1980, Gene 11,
291-298 I.B.R.).
[0057] To prepare a gene library of C. glutamicum in E. coli it is
also possible to use plasmids such as pBR322 (Bolivar, 1979, Life
Sciences, 25, 807-818 I.B.R.) or pUC9 (Vieira et al., 1982, Gene,
19:259-268 I.B.R.). Suitable hosts are, in particular, those E.
coli strains which are restriction- and recombination-defective,
such as, for example, the strain DH5.alpha.mcr, which has been
described by Grant et al. (Proceedings of the National Academy of
Sciences USA, 87 (1990) 4645-4649 I.B.R.). The long DNA fragments
cloned with the aid of cosmids or other .lamda. vectors can then in
turn be subcloned and subsequently sequenced in the usual vectors
which are suitable for DNA sequencing, such as is described e. g.
by Sanger et al. (Proceedings of the National Academy of Sciences
of the United States of America, 74:5463-5467, 1977) I.B.R.
[0058] The resulting DNA sequences can then be investigated with
known algorithms or sequence analysis programs, such as e.g. that
of Staden (Nucleic Acids Research 14, 217-23.2(1986)) I.B.R., that
of Marck (Nucleic Acids Research 16, 1829-1836 (1988).) I.B.R. or
the GCG program of Butler (Methods of Biochemical Analysis 39,
74-97 (1998)) I.B.R.
[0059] The new DNA sequence of C. glutamicum which codes for the
dep34 gene and which, as SEQ ID No. 1, is a constituent of the
present invention has been found. The amino acid sequence of the
corresponding protein has furthermore been derived from the present
DNA sequence by the methods described above. The resulting amino
acid sequence of the dep34 gene product is shown in SEQ ID No.
2.
[0060] Coding DNA sequences which result from SEQ ID No. 1 by the
degeneracy of the genetic code are also a constituent of the
invention. In the same way, DNA sequences which hybridize with SEQ
ID No. 1 or parts of SEQ ID No. 1 are a constituent of the
invention. Conservative amino acid exchanges, such as e.g. exchange
of glycine for alanine or of aspartic acid for glutamic acid in
proteins, are furthermore known among experts as "sense mutations"
which do not lead to a fundamental change in the activity of the
protein, i.e. are of neutral function. It is furthermore known that
changes on the N and/or C terminus of a protein cannot
substantially impair or can even stabilize the function thereof.
Information in this context can be found by the expert, inter alia,
in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987))
I.B.R., in. O'Regan et al. (Gene 77:237-251 (1989)) I.B.R., in
Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)) I.B.R., in
Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) I.B.R. and in
known textbooks of genetics and molecular biology. Amino acid
sequences which result in a corresponding manner from SEQ ID No. 2
are also a constituent of the invention.
[0061] In the same way, DNA sequences which hybridize with SEQ ID
No. 1 or parts of SEQ ID No. 1 are a constituent of the invention.
Finally, DNA sequences which are prepared by the polymerase chain
reaction (PCR) using primers which result from SEQ ID No. 1 are a
constituent of the invention. Such oligonucleotides typically have
a length of at least 15 nucleotides.
[0062] Instructions for identifying DNA sequences by means of
hybridization can be found by the expert, inter alia, in the
handbook "The DIG System Users Guide for Filter Hybridization" from
Boehringer Mannheim GmbH (Mannheim, Germany, 1993) I.B.R. and in
Liebl et al. (International Journal of Systematic Bacteriology 41:
255-260 (1991)) I.B.R. The hybridization takes place under
stringent conditions, that is to say only hybrids in which the
probe and target sequence, i. e. the polynucleotides treated with
the probe, are at least 70% identical are formed. It is known that
the stringency of the hybridization, including the washing steps,
is influenced or determined by varying the buffer composition, the
temperature and the salt concentration. The hybridization reaction
is preferably carried out under a relatively low stringency
compared with the washing steps (Hybaid Hybridisation Guide, Hybaid
Limited, Teddington, UK, 1996 I.B.R.).
[0063] A 5.times.SSC buffer at a temperature of approx. 50.degree.
C.-68.degree. C., for example, can be employed for the
hybridization reaction. Probes can also hybridize here with
polynucleotides which are less than 70% identical to the sequence
of the probe. Such hybrids are less stable and are removed by
washing under stringent conditions. This can be achieved, for
example, by lowering the salt concentration to 2.times.SSC and
optionally subsequently 0.5.times.SSC (The DIG System User's Guide
for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany,
1995 I.B.R.) a temperature of approx. 50.degree. C.-68.degree. C.
being established. It is optionally possible to lower the salt
concentration to 0.1.times.SSC. Polynucleotide fragments which are,
for example, at least 70% or at least 80% or at least 90% to 95%
identical to the sequence of the probe employed can be isolated by
increasing the hybridization temperature stepwise from 50.degree.
C. to 68.degree. C. in steps of approx. 1-2.degree. C. Further
instructions on hybridization are obtainable on the market in the
form of so-called kits (e.g. DIG Easy Hyb from Roche Diagnostics
GmbH, Mannheim, Germany, Catalogue No. 1603558).
[0064] Instructions for amplification of DNA sequences with the aid
of the polymerase chain reaction (PCR) can be found by the expert,
inter alia, in the handbook by Gait: Oligonucleotide Synthesis: A
Practical Approach (IRL Press, Oxford, UK, 1984 I.B.R.) and in
Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg,
Germany, 1994 I.B.R.).
[0065] It has been found that coryneform bacteria produce amino
acids in an improved manner after attenuation of the dep34
gene.
[0066] To achieve an attenuation, either the expression of the
dep34 gene or the catalytic properties of the enzyme protein can be
reduced or eliminated. The two measures can optionally be
combined.
[0067] The reduction in gene expression can take place by suitable
culturing or by genetic modification (mutation) of the signal
structures of gene expression. Signal structures of gene expression
are, for example, repressor genes, activator genes, operators,
promoters, attenuators, ribosome binding sites, the start codon and
terminators. The expert can find information on this e.g.. in the
patent application WO 96/15246 I.B.R., in Boyd and Murphy (Journal
of Bacteriology 170: 5949 (1988)) I.B.R., in Voskuil and Chambliss
(Nucleic Acids Research 26: 3548 (1998) I.B.R., in Jensen and
Hammer (Biotechnology and Bioengineering 58: 191 (1998)) I.B.R., in
Patek et al. (Microbiology 142: 1297 (1996)) I.B.R., Vasicova et
al. (Journal of Bacteriology 181: 6188 (1999)) I.B.R. and in known
textbooks of genetics and molecular biology, such as e.g. the
textbook by Knippers ("Molekulare Genetik [Molecular Genetics]",
6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) I.B.R.
or that by Winnacker ("Gene und Klone [Genes and Clones]", VCH
Verlagsgesellschaft, Weinheim, Germany, 1990) I.B.R.
[0068] Mutations which lead to a change or reduction in the
catalytic properties of enzyme proteins are known from the prior
art; examples which may be mentioned are the works by Qiu and
Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997))
I.B.R., Sugimoto et al. (Bioscience Biotechnology and Biochemistry
61: 1760-1762 (1997)) I.B.R. and Mockel ("Die Threonindehydratase
aus Corynebacterium glutamicum: Aufhebung der allosterischen
Regulation und Struktur des Enzyms [Threonine dehydratase from
Corynebacterium glutamicum: Canceling the allosteric regulation and
structure of the enzyme]", Reports from the Julich Research Center,
Jul-2906, ISSN09442952, Julich, Germany, 1994) I.B.R. Summarizing
descriptions can be found in known textbooks of genetics and
molecular biology, such as e.g. that by Hagemann ("Allgemeine
Genetik [General Genetics]", Gustav Fischer Verlag, Stuttgart,
1986) I.B.R.
[0069] Possible mutations are transitions, transversions,
insertions and deletions. Depending on the effect of the amino acid
exchange on the enzyme activity, "missense mutations" or "nonsense
mutations" are referred to. Insertions or deletions of at least one
base pair (bp) in a gene lead to frame shift mutations, as a
consequence of which incorrect amino acids are incorporated or
translation is interrupted prematurely. Deletions of several codons
typically lead to a complete loss of the enzyme activity.
Instructions on generation of such mutations are prior art and can
be found in known textbooks of genetics and molecular biology, such
as e.g. the textbook by Knippers ("Molekulare Genetik [Molecular
Genetics]", 6th edition, Georg Thieme Verlag, Stuttgart, Germany,
1995) I.B.R., that by Winnacker ("Gene und Klone [Genes and
Clones]", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) I.B.R.
or that by Hagemann ("Allgemeine Genetik [General Genetics]",
Gustav Fischer Verlag, Stuttgart, 1986) I.B.R.
[0070] A common method of mutating genes of C. glutamicum is the
method of "gene disruption" and "gene replacement" described by
Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991)) I.B.R.
[0071] In the method of gene disruption a central part of the
coding region of the gene of interest is cloned in a plasmid vector
which can replicate in a host (typically E. coli), but not in C.
glutamicum. Possible vectors are, for example, pSUP301 (Simon et
al., Bio/Technology 1, 784-791 (1983)) I.B.R., pK18mob or pK19mob
(Schafer et al., Gene 145, 69-73 (1994) I.B.R.), pK18mobsacB or
pK19mobsacB (Jager et al., Journal of Bacteriology 174: 5462-65
(1992) I.B.R.), pGEM-T (Promega corporation., Madison, Wis., USA),
pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry
269:32678-84 I.B.R.; U.S. Pat. No. 5,487,993 I.B.R.), pCR.RTM.Blunt
(Invitrogen, Groningen, Holland; Bernard et al., Journal of
Molecular Biology, 234: 534-541 (1993) I.B.R.) or pEM1 (Schrumpf et
al, 1991, Journal of Bacteriology 173:4510-4516 I.B.R.). The
plasmid vector which contains the central part of the coding region
of the gene is then transferred into the desired strain of C.
glutamicum by conjugation or transformation. The method of
conjugation is described, for example, by Schafer et al. (Applied
and Environmental Microbiology 60, 756-759 (1994)) I.B.R. Methods
for transformation are described, for example, by Thierbach et al.
(Applied Microbiology and Biotechnology 29, 356-362 (1988)) I.B.R.,
Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) I.B.R. and
Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994))
I.B.R. After homologous recombination by means of a "cross-over"
event, the coding region of the gene in question is interrupted by
the vector sequence and two incomplete alleles are obtained, one
lacking the 3' end and one lacking the 5' end. This method has been
used, for example, by Fitzpatrick et al. (Applied Microbiology and
Biotechnology 42, 575-580 (1994)) I.B.R. to eliminate the recA gene
of C. glutamicum.
[0072] In the method of "gene replacement", a mutation, such as
e.g. a deletion, insertion or base exchange, is established in
vitro in the gene of interest. The allele prepared is in turn
cloned in a vector which is not replicative for C. glutamicum and
this is then transferred into the desired host of C. glutamicum by
transformation or conjugation. After homologous recombination by
means of a first "cross-over" event which effects integration and a
suitable second "cross-over" event which effects excision in the
target gene or in the target sequence, the incorporation of the
mutation or of the allele is achieved. This method was used, for
example, by Peters-Wendisch et al. (Microbiology 144, 915-927
(1998)) I.B.R. to eliminate the pyc gene of C. glutamicum by a
deletion.
[0073] A deletion, insertion or a base exchange can be incorporated
into the dep34 gene in this manner.
[0074] In addition, it may be advantageous for the production of
L-amino acids to enhance, in particular over-express, one or more
enzymes of the particular biosynthesis pathway, of glycolysis, of
anaplerosis, of the citric acid cycle, of the pentose phosphate
cycle, of amino acid export and optionally regulatory proteins, in
addition to the attenuation of the dep34 gene.
[0075] The term "enhancement" in this connection describes the
increase in the intracellular activity of one or more enzymes
(proteins) in a microorganism which are coded by the corresponding
DNA, for example by increasing the number of copies of the gene or
genes, using a potent promoter or using a gene or allele which
codes for a corresponding enzyme (protein) having a high activity,
and optionally combining these measures.
[0076] By enhancement measures, in particular over-expression, the
activity or concentration of the corresponding protein is in
general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%,
300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on
that of the wild-type protein or the activity or concentration of
the protein in the starting microorganism.
[0077] Thus, for the preparation of L-amino acids, in addition to
attenuation of the dep34 gene, at the same time one or more of the
genes chosen from the group consisting of [0078] the dapA gene
which codes for dihydrodipicolinate. synthase (EP-B 0 197 335
I.B.R.), [0079] the gap gene which codes for glyceraldehyde
3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology
174:6076-6086 I.B.R.), [0080] the tpi gene which codes for triose
phosphate isomerase (Eikmanns (1992), Journal of Bacteriology
174:6076-6086 I.B.R.), [0081] the pgk gene which codes for
3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology
174:6076-6086 I.B.R.), [0082] the zwf gene which codes for glucose
6-phosphate dehydrogenase (JP-A-09224661 I.B.R.), [0083] the pyc
gene which codes for pyruvate carboxylase (DE-A-198 31 609 I.B.R.),
[0084] the mqo gene which codes for malate-quinone oxidoreductase
(Molenaar et al., European Journal of Biochemistry 254, 395-403
(1998) I.B.R.), [0085] the lysC gene which codes for a feed-back
resistant aspartate kinase (Accession No. P26512; EP-B-0387527
I.B.R.; EP-A-0699759 I.B.R.; WO 00/63388 I.B.R.), [0086] the lysE
gene which codes for lysine export (DE-A-195 48 222 I.B.R.), [0087]
the hom gene which codes for homoserine dehydrogenase (EP-A 0131171
I.B.R.), [0088] the ilvA gene which codes for threonine dehydratase
(Mockel et al., Journal of Bacteriology (1992) 8065-8072) I.B.R.)
or the ilvA(Fbr) allele which codes for a "feed back resistant"
threonine dehydratase (Mockel et al., (1994) Molecular Microbiology
13: 833-842 I.B.R.), [0089] the ilvBN gene which codes for
acetohydroxy-acid synthase (EP-B 0356739 I.B.R.), [0090] the ilvD
gene which codes for dihydroxy-acid dehydratase (Sahm and Eggeling
(1999) Applied and Environmental Microbiology 65: 1973-1979
I.B.R.), [0091] the zwa1 gene which codes for the Zwa1 protein (DE:
19959328.0 I.B.R., DSM 13115) can be enhanced, in particular
over-expressed.
[0092] It may furthermore be advantageous for the production of
amino acids, in addition to attenuation of the dep34 gene, at the
same time for one or more of the genes chosen from the group
consisting of [0093] the pck gene which codes for phosphoenol
pyruvate carboxykinase (DE 199 50 409.1 I.B.R., DSM 13047), [0094]
the pgi gene which codes for glucose 6-phosphate isomerase (U.S.
Ser. No. 09/396,478 I.B.R., DSM 12969), [0095] the poxB gene which
codes for pyruvate oxidase (DE:1995 1975.7 I.B.R., DSM 13114),
[0096] the zwa2 gene which codes for the Zwa2 protein (DE:
19959327.2 I.B.R., DSM 13113) to be attenuated, in particular for
the expression thereof to be reduced.
[0097] In addition to the attenuation of the dep34 gene it may
furthermore be advantageous for the production of amino acids to
eliminate undesirable side reactions (Nakayama: "Breeding of Amino
Acid Producing Microorganisms", in: Overproduction of Microbial
Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London,
UK, 1982 I.B.R.).
[0098] The invention also provides the microorganisms prepared
according to the invention, and these can be cultured continuously
or discontinuously in the batch process (batch culture) or in the
fed batch (feed process) or repeated fed batch process (repetitive
feed process) for the purpose of production of L-amino acids. A
summary of known culture methods is described in the textbook by
Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik
[Bioprocess Technology 1. Introduction to Bioprocess Technology
(Gustav Fischer Verlag, Stuttgart, 1991) I.B.R.) or in the textbook
by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors
and Peripheral Equipment] (Vieweg Verlag, Braunschweig/Wiesbaden,
1994) I.B.R.).
[0099] The culture medium to be used must meet the requirements of
the particular strains in a suitable manner. Descriptions of
culture media for various microorganisms are contained in the
handbook "Manual of Methods for General Bacteriology" of the
American Society for Bacteriology (Washington D.C., USA, 1981)
I.B.R..
[0100] Sugars and carbohydrates, such as e.g. glucose, sucrose,
lactose, fructose, maltose, molasses, starch and cellulose, oils
and fats, such as, for example, soya oil, sunflower oil, groundnut
oil and coconut fat, fatty acids, such as, for example, palmitic
acid, stearic acid and linoleic acid, alcohols, such as, for
example, glycerol and ethanol, and organic acids, such as, for
example, acetic acid, can be used as the source of carbon. These
substances can be used individually or as a mixture.
[0101] Organic nitrogen-containing compounds, such as peptones,
yeast extract, meat extract, malt extract, corn steep liquor, soya
bean flour and urea, or inorganic compounds, such as ammonium
sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate
and ammonium nitrate, can be used as the source of nitrogen. The
sources of nitrogen can be used individually or as a mixture.
[0102] Phosphoric acid, potassium dihydrogen phosphate or
dipotassium hydrogen phosphate or the corresponding
sodium-containing salts can be used as the source of phosphorus.
The culture medium must furthermore comprise salts of metals, such
as, for example, magnesium sulfate or iron sulfate, which are
necessary for growth. Finally, essential growth substances, such as
amino acids and vitamins, can be employed in addition to the
above-mentioned substances. Suitable precursors can moreover be
added to the culture medium. The starting substances mentioned can
be added to the culture in the form of a single batch, or can be
fed in during the culture in a suitable manner.
[0103] Basic compounds, such as sodium hydroxide, potassium
hydroxide, ammonia or aqueous ammonia, or acid compounds, such as
phosphoric acid or sulfuric acid, can be employed in a suitable
manner to control the pH of the culture. Antifoams, such as, for
example, fatty acid polyglycol esters, can be employed to control
the development of foam. Suitable substances having a selective
action, such as, for example, antibiotics, can be added to the
medium to maintain the stability of plasmids. To maintain aerobic
conditions, oxygen or oxygen-containing gas mixtures, such as, for
example, air, are introduced into the culture. The temperature of
the culture is usually 20.degree. C. to 45.degree. C., and
preferably 25.degree. C. to 40.degree. C. Culturing is continued
until a maximum of the desired product has formed. This target is
usually reached within 10 hours to 160 hours.
[0104] Methods for the determination of L-amino acids are known
from the prior art. The analysis can thus be carried out, for
example, as described by Spackman et al. (Analytical Chemistry, 30,
(1958), 1190) I.B.R. by anion exchange chromatography with
subsequent ninhydrin derivation, or it can be carried out by
reversed phase HPLC, for example as described by Lindroth et al.
(Analytical Chemistry (1979) 51: 1167-1174) I.B.R.
[0105] The process according to the invention is used for
fermentative preparation of amino acids.
[0106] The following microorganism was deposited on May 3, 2001 as
a pure culture at the Deutsche Sammlung fur Mikroorganismen und
Zellkulturen (DSMZ=German Collection of Microorganisms and Cell
Cultures, Braunschweig, Germany) in accordance with the Budapest
Treaty: [0107] Escherichia coli top10/pCR2.1dep34int as DSM
14144.
[0108] The present invention is explained in more detail in the
following with the aid of embodiment examples.
[0109] The isolation of plasmid DNA from Escherichia coli and all
techniques of restriction, Klenow and alkaline phosphatase
treatment were carried out by the method of Sambrook et al.
(Molecular Cloning. A Laboratory Manual, 1989, Cold Spring Harbour
Laboratory Press, Cold Spring Harbor, N.Y., USA) I.B.R. Methods for
transformation of Escherichia coli are also described in this
handbook.
[0110] The composition of the usual nutrient media, such as LB or
TY medium, can also be found in the handbook by Sambrook et al.
EXAMPLE 1
Preparation of a Genomic Cosmid Gene Library from C. glutamicum
ATCC 13032
[0111] Chromosomal DNA from C. glutamicum ATCC 13032 was isolated
as described by Tauch et al. (1995, Plasmid 33:168-179) I.B.R. and
partly cleaved with the restriction enzyme Sau3AI (Amersham
Pharmacia, Freiburg, Germany, Product Description Sau3AI, Code no.
27-0913-02 I.B.R.). The DNA fragments were dephosphorylated with
shrimp alkaline phosphatase (Roche Molecular Biochemicals,
Mannheim, Germany, Product Description SAP, Code no. 1758250
I.B.R.). The DNA of the cosmid vector SuperCos1 (Wahl et al.
(1987), Proceedings of the National Academy of Sciences, USA
84:2160-2164 I.B.R.), obtained from Stratagene (La Jolla, USA,
Product Description SuperCos1 Cosmid Vector Kit, Code no. 251301
I.B.R.) was cleaved with the restriction enzyme XbaI (Amersham
Pharmacia, Freiburg, Germany, Product Description XbaI, Code no.
27-0948-02 I.B.R.) and likewise dephosphorylated with shrimp
alkaline phosphatase.
[0112] The cosmid DNA was then cleaved with the restriction enzyme
BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description
BamHI, Code no. 27-0868-04 I.B.R.). The cosmid DNA treated in this
manner was mixed with the treated ATCC13032 DNA and the batch was
treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany,
Product Description T4-DNA-Ligase, Code no. 27-0870-04 I.B.R.). The
ligation mixture was then packed in phages with the aid of Gigapack
II XL Packing Extract (Stratagene, La Jolla, USA, Product
Description Gigapack II XL Packing Extract, Code no. 200217
I.B.R.).
[0113] For infection of the E. coli strain NM554 (Raleigh et al.
1988, Nucleic Acid Res. 16:1563-1575 I.B.R.) the cells were taken
up in 10 mM MgSO.sub.4 and mixed with an aliquot of the phage
suspension. The infection and titering of the cosmid library were
carried out as described by Sambrook et al. (1989, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor I.B.R.), the cells
being plated but on LB agar (Lennox, 1955, Virology, 1:190
I.B.R.)+100 .mu.g/ml ampicillin. After incubation overnight at
37.degree. C., recombinant individual clones were selected.
EXAMPLE 2
Isolation and Sequencing of the dep34 Gene
[0114] The cosmid DNA of an individual colony was isolated with the
Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden,
Germany) in accordance with the manufacturer's instructions and
partly cleaved with the restriction enzyme Sau3AI (Amersham
Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product
No. 27-0913-02 I.B.R.). The DNA fragments were dephosphorylated
with shrimp alkaline phosphatase (Roche Molecular Biochemicals,
Mannheim, Germany, Product Description SAP, Product No. 1758250
I.B.R.). After separation by gel electrophoresis, the cosmid
fragments in the size range of 1500 to 2000 bp were isolated with
the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden,
Germany).
[0115] The DNA of the sequencing vector pZero-1, obtained from
Invitrogen (Groningen, The Netherlands, Product Description Zero
Background. Cloning Kit, Product No. K2500-01 I.B.R.) was cleaved
with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg,
Germany, Product Description BamHI, Product No. 27-0868-04 I.B.R.).
The ligation of the cosmid fragments in the sequencing vector
pZero-1 was carried out as described by Sambrook et al. (1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor) I.B.R.,
the DNA mixture being incubated overnight with T4 ligase (Pharmacia
Biotech, Freiburg, Germany). This ligation mixture was then
electroporated (Tauch et al. 1994, FEMS Microbiol. Letters,
123:343-7 I.B.R.) into the E. coli strain DH5.alpha.mcr (Grant,
1990, Proceedings of the National Academy of Sciences, U.S.A.,
87:4645-4649 I.B.R.). Letters, 123:343-7) and plated out on LB agar
(Lennox, 1955, Virology, 1:190 I.B.R.) with 50 .mu.g/ml zeocin.
[0116] The plasmid preparation of the recombinant clones was
carried out with the Biorobot 9600 (Product No. 900200, Qiagen,
Hilden, Germany). The sequencing was carried out by the dideoxy
chain termination method of Sanger et al. (1977, Proceedings of the
National Academies of Sciences, U.S.A., 74:5463-5467 I.B.R.) with
modifications according to Zimmermann et al. (1990, Nucleic Acids
Research, 18:1067 I.B.R.). The "RR dRhodamin Terminator Cycle
Sequencing Kit" from PE Applied Biosystems (Product No. 403044,
Weiterstadt, Germany) was used. The separation by gel
electrophoresis and analysis of the sequencing reaction were
carried out in a "Rotiphoresis NF Acrylamide/Bisacrylamide" Gel
(29:1) (Product No. A124.1, Roth, Karlsruhe, Germany) with the "ABI
Prism 377" sequencer from PE Applied Biosystems (Weiterstadt,
Germany).
[0117] The raw sequence data obtained were then processed using the
Staden program package (1986, Nucleic Acids Research, 14:217-231
I.B.R.) version 97-0. The individual sequences of the pZero1
derivatives were assembled to a continuous contig. The
computer-assisted coding region analyses were prepared with the
XNIP program (Staden, 1986, Nucleic Acids Research, 14:217-231
I.B.R.). Further analyses were carried out with the "BLAST search
program" (Altschul et al., 1997, Nucleic Acids Research,
25:3389-3402 I.B.R.) against the non-redundant databank of the
"National Center for Biotechnology Information" (NCBI, Bethesda,
Md., USA).
[0118] The resulting nucleotide sequence is shown in SEQ ID No. 1.
Analysis of the nucleotide sequence showed an open reading frame of
1650 bp, which was called the dep34 gene. The dep34 gene codes for
a polypeptide of 549 amino acids.
EXAMPLE 3
Preparation of an Integration Vector for Integration Mutagenesis of
the dep34 Gene
[0119] From the strain ATCC 13032, chromosomal DNA was isolated by
the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994))
I.B.R. On the basis of the sequence of the dep34 gene known for C.
glutamicum from example 2, the following oligonucleotides were
chosen for the polymerase chain reaction (see SEQ ID No. 3 and SEQ
ID No. 4): TABLE-US-00001 dep34-int1: 5' CTG TGC TGC TGA AAC TTC C
3' SEQ ID NO:3 dep34-int2: 5' AGT CCA ATG AGA GCC AAG C 3' SEQ ID
NO:4
[0120] The primers shown were synthesized by MWG Biotech
(Ebersberg, Germany) and the PCR reaction was carried out by the
standard PCR method of Innis et al. (PCR protocols. A guide to
methods and applications, 1990, Academic Press I.B.R.) with the
Taq-polymerase from Boehringer Mannheim (Germany, Product
Description Taq DNA polymerase, Product No. 1 146 165). With the
aid of the polymerase chain reaction, the primers allow
amplification of an internal fragment of the dep34 gene 541 bp in
size. The product amplified in this way was tested
electrophoretically in a 0.8% agarose gel.
[0121] The amplified DNA fragment was ligated with the TOPO TA
Cloning Kit from Invitrogen Corporation (Carlsbad, Calif., USA;
Catalogue Number K4500-01) in the vector pCR2.1-TOPO (Mead at al.
(1991) Bio/Technology 9:657-663 I.B.R.).
[0122] The E. coli strain TOP10 was then electroporated with the
ligation batch (Hanahan, In: DNA cloning. A practical approach.
Vol. I. IRL-Press, Oxford, Washington D.C., USA, 1985 I.B.R.).
Selection of plasmid-carrying cells was carried out by plating out
the transformation batch on LB Agar (Sambrook et al., Molecular
cloning: a laboratory manual. 2.sup.nd Ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989 I.B.R.), which had
been supplemented with 50 mg/l kanamycin. Plasmid DNA was isolated
from a transformant with the aid of the QIAprep Spin Miniprep Kit
from Qiagen and checked by restriction with the restriction enzyme
EcoRI and subsequent agarose gel electrophoresis (0.8%). The
plasmid was called pCR2.1dep34int and is shown in FIG. 1.
EXAMPLE 4
Integration Mutagenesis of the dep34 Gene in the Strain DSM
5715
[0123] The vector pCR2.1dep34int mentioned in example 3 was
electroporated by the electroporation method of Tauch et al. (FEMS
Microbiological Letters, 123:343-347 (1994) I.B.R.) in
Corynebacterium glutamicum DSM 5715. The strain DSM 5715 is an
AEC-resistant lysine producer. The vector pCR2.1dep34int cannot
replicate independently in DSM5715 and is retained in the cell only
if it has integrated into the chromosome of DSM 5715. Selection of
clones with pCR2.1dep34int integrated into the chromosome was
carried out by plating out the electroporation batch on LB agar
(Sambrook et al., Molecular cloning: a laboratory manual. 2.sup.nd
Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
I.B.R.), which had been supplemented with 15 mg/l kanamycin.
[0124] For detection of the integration, the dep34int fragment was
labeled with the Dig hybridization kit from Boehringer by the
method of "The DIG System Users Guide for Filter Hybridization" of
Boehringer Mannheim GmbH (Mannheim, Germany, 1993 I.B.R.).
Chromosomal DNA of a potential integrant was isolated by the method
of Eikmanns et al. (Microbiology 140: 1817-1828 (1994) I.B.R.) and
in each case cleaved with the restriction enzymes KpnI, EcoRI and
PstI. The fragments formed were separated by means of agarose gel
electrophoresis and hybridized at 68.degree. C. with the Dig
hybridization kit from Boehringer. The plasmid pCR2.1dep34int
mentioned in example 3 had been inserted into the chromosome of
DSM5715 within the chromosomal dep34 gene. The strain was called
DSM5715::pCR2.1dep34int.
EXAMPLE 5
Preparation of Lysine
[0125] The C. glutamicum strain DSM5715::pCR2.1dep34int obtained in
example 4 was cultured in a nutrient medium suitable for the
production of lysine and the lysine content in the culture
supernatant was determined.
[0126] For this, the strain was first incubated on an agar plate
with the corresponding antibiotic (brain-heart agar with kanamycin
(25 mg/l)) for 24 hours at 33.degree. C. Starting from this agar
plate culture, a preculture was seeded (10 ml medium in a 100 ml
conical flask). The complete medium CgIII was used as the medium
for the preculture. TABLE-US-00002 Medium Cg III NaCl 2.5 g/l
Bacto-Peptone 10 g/l Bacto-Yeast extract 10 g/l Glucose (autoclaved
separately) 2% (w/v) The pH was brought to pH 7.4
[0127] Kanamycin (25 mg/1) was added to this. The preculture was
incubated for 16 hours at 33.degree. C. at 240 rpm on a shaking
machine. A main culture was seeded from this preculture such that
the initial OD (660 nm) of the main culture was 0.1 OD. Medium MM
was used for the main culture. TABLE-US-00003 Medium MM CSL (corn
steep liquor) 5 g/l MOPS (morpholinopropanesulfonic 20 g/l acid)
Glucose (autoclaved separately) 50 g/l Salts:
(NH.sub.4).sub.2SO.sub.4 25 g/l KH.sub.2PO.sub.4 0.1 g/l MgSO.sub.4
* 7H.sub.2O 1.0 g/l CaCl.sub.2 * 2H.sub.2O 10 mg/l FeSO.sub.4 *
7H.sub.2O 10 mg/l MnSO.sub.4 * H.sub.2O 5.0 mg/l Biotin
(sterile-filtered) 0.3 mg/l Thiamine * HCl (sterile-filtered) 0.2
mg/l Leucine (sterile-filtered) 0.1 g/l CaCO.sub.3 25 g/l
The CSL, MOPS and the salt solution are brought to pH 7 with
aqueous ammonia and autoclaved. The sterile substrate and vitamin
solutions are then added, and the CaCO.sub.3 autoclaved in the dry
state is added.
[0128] Culturing is carried out in a 10 ml volume in a 100 ml
conical flask with baffles. Kanamycin (25 mg/l) was added.
Culturing was carried out at 33.degree. C. and 80% atmospheric
humidity.
[0129] After 72 hours, the OD was determined at a measurement
wavelength of 660 nm with a Biomek 1000 (Beckmann Instruments GmbH,
Munich). The amount of lysine formed was determined with an amino
acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion
exchange chromatography and post-column derivation with ninhydrin
detection.
[0130] The result of the experiment is shown in Table 1.
TABLE-US-00004 TABLE 1 OD Lysine HCl Strain (660 nm) g/l DSM5715
8.7 12.64 DSM5715::pCR2.1dep34int 9.1 14.14
[0131] This application claims priority to German Priority Document
Application No. 100 44 708.2, filed on Sep. 9, 2000 and to German
Priority Document Application No. 101 12 429.5, filed on Mar. 15,
2001. Both German Priority Documents are hereby incorporated by
reference in their entirety.
Sequence CWU 1
1
4 1 2120 DNA Corynebacterium glutamicum CDS (259)..(1905) 1
acatttcgcc aggttccacc aagcacgcga agggctagaa cacctaattg ttgagtactt
60 cgaaaaatgg ccaggctccc aacatctaga tgagcctgca gatcgagaag
caatcgccat 120 agttggcctg ctgatctcgg tcatgcttca aggttctcgt
gaatggcacg acatgccaca 180 aggcacgcaa gctgatttcc aagcctgctg
tcgcaaagca attaaaaata cttttcttct 240 tagaggtgga ttttcaga atg aca
tca cag gtc aag ccg gac gac gaa cgt 291 Met Thr Ser Gln Val Lys Pro
Asp Asp Glu Arg 1 5 10 ccg gta aca aca att tca aaa agt ggt gca cct
tcg gcc cac acc tca 339 Pro Val Thr Thr Ile Ser Lys Ser Gly Ala Pro
Ser Ala His Thr Ser 15 20 25 gca cca tat ggt gca gca gca act gaa
gaa gct gtc gag gaa aaa acc 387 Ala Pro Tyr Gly Ala Ala Ala Thr Glu
Glu Ala Val Glu Glu Lys Thr 30 35 40 aaa ggt cgc gtt gga ttt atc
atc gca gcc ctc atg ttg gcg atg ctt 435 Lys Gly Arg Val Gly Phe Ile
Ile Ala Ala Leu Met Leu Ala Met Leu 45 50 55 ctt agc tcc ttg ggt
cag acc att ttc ggt tct gcc ctg cca acg att 483 Leu Ser Ser Leu Gly
Gln Thr Ile Phe Gly Ser Ala Leu Pro Thr Ile 60 65 70 75 gtt ggt gag
ctt ggc ggc gtt aac cac atg acc tgg gtg att acc gcc 531 Val Gly Glu
Leu Gly Gly Val Asn His Met Thr Trp Val Ile Thr Ala 80 85 90 ttc
ctc ttg ggc cag acc att tca ttg cct att ttc ggc aag ttg ggt 579 Phe
Leu Leu Gly Gln Thr Ile Ser Leu Pro Ile Phe Gly Lys Leu Gly 95 100
105 gac cag ttt ggt cgc aaa tac ctc ttc atg ttt gcc atc gca ctg ttc
627 Asp Gln Phe Gly Arg Lys Tyr Leu Phe Met Phe Ala Ile Ala Leu Phe
110 115 120 gtg gtg ggt tcc atc atc ggt gct ttg gct cag aac atg acc
acc ttg 675 Val Val Gly Ser Ile Ile Gly Ala Leu Ala Gln Asn Met Thr
Thr Leu 125 130 135 att gtg gct cgt gca ctg cag ggt atc gcc ggt ggt
ggc ttg atg att 723 Ile Val Ala Arg Ala Leu Gln Gly Ile Ala Gly Gly
Gly Leu Met Ile 140 145 150 155 ctt tct cag gca att acc gct gat gtc
acc acc gcc cgt gag cgt gca 771 Leu Ser Gln Ala Ile Thr Ala Asp Val
Thr Thr Ala Arg Glu Arg Ala 160 165 170 aag tac atg ggc atc atg ggt
tcc gtt ttc gga ctg tcc tcc atc ctt 819 Lys Tyr Met Gly Ile Met Gly
Ser Val Phe Gly Leu Ser Ser Ile Leu 175 180 185 ggc cca ttg ctt ggt
ggc tgg ttc act gac ggt cca ggc tgg cgt tgg 867 Gly Pro Leu Leu Gly
Gly Trp Phe Thr Asp Gly Pro Gly Trp Arg Trp 190 195 200 ggt ctg tgg
ttg aac gtt cca atc ggc atc atc gca ctg gtt gct atc 915 Gly Leu Trp
Leu Asn Val Pro Ile Gly Ile Ile Ala Leu Val Ala Ile 205 210 215 gct
gtg ctg ctg aaa ctt cca gct cgt gaa cgt ggc aag gtc tcc gtt 963 Ala
Val Leu Leu Lys Leu Pro Ala Arg Glu Arg Gly Lys Val Ser Val 220 225
230 235 gac tgg ttg gga agc atc ttc atg gct atc gcc acc acc gca ttt
gtc 1011 Asp Trp Leu Gly Ser Ile Phe Met Ala Ile Ala Thr Thr Ala
Phe Val 240 245 250 ctc gca gtg acc tgg ggt ggc aat gaa tat gag tgg
gca tca cca atg 1059 Leu Ala Val Thr Trp Gly Gly Asn Glu Tyr Glu
Trp Ala Ser Pro Met 255 260 265 atc atc ggt ttg ttc atc acg aca ttg
gtc gct gcg ata gtg ttc gtt 1107 Ile Ile Gly Leu Phe Ile Thr Thr
Leu Val Ala Ala Ile Val Phe Val 270 275 280 ttc gtc gaa aag cgt gct
gtt gac cca ctg gtc ccc atg ggc ctt ttc 1155 Phe Val Glu Lys Arg
Ala Val Asp Pro Leu Val Pro Met Gly Leu Phe 285 290 295 tcg aac cgc
aac ttc gtg ctc acc gcc gtc gcc ggt atc ggc gta ggc 1203 Ser Asn
Arg Asn Phe Val Leu Thr Ala Val Ala Gly Ile Gly Val Gly 300 305 310
315 ctg ttt atg atg ggc acc atc gcg tac atg cct acc tac ctg cag atg
1251 Leu Phe Met Met Gly Thr Ile Ala Tyr Met Pro Thr Tyr Leu Gln
Met 320 325 330 gtt cat ggt ctg aac cca acg caa gct ggt ctg atg ctg
atc cca atg 1299 Val His Gly Leu Asn Pro Thr Gln Ala Gly Leu Met
Leu Ile Pro Met 335 340 345 atg atc ggc ctg att ggt aca tcc act gtg
gtg ggc aac atc gtg tcc 1347 Met Ile Gly Leu Ile Gly Thr Ser Thr
Val Val Gly Asn Ile Val Ser 350 355 360 aag act ggc aag tac aag tgg
tac cca ttc atc ggc atg ctc atc atg 1395 Lys Thr Gly Lys Tyr Lys
Trp Tyr Pro Phe Ile Gly Met Leu Ile Met 365 370 375 gtc ctt gcc cta
gta ctg cta tcg acg ctg aca cct tcg gca agc ttg 1443 Val Leu Ala
Leu Val Leu Leu Ser Thr Leu Thr Pro Ser Ala Ser Leu 380 385 390 395
gct ctc att gga ctg tac ttc ttc gtc ttc gga ttc ggc ctg ggc tgt
1491 Ala Leu Ile Gly Leu Tyr Phe Phe Val Phe Gly Phe Gly Leu Gly
Cys 400 405 410 gca atg cag att ttg gtt ctc atc gtg cag aac tcc ttc
cca atc acc 1539 Ala Met Gln Ile Leu Val Leu Ile Val Gln Asn Ser
Phe Pro Ile Thr 415 420 425 atg gtt ggc acc gcg acc ggt tcc aac aac
ttc ttc cgc caa atc ggt 1587 Met Val Gly Thr Ala Thr Gly Ser Asn
Asn Phe Phe Arg Gln Ile Gly 430 435 440 gga gca gta ggt tcc gca ctg
atc ggt ggc ctg ttt atc tcc aac ctg 1635 Gly Ala Val Gly Ser Ala
Leu Ile Gly Gly Leu Phe Ile Ser Asn Leu 445 450 455 tcc gac cga ttc
acc gaa aac gtc ccc gca gca gtg gct tcc atg ggt 1683 Ser Asp Arg
Phe Thr Glu Asn Val Pro Ala Ala Val Ala Ser Met Gly 460 465 470 475
gaa gaa ggc gca caa tac gcc tca gca atg tcc gat ttc tcc ggt gca
1731 Glu Glu Gly Ala Gln Tyr Ala Ser Ala Met Ser Asp Phe Ser Gly
Ala 480 485 490 tcc aac ctc act cca cac ctt gtt gaa tca ctt cca caa
gca ctc cgt 1779 Ser Asn Leu Thr Pro His Leu Val Glu Ser Leu Pro
Gln Ala Leu Arg 495 500 505 gaa gca att caa ctt tct tac aac gac gcc
ctg aca cca atc ttc ttg 1827 Glu Ala Ile Gln Leu Ser Tyr Asn Asp
Ala Leu Thr Pro Ile Phe Leu 510 515 520 gcg ctc acc ccg atc gca gta
gtc gcc gcg atc ctc ctc ttt ttc atc 1875 Ala Leu Thr Pro Ile Ala
Val Val Ala Ala Ile Leu Leu Phe Phe Ile 525 530 535 cgt gaa gat cac
ctc aag gaa acg cac gaa taatgacaca cgaaacttcc 1925 Arg Glu Asp His
Leu Lys Glu Thr His Glu 540 545 gtccccggac ctgccgacgc gcaggtcgca
ggagatacga agctgcgcaa aggccgcgcg 1985 aagaaggaaa aaactccttc
atcaatgacg cctgaacaac aaaagaaagt ctggtgggtc 2045 ctcagcgcgc
tgatggtcgc catgatgatg gcctcccttg accagatgat tttcggcaca 2105
gccctgccaa caatc 2120 2 549 PRT Corynebacterium glutamicum 2 Met
Thr Ser Gln Val Lys Pro Asp Asp Glu Arg Pro Val Thr Thr Ile 1 5 10
15 Ser Lys Ser Gly Ala Pro Ser Ala His Thr Ser Ala Pro Tyr Gly Ala
20 25 30 Ala Ala Thr Glu Glu Ala Val Glu Glu Lys Thr Lys Gly Arg
Val Gly 35 40 45 Phe Ile Ile Ala Ala Leu Met Leu Ala Met Leu Leu
Ser Ser Leu Gly 50 55 60 Gln Thr Ile Phe Gly Ser Ala Leu Pro Thr
Ile Val Gly Glu Leu Gly 65 70 75 80 Gly Val Asn His Met Thr Trp Val
Ile Thr Ala Phe Leu Leu Gly Gln 85 90 95 Thr Ile Ser Leu Pro Ile
Phe Gly Lys Leu Gly Asp Gln Phe Gly Arg 100 105 110 Lys Tyr Leu Phe
Met Phe Ala Ile Ala Leu Phe Val Val Gly Ser Ile 115 120 125 Ile Gly
Ala Leu Ala Gln Asn Met Thr Thr Leu Ile Val Ala Arg Ala 130 135 140
Leu Gln Gly Ile Ala Gly Gly Gly Leu Met Ile Leu Ser Gln Ala Ile 145
150 155 160 Thr Ala Asp Val Thr Thr Ala Arg Glu Arg Ala Lys Tyr Met
Gly Ile 165 170 175 Met Gly Ser Val Phe Gly Leu Ser Ser Ile Leu Gly
Pro Leu Leu Gly 180 185 190 Gly Trp Phe Thr Asp Gly Pro Gly Trp Arg
Trp Gly Leu Trp Leu Asn 195 200 205 Val Pro Ile Gly Ile Ile Ala Leu
Val Ala Ile Ala Val Leu Leu Lys 210 215 220 Leu Pro Ala Arg Glu Arg
Gly Lys Val Ser Val Asp Trp Leu Gly Ser 225 230 235 240 Ile Phe Met
Ala Ile Ala Thr Thr Ala Phe Val Leu Ala Val Thr Trp 245 250 255 Gly
Gly Asn Glu Tyr Glu Trp Ala Ser Pro Met Ile Ile Gly Leu Phe 260 265
270 Ile Thr Thr Leu Val Ala Ala Ile Val Phe Val Phe Val Glu Lys Arg
275 280 285 Ala Val Asp Pro Leu Val Pro Met Gly Leu Phe Ser Asn Arg
Asn Phe 290 295 300 Val Leu Thr Ala Val Ala Gly Ile Gly Val Gly Leu
Phe Met Met Gly 305 310 315 320 Thr Ile Ala Tyr Met Pro Thr Tyr Leu
Gln Met Val His Gly Leu Asn 325 330 335 Pro Thr Gln Ala Gly Leu Met
Leu Ile Pro Met Met Ile Gly Leu Ile 340 345 350 Gly Thr Ser Thr Val
Val Gly Asn Ile Val Ser Lys Thr Gly Lys Tyr 355 360 365 Lys Trp Tyr
Pro Phe Ile Gly Met Leu Ile Met Val Leu Ala Leu Val 370 375 380 Leu
Leu Ser Thr Leu Thr Pro Ser Ala Ser Leu Ala Leu Ile Gly Leu 385 390
395 400 Tyr Phe Phe Val Phe Gly Phe Gly Leu Gly Cys Ala Met Gln Ile
Leu 405 410 415 Val Leu Ile Val Gln Asn Ser Phe Pro Ile Thr Met Val
Gly Thr Ala 420 425 430 Thr Gly Ser Asn Asn Phe Phe Arg Gln Ile Gly
Gly Ala Val Gly Ser 435 440 445 Ala Leu Ile Gly Gly Leu Phe Ile Ser
Asn Leu Ser Asp Arg Phe Thr 450 455 460 Glu Asn Val Pro Ala Ala Val
Ala Ser Met Gly Glu Glu Gly Ala Gln 465 470 475 480 Tyr Ala Ser Ala
Met Ser Asp Phe Ser Gly Ala Ser Asn Leu Thr Pro 485 490 495 His Leu
Val Glu Ser Leu Pro Gln Ala Leu Arg Glu Ala Ile Gln Leu 500 505 510
Ser Tyr Asn Asp Ala Leu Thr Pro Ile Phe Leu Ala Leu Thr Pro Ile 515
520 525 Ala Val Val Ala Ala Ile Leu Leu Phe Phe Ile Arg Glu Asp His
Leu 530 535 540 Lys Glu Thr His Glu 545 3 19 DNA Corynebacterium
glutamicum 3 ctgtgctgct gaaacttcc 19 4 19 DNA Corynebacterium
glutamicum 4 agtccaatga gagccaagc 19
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