U.S. patent application number 11/155656 was filed with the patent office on 2005-10-20 for nucleotide sequence which code for the meth gene.
This patent application is currently assigned to DEGUSSA AG. Invention is credited to Bathe, Brigitte, Binder, Michael, Greissinger, Dieter, Huthmacher, Klaus, Kalinowski, Joern, Moeckel, Bettina, Pfefferle, Walter, Puehler, Alfred, Rueckert, Christian, Thierbach, Georg.
Application Number | 20050233373 11/155656 |
Document ID | / |
Family ID | 27437858 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050233373 |
Kind Code |
A1 |
Bathe, Brigitte ; et
al. |
October 20, 2005 |
Nucleotide sequence which code for the metH gene
Abstract
An isolated polynucleotide comprising a polynucleotide sequence
selected from the group consisting of a) polynucleotide which is at
least 70% identical to a polynucleotide that codes for a
polypeptide which comprises the amino acid sequence of SEQ ID No.
2, b) polynucleotide which codes for a polypeptide that comprises
an amino acid sequence which is at least 70% identical to the amino
acid sequence of SEQ ID No. 2, c) polynucleotide which is
complementary to the polynucleotides of a) or b), and d)
polynucleotide comprising at least 15 successive nucleotides of the
polynucleotide sequence of a), b) or c), and processes for the
fermentative preparation of L-amino acids using coryneform bacteria
in which at least the metH gene is present in enhanced form, and
use of the polynucleotide sequences as hybridization probes.
Inventors: |
Bathe, Brigitte;
(Salzkotten, DE) ; Moeckel, Bettina; (Duesseldorf,
DE) ; Pfefferle, Walter; (Halle (Westf.), DE)
; Huthmacher, Klaus; (Gelnhausen, DE) ; Rueckert,
Christian; (Guetersloh, DE) ; Kalinowski, Joern;
(Bielefeld, DE) ; Puehler, Alfred; (Bielefeld,
DE) ; Binder, Michael; (Steinhagen (Westf.), DE)
; Greissinger, Dieter; (Niddatal, DE) ; Thierbach,
Georg; (Bielefeld, DE) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND, MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
DEGUSSA AG
Duesseldorf
DE
|
Family ID: |
27437858 |
Appl. No.: |
11/155656 |
Filed: |
June 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11155656 |
Jun 20, 2005 |
|
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09919891 |
Aug 2, 2001 |
|
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60294251 |
May 31, 2001 |
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Current U.S.
Class: |
435/6.13 ;
435/106; 435/193; 435/252.3; 435/471; 435/6.15; 536/23.2 |
Current CPC
Class: |
A23K 20/142 20160501;
C12N 1/205 20210501; C12R 2001/15 20210501; C12N 9/1007 20130101;
C12P 13/12 20130101 |
Class at
Publication: |
435/006 ;
435/106; 435/193; 435/252.3; 435/471; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 013/04; C12N 009/10; C12N 015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 2, 2000 |
DE |
100 38 050.6 |
Feb 28, 2001 |
DE |
101 09 687.9 |
Claims
1-34. (canceled)
35. A nucleic acid comprising at least 15 consecutive nucleotides
of a polynucleotide encoding SEQ ID NO: 2 or its full complement,
and which binds to a nucleic acid which encodes a polypeptide
having homocysteine methyl transferase activity.
36. The nucleic acid of claim 35, which comprises at least 20
consecutive nucleotides of a polynucleotide encoding SEQ ID NO: 2
or its full complement, and which binds to a nucleic acid which
encodes a polypeptide having homocysteine methyl transferase
activity.
37. The nucleic acid of claim 35, which comprises at least 30
consecutive nucleotides of a polynucleotide encoding SEQ ID NO: 2
or its full complement, and which binds to a nucleic acid which
encodes a polypeptide having homocysteine methyl transferase
activity.
38. The nucleic acid of claim 35, which comprises at least 50
consecutive nucleotides of a polynucleotide encoding SEQ ID NO: 2
or its full complement, and which binds to a nucleic acid which
encodes a polypeptide having homocysteine methyl transferase
activity.
39. The nucleic acid of claim 35 which comprises at least 15
consecutive nucleotides of a polynucleotide encoding SEQ ID NO:
2.
40. The nucleic acid of claim 35 which comprises at least 15
consecutive nucleotides of the full complement of a polynucleotide
encoding SEQ ID NO: 2.
41. A method of identifying a nucleic acid which encodes a
polypeptide having homocysteine methyltransferase II activity,
comprising probing a nucleic acid sample with the nucleic acid of
claim 35.
42. The method of claim 41, wherein said nucleic acid sample
comprises RNA, DNA or cDNA.
43. A method for producing a nucleic acid which encodes a
polypeptide having homocysteine methyltransferase II activity,
comprising priming a nucleic acid sample with the nucleic acid of
claim 35.
44. The method of claim 43, wherein said nucleic acid sample
comprises RNA, DNA or cDNA.
45. A process for preparing an L-methionine-containing animal
feedstuffs additive, comprising: a) culturing an
L-methionine-producing microorganism in a fermentation medium to
produce a fermented medium containing L-methionine, b) removing
water from the fermented medium (concentration); c) removing 0 to
100 wt. % of the biomass from the fermented medium; and/or d)
drying the fermented medium, optionally to form a powder or
granules.
46. The process of claim 45, wherein the expression of at least one
gene in the biosynthesis pathway for L-methionine in said
microorganism is enhanced.
47. The process of claim 46, wherein said gene is selected from the
group consisting of: the lysC gene which codes for a feed back
resistant aspartate kinase, the gap gene which codes for
glycerolaldehyde 3-phosphate dehydrogenase, the pgk gene which
codes for 3-phosphoglycerate kinase, the pyc gene which codes for
pyruvate carboxylase, the tpi gene which codes for triose phosphate
isomerase the metA gene which codes for homoserine
0-acetyltransferase the metB gene which codes for cystathionine
gamma-synthase aecD gene which codes for cystathionine gamma-lyase
glyA gene which codes for serine hydroxymethyltransferase and metY
gene which codes for 0-acetylhomoserine-sulfhydrylase.
48. The process of claim 45, wherein the expression of at least one
gene which decreases L-methionine levels in said microorganism is
reduced or eliminated.
49. The process of claim 48, wherein said gene is selected from the
group consisting of: the thrB gene which codes for homoserine
kinase the ilvA gene which codes for threonine dehydratase the thrC
gene which codes for threonine synthase the ddh gene which codes
for meso-diaminopimelate D-dehydrogenase the pck gene which codes
for phosphoenol pyruvate carboxykinase the pgi gene which codes for
glucose 6-phosphate isomerase and the poxB gene which codes for
pyruvate oxidase.
50. The process of claim 45, wherein expression of the
polynucleotide encoding the metF gene product is enhanced.
51. The process of claim 45, wherein microorganisms of the species
Corynebacterium glutamicum are employed.
52. The process of claim 51, wherein the Corynebacterium glutamicum
strain ATCC13032/pCREmetF is employed.
53. The process of claim 45, further comprising at least one of the
following steps: e) adding at least one organic substance to the
product obtained by steps b), c) and/or d); f) adding at least one
auxiliary substance selected from the group consisting of silicas,
silicates, stearates, grits and bran to the product obtained by
steps to b), c), d) and/or e); and/or g) coating the product
obtained by steps b), c), d), e) and/or f) with a film-forming
agent.
54. The process of claim 53, wherein the film-forming agent is at
least one selected from the group consisting of metal carbonates,
silicas, silicates, alginates, stearates, starches, gums and
cellulose ethers.
55. The process of claim 45, wherein a portion of the biomass is
removed.
56. The process of claim 45, wherein essentially 100% of the
biomass is removed.
57. The process as claimed in claim 45, wherein the water content
in said feedstuff additive is up to 5 wt. %.
58. The process of claim 45, wherein the water content in said
feedstuff additive is less than 2 wt. %.
59. An animal feedstuffs additive prepared by the process of claim
45.
60. The animal feedstuffs additive of claim 59, which comprises 1
wt. % to 80 wt. % L-methionine, D-methionine, D,L-methionine or a
mixture thereof, based on its dry weight.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention provides nucleotide sequences from coryneform
bacteria which code for the metH gene and a process for the
fermentative preparation of amino acids, in particular
L-methionine, using bacteria in which the metH gene is
enhanced.
[0003] 2. Description of the Related Art
[0004] L-Amino acids, in particular L-methionine, are used in human
medicine and in the pharmaceuticals industry, in the foodstuffs
industry and very particularly in animal nutrition.
[0005] 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 process. Improvements
to the process can relate to fermentation measures, such as,
stirring and supply of oxygen, or to the composition of the
nutrient media, such as, the sugar concentration during the
fermentation, or to the working up of the product by, for example,
ion exchange chromatography, or to the intrinsic output properties
of the microorganism itself.
[0006] Methods of mutagenesis, selection and mutant selection are
used to improve the output properties of these microorganisms.
Strains which are resistant to antimetabolites, such as e.g. the
methionine analogue .alpha.-methyl-methionine, ethionine,
norleucine, N-acetylnorleucine, S-trifluoromethylhombcysteine,
2-amino-5-heprenoitic acid, seleno-methionine,
methionine-sulfoximine, methoxine, 1-aminocyclopentane-carboxylic
acid, or are auxotrophic for metabolites of regulatory importance
and produce amino acids, such as e.g. L-methionine, are obtained in
this manner.
[0007] Recombinant DNA techniques have also been employed for some
years for improving Corynebacterium strains which produce L-amino
acids, by amplifying individual amino acid biosynthesis genes and
investigating their effect on the amino acid production.
SUMMARY OF THE INVENTION
[0008] One object of the present invention is to provide new
measures for improved fermentative preparation of amino acids, in
particular L-methionine.
[0009] When L-methionine or methionine are mentioned in the
following, the salts, such as methionine hydrochloride or
methionine sulfate are also meant.
[0010] The invention provides an isolated polynucleotide from
coryneform bacteria, comprising a polynucleotide sequence which
codes for the metH gene, chosen from the group consisting of
[0011] a) polynucleotide which is at least 70% identical to a
polynucleotide that codes for a polypeptide which comprises the
amino acid sequence of SEQ ID No. 2,
[0012] b) polynucleotide which codes for a polypeptide that
comprises an amino acid sequence which is at least 70% identical to
the amino acid sequence of SEQ ID No. 2,
[0013] c) polynucleotide which is complementary to the
polynucleotides of a) or b), and
[0014] d) polynucleotide comprising at least 15 successive
nucleotides of the polynucleotide sequence of a), b) or c), and the
corresponding polypeptides having the enzymatic activity of
homocysteine methyltransferase II.
[0015] The invention also provides the above-mentioned
polynucleotides, as DNA which is capable of replication,
comprising:
[0016] (i) the nucleotide sequence shown in SEQ ID No. 1, or
[0017] (ii) at least one sequence which corresponds to sequence (i)
within the range of the degeneration of the genetic code, or
[0018] (iii) at least one sequence which hybridizes with the
sequence complementary to sequence (i) or (ii), and optionally
[0019] (iv) sense mutations of neutral function in (i).
[0020] The invention also provides
[0021] a polynucleotide comprising the nucleotide sequence as shown
in SEQ ID No. 1;
[0022] a polynucleotide that codes for a polypeptide which
comprises the amino acid sequence as shown in SEQ ID No. 2,
[0023] a vector containing the polynucleotide according to the
invention, in particular a shuttle vector or plasmid vector,
and
[0024] and coryneform bacteria serving as the host cell, which
contain the vector or in which the metH gene is enhanced.
[0025] The invention also provides polynucleotides which are
obtained by screening a corresponding gene library, which comprises
the complete gene having the polynucleotide sequence corresponding
to SEQ ID No. 1, by means of hybridization with a probe which
comprises the sequence of the polynucleotide mentioned, according
to SEQ ID No. 1 or a fragment thereof, and isolation of the DNA
sequence mentioned.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1 shows plasmid pCREmetH.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] Polynucleotides 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 homocysteine methyltransferase II or to isolate those
nucleic acids or polynucleotides or genes which have a high
similarity of sequence or homology with that of the homocysteine
methyltransferase II gene.
[0028] Polynucleotides according to the invention are furthermore
suitable as primers with the aid of which DNA of genes that code
for homocysteine methyltransferase II can be prepared by the
polymerase chain reaction (PCR).
[0029] Such oligonucleotides that serve as probes or primers
comprise at least 30, preferably at least 20, very particularly at
least 15 successive nucleotides. oligonucleotides which have a
length of at least 40 or 50 nucleotides are also suitable.
Oligonucleotides with a length of at least 100, 150, 200, 250 or
300 nucleotides are optionally also suitable.
[0030] "Isolated" means separated out of its natural
environment.
[0031] "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.
[0032] "Polypeptides" are understood as meaning peptides or
proteins which comprise two or more amino acids bonded via peptide
bonds.
[0033] The polypeptides according to the invention include a
polypeptide according to SEQ ID No. 2, in particular those with the
biological activity of homocysteine methyltransferase II, and also
those which are at least 70%, preferably at least 80% and in
particular which are at least 90% to 95% identical to the
polypeptide according to SEQ ID No. 2 and have the activity
mentioned.
[0034] The invention moreover provides a process for the
fermentative preparation of amino acids, in particular
L-methionine, using coryneform bacteria which in particular already
produce amino acids, and in which the nucleotide sequences which
code for the metH gene are enhanced, in particular
over-expressed.
[0035] 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.
[0036] 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 the
starting microorganism.
[0037] The microorganisms which the present invention provides can
prepare L-amino acids, in particular L-methionine, 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.
[0038] Suitable strains of the genus Corynebacterium, in particular
of the species Corynebacterium glutamicum (C. glutamicum), are in
particular the known wild-type strains
[0039] Corynebacterium glutamicum ATCC13032
[0040] Corynebacterium acetoglutamicum ATCC15806
[0041] Corynebacterium acetoacidophilum ATCC13870
[0042] Corynebacterium thermoaminogenes FERM BP-1539
[0043] Corynebacterium melassecola ATCC17965
[0044] Brevibacterium flavum ATCC14067
[0045] Brevibacterium lactofermentum ATCC13869 and
[0046] Brevibacterium divaricatum ATCC14020
[0047] or L-amino acid-producing mutants or strains prepared
therefrom, such as, for example, the L-methionine-producing
strain
[0048] Corynebacterium glutamicum ATCC21608.
[0049] The new metH gene from C. glutamicum which codes for the
enzyme homocysteine methyltransferase II (EC 2.1.1.13) has been
isolated.
[0050] To isolate the metH 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
(Verlag Chemie, Weinheim, Germany, 1990), or the handbook by
Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold
Spring Harbor Laboratory Press, 1989) may be mentioned as examples.
A well-known gene library is that of the E. coli K-12 strain W3110
set up in .lambda. vectors by Kohara et al. (Cell 50, 495-508
(1987)). Bathe et al. (Molecular and General Genetics, 252:255-265,
1996) 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) in the E. coli K-12 strain NM554 (Raleigh et al.,
1988, Nucleic Acids Research 16:1563-1575).
[0051] Bormann et al. (Molecular Microbiology 6(3), 317-326)
(1992)) in turn describe a gene library of C. glutamicum ATCC13032
using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)).
To prepare a gene library of C. glutamicum in E. coli it is also
possible to use plasmids such as pBR322 (Bolivar, Life Sciences,
25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene,
19:259-268). Suitable hosts are, in particular, those E. coli
strains which are restriction- and recombination-defective. An
example of these is 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). The long DNA fragments cloned
with the aid of cosmids can in turn be subcloned in the usual
vectors suitable for sequencing and then sequenced, 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).
[0052] The resulting DNA sequences can then be investigated with
known algorithms or sequence analysis programs, such as that of
Staden (Nucleic Acids Research 14, 217-232(1986)), that of Marck
(Nucleic Acids Research 16, 1829-1836 (1988)) or the GCG program of
Butler (Methods of Biochemical Analysis 39, 74-97 (1998)).
[0053] The new DNA sequence of C. glutamicum which codes for the
metH 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 metH gene product is shown in SEQ ID No.
2.
[0054] 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. they are of neutral function.
[0055] It is furthermore known that changes at the N and/or C
terminus of a protein must not substantially impair and may even
stabilize the function thereof. Information in this context can be
found in Ben-Bassat et al. (Journal of Bacteriology 169:751-757
(1987)), in O'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth
et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al.
(Bio/Technology 6:1321-1325 (1988)) 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.
[0056] 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.
[0057] Instructions for identifying DNA sequences by means of
hybridization can be found in the handbook "The DIG System Users
Guide for Filter Hybridization" from Boehringer Mannheim GmbH
(Mannheim, Germany, 1993) and in Liebl et al. (International
Journal of Systematic Bacteriology (1991) 41: 255-260).
Instructions for amplification of DNA sequences with the aid of the
polymerase chain reaction (PCR) can be found in the handbook by
Gait: Oligonucleotide Synthesis: A Practical Approach (IRL Press,
Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum
Akademischer verlag, Heidelberg, Germany, 1994).
[0058] It has been found that coryneform bacteria produce amino
acids, in particular L-methionine, in an improved manner after
over-expression of the metH gene.
[0059] To achieve an over-expression, the number of copies of the
corresponding genes can be increased, or the promoter and
regulation region or the ribosome binding site upstream of the
structural gene can be mutated. Expression cassettes which are
incorporated upstream of the structural gene act in the same way.
By inducible promoters, it is additionally possible to increase the
expression in the course of fermentative L-methionine production.
The expression is likewise improved by measures to prolong the life
of the m-RNA. Furthermore, the enzyme activity is also increased by
preventing the degradation of the enzyme protein. The genes or gene
constructs can either be present in plasmids with a varying number
of copies, or can be integrated and amplified in the chromosome.
Alternatively, an over-expression of the genes in question can
furthermore be achieved by changing the composition of the media
and the culture procedure.
[0060] Instructions in this context can be found in Martin et al.
(Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138,
35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430
(1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in European
Patent Specification 0 472 869, in U.S. Pat. No. 4,601,893, in
Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991), in Reinscheid
et al. (Applied and Environmental Microbiology 60, 126-132 (1994)),
in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)),
in Patent Application WO 96/15246, in Malumbres et al. (Gene 134,
15- 24 (1993)), in Japanese Laid-Open Specification JP-A-10-229891,
in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195
(1998)), in Makrides (Microbiological Reviews 60:512-538 (1996))
and in known textbooks of genetics and molecular biology.
[0061] By way of example, for enhancement the metH gene according
to the invention was over-expressed with the aid of episomal
plasmids. Suitable plasmids are those which are replicated in
coryneform bacteria. Numerous known plasmid vectors, such as e.g.
pZl (Menkel et al., Applied and Environmental Microbiology (1989)
64: 549-554), pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or
pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) are based on the
cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors, such
as those based on pCG4 (U.S. Pat. No. 4,489,160), or pNG2
(Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124
(1990)), or pAG1 (U.S. Pat. No. 5,158,891), can be used in the same
manner. Plasmid vectors which are furthermore suitable are also
those with the aid of which the process of gene amplification by
integration into the chromosome can be used, as has been described,
for example, by Reinscheid et al. (Applied and Environmental
Microbiology 60, 126-132 (1994)) for duplication or amplification
of the hom-thrB operon. In this method, the complete gene 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)), pK18mob
or pK19mob (Schafer et al., Gene 145, 69-73 (1994)), pGEM-T
(Promega corporation, Madison, Wis., U.S.A.), pCR2.1-TOPO (Shuman
(1994). Journal of Biological Chemistry 269:32678-84; U.S. Pat. No.
5,487,993), pCR.RTM.Blunt (Invitrogen, Groningen, Holland; Bernard
et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1
(Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) or
pBGS8 (Spratt et al., 1986, Gene 41: 337-342). The plasmid vector
which contains the gene to be amplified 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)). Methods for transformation are described, for
example, by Thierbach et al. (Applied Microbiology and
Biotechnology 29, 356-362 (1988)), Dunican and Shivnan
(Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS
Microbiological Letters 123, 343-347 (1994)). After homologous
recombination by means of a "cross over" event, the resulting
strain contains at least two copies of the gene in question.
[0062] In addition, it may be advantageous for the production of
amino acids, in particular L-methionine, to enhance one or more
enzymes of the particular biosynthesis pathway, of glycolysis, of
anaplerosis, of the citric acid cycle or of amino acid export, in
addition to the metH gene.
[0063] Thus for the preparation of amino acids, in particular
L-methionine, one or more genes chosen from the group consisting
of
[0064] the gap gene which codes for glyceraldehyde 3-phosphate
dehydrogenase (Eikmanns (1992), Journal of Bacteriology
174:6076-6086),
[0065] the tpi gene which codes for triose phosphate isomerase
(Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
[0066] the pgk gene which codes for 3-phosphoglycerate kinase
(Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
[0067] the pyc gene which codes for pyruvate carboxylase (Eikmanns
(1992), Journal of Bacteriology 174:6076-6086),
[0068] the lysC gene which codes for a feed-back resistant
aspartate kinase (ACCESSION NUMBER P26512; EP-B-0387527;
EP-A-0699759),
[0069] the metA gene which codes for homoserine O-acetyltransferase
(ACCESSION Number AF052652),
[0070] the metB gene which codes for cystathionine gamma-synthase
(ACCESSION Number AF126953),
[0071] the aecD gene which codes for cystathionine gamma-lyase
(ACCESSION Number M89931)
[0072] the glyA gene which codes for serine
hydroxymethyltransferase (JP-A-08107788),
[0073] the metY gene which codes for
O-acetylhomoserine-sulfhydrylase (DSM 13556)
[0074] can be enhanced, in particular over-expressed.
[0075] It may furthermore be advantageous for the production of
amino acids, in particular L-methionine, in addition to the
enhancement of the metH gene, for one or more genes chosen from the
group consisting of
[0076] thrB gene which codes for homoserine kinase (ACCESSION
Number P08210),
[0077] the ilvA gene which codes for threonine dehydratase
(ACCESSION Number Q04513),
[0078] the thrC gene which codes for threonine synthase (ACCESSION
Number P23669),
[0079] the ddh gene which codes for meso-diaminopimelate
D-dehydrogenase (ACCESSION Number Y00151),
[0080] the pck gene which codes for phosphoenol pyruvate
carboxykinase (DE 199 50 409.1; DSM 13047),
[0081] the pgi gene which codes for glucose 6-phosphate isomerase
(U.S. Pat. No. 09/396,478; DSM 12969),
[0082] the poxB gene which codes for pyruvate oxidase (DE: 1995
1975.7; DSM 13114)
[0083] to be attenuated, in particular for the expression thereof
to be reduced.
[0084] 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.
[0085] By attenuation measures, the activity or concentration of
the corresponding protein is in general reduced to 0 to 50%, 0 to
25%, 0 to 10% or 0 to 5% of the activity or concentration of the
wild-type protein.
[0086] In addition to over-expression of the metH gene it may
furthermore be advantageous for the production of amino acids, in
particular L-methionine, to eliminate undesirable side reactions,
(Nakayama: "Breeding of Amino Acid Producing Micro-organisms", in:
Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek
(eds.), Academic Press, London, UK, 1982).
[0087] The microorganisms prepared according to the invention 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 amino acids, in particular L-methionine. A summary of
known culture methods is described in the textbook by Chmiel
(Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik
(Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by
Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag,
Braunschweig/Wiesbaden, 1994)).
[0088] 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).
[0089] Sugars and carbohydrates, such as e.g. glucose, sucrose,
lactose, fructose, maltose, molasses, starch and cellulose, oils
and fats, such as e.g. soya oil, sunflower oil, groundnut oil and
coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid
and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and
organic acids, such as e.g. acetic acid, can be used as the source
of carbon. These substances can be used individually or as a
mixture.
[0090] 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.
[0091] Organic and inorganic sulfur-containing compounds, such as,
for example, sulfides, sulfites, sulfates and thiosulfates, can be
used as a source of sulfur, in particular for the preparation of
methionine.
[0092] 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 e. g. 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.
[0093] 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 e.g.
fatty acid polyglycol esters, can be employed to control the
development of foam. Suitable substances having a selective action,
such as e.g. 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 e.g. 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.
[0094] The fermentation broths obtained in this way, in particular
containing L-methionine, usually have a dry weight of 7.5 to 25 wt.
% and contain L-methionine. It is furthermore also advantageous if
the fermentation is conducted in a sugar-limited procedure at least
at the end, but in particular over at least 30% of the duration of
the fermentation. That is to say, the concentration of utilizable
sugar in the fermentation medium is reduced to .gtoreq.0 to 3 g/l
during this period.
[0095] The fermentation broth prepared in this manner, in
particular containing L-methionine, is then further processed.
Depending on requirements all or some of the biomass can be removed
from the fermentation broth by separation methods, such as
centrifugation, filtration, decanting or a combination thereof, or
it can be left completely in. This broth is then thickened or
concentrated by known methods, such as with the aid of a rotary
evaporator, thin film evaporator, falling film evaporator, by
reverse osmosis, or by nanofiltration. This concentrated
fermentation broth can then be worked up by methods of freeze
drying, spray drying, spray granulation or by other processes to
give a preferably free-flowing, finely divided powder.
[0096] This free-flowing, finely divided powder can then in turn by
converted by suitable compacting or granulating processes into a
coarse-grained, readily free-flowing, storable and largely
dust-free product. In the granulation or compacting it is
advantageous to employ conventional organic or inorganic auxiliary
substances or carriers, such as starch, gelatin, cellulose
derivatives or similar substances, such as are conventionally used
as binders, gelling agents or thickeners in foodstuffs or
feedstuffs processing, or further substances, such as, for example,
silicas, silicates or stearates.
[0097] "Free-flowing" is understood as meaning powders which flow
unimpeded out of the vessel with the opening of 5 mm (millimeters)
of a series of glass outflow vessels with outflow openings of
various sizes (Klein, Seifen, le, Fette, Wachse 94, 12 (1968)).
[0098] As described here, "finely divided" means a powder with a
predominant content (>50%) having a particle size of 20 to 200
.mu.m diameter. "Coarse-grained" means products with a predominant
content (>50%) having a particle size of 200 to 2000 .mu.m
diameter. In this context, "dust-free" means that the product
contains only small contents (<5%) having particle sizes of less
than 20 .mu.m diameter. The particle size determination can be
carried out with methods of laser diffraction spectrometry. The
corresponding methods are described in the textbook on
"Teilchengro.beta.enmessung in der Laborpraxis" by R. H. Muller and
R. Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart
(1996) or in the textbook "Introduction to Particle Technology" by
M. Rhodes, Verlag Wiley & Sons (1998).
[0099] "Storable" in the context of this invention means a product
which can be stored for up to 120 days, preferably up to 52 weeks,
particularly preferably 60 months, without a substantial loss
(<5%) of methionine occurring.
[0100] Alternatively, however, the product can be absorbed on to an
organic or inorganic carrier substance which is known and
conventional in feedstuffs processing, for example, silicas,
silicates, grits, brans, meals, starches, sugars or others, and/or
mixed and stabilized with conventional thickeners or binders. Use
examples and processes in this context are described in the
literature (Die Muhle+Mischfuttertechnik 132 (1995) 49, page
817).
[0101] Finally, the product can be brought into a state in which it
is stable to digestion by animal stomachs, in particular the
stomach of ruminants, by coating processes ("coating") using
film-forming agents, such as, for example, metal carbonates,
silicas, silicates, alginates, stearates, starches, gums and
cellulose ethers, as described in DE-C-4100920.
[0102] If the biomass is separated off during the process, further
inorganic solids, for example added during the fermentation, are in
general removed. In addition,. the animal feedstuffs additive
according to the invention comprises at least the predominant
proportion of the further substances, in particular organic
substances, which are formed or added and are present in solution
in the fermentation broth, where these have not been separated off
by suitable processes.
[0103] In one aspect of the invention, the biomass can be separated
off to the extent of up to 70%, preferably up to 80%, preferably up
to 90%, preferably up to 95%, and particularly preferably up to
100%. In another aspect of the invention, up to 20% of the biomass,
preferably up to 15%, preferably up to 10%, preferably up to 5%,
particularly preferably no biomass is separated off.
[0104] These organic substances include organic by-products which
are optionally produced, in addition to the L-methionine, and
optionally discharged by the microorganisms employed in the
fermentation. These include L-amino acids chosen from the group
consisting of L-lysine, L-valine, L-threonine, L-alanine or
L-tryptophan. They include vitamins chosen from the group
consisting of vitamin B1 (thiamine), vitamin B2
(riboflavin),vitamin B5 (pantothenic acid), vitamin B6
(pyridoxine), vitamin B12 (cyanocobalamin), nicotinic
acid/nicotinamide and vitamin E (tocopherol). They also include
organic acids which carry one to three carboxyl groups, such as
acetic acid, lactic acid, citric acid, malic acid or fumaric acid.
Finally, they also include sugars, for example, trehalose. These
compounds are optionally desired if they improve the nutritional
value of the product.
[0105] These organic substances, including L-methionine and/or
D-methionine and/or the racemic mixture D,L-methionine, can also be
added, depending on requirements, as a concentrate or pure
substance in solid or liquid form during a suitable process step.
These organic substances mentioned can be added individually or as
mixtures to the resulting or concentrated fermentation broth, or
also during the drying or granulation process. It is likewise
possible to add an organic substance or a mixture of several
organic substances to the fermentation broth and a further organic
substance or a further mixture of several organic substances during
a later process step, for example granulation.
[0106] The product described above is suitable as a feedstuffs
additive, i.e. feed additive, for animal nutrition.
[0107] The L-methionine content of the animal feedstuffs additive
is conventionally 1 wt. % to 80 wt. %, preferably 2 wt. % to 80 wt.
%, particularly preferably 4 wt. % to 80 wt. %, and very
particularly preferably 8 wt. % to 80 wt. %, based on the dry
weight of the animal feedstuffs additive. Contents of 1 wt. % to 60
wt. %, 2 wt. % to 60 wt. %, 4 wt. % to 60 wt. %, 6 wt. % to 60 wt.
%, 1 wt. % to 40 wt. %, 2 wt. % to 40 wt. % or 4 wt. % to 40 wt. %
are likewise possible. The water content of the feedstuffs additive
is conventionally up to 5 wt. %, preferably up to 4 wt. %, and
particularly preferably less than 2 wt. %.
[0108] The invention also provides a process for the preparation of
an L-methionine-containing animal feedstuffs additive from
fermentation broths, which comprises the steps
[0109] a) culture and fermentation of an L-methionine-producing
microorganism in a fermentation medium;
[0110] b) removal of water from the L-methionine-containing
fermentation broth (concentration);
[0111] c) removal of an amount of 0 to 100 wt. % of the biomass
formed during the fermentation; and
[0112] d) drying of the fermentation broth obtained according to a)
and/or b) to obtain the animal feedstuffs additive in the desired
powder or granule form.
[0113] If desired, one or more of the following steps can
furthermore be carried out in the process according to the
invention:
[0114] e) addition of one or more organic substances, including
L-methionine and/or D-methionine and/or the racemic mixture
D,L-methionine, to the products obtained according to a), b) and/or
c);
[0115] f) addition of auxiliary substances chosen from the group
consisting of silicas, silicates, stearates, grits and bran to the
substances obtained according to a) to d) for stabilization and to
increase the storability; or
[0116] g) conversion of the substances obtained according to a) to
e) into a form stable to the animal stomach, in particular rumen,
by coating with film-forming agents.
[0117] The analysis of L-methionine can be carried out by ion
exchange chromatography with subsequent ninhydrin derivation, as
described by Spackman et al. (Analytical Chemistry, 30, (1958) ,
1190).
[0118] The process according to the invention is used for the
fermentative preparation of amino acids, in particular
L-methionine.
[0119] The following microorganism was deposited as a pure culture
on 14th Jun. 2001 at the Deutsche Sammlung fur Mikroorganismen und
Zellkulturen (DSMZ=German Collection of Microorganisms and Cell
Cultures, Braunschweig, Germany) in accordance with the Budapest
Treaty:
[0120] Escherichia coli DH5.alpha.mcr/pCREmetH as DSM 14354.
[0121] The present invention is explained in more detail in the
following with the aid of embodiment examples.
EXAMPLE 1
[0122] Preparation of a Genomic Cosmid Gene Library from
Corynebacterium glutamicum ATCC 13032
[0123] Chromosomal DNA from Corynebacterium glutamicum ATCC 13032
was isolated as described by Tauch et al. (1995, Plasmid
33:168-179) and partly cleaved with the restriction enzyme Sau3AI
(Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI,
Code no. 27-0913-02). The DNA fragments were dephosphorylated with
shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim,
Germany, Product Description SAP, Code no. 1758250).
[0124] The DNA of the cosmid vector SuperCosl (Wahl et al. (1987)
Proceedings of the National Academy of Sciences USA 84:2160-2164),
obtained from Stratagene (La Jolla, USA, Product Description
SuperCos1 Cosmid Vector Kit, Code no. 251301) was cleaved with the
restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany,
Product Description XbaI, Code no. 27-0948-02) and likewise
dephosphorylated with shrimp alkaline phosphatase. The cosmid DNA
was then cleaved with the restriction enzyme BamHI (Amersham
Pharmacia, Freiburg, Germany, Product Description BamHI, Code no.
27-0868-04). 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). 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).
[0125] For infection of the E. coli strain NM554 (Raleigh et al.
1988, Nucleic Acid Research 16:1563-1575) 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), the cells being
plated out on LB agar (Lennox, 1955, Virology, 1:190) with 100 mg/l
ampicillin. After incubation overnight at 37.degree. C.,
recombinant individual clones were selected.
EXAMPLE 2
[0126] Isolation and Sequencing of the metH Gene
[0127] 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). The DNA fragments were dephosphorylated with
shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim,
Germany, Product Description SAP, Product No. 1758250). 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).
[0128] The DNA of the sequencing vector pZero-1, obtained from
Invitrogen (Groningen, The Netherlands, Product Description Zero
Background Cloning Kit, Product No. K2500-01) was cleaved with the
restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany,
Product Description BamHI, Product No. 27-0868-04). 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), 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) into the E. coli
strain DH5.alpha.mcr (Grant, 1990, Proceedings of the National
Academy of Sciences U.S.A., 87:4645-4649) and plated out on LB agar
(Lennox, 1955, Virology, 1:190) with 50 mg/l zeocin.
[0129] The plasmid preparation of the recombinant clones was
carried out with 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 Academy of Sciences U.S.A., 74:5463-5467) with
modifications according to Zimmermann et al. (1990, Nucleic Acids
Research, 18:1067). 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).
[0130] The raw sequence data obtained were then processed using the
Staden program package (1986, Nucleic Acids Research, 14:217-231)
version 97-0. The individual sequences of the pZero1 derivatives
were assembled to a continuous contig. The computer-assisted coding
region analysis was prepared with the XNIP program (Staden, 1986,
Nucleic Acids Research, 14:217-231).
[0131] The resulting nucleotide sequence is shown in SEQ ID No. 1.
Analysis of the nucleotide sequence showed an open reading frame of
3662 base pairs, which was called the metH gene. The metH gene
codes for a protein of 1221 amino acids.
EXAMPLE 3
[0132] Preparation of the Strain C. glutamicum
ATCC13032/pCREmetH
[0133] 3.1 Amplification of the metH Gene
[0134] From the strain ATCC13032, chromosomal DNA was isolated by
the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)).
Starting from the nucleotide sequences of the methionine
biosynthesis genes metH (SEQ ID No. 1) of C. glutamicum ATCC13032,
the following oligonucleotides were chosen for the polymerase chain
reaction (PCR) (see SEQ ID No. 3 and SEQ ID No. 4):
1 metH-EVP5: 5'-GATCTAAGATCTAAAGGAGGACAACCATGTCTACTTCAGTTAC-
TTCACCAGC-3' metH-EVP3: 5'-GATCTAGTCGACCCCTCTCAAAGG-
TGTTAGAC-3'
[0135] The primers shown were synthesized by MWG-Biotech AG
(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) with Pwo-Polymerase
from Roche Diagnostics GmbH (Mannheim, Germany). With the aid of
the polymerase chain reaction, the primers allow amplification of a
DNA fragment 3718 bp in size, which carries the metH gene.
[0136] Furthermore, the primer metH-EVP5 contains the sequence for
the cleavage site of the restriction endonuclease BglII and the
primer metH-EVP3 the cleavage site of the restriction endonuclease
SalI, which are marked by underlining in the nucleotide sequence
shown above.
[0137] The metH fragment 3718 bp in size was cleaved with the
restriction endonucleases BglII and SalI. The batch was separated
by gel electrophoresis and the metH fragment (approx. 3700 bp) was
then isolated from the agarose gel with the QiaExII Gel Extraction
Kit (Product No. 20021, Qiagen, Hilden, Germany).
[0138] 3.2 Cloning of metH in the Vector pZ8-1
[0139] The E. coli -C. glutamicum shuttle expression vector pZ8-1
(EP 0 375 889) was used as the base vector for the expression.
[0140] DNA of the plasmid pZ8-1 was cleaved completely with the
restriction enzymes BamHI and SalI and then dephosphorylated with
shrimp alkaline phosphatase (Roche Diagnostics GmbH, Mannheim,
Germany, Product Description SAP, Product No. 1758250).
[0141] The metH fragment approx. 3700 bp in size isolated from the
agarose gel in example 3.1 and cleaved with the restriction
endonucleases BglII and SalI was mixed with the vector pZ8-1
prepared in this way and the batch was treated with T4 DNA ligase
(Amersham Pharmacia, Freiburg, Germany, Product Description
T4-DNA-Ligase, Code no.27-0870-04).
[0142] The ligation batch was transformed in the E. coli strain
DH5.alpha.mcr (Hanahan, In: DNA cloning. A Practical Approach. Vol.
I. IRL-Press, Oxford, Washington DC, U.S.A.). Selection of
plasmid-carrying cells was made by plating out the transformation
batch on LB agar (Lennox, 1955, Virology, 1:190) with 50 mg/l
kanamycin. After incubation overnight at 37.degree. C., recombinant
individual clones were selected. Plasmid DNA was isolated from a
transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106,
Qiagen, Hilden, Germany) in accordance with the manufacturer's
instructions and checked by restriction cleavage. The resulting
plasmid was called pCREmetH. The strain E. coli
DH5.alpha.mcr/pCREmetH was deposited as a pure culture on 14th Jun.
2001 at the Deutsche Sammlung fur Mikroorganismen und Zellkulturen
(DSMZ=German Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany) in accordance with the Budapest Treaty as
DSM 14354.
[0143] 3.3 Preparation of the Strain C. glutamicum
ATCC13032/pCREmetH
[0144] The vector pCREmetH obtained in example 3.2 was
electroporated in the strain C. glutamicum ATCC13032 using the
electroporation method described by Liebl et al. (FEMS Microbiology
Letters, 53:299-303 (1989)). Selection of the plasmid-carrying
cells took place on LBHIS agar comprising 18.5 g/l brain-heart
infusion broth, 0.5 M sorbitol, 5 g/l Bacto-tryptone, 2.5 g/l
Bacto-yeast extract, 5 g/l NaCl and 18 g/l Bacto-agar, which had
been supplemented with 25 mg/l kanamycin. Incubation was carried
out for 2 days at 33.degree. C.
[0145] Plasmid DNA was isolated from a transformant by conventional
methods (Peters-Wendisch et al., 1998, Microbiology 144, 915-927)
and checked by restriction cleavage. The resulting strain was
called ATCC13032pCREmetH.
EXAMPLE 4
[0146] Preparation of Methionine with the strain C. glutamicum
ATCC13032/pCREmetH
[0147] The C. glutamicum strain ATCC13032/pCREmetH obtained in
example 3 was cultured in a nutrient medium suitable for the
production of methionine and the methionine content in the culture
supernatant was determined.
[0148] 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 medium MM was used as the medium for the
preculture.
2 Medium MM CSL (corn steep liquor) 5 g/l MOPS
(morpholinopropanesulfonic acid) 20 g/l 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 * 7 H.sub.2O 1.0 g/l CaCl.sub.2
* 2 H.sub.2O 10 mg/l FeSO.sub.4 * 7 H.sub.2O 10 mg/l MnSO.sub.4 *
H.sub.2O 5.0 mg/l Biotin (sterile-filtered) 0.01 mg/l Vitamin B12
(sterile-filtered) 0.02 mg/l Thiamine * HCl (sterile-filtered) 0.2
mg/l CaCO.sub.3 25 g/l
[0149] The CSL, MOPS and the salt solution were brought to pH 7
with aqueous ammonia and autoclaved. The sterile substrate and
vitamin solutions were then added, as well as the CaCO.sub.3
autoclaved in the dry state.
[0150] Kanamycin (25 mg/l) 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. Medium MM was
also used for the main culture.
[0151] 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.
[0152] 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 methionine formed was determined with an
amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by
ion exchange chromatography and post-column derivation with
ninhydrin detection.
[0153] The result of the experiment is shown in table 1.
3 TABLE 1 OD Methionine Strain (660 nm) mg/l ATCC13032 12.3 1.4
ATCC13032/pCREmetH 14.3 5.3
BRIEF DESCRIPTION OF THE FIGURE
[0154] FIG. 1: Plasmid pCREmetH
[0155] The abbreviations used in the figures have the following
meaning:
4 Km: Resistance gene for kanamycin metH: metH gene of C.
glutamicum Ptac: tac promoter T1 T2: Terminator T1T2 of the rrnB
gene of E. coli rep: Plasmid-coded replication origin for C.
glutamicum (of pHM1519) EcoRI: Cleavage site of the restriction
enzyme EcoRI SalI: Cleavage site of the restriction enzyme SalI
[0156] This disclosure is based on priority documents DE 100 38
050.6, DE 101 09 687.9 and U.S. Pat. No. 60/294,251, each
incorporated by reference.
[0157] Obviously, numerous modifications of the invention are
possible in view of the above teachings. Therefore, within the
scope of the appended claims, the invention may be practiced
otherwise than as specifically described herein.
Sequence CWU 1
1
4 1 4301 DNA Corynebacterium glutamicum CDS (385)..(4047) 1
taagggtttt ggaggcattg gccgcgaacc catcgctggt catcccgggt ttgcgcatgc
60 cacgttcgta ttcataacca atcgcgatgc cttgagccca ccagccactg
acatcaaagt 120 tgtccacgat gtgctttgcg atgtgggtgt gagtccaaga
ggtggctttt acgtcgtcaa 180 gcaattttag ccactcttcc cacggctttc
cggtgccgtt gaggatagct tcaggggaca 240 tgcctggtgt tgagccttgc
ggagtggagt cagtcatgcg accgagacta gtggcgcttt 300 gcctgtgttg
cttaggcggc gttgaaaatg aactacgaat gaaaagttcg ggaattgtct 360
aatccgtact aagctgtcta caca atg tct act tca gtt act tca cca gcc 411
Met Ser Thr Ser Val Thr Ser Pro Ala 1 5 cac aac aac gca cat tcc tcc
gaa ttt ttg gat gcg ttg gca aac cat 459 His Asn Asn Ala His Ser Ser
Glu Phe Leu Asp Ala Leu Ala Asn His 10 15 20 25 gtg ttg atc ggc gac
ggc gcc atg ggc acc cag ctc caa ggc ttt gac 507 Val Leu Ile Gly Asp
Gly Ala Met Gly Thr Gln Leu Gln Gly Phe Asp 30 35 40 ctg gac gtg
gaa aag gat ttc ctt gat ctg gag ggg tgt aat gag att 555 Leu Asp Val
Glu Lys Asp Phe Leu Asp Leu Glu Gly Cys Asn Glu Ile 45 50 55 ctc
aac gac acc cgc cct gat gtg ttg agg cag att cac cgc gcc tac 603 Leu
Asn Asp Thr Arg Pro Asp Val Leu Arg Gln Ile His Arg Ala Tyr 60 65
70 ttt gag gcg gga gct gac ttg gtt gag acc aat act ttt ggt tgc aac
651 Phe Glu Ala Gly Ala Asp Leu Val Glu Thr Asn Thr Phe Gly Cys Asn
75 80 85 ctg ccg aac ttg gcg gat tat gac atc gct gat cgt tgc cgt
gag ctt 699 Leu Pro Asn Leu Ala Asp Tyr Asp Ile Ala Asp Arg Cys Arg
Glu Leu 90 95 100 105 gcc tac aag ggc act gca gtg gct agg gaa gtg
gct gat gag atg ggg 747 Ala Tyr Lys Gly Thr Ala Val Ala Arg Glu Val
Ala Asp Glu Met Gly 110 115 120 ccg ggc cga aac ggc atg cgg cgt ttc
gtg gtt ggt tcc ctg gga cct 795 Pro Gly Arg Asn Gly Met Arg Arg Phe
Val Val Gly Ser Leu Gly Pro 125 130 135 gga acg aag ctt cca tcg ctg
ggc cat gca ccg tat gca gat ttg cgt 843 Gly Thr Lys Leu Pro Ser Leu
Gly His Ala Pro Tyr Ala Asp Leu Arg 140 145 150 ggg cac tac aag gaa
gca gcg ctt ggc atc atc gac ggt ggt ggc gat 891 Gly His Tyr Lys Glu
Ala Ala Leu Gly Ile Ile Asp Gly Gly Gly Asp 155 160 165 gcc ttt ttg
att gag act gct cag gac ttg ctt cag gtc aag gct gcg 939 Ala Phe Leu
Ile Glu Thr Ala Gln Asp Leu Leu Gln Val Lys Ala Ala 170 175 180 185
gtt cac ggc gtt caa gat gcc atg gct gaa ctt gat aca ttc ttg ccc 987
Val His Gly Val Gln Asp Ala Met Ala Glu Leu Asp Thr Phe Leu Pro 190
195 200 att att tgc cac gtc acc gta gag acc acc ggc acc atg ctc atg
ggt 1035 Ile Ile Cys His Val Thr Val Glu Thr Thr Gly Thr Met Leu
Met Gly 205 210 215 tct gag atc ggt gcc gcg ttg aca gcg ctg cag cca
ctg ggt atc gac 1083 Ser Glu Ile Gly Ala Ala Leu Thr Ala Leu Gln
Pro Leu Gly Ile Asp 220 225 230 atg att ggt ctg aac tgc gcc acc ggc
cca gat gag atg agc gag cac 1131 Met Ile Gly Leu Asn Cys Ala Thr
Gly Pro Asp Glu Met Ser Glu His 235 240 245 ctg cgt tac ctg tcc aag
cac gcc gat att cct gtg tcg gtg atg cct 1179 Leu Arg Tyr Leu Ser
Lys His Ala Asp Ile Pro Val Ser Val Met Pro 250 255 260 265 aac gca
ggt ctt cct gtc ctg ggt aaa aac ggt gca gaa tac cca ctt 1227 Asn
Ala Gly Leu Pro Val Leu Gly Lys Asn Gly Ala Glu Tyr Pro Leu 270 275
280 gag gct gag gat ttg gcg cag gcg ctg gct gga ttc gtc tcc gaa tat
1275 Glu Ala Glu Asp Leu Ala Gln Ala Leu Ala Gly Phe Val Ser Glu
Tyr 285 290 295 ggc ctg tcc atg gtg ggt ggt tgt tgt ggc acc aca cct
gag cac atc 1323 Gly Leu Ser Met Val Gly Gly Cys Cys Gly Thr Thr
Pro Glu His Ile 300 305 310 cgt gcg gtc cgc gat gcg gtg gtt ggt gtt
cca gag cag gaa acc tcc 1371 Arg Ala Val Arg Asp Ala Val Val Gly
Val Pro Glu Gln Glu Thr Ser 315 320 325 aca ctg acc aag atc cct gca
ggc cct gtt gag cag gcc tcc cgc gag 1419 Thr Leu Thr Lys Ile Pro
Ala Gly Pro Val Glu Gln Ala Ser Arg Glu 330 335 340 345 gtg gag aaa
gag gac tcc gtc gcg tcg ctg tac acc tcg gtg cca ttg 1467 Val Glu
Lys Glu Asp Ser Val Ala Ser Leu Tyr Thr Ser Val Pro Leu 350 355 360
tcc cag gaa acc ggc att tcc atg atc ggt gag cgc acc aac tcc aac
1515 Ser Gln Glu Thr Gly Ile Ser Met Ile Gly Glu Arg Thr Asn Ser
Asn 365 370 375 ggt tcc aag gca ttc cgt gag gca atg ctg tct ggc gat
tgg gaa aag 1563 Gly Ser Lys Ala Phe Arg Glu Ala Met Leu Ser Gly
Asp Trp Glu Lys 380 385 390 tgt gtg gat att gcc aag cag caa acc cgc
gat ggt gca cac atg ctg 1611 Cys Val Asp Ile Ala Lys Gln Gln Thr
Arg Asp Gly Ala His Met Leu 395 400 405 gat ctt tgt gtg gat tac gtg
gga cga gac ggc acc gcc gat atg gcg 1659 Asp Leu Cys Val Asp Tyr
Val Gly Arg Asp Gly Thr Ala Asp Met Ala 410 415 420 425 acc ttg gca
gca ctt ctt gct acc agc tcc act ttg cca atc atg att 1707 Thr Leu
Ala Ala Leu Leu Ala Thr Ser Ser Thr Leu Pro Ile Met Ile 430 435 440
gac tcc acc gag cca gag gtt att cgc aca ggc ctt gag cac ttg ggt
1755 Asp Ser Thr Glu Pro Glu Val Ile Arg Thr Gly Leu Glu His Leu
Gly 445 450 455 gga cga agc atc gtt aac tcc gtc aac ttt gaa gac ggc
gat ggc cct 1803 Gly Arg Ser Ile Val Asn Ser Val Asn Phe Glu Asp
Gly Asp Gly Pro 460 465 470 gag tcc cgc tac cag cgc atc atg aaa ctg
gta aag cag cac ggt gcg 1851 Glu Ser Arg Tyr Gln Arg Ile Met Lys
Leu Val Lys Gln His Gly Ala 475 480 485 gcc gtg gtt gcg ctg acc att
gat gag gaa ggc cag gca cgt acc gct 1899 Ala Val Val Ala Leu Thr
Ile Asp Glu Glu Gly Gln Ala Arg Thr Ala 490 495 500 505 gag cac aag
gtg cgc att gct aaa cga ctg att gac gat atc acc ggc 1947 Glu His
Lys Val Arg Ile Ala Lys Arg Leu Ile Asp Asp Ile Thr Gly 510 515 520
agc tac ggc ctg gat atc aaa gac atc gtt gtg gac tgc ctg acc ttc
1995 Ser Tyr Gly Leu Asp Ile Lys Asp Ile Val Val Asp Cys Leu Thr
Phe 525 530 535 ccg atc tct act ggc cag gaa gaa acc agg cga gat ggc
att gaa acc 2043 Pro Ile Ser Thr Gly Gln Glu Glu Thr Arg Arg Asp
Gly Ile Glu Thr 540 545 550 atc gaa gcc atc cgc gag ctg aag aag ctc
tac cca gaa atc cac acc 2091 Ile Glu Ala Ile Arg Glu Leu Lys Lys
Leu Tyr Pro Glu Ile His Thr 555 560 565 acc ctg ggt ctg tcc aat att
tcc ttc ggc ctg aac cct gct gca cgc 2139 Thr Leu Gly Leu Ser Asn
Ile Ser Phe Gly Leu Asn Pro Ala Ala Arg 570 575 580 585 cag gtt ctt
aac tct gtg ttc ctc aat gag tgc att gag gct ggt ctg 2187 Gln Val
Leu Asn Ser Val Phe Leu Asn Glu Cys Ile Glu Ala Gly Leu 590 595 600
gac tct gcg att gcg cac agc tcc aag att ttg ccg atg aac cgc att
2235 Asp Ser Ala Ile Ala His Ser Ser Lys Ile Leu Pro Met Asn Arg
Ile 605 610 615 gat gat cgc cag cgc gaa gtg gcg ttg gat atg gtc tat
gat cgc cgc 2283 Asp Asp Arg Gln Arg Glu Val Ala Leu Asp Met Val
Tyr Asp Arg Arg 620 625 630 acc gag gat tac gat ccg ctg cag gaa ttc
atg cag ctg ttt gag ggc 2331 Thr Glu Asp Tyr Asp Pro Leu Gln Glu
Phe Met Gln Leu Phe Glu Gly 635 640 645 gtt tct gct gcc gat gcc aag
gat gct cgc gct gaa cag ctg gcc gct 2379 Val Ser Ala Ala Asp Ala
Lys Asp Ala Arg Ala Glu Gln Leu Ala Ala 650 655 660 665 atg cct ttg
ttt gag cgt ttg gca cag cgc atc atc gac ggc gat aag 2427 Met Pro
Leu Phe Glu Arg Leu Ala Gln Arg Ile Ile Asp Gly Asp Lys 670 675 680
aat ggc ctt gag gat gat ctg gaa gca ggc atg aag gag aag tct cct
2475 Asn Gly Leu Glu Asp Asp Leu Glu Ala Gly Met Lys Glu Lys Ser
Pro 685 690 695 att gcg atc atc aac gag gac ctt ctc aac ggc atg aag
acc gtg ggt 2523 Ile Ala Ile Ile Asn Glu Asp Leu Leu Asn Gly Met
Lys Thr Val Gly 700 705 710 gag ctg ttt ggt tcc gga cag atg cag ctg
cca ttc gtg ctg caa tcg 2571 Glu Leu Phe Gly Ser Gly Gln Met Gln
Leu Pro Phe Val Leu Gln Ser 715 720 725 gca gaa acc atg aaa act gcg
gtg gcc tat ttg gaa ccg ttc atg gaa 2619 Ala Glu Thr Met Lys Thr
Ala Val Ala Tyr Leu Glu Pro Phe Met Glu 730 735 740 745 gag gaa gca
gaa gct acc gga tct gcg cag gca gag ggc aag ggc aaa 2667 Glu Glu
Ala Glu Ala Thr Gly Ser Ala Gln Ala Glu Gly Lys Gly Lys 750 755 760
atc gtc gtg gcc acc gtc aag ggt gac gtg cac gat atc ggc aag aac
2715 Ile Val Val Ala Thr Val Lys Gly Asp Val His Asp Ile Gly Lys
Asn 765 770 775 ttg gtg gac atc att ttg tcc aac aac ggt tac gac gtg
gtg aac ttg 2763 Leu Val Asp Ile Ile Leu Ser Asn Asn Gly Tyr Asp
Val Val Asn Leu 780 785 790 ggc atc aag cag cca ctg tcc gcc atg ttg
gaa gca gcg gaa gaa cac 2811 Gly Ile Lys Gln Pro Leu Ser Ala Met
Leu Glu Ala Ala Glu Glu His 795 800 805 aaa gca gac gtc atc ggc atg
tcg gga ctt ctt gtg aag tcc acc gtg 2859 Lys Ala Asp Val Ile Gly
Met Ser Gly Leu Leu Val Lys Ser Thr Val 810 815 820 825 gtg atg aag
gaa aac ctt gag gag atg aac aac gcc ggc gca tcc aat 2907 Val Met
Lys Glu Asn Leu Glu Glu Met Asn Asn Ala Gly Ala Ser Asn 830 835 840
tac cca gtc att ttg ggt ggc gct gcg ctg acg cgt acc tac gtg gaa
2955 Tyr Pro Val Ile Leu Gly Gly Ala Ala Leu Thr Arg Thr Tyr Val
Glu 845 850 855 aac gat ctc aac gag gtg tac acc ggt gag gtg tac tac
gcc cgt gat 3003 Asn Asp Leu Asn Glu Val Tyr Thr Gly Glu Val Tyr
Tyr Ala Arg Asp 860 865 870 gct ttc gag ggc ctg cgc ctg atg gat gag
gtg atg gca gaa aag cgt 3051 Ala Phe Glu Gly Leu Arg Leu Met Asp
Glu Val Met Ala Glu Lys Arg 875 880 885 ggt gaa gga ctt gat ccc aac
tca cca gaa gct att gag cag gcg aag 3099 Gly Glu Gly Leu Asp Pro
Asn Ser Pro Glu Ala Ile Glu Gln Ala Lys 890 895 900 905 aag aag gcg
gaa cgt aag gct cgt aat gag cgt tcc cgc aag att gcc 3147 Lys Lys
Ala Glu Arg Lys Ala Arg Asn Glu Arg Ser Arg Lys Ile Ala 910 915 920
gcg gag cgt aaa gct aat gcg gct ccc gtg att gtt ccg gag cgt tct
3195 Ala Glu Arg Lys Ala Asn Ala Ala Pro Val Ile Val Pro Glu Arg
Ser 925 930 935 gat gtc tcc acc gat act cca acc gcg gca cca ccg ttc
tgg gga acc 3243 Asp Val Ser Thr Asp Thr Pro Thr Ala Ala Pro Pro
Phe Trp Gly Thr 940 945 950 cgc att gtc aag ggt ctg ccc ttg gcg gag
ttc ttg ggc aac ctt gat 3291 Arg Ile Val Lys Gly Leu Pro Leu Ala
Glu Phe Leu Gly Asn Leu Asp 955 960 965 gag cgc gcc ttg ttc atg ggg
cag tgg ggt ctg aaa tcc acc cgc ggc 3339 Glu Arg Ala Leu Phe Met
Gly Gln Trp Gly Leu Lys Ser Thr Arg Gly 970 975 980 985 aac gag ggt
cca agc tat gag gat ttg gtg gaa act gaa ggc cga cca 3387 Asn Glu
Gly Pro Ser Tyr Glu Asp Leu Val Glu Thr Glu Gly Arg Pro 990 995
1000 cgc ctg cgc tac tgg ctg gat cgc ctg aag tct gag ggc att ttg
3432 Arg Leu Arg Tyr Trp Leu Asp Arg Leu Lys Ser Glu Gly Ile Leu
1005 1010 1015 gac cac gtg gcc ttg gtg tat ggc tac ttc cca gcg gtc
gcg gaa 3477 Asp His Val Ala Leu Val Tyr Gly Tyr Phe Pro Ala Val
Ala Glu 1020 1025 1030 ggc gat gac gtg gtg atc ttg gaa tcc ccg gat
cca cac gca gcc 3522 Gly Asp Asp Val Val Ile Leu Glu Ser Pro Asp
Pro His Ala Ala 1035 1040 1045 gaa cgc atg cgc ttt agc ttc cca cgc
cag cag cgc ggc agg ttc 3567 Glu Arg Met Arg Phe Ser Phe Pro Arg
Gln Gln Arg Gly Arg Phe 1050 1055 1060 ttg tgc atc gcg gat ttc att
cgc cca cgc gag caa gct gtc aag 3612 Leu Cys Ile Ala Asp Phe Ile
Arg Pro Arg Glu Gln Ala Val Lys 1065 1070 1075 gac ggc caa gtg gac
gtc atg cca ttc cag ctg gtc acc atg ggt 3657 Asp Gly Gln Val Asp
Val Met Pro Phe Gln Leu Val Thr Met Gly 1080 1085 1090 aat cct att
gct gat ttc gcc aac gag ttg ttc gca gcc aat gaa 3702 Asn Pro Ile
Ala Asp Phe Ala Asn Glu Leu Phe Ala Ala Asn Glu 1095 1100 1105 tac
cgc gag tac ttg gaa gtt cac ggc atc ggc gtg cag ctc acc 3747 Tyr
Arg Glu Tyr Leu Glu Val His Gly Ile Gly Val Gln Leu Thr 1110 1115
1120 gaa gca ttg gcc gag tac tgg cac tcc cga gtg cgc agc gaa ctc
3792 Glu Ala Leu Ala Glu Tyr Trp His Ser Arg Val Arg Ser Glu Leu
1125 1130 1135 aag ctg aac gac ggt gga tct gtc gct gat ttt gat cca
gaa gac 3837 Lys Leu Asn Asp Gly Gly Ser Val Ala Asp Phe Asp Pro
Glu Asp 1140 1145 1150 aag acc aag ttc ttc gac ctg gat tac cgc ggc
gcc cgc ttc tcc 3882 Lys Thr Lys Phe Phe Asp Leu Asp Tyr Arg Gly
Ala Arg Phe Ser 1155 1160 1165 ttt ggt tac ggt tct tgc cct gat ctg
gaa gac cgc gca aag ctg 3927 Phe Gly Tyr Gly Ser Cys Pro Asp Leu
Glu Asp Arg Ala Lys Leu 1170 1175 1180 gtg gaa ttg ctc gag cca ggc
cgt atc ggc gtg gag ttg tcc gag 3972 Val Glu Leu Leu Glu Pro Gly
Arg Ile Gly Val Glu Leu Ser Glu 1185 1190 1195 gaa ctc cag ctg cac
cca gag cag tcc aca gac gcg ttt gtg ctc 4017 Glu Leu Gln Leu His
Pro Glu Gln Ser Thr Asp Ala Phe Val Leu 1200 1205 1210 tac cac cca
gag gca aag tac ttt aac gtc taacaccttt gagagggaaa 4067 Tyr His Pro
Glu Ala Lys Tyr Phe Asn Val 1215 1220 actttcccgc acattgcaga
tcgtgccact ttaactaagg ttgacggcat gattaaggcg 4127 attttctggg
acatggacgg cacgatggtg gactctgagc cacagtgggg cattgctacc 4187
tacgagctca gcgaagccat gggccgccgc ctcaccccgg agctccggga actcaccgtc
4247 ggctcgagcc tgccgcgcac catgcgctta tgcgcagagc acgcaggcat taca
4301 2 1221 PRT Corynebacterium glutamicum 2 Met Ser Thr Ser Val
Thr Ser Pro Ala His Asn Asn Ala His Ser Ser 1 5 10 15 Glu Phe Leu
Asp Ala Leu Ala Asn His Val Leu Ile Gly Asp Gly Ala 20 25 30 Met
Gly Thr Gln Leu Gln Gly Phe Asp Leu Asp Val Glu Lys Asp Phe 35 40
45 Leu Asp Leu Glu Gly Cys Asn Glu Ile Leu Asn Asp Thr Arg Pro Asp
50 55 60 Val Leu Arg Gln Ile His Arg Ala Tyr Phe Glu Ala Gly Ala
Asp Leu 65 70 75 80 Val Glu Thr Asn Thr Phe Gly Cys Asn Leu Pro Asn
Leu Ala Asp Tyr 85 90 95 Asp Ile Ala Asp Arg Cys Arg Glu Leu Ala
Tyr Lys Gly Thr Ala Val 100 105 110 Ala Arg Glu Val Ala Asp Glu Met
Gly Pro Gly Arg Asn Gly Met Arg 115 120 125 Arg Phe Val Val Gly Ser
Leu Gly Pro Gly Thr Lys Leu Pro Ser Leu 130 135 140 Gly His Ala Pro
Tyr Ala Asp Leu Arg Gly His Tyr Lys Glu Ala Ala 145 150 155 160 Leu
Gly Ile Ile Asp Gly Gly Gly Asp Ala Phe Leu Ile Glu Thr Ala 165 170
175 Gln Asp Leu Leu Gln Val Lys Ala Ala Val His Gly Val Gln Asp Ala
180 185 190 Met Ala Glu Leu Asp Thr Phe Leu Pro Ile Ile Cys His Val
Thr Val 195 200 205 Glu Thr Thr Gly Thr Met Leu Met Gly Ser Glu Ile
Gly Ala Ala Leu 210 215 220 Thr Ala Leu Gln Pro Leu Gly Ile Asp Met
Ile Gly Leu Asn Cys Ala 225 230 235 240 Thr Gly Pro Asp Glu Met Ser
Glu His Leu Arg Tyr Leu Ser Lys His 245 250 255 Ala Asp Ile Pro Val
Ser Val Met Pro Asn Ala Gly Leu Pro Val Leu 260 265 270 Gly Lys Asn
Gly Ala Glu Tyr Pro Leu Glu Ala Glu Asp Leu Ala Gln 275 280 285 Ala
Leu Ala Gly Phe Val Ser Glu Tyr Gly Leu Ser Met Val Gly Gly 290 295
300 Cys Cys Gly Thr Thr Pro Glu His Ile Arg Ala Val Arg Asp Ala Val
305 310 315 320 Val Gly Val Pro Glu Gln Glu Thr Ser Thr Leu Thr Lys
Ile Pro Ala
325 330 335 Gly Pro Val Glu Gln Ala Ser Arg Glu Val Glu Lys Glu Asp
Ser Val 340 345 350 Ala Ser Leu Tyr Thr Ser Val Pro Leu Ser Gln Glu
Thr Gly Ile Ser 355 360 365 Met Ile Gly Glu Arg Thr Asn Ser Asn Gly
Ser Lys Ala Phe Arg Glu 370 375 380 Ala Met Leu Ser Gly Asp Trp Glu
Lys Cys Val Asp Ile Ala Lys Gln 385 390 395 400 Gln Thr Arg Asp Gly
Ala His Met Leu Asp Leu Cys Val Asp Tyr Val 405 410 415 Gly Arg Asp
Gly Thr Ala Asp Met Ala Thr Leu Ala Ala Leu Leu Ala 420 425 430 Thr
Ser Ser Thr Leu Pro Ile Met Ile Asp Ser Thr Glu Pro Glu Val 435 440
445 Ile Arg Thr Gly Leu Glu His Leu Gly Gly Arg Ser Ile Val Asn Ser
450 455 460 Val Asn Phe Glu Asp Gly Asp Gly Pro Glu Ser Arg Tyr Gln
Arg Ile 465 470 475 480 Met Lys Leu Val Lys Gln His Gly Ala Ala Val
Val Ala Leu Thr Ile 485 490 495 Asp Glu Glu Gly Gln Ala Arg Thr Ala
Glu His Lys Val Arg Ile Ala 500 505 510 Lys Arg Leu Ile Asp Asp Ile
Thr Gly Ser Tyr Gly Leu Asp Ile Lys 515 520 525 Asp Ile Val Val Asp
Cys Leu Thr Phe Pro Ile Ser Thr Gly Gln Glu 530 535 540 Glu Thr Arg
Arg Asp Gly Ile Glu Thr Ile Glu Ala Ile Arg Glu Leu 545 550 555 560
Lys Lys Leu Tyr Pro Glu Ile His Thr Thr Leu Gly Leu Ser Asn Ile 565
570 575 Ser Phe Gly Leu Asn Pro Ala Ala Arg Gln Val Leu Asn Ser Val
Phe 580 585 590 Leu Asn Glu Cys Ile Glu Ala Gly Leu Asp Ser Ala Ile
Ala His Ser 595 600 605 Ser Lys Ile Leu Pro Met Asn Arg Ile Asp Asp
Arg Gln Arg Glu Val 610 615 620 Ala Leu Asp Met Val Tyr Asp Arg Arg
Thr Glu Asp Tyr Asp Pro Leu 625 630 635 640 Gln Glu Phe Met Gln Leu
Phe Glu Gly Val Ser Ala Ala Asp Ala Lys 645 650 655 Asp Ala Arg Ala
Glu Gln Leu Ala Ala Met Pro Leu Phe Glu Arg Leu 660 665 670 Ala Gln
Arg Ile Ile Asp Gly Asp Lys Asn Gly Leu Glu Asp Asp Leu 675 680 685
Glu Ala Gly Met Lys Glu Lys Ser Pro Ile Ala Ile Ile Asn Glu Asp 690
695 700 Leu Leu Asn Gly Met Lys Thr Val Gly Glu Leu Phe Gly Ser Gly
Gln 705 710 715 720 Met Gln Leu Pro Phe Val Leu Gln Ser Ala Glu Thr
Met Lys Thr Ala 725 730 735 Val Ala Tyr Leu Glu Pro Phe Met Glu Glu
Glu Ala Glu Ala Thr Gly 740 745 750 Ser Ala Gln Ala Glu Gly Lys Gly
Lys Ile Val Val Ala Thr Val Lys 755 760 765 Gly Asp Val His Asp Ile
Gly Lys Asn Leu Val Asp Ile Ile Leu Ser 770 775 780 Asn Asn Gly Tyr
Asp Val Val Asn Leu Gly Ile Lys Gln Pro Leu Ser 785 790 795 800 Ala
Met Leu Glu Ala Ala Glu Glu His Lys Ala Asp Val Ile Gly Met 805 810
815 Ser Gly Leu Leu Val Lys Ser Thr Val Val Met Lys Glu Asn Leu Glu
820 825 830 Glu Met Asn Asn Ala Gly Ala Ser Asn Tyr Pro Val Ile Leu
Gly Gly 835 840 845 Ala Ala Leu Thr Arg Thr Tyr Val Glu Asn Asp Leu
Asn Glu Val Tyr 850 855 860 Thr Gly Glu Val Tyr Tyr Ala Arg Asp Ala
Phe Glu Gly Leu Arg Leu 865 870 875 880 Met Asp Glu Val Met Ala Glu
Lys Arg Gly Glu Gly Leu Asp Pro Asn 885 890 895 Ser Pro Glu Ala Ile
Glu Gln Ala Lys Lys Lys Ala Glu Arg Lys Ala 900 905 910 Arg Asn Glu
Arg Ser Arg Lys Ile Ala Ala Glu Arg Lys Ala Asn Ala 915 920 925 Ala
Pro Val Ile Val Pro Glu Arg Ser Asp Val Ser Thr Asp Thr Pro 930 935
940 Thr Ala Ala Pro Pro Phe Trp Gly Thr Arg Ile Val Lys Gly Leu Pro
945 950 955 960 Leu Ala Glu Phe Leu Gly Asn Leu Asp Glu Arg Ala Leu
Phe Met Gly 965 970 975 Gln Trp Gly Leu Lys Ser Thr Arg Gly Asn Glu
Gly Pro Ser Tyr Glu 980 985 990 Asp Leu Val Glu Thr Glu Gly Arg Pro
Arg Leu Arg Tyr Trp Leu Asp 995 1000 1005 Arg Leu Lys Ser Glu Gly
Ile Leu Asp His Val Ala Leu Val Tyr 1010 1015 1020 Gly Tyr Phe Pro
Ala Val Ala Glu Gly Asp Asp Val Val Ile Leu 1025 1030 1035 Glu Ser
Pro Asp Pro His Ala Ala Glu Arg Met Arg Phe Ser Phe 1040 1045 1050
Pro Arg Gln Gln Arg Gly Arg Phe Leu Cys Ile Ala Asp Phe Ile 1055
1060 1065 Arg Pro Arg Glu Gln Ala Val Lys Asp Gly Gln Val Asp Val
Met 1070 1075 1080 Pro Phe Gln Leu Val Thr Met Gly Asn Pro Ile Ala
Asp Phe Ala 1085 1090 1095 Asn Glu Leu Phe Ala Ala Asn Glu Tyr Arg
Glu Tyr Leu Glu Val 1100 1105 1110 His Gly Ile Gly Val Gln Leu Thr
Glu Ala Leu Ala Glu Tyr Trp 1115 1120 1125 His Ser Arg Val Arg Ser
Glu Leu Lys Leu Asn Asp Gly Gly Ser 1130 1135 1140 Val Ala Asp Phe
Asp Pro Glu Asp Lys Thr Lys Phe Phe Asp Leu 1145 1150 1155 Asp Tyr
Arg Gly Ala Arg Phe Ser Phe Gly Tyr Gly Ser Cys Pro 1160 1165 1170
Asp Leu Glu Asp Arg Ala Lys Leu Val Glu Leu Leu Glu Pro Gly 1175
1180 1185 Arg Ile Gly Val Glu Leu Ser Glu Glu Leu Gln Leu His Pro
Glu 1190 1195 1200 Gln Ser Thr Asp Ala Phe Val Leu Tyr His Pro Glu
Ala Lys Tyr 1205 1210 1215 Phe Asn Val 1220 3 52 DNA Artificial
sequence Synthetic DNA 3 gatctaagat ctaaaggagg acaaccatgt
ctacttcagt tacttcacca gc 52 4 32 DNA Artificial sequence Synthetic
DNA 4 gatctagtcg acccctctca aaggtgttag ac 32
* * * * *