U.S. patent application number 09/725898 was filed with the patent office on 2002-04-04 for nucleotide sequences encoding the glk-gene.
Invention is credited to Mockel, Bettina, Pfefferle, Walter.
Application Number | 20020040129 09/725898 |
Document ID | / |
Family ID | 7931209 |
Filed Date | 2002-04-04 |
United States Patent
Application |
20020040129 |
Kind Code |
A1 |
Mockel, Bettina ; et
al. |
April 4, 2002 |
Nucleotide sequences encoding the glk-gene
Abstract
The invention relates to an isolated polynucleotide containing a
polynucleotide sequence selected from the group comprising: a) a
polynucleotide that is at least 70% identical to a polynucleotide
encoding a polypeptide that contains the amino acid sequence of SEQ
ID NO:2, b) a polynucleotide encoding a polypeptide that contains
an amino acid sequence that is at least 70% identical to the amino
acid sequence of SEQ ID NO:2, c) a polynucleotide that is
complementary to the polynucleotides of a) or b), and d) a
polynucleotide containing at least 15 successive bases of the
polynucleotide sequence of a), b) or c), and processes for the
fermentative production of L-amino acids by enhancement of the
glk-gene coding for the enzyme glucokinase.
Inventors: |
Mockel, Bettina;
(Dusseldorf, DE) ; Pfefferle, Walter; (Halle,
DE) |
Correspondence
Address: |
PILLSBURY WINTHROP LLP
9TH FLOOR
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Family ID: |
7931209 |
Appl. No.: |
09/725898 |
Filed: |
November 30, 2000 |
Current U.S.
Class: |
536/23.1 ;
435/106 |
Current CPC
Class: |
C12P 13/04 20130101;
C12P 13/08 20130101; C12N 9/1205 20130101 |
Class at
Publication: |
536/23.1 ;
435/106 |
International
Class: |
C07H 021/02; C07H
021/04; C12P 013/04 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 2, 1999 |
DE |
199 58 159.2 |
Claims
What is claimed is:
1. An isolated polynucleotide obtained from coryneform bacteria,
containing a polynucleotide sequence selected from the group
consisting of a) a polynucleotide that is at least 70% identical to
a polynucleotide coding for a polypeptide that contains the amino
acid sequence of SEQ ID NO:2, b) a polynucleotide encoding a
polypeptide, that contains an amino acid sequence that is at least
70% identical to the amino acid sequence of SEQ ID NO:2, c) a
polynucleotide that is complementary to the polynucleotides of a)
or b), and d) a polynucleotide containing at least 15 successive
nucleotides of the polynucleotide sequence of a), b) or c).
2. The polynucleotide according to claim 1, wherein the
polynucleotide is a DNA replicable in coryneform bacteria.
3. The polynucleotide according to claim 2 that is a recombinant
DNA.
4. The polynucleotide according to claim 1, wherein the
polynucleotide is an RNA.
5. The replicable DNA according to claim 2, containing (i) the
nucleotide sequence shown in SEQ ID NO:1, or (ii) at least one
sequence that corresponds to the sequence (i) within the region of
degeneracy of the genetic code, or (iii) at least one sequence that
hybridises with the sequence that is complementary to the sequence
(i) or (ii), and optionally (iv) functionally neutral sense
mutations in (i).
6. The polynucleotide sequence according to claim 2, encoding a
polypeptide that contains the amino acid sequence shown in SEQ ID
NO:2.
7. A vector containing a polynucleotide sequence according to claim
1.
8. Coryneform bacteria containing a vector according to claim
7.
9. A process for the fermentative production of L-amino acids,
comprising the following steps: a) fermentation of the coryneform
bacteria producing the L-amino acid, in which at least the gene
coding for the enzyme glucokinase is enhanced, b) enrichment of the
L-amino acid in the medium or in the cells of the bacteria, and c)
isolation of the L-amino acid.
10. The process according to claim 9 in which the gene is enhanced
by overexpression.
11. The process according to claim 9, wherein bacteria are used in
which, additionally, further genes of the biosynthesis pathway of
the desired L-amino acid are enhanced.
12. The process according to claim 9, wherein bacteria are used in
which the metabolic pathways that reduce the formation of the
L-amino acid are at least partially switched off.
13. The process according to claim 9, wherein a strain transformed
with a plasmid vector is used, and said plasmid vector carries the
nucleotide sequence of the gene encoding the enzyme
glucokinase.
14. The process according to one of claims 9 to 13, wherein
coryneform bacteria are used that produce L-lysine.
15. The process according to claim 11, wherein the dapA-gene
encoding dihydrodipicolinate synthase is simultaneously
overexpressed.
16. The process according to claim 11, wherein a lysC-gene encoding
a feedback-resistant aspartate kinase is simultaneously
overexpressed.
17. The process according to claim 11, wherein the gap-gene
encoding gyceraldehyde-3-phosphate dehydrogenase is simultaneously
overexpressed.
18. The process according to claim 11, wherein the tpi-gene
encoding triosephosphate isomerase is simultaneously
overexpressed.
19. The process according to claim 11, wherein the pgk-gene
encoding 3-phosphate glycerate kinase is simultaneously
overexpressed.
20. The process according to claim 11, wherein the pyc-gene coding
for pyruvate carboxylase is simultaneously overexpressed.
21. The process according to claim 11, wherein the mqo-gene
encoding malate-quinone oxidoreductase is simultaneously
overexpressed.
22. The process according to claim 11, wherein the lysE-gene
encoding lysine export is simultaneously overexpressed.
23. The process according to claim 12, wherein the pgi-gene
encoding glucose-6-phosphate isomerase is simultaneously
attenuated.
24. The process according to claim 12, wherein the pck-gene
encoding phosphoenolpyruvate carboxykinase is simultaneously
attenuated.
Description
[0001] This application claims priority from German Application No.
199 58 159.2, filed on Dec. 2, 1999, the subject matter of which is
hereby incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention provides nucleotide sequences encoding the
glk-gene and processes for the fermentative production of L amino
acids, in particular L-lysine, using coryneform bacteria in which
the glk-gene is enhanced.
[0004] 2. Background Information
[0005] L-amino acids, in particular L-lysine, are used in human
medicine and in the pharmaceutical industry, but especially in
animal nutrition.
[0006] It is known that L-amino acids can be produced by
fermentation of strains of coryneform bacteria, in particular
Corynebacterium glutamicum. On account of the great importance of
amino acids efforts are constantly being made to improve the
production processes. Improvements in production processes may
involve fermentation technology measures, such as for example
stirring and provision of oxygen, or the composition of the
nutrient media, such as for example the sugar concentration during
fermentation, or the working-up to the product form by for example
ion exchange chromatography, or the intrinsic output properties of
the microorganism itself.
[0007] Methods involving mutagenesis, selection and choice of
mutants are used to improve the output properties of these
microorganisms. In this way strains are obtained that are resistant
to antimetabolites, such as for example the lysine-analogon
S-(2-aminoethyl)-cysteine or that are auxotrophic for regulatorily
important metabolites and produce L-amino acids such as for example
L-lysine.
[0008] For some years recombinant DNA technology methods have also
been used to improve strains of Corynebacterium producing L-amino
acids, by amplifying individual biosynthesis genes for L-amino
acids and investigating the effect on the production of L-amino
acids. Review articles on this topic may be found in, inter alia,
Kinoshita ("Glutamic Acid Bacteria", in: Biology of Industrial
Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings,
London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)),
Eggeling (Amino Acids 6:261-272 (1994)), Jetten and Sinskey
(Critical Reviews in Biotechnology 15, 73-103 (1995)) and Sahm et
al. (Annuals of the New York Academy of Science 782, 25-39
(1996)).
SUMMARY OF THE INVENTION
OBJECT OF THE INVENTION
[0009] It is an object of the invention to provide new means for
improving the fermentative production of L-amino acids, in
particular L-lysine.
DESCRIPTION OF THE INVENTION
[0010] L-amino acids, in particular L-lysine, are used in human
medicine, in the pharmaceutical industry and in particular in
animal nutrition. It is therefore of general interest to provide
new improved processes for the production of L-amino acids, in
particular L-lysine.
[0011] Wherever L-lysine or lysine are mentioned hereinafter, this
should be understood to mean not only the bases per se but also the
salts, for example lysine monohydrochloride or lysine sulfate.
[0012] The invention provides an isolated polynucleotide obtained
from coryneform bacteria, containing a polynucleotide sequence
selected from the following group
[0013] a) a polynucleotide that is at least 70% identical to a
polynucleotide encoding a polypeptide that contains the amino acid
sequence of SEQ ID NO:2,
[0014] b) a polynucleotide encoding a polypeptide, that contains an
amino acid sequence that is at least 70% identical to the amino
acid sequence of SEQ ID NO:2,
[0015] c) a polynucleotide that is complementary to the
polynucleotides of a) or b), and
[0016] d) a polynucleotide containing at least 15 successive
nucleotides of the polynucleotide sequence of a), b) or c).
[0017] The invention also provides a polynucleotide that is
preferably a recombinant DNA replicable in coryneform bacteria.
[0018] The invention likewise provides a polynucleotide that is an
RNA.
[0019] The invention moreover provides a polynucleotide with the
aforementioned features which polynucleotide is preferably a
replicable DNA, containing: (i)
[0020] the nucleotide sequence shown in SEQ ID NO:1, or
[0021] (ii) at least one sequence that corresponds to the sequence
(i) within the region of degeneration of the genetic code, or
[0022] (iii) at least one sequence that hybridises with the
sequence that is complementary to the sequence (i) or (ii), and
optionally
[0023] (iv) functionally neutral sense mutations in (i).
[0024] The invention in addition provides:
[0025] a vector containing a polypeptide with features described
above, and coryneform bacteria serving as host cell that contain
the said vector.
[0026] The invention moreover provides polynucleotides that
substantially comprise a polynucleotide sequence, that can be
obtained by screening by means of hybridisation of a corresponding
gene library that contains the complete gene with the
polynucleotide sequence corresponding to SEQ ID NO:1 with a probe
that contains the sequence of the aforementioned polynucleotide
according to SEQ ID NO:1 or a fragment thereof, and isolation of
the aforementioned DNA sequence.
[0027] Polynucleotide sequences according to the invention are
suitable as hybridisation probes for RNA, cDNA and DNA in order to
isolate in full length cDNA that code for glucokinase and to
isolate such cDNA or genes that have a high degree of similarity to
the sequence of the glucokinase gene.
[0028] Polynucleotide sequences according to the invention are
furthermore suitable as primers for producing DNA of genes that
code for glucokinase, by the polymerase chain reaction (PCR).
[0029] Such oligonucleotides serving as probes or primers contain
at least 30, preferably at least 20, and most particularly
preferably at least 15 successive bases. Oligonucleotides with a
length of at least 40 or 50 nucleotides are also suitable.
[0030] "Isolated" means separated from its natural environment.
[0031] "Polynucleotide" refers in general to polyribonucleotides
and polydeoxyribonucleotides, in which connection these terms may
refer to unmodified RNA or DNA or modified RNA or DNA.
[0032] By the term "polypeptides" are understood peptides or
proteins that contain two or more amino acids bound via peptide
bonds.
[0033] The polypeptides according to the invention include a
polypeptide according to SEQ ID NO:2, in particular those having
the biological activity of glucokinase as well as those that are at
least 70% identical to the polypeptide according to SEQ ID NO:2,
preferably at least 80% and particularly at least 90% to 95%
identical to the polypeptide according to SEQ ID NO:2 and that have
the aforementioned activity.
[0034] The invention furthermore relates to a process for the
fermentative production of L-amino acids, in particular L-lysine,
using coryneform bacteria that in particular already produce an
L-amino acid, and in which the nucleotide sequences coding for the
glk-gene are enhanced, in particular are overexpressed.
[0035] The term "enhancement" describes in this connection
increasing the intracellular activity of one or more enzymes in a
microorganism that are coded by the corresponding DNA, by for
example increasing the number of copies of the gene and/or genes,
using a strong promoter or using a gene that codes for a
corresponding enzyme having a high activity, and optionally
combining these measures.
[0036] The microorganisms that are the subject of the present
invention can produce amino acids, in particular L-lysine, from
glucose, sucrose, lactose, fructose, maltose, molasses, starch,
cellulose or from glycerol and ethanol. The microorganisms may be
types of coryneform bacteria, in particular of the genus
Corynebacterium. In the genus Corynebacterium there should in
particular be mentioned the type Corynebacterium glutamicum, which
is known to those in the specialist field for its ability to
produce L-amino acids.
[0037] Suitable strains of the genus Corynebacterium, in particular
of the type Corynebacterium glutamicum, are for example the
following known wild type strains:
[0038] Corynebacterium glutamicum ATCC13032
[0039] Corynebacterium acetoglutamicum ATCC15806
[0040] Corynebacterium acetoacidophilum ATCC13870
[0041] Corynebacterium thermoaminogenes FERM BP-1539
[0042] Corynebacterium melassecola ATCC17965
[0043] Brevibacterium flavum ATCC14067
[0044] Brevibacterium lactofermentum ATCC13869 and
[0045] Brevibacterium divaricatum ATCC14020
[0046] and mutants and/or strains obtained therefrom that produce
L-amino acids, such as for example
[0047] Corynebacterium glutamicum FERM-P 1709
[0048] Brevibacterium flavum FERM-P 1708
[0049] Brevibacterium lactofermentum FERM-P 1712
[0050] Corynebacterium glutamicum FERM-P 6463
[0051] Corynebacterium glutamicum FERM-P 6464 and
[0052] Corynebacterium glutamicum DSM5715.
[0053] The inventors have succeeded in isolating the new glk-gene
from C. glutamicum coding for the enzyme glucokinase (EC
2.7.1.2).
[0054] In order to isolate the glk-gene or also other genes from C.
glutamicum, a gene library of this microorganism is first of all
cultivated in E. coli. The cultivation of gene libraries is
described in generally known textbooks and handbooks. By way of
example there may be mentioned the textbook by Winnacker: Gene und
Klone, Eine Einfuthrung in die Gentechnologie (Genes and Clones, An
Introduction to Gene Technology) (Verlag Chemie, Weinheim, Germany,
1990) or the handbook by Sambrook et al.: Molecular Cloning, A
Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A
very well-known gene library is that of the E. coli K-12 strain
W3110, which has been cultivated by Kohara et al. (Cell 50, 495-508
(1987)) in k-vectors. Bathe et al. (Molecular and General Genetics,
252:255-265, 1996) describe a gene library from C. glutamicum
ATCC13032 that has been cultivated 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).
Bormann et al. (Molecular Microbiology 6(3), 317-326 (1992)) in
turn describe a gene library obtained from C. glutamicum ATCC13032
using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)).
In order to produce a gene library from C. glutamicum in E. coli,
plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818
(1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268) may also be
used. Particularly suitable as hosts are those E. coli strains that
are restriction-defective and recombinant-defective. An example of
such strains is the strain DH5.alpha.mcr that 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 may then be sub-cloned in turn in suitable vectors
available for the sequencing and finally sequenced, such as is
described for example by Sanger et al. (Proceedings of the National
Academy of Sciences of the United States of America, 74:5463-5467,
1977).
[0055] In this way the new DNA sequence of C. glutamicum encoding
the gene glk was obtained which, as SEQ ID NO:1, is a subject of
the present invention. Furthermore the amino acid sequence of the
corresponding protein was derived from the present DNA sequence
using the methods described above. The amino acid sequence of the
glk-gene product that is obtained is shown in SEQ ID NO:2.
[0056] Coding DNA sequences that are obtained from SEQ ID NO:1 due
to the degeneracy of the genetic code are likewise included in the
invention. Conservative amino acid exchanges, such as, for example,
the exchange of glycine by alanine or of aspartic acid by glutamic
acid in proteins are known in the art as sense mutations that do
not lead to any fundamental change in the activity of the protein,
i.e. that are functionally neutral. It is furthermore known that
changes at the N- and/or C-terminus of a protein do not
substantially affect, or may even stabilise, its function. The
person skilled in the art may find information on this in, inter
alia, 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 that are
obtained in a corresponding manner from SEQ ID NO:2 and DNA
sequences coding for these amino acid sequences are similarly a
subject of the invention.
[0057] Similarly, DNA sequences that hybridise with SEQ ID NO:1 or
portions of SEQ ID NO:1 are included in the invention. Finally, DNA
sequences that are produced by the polymerase chain reaction (PCR)
using primers that are formed from SEQ ID NO:1 are also intended to
be included in the invention. Such oligonucleotides typically have
a length of at least 15 nucleotides.
[0058] The person skilled in the art will find information on
identifying DNA sequences by means of hybridisation in, inter alia,
the handbook "The DIG System User's Guide for Filter Hybridization"
published by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and
in Liebl et al. (International Journal of Systematic Bacteriology
(1991) 41: 255-260). The person skilled in the art can find details
of the amplification of DNA sequences by means of the polymerase
chain reaction (PCR) in, inter alia, 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).
[0059] The inventors have found that coryneform bacteria produce
L-amino acids, in particular L-lysine, in an improved manner after
overexpression of the glk-gene.
[0060] In order to achieve overexpression, the number of copies of
the corresponding genes can be increased, or the promoter and
regulation region or the ribosome binding site that is located
upstream of the structure gene can be mutated. Expression cassettes
that are incorporated upstream to the structure gene act in the
same way. It is in addition possible by means of inducible
promoters to increase the expression during the course of the
fermentative production of L-amino acid. The expression is
similarly improved by measures adopted to increase the lifetime of
the m-RNA. Furthermore, the enzyme activity may be increased by
preventing the decomposition of the enzyme protein. The genes or
gene constructs may be present either in plasmids with different
numbers of copies or may be integrated and amplified in the
chromosome. Alternatively, overexpression of the relevant genes may
be achieved by changing the composition of the medium and
cultivation conditions.
[0061] The person skilled in the art may find details of this in,
inter alia, 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 EPS 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
published patent application 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 standard
textbooks on genetics and molecular biology.
[0062] For example, the glk-gene according to the invention was
overexpressed by means of plasmids.
[0063] Suitable plasmids are those that are replicated in
coryneform bacteria. Numerous known plasmid vectors, such as, for
example, pZ1 (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, for example, those that are 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) may be used in the same way.
[0064] Also suitable are those plasmid vectors by means of which
the process of gene amplification by integration into the
chromosome can be employed, as has been described for example by
Reinscheid et al. (Applied and Environmental Microbiology 60,
126-132 (1994)) for the duplication and/or amplification of the
hom-thrB-operon. In this method, the full gene is cloned in a
plasmid vector that can replicate in a host (typically E. coli) but
not in C. glutamicum. Suitable vectors that may be mentioned are
for example pSUP301 (Simon et al., Bio/Technology 1, 784-791
(1983)), pK18mob or pK19mob (Schfer et al., Gene 145, 69-73
(1994)), pGEM-T (Promega corporation, Madison, Wis., USA),
pCR2.1-TOPO (Shuman (1994), Journal of Biological Chemistry
269:32678-84; U.S. Pat. No. 5,487,993), pCR.RTM.Blunt (Firma
Invitrogen, Groningen, Netherlands; Bernard et al., Journal of
Molecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al,
1991, Journal of Bacteriology 173:4510-4516) . The plasmid vector
that contains the gene to be amplified is then transferred by
conjugation or transformation into the desired strain of C.
glutamicum. The method of conjugation is described, for example, by
Schfer et al. (Applied and Environmental Microbiology 60, 756-759
(1994)). Methods of transformation are described, for example, in
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 crossover
event, the resulting strain contains at least two copies of the
relevant gene.
[0065] The invention accordingly also provides a process for the
fermentative production of L-amino acids, in particular L-lysine,
in which a strain transformed with a plasmid vector is used, and
the plasmid vector carries the nucleotide sequence of the gene
coding for the enzyme glucokinase.
[0066] In addition it may be advantageous for the production of
L-amino acids, in particular L-lysine, to enhance, as well as the
glk-gene, further genes of the biosynthesis pathway of the desired
L-amino acid so that one or more enzymes of the relevant
biosynthesis pathway, glycolysis, anaplerotic, citric acid cycle or
of the amino acid export is overexpressed.
[0067] Thus, the following may for example be overexpressed for the
production of L-lysine:
[0068] at the same time the dapA-gene encoding dihydrodipicolinate
synthase (EP-B 0 197 335), or
[0069] at the same time an lysC-gene encoding a feedback-resistant
aspartate kinase (Kalinowski et al. (1990), Molecular and General
Genetics 224: 317-324), or
[0070] at the same time the gap-gene encoding
glyceraldehyde-3-phosphate-d- ehydrogenase (Eikmanns (1992),
Journal of Bacteriology 174:6076-6086), or
[0071] at the same time the tpi-gene encoding triosephosphate
isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
or
[0072] at the same time the pgk-gene encoding 3-phosphate glycerate
kinase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086),
or
[0073] at the same time the pyc-gene encoding pyruvate carboxylase
(Eikmanns (1992), Journal of Bacteriology 174:6076-6086), or
[0074] at the same time the mqo-gene encoding
malate-quinone-oxidoreduktas- e (Molenaar et al., European Journal
of Biochemistry 254, 395 -403 (1998)), or
[0075] at the same time the lysE-gene encoding lysine export
(DE-A-195 48 222).
[0076] Furthermore, it may be advantageous for the production of
L-amino acids, in particular L-lysine, to attenuate in addition to
the glk-gene the following at the same time:
[0077] the pck-gene encoding phosphoenolpyruvate carboxykinase (DE
199 50 409.1; DSM 13047) and/or
[0078] the pgi-gene encoding glucose-6-phosphate isomerase (U.S.
Ser. No. 09/396,478; DSM 12969).
[0079] Furthermore it may be advantageous for the production of
L-amino acids, in particular L-lysine, in addition to the
overexpression of the glk-gene, to switch off undesirable secondary
reactions (Nakayama: "Breeding of Amino Acid Producing
Micro-organisms", in: Overproduction of Microbial Products,
Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK,
1982).
[0080] The microorganisms produced according to the invention may
be cultured continuously or batchwise in a batch process (batch
cultivation) or in a fed batch or repeated fed batch process in
order to produce L-amino acids, in particular L-lysine. An overview
of known cultivation methods is given 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, Brunswick/ Wiesbaden, 1994)).
[0081] The culture medium to be used must suitably satisfy the
requirements of the relevant strains. Descriptions of culture media
for various microorganisms are given in the handbook "Manual of
Methods for General Bacteriology" of the American Society for
Bacteriology (Washington D.C., USA, 1981). As a carbon source,
sugars and carbohydrates, such as for example glucose, sucrose,
lactose, fructose, maltose, molasses, starch and cellulose, oils
and fats such as for example soya bean oil, sunflower oil,
groundnut oil and coconut oil, fatty acids such as for example
palmitic acid, stearic acid and linoleic acid, alcohols such as for
example glycerol, ethanol, and organic acids such as for example
acetic acid, may be used. These substances may be used individually
or as a mixture. As a nitrogen source, 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, may be used.
The nitrogen sources may be used individually or as a mixture. As a
phosphorus source, phosphoric acid, potassium dihydrogen phosphate
or dipotassium hydrogen phosphate, or the corresponding
sodium-containing salts, may be used. The culture medium must
furthermore contain salts of metals that are necessary for growth
such as, for example, magnesium sulfate or iron sulfate. Finally,
essential growth substances such as amino acids and vitamins may be
used in addition to the substances mentioned above. Apart from
this, suitable precursors may be added to the culture medium. The
aforementioned feedstock substances may be added to the culture in
the form of a single batch, or may be metered in in a suitable way
during the cultivation.
[0082] Alkaline compounds such as sodium hydroxide, potassium
hydroxide, ammonia or ammonia water or acidic compounds such as
phosphoric acid or sulfuric acid may be used in an appropriate
manner in order to regulate the pH of the culture. Antifoaming
agents such as, for example, fatty acid polyglycol esters may be
used to regulate foam formation. Suitable selectively acting
substances such as, for example, antibiotics may be added to the
medium in order to maintain the stability of plasmids. Oxygen or
oxygen-containing gas mixtures such as, for example, air are
introduced into the culture in order to maintain aerobic
conditions. The temperature of the culture is normally 20.degree.
C. to 45.degree. C. and preferably 25.degree. C. to 40.degree. C.
The culture is continued until a maximum yield of lysine has been
formed. This target is normally achieved within 10 hours to 160
hours.
[0083] The invention accordingly also provides a process for the
fermentative production of L-amino acids, in particular L-lysine,
in which the following steps are carried out:
[0084] a) fermentation of coryneform bacteria producing the L-amino
acid, in which at least the gene encoding the enzyme glucokinase is
enhanced, in particular is overexpressed,
[0085] b) enrichment of the L-amino acid in the medium or in the
cells of the bacteria, and
[0086] c) isolation of the L-amino acid.
[0087] The analysis of the L-lysine may be carried out by anion
exchange chromatography followed by ninhydrin derivatisation, as
described for example by Spackman et al. (Analytical Chemistry, 30,
(1958), 1190).
[0088] The process according to the invention allows the
fermentative production of L-amino acids, in particular
L-lysine.
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] FIG. 1: Map of the plasmid pEC-K18mob2
[0090] FIG. 2: Map of the plasmid pEC-K18mob2glkexp
[0091] The acronyms and abbreviations used have the following
meanings:
[0092] per: Gene for controlling the number of copies of pGA1
[0093] oriV: ColE1-like origin of pMB1
[0094] rep: Plasmid-coded replication region of C. glutamicum
plasmid pGA1
[0095] RP4mob: RP4-mobilisation site
[0096] Kan: Resistance gene for kanamycin
[0097] glk: glk-gene of C.glutamicum
[0098] EcoRI: Cleavage site of the restriction enzyme EcoRI
[0099] HindIII: Cleavage site of the restriction enzyme HindIII
[0100] Ecl136II: Cleavage site of the restriction enzyme
Ecl136II
DETAILED DESCRIPTION OF THE INVENTION
[0101] The present invention is illustrated in more detail
hereinafter with the aid of embodiment examples.
EXAMPLE 1
[0102] Production of a Genomic Cosmid Gene Library From
Corynebacterium glutamicum ATCC 13032
[0103] Chromosomal DNA from Corynebacterium glutamicum ATCC 13032
was isolated as described by Tauch et al. (1995, Plasmid
33:168-179) and partially 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 Molecular
Biochemicals, Mannheim, Germany, Product Description SAP, Code
no.1758250). The DNA of the cosmid vector SuperCos1 (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 way 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 the Gigapack II
XL Packing Extracts (Stratagene, La Jolla, USA, Product Description
Gigapack II XL Packing Extract, Code no. 200217). In order to
infect 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. Infection and
titration of the cosmid bank were carried out as described by
Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor), the cells having been plated out on LB-agar
(Lennox, 1955, Virology, 1:190) with 100 .mu.g/ml ampicillin.
Recombinant individual clones were selected after incubation
overnight at 37.degree. C.
EXAMPLE2
[0104] Isolation and Sequencing of the glk-gene
[0105] The cosmid-DNA of an individual colony was isolated using
the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden,
Germany) according to the manufacturer's instructions and partially
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 Molecular Biochemicals, Mannheim,
Germany, Product Description SAP, Product No. 1758250). After gel
electrophoresis separation the cosmid fragments were isolated in
the large region from 1500 to 2000 bp using the QiaExII Gel
Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany). The
DNA of the sequencing vector pzero-1 obtained from Invitrogen
(Groningen, 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 having been
incubated overnight with T4-ligase (Pharmacia Biotech, Freiburg,
Germany). This ligation mixture was electroporated into the E. coli
strain DHSMCR (Grant, 1990, Proceedings of the National Academy of
Sciences U.S.A., 87:4645-4649) (Tauch et al. 1994, FEMS Microbiol
Letters, 123:343-7) and was plated out on LB-agar (Lennox, 1955,
Virology, 1:190) with 50 .mu.g/ml zeocin. The plasmid preparation
of the recombinant clones was performed with Biorobot 9600 (Product
No. 900200, Qiagen, Hilden, Germany). The sequencing was carried
out according to the dideoxy chain termination method of Sanger et
al. (1977, Proceedings of the National Academies of Sciences
U.S.A., 74:5463-5467) as modified by Zimmermann et al. (1990,
Nucleic Acids Research, 18:1067). The RR dRhodamine Terminator
Cycle Sequencing Kit from PE Applied Biosystems(Product No. 403044,
Weiterstadt, Germany) was used. The gel electrophoresis separation
and analysis of the sequencing reaction was performed in a
rotiphoresis NF acrylamide/bisacrylamide gel (29:1) (Product No.
A124.1, Roth, Karlsruhe, Germany) together with the ABI Prism 377
sequencing equipment from PE Applied Biosystems (Weiterstadt,
Germany).
[0106] 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 pZerol derivates were
assembled into a coherent Contig. The computer-assisted analysis of
the coding region was performed with the program XNIP (Staden,
1986, Nucleic Acids Research, 14:217-231). Further analyses were
carried out with the BLAST search programs (Altschul et al., 1997,
Nucleic Acids Research, 25:3389-3402), against the non-redundant
data bank of the National Center for Biotechnology Information
(NCBI, Bethesda, Md., USA).
[0107] The nucleotide sequence obtained is shown in SEQ ID NO:1.
Analysis of the nucleotide sequence revealed an open reading frame
of 969 base pairs, which was termed the glk-gene. The glk-gene
codes for a protein of 323 amino acids.
EXAMPLE 3
[0108] Production of a Shuttle Vector pEC-K18mob2glkexp for
Enhancing the glk-gene in C. glutamicum
[0109] 3.1. Cloning of the glk-gene
[0110] Chromosomal DNA was isolated from the strain ATCC 13032
according to the method of Eikmanns et al. (Microbiology 140:
1817-1828 (1994)). On the basis of the sequence of the glk-gene
known for C. glutamicum from Example 2 the following
oligonucleotides were selected for the polymerase chain
reaction:
[0111] glk-ex1:
[0112] 5' ACT GAC GTG AGC CAG AAC 3'
[0113] glk-ex2:
[0114] 5' GAT CTA TCT AGA CAC CTA GTT GGC TTC CAC 3'
[0115] The illustrated primers were synthesised by ARK Scientific
GmbH Biosystems (Darmstadt, Germany) and the PCR reaction was
carried out according to 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
permit the amplification of a ca. 1.45 kb size DNA fragment which
carries the glk-gene with the potential promoter region. The DNA
sequence of the amplified DNA fragment was checked by
sequencing.
[0116] 3.2. Production of the E. coli -C. glutamicum Shuttle Vector
pEC-K18mob2
[0117] The E. coli -C. glutamicum shuttle vector was constructed
according to the prior art. The vector contains the replication
region rep of the plasmid pGA1 including the replication effector
per (U.S. Pat. No. 5,175,108; Nesvera et al., Journal of
Bacteriology 179, 1525-1532 (1997)), the aph(3')-IIa-gene of the
transposon Tn5 imparting resistance to kanamycin (Beck et al., Gene
19, 327-336 (1982)), the replication region oriV of the plasmid
pMB1 (Sutcliffe, Cold Spring Harbor Symposium on Quantitative
Biology 43, 77-90 (1979)), the lacZ.alpha. gene fragment including
the lac promoter and a multiple cloning site (mcs) (Norrander, J.
M. et al., Gene 26, 101-106 (1983)) and the mob region of the
plasmid RP4 (Simon et al., Bio/Technology 1:784-791 (1983)). The
constructed vector was transformed into the E. coli strain
DH5.alpha. (Hanahan, In: DNA Cloning. A Practical Approach. Vol. I,
IRL-Press, Oxford, Washington D.C., USA). The selection of
plasmid-carrying cells was made by plating out the transformation
batch onto LB agar (Sambrook et al., Molecular cloning: A
Laboratory Manual. 2.sup.nd Ed. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.) that had been supplemented with 25
mg/l kanamycin. Plasmid DNA was isolated from a transformant using
the QIAprep Spin Miniprep Kit from Qiagen and was checked by
restriction with the restriction enzyme EcoRI and HindIII followed
by agarose gel electrophoresis (0.8%). The plasmid was named
pEC-K18mob2 and is shown in FIG. 1.
[0118] The following microorganism was filed at the German
Collection of Microorganisms and Cell Cultures (DSMZ, Brunswick,
Germany) according to the Budapest Convention:
[0119] C. glutamicum strain DSM 5715/pEC-K18mob2 as DSM 13245
[0120] 3.3. Cloning of glk in the E. coli -C. glutamicum Shuttle
Vector pEC-K18mob2
[0121] The E. coli -C. glutamicum shuttle vector pEC-K18mob2
described in Example 3.2 was used as vector. DNA of this plasmid
was completely cleaved with the restriction enzyme Ecl136II and
then dephosphorylated with shrimp alkaline phosphatase (Roche
Diagnostics GmbH, Mannheim, Germany, Product Description SAP,
Product No. 1758250).
[0122] The glk fragment obtained as described in Example 3.1 was
mixed with the prepared vector pEC-K18mob2 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 batch was transformed into the E. coli strain DH5.alpha.
(mcr (Grant, 1990, Proceedings of the National Academy of Sciences
U.S.A., 87:4645-4649). The selection of plasmid-carrying cells was
made by plating out the transformation batch onto LB-agar (Lennox,
1955, Virology, 1:190) with 25 mg/l of kanamycin. Recombinant
individual cells were selected after incubation overnight at
37.degree. C. Plasmid DNA was isolated from a transformant using
the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden,
Germany) according to the manufacturer's instructions and cleaved
with the restriction enzymes EcoRI and XbaI in order to check the
plasmid by subsequent agarose gel electrophoresis. The plasmid
obtained was named pEC-K18mob2glkexp and is shown in FIG. 2.
EXAMPLE 4
[0123] Transformation of the Strain Corynebacterium glutamicum
RES167 with the Plasmid pEC-K18mob2glkexp.
[0124] The strain C. glutamicum RES167 (Schfer, A. et al., Journal
Bacteriological 176: 7309-731(1994)) was transformed with the
plasmid pEC-K18mob2glkexp using the electroporation method
described by Liebl et al., (FEMS Microbiology Letters, 53:299-303
(1989)). The selection of the tranformants was made on LBHIS agar
consisting of 18.5 g/l brain-heart infusion broth, 0.5 M sorbitol,
5 g/l Bacto-Trypton, 2,5 g/l Bacto-Yeast Extract, 5 g/l NaCl and 18
g/l Bacto-Agar, that had been supplemented with 25 mg/l kanamycin.
Incubation was carried out at 33.degree. C. for two days.
[0125] Plasmid DNA was isolated from a transformant by the usual
methods (Peters-Wendisch et al., Microbiology, 144:915-927 (1998)),
cleaved with the restriction endonucleases EcoRI and XbaI, and the
plasmid was checked by subsequent agarose gel electrophoresis. The
strain obtained was named C. glutamicum
RES167/pEC-K18mob2glkexp.
EXAMPLE 5
[0126] Production of Lysine
[0127] The strain C. glutamicum RES167/pEC-K18mob2glkexp obtained
in Example 4 was cultivated in a nutrient medium suitable for
producing lysine, and the lysine content in the culture supernatant
was determined.
[0128] For this purpose the strain was first incubated at
33.degree. C. for 24 hours on agar plates with the appropriate
antibiotic (brain-heart agar with kanamycin (25 mg/l)). A
preculture was inoculated using this agar plate culture (10 ml
medium in 100 ml Erlenmeyer flask). The full medium CgIII was used
as medium for the preculture.
1 Medium Cg III NaCl 2.5 g/l Bacto-Pepton 10 g/l Bacto-Yeast
Extract 10 g/l Glucose (separately autoclaved) 2% (w/v) The pH was
adjusted to pH 7.4
[0129] Kanamycin (25 mg/l) was added to this medium. The
pre-culture was incubated on a shaker for 16 hours at 33.degree. C.
and 240 rpm. A main culture was inoculated from this pre-culture so
that the initial optical density (660 nm) of the main culture was
0.05. The medium MM was used for the main culture.
2 Medium MM CSL (Corn Steep Liquor) 5 g/l MOPS
(Morpholinopropanesulfonic 20 g/l acid) Glucose (separately
autoclaved) 50 g/l (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) 0.3 mg/l Thiamine.HCl (sterile filtered) 0.2
mg/l CaCO.sub.3 25 g/l
[0130] CSL, MOPS and the salt solution were adjusted with ammonia
water to pH 7 and autoclaved. The sterile substrate and vitamin
solutions as well as the dry autoclaved CaCO.sub.3 were then
added.
[0131] Cultivation takes place in a 10 ml volume in a 100 ml
Erlenmeyer flask with baffles. Kanamycin (25 mg/l) was added.
Cultivation was performed at 33.degree. C. and 80% atmospheric
humidity.
[0132] After 72 hours the OD was measured at a measurement
wavelength of 660 nm using the Biomek 1000 (Beckmann Instruments
GmbH, Munich). The amount of lysine formed was measured with an
amino acid analyser from Eppendorf-BioTronik (Hamburg, Germany) by
ion exchange chromatography and post-column derivatisation with
ninhydrin detection.
[0133] The result of the test is shown in Table 1.
3 TABLE 1 Lysine-HCl Strain OD (660) mg/l C. glutamicum RES167 13.8
287 C. glutamicum RES167/ 15 345 pEC-K18mob2glkexp
[0134]
Sequence CWU 1
1
4 1 1540 DNA Corynebacterium glutamicum CDS (461)..(1429) 1
aaattccttg ggcgccttga atatcaagat atgatcaaca cgcttgccgc cgcagatatt
60 ttcgcgatgc cagcgcgcac ccgcggtggc ggacttgatg ttgaaggctt
gggcattgtc 120 tatctcgagg cacaagcctg cggagtgccg gtgatagccg
gcacctctgg cggcgcgcca 180 gagacggtga ctccggcaac tggcctggtt
gtggaggggt cggacgtcga taagctgtct 240 gagcttttaa ttgagcttct
cgacgatccg atccgccgcg ccgcgatggg cgctgcaggt 300 agggcgcatg
tggaggccga atggtcgtgg gaaatcatgg gggagcggtt gaccaatatt 360
ttgcagagtg aaccacgatg atggttggac agctgttgat agctatactt tgaaagatta
420 aattcaccta aatcctgtgt agaacgcgag gggcactctt atg cca caa aaa ccg
475 Met Pro Gln Lys Pro 1 5 gcc agt ttc gcg gtg ggc ttt gac atc ggc
ggc acc aac atg cga gcc 523 Ala Ser Phe Ala Val Gly Phe Asp Ile Gly
Gly Thr Asn Met Arg Ala 10 15 20 ggg ctt gtc gac gaa tcc ggg cgc
atc gtg acc agt ttg tcg gcg ccg 571 Gly Leu Val Asp Glu Ser Gly Arg
Ile Val Thr Ser Leu Ser Ala Pro 25 30 35 tcg ccg cgc acg acg cag
gca atg gaa cag ggg att ttt gat cta gtc 619 Ser Pro Arg Thr Thr Gln
Ala Met Glu Gln Gly Ile Phe Asp Leu Val 40 45 50 gaa cag ctc aag
gcc gaa tac ccg gtt ggt gct gtg gga ctt gcc gtc 667 Glu Gln Leu Lys
Ala Glu Tyr Pro Val Gly Ala Val Gly Leu Ala Val 55 60 65 gcg gga
ttt ttg gat cct gag tgc gag gtt gtt cga ttt gcc ccg cac 715 Ala Gly
Phe Leu Asp Pro Glu Cys Glu Val Val Arg Phe Ala Pro His 70 75 80 85
ctt cct tgg cgc gat gag cca gtg cgt gaa aag ttg gaa aac ctt ttg 763
Leu Pro Trp Arg Asp Glu Pro Val Arg Glu Lys Leu Glu Asn Leu Leu 90
95 100 ggc ctg cct gtt cgt ttg gaa cat gat gcc aac tca gca gcg tgg
ggt 811 Gly Leu Pro Val Arg Leu Glu His Asp Ala Asn Ser Ala Ala Trp
Gly 105 110 115 gag cat cgt ttt ggt gca gct caa ggc gct gac aac tgg
gtt ttg ttg 859 Glu His Arg Phe Gly Ala Ala Gln Gly Ala Asp Asn Trp
Val Leu Leu 120 125 130 gca ctc ggc act gga att ggt gca gcg ctg att
gaa aaa ggc gaa att 907 Ala Leu Gly Thr Gly Ile Gly Ala Ala Leu Ile
Glu Lys Gly Glu Ile 135 140 145 tac cgt ggt gca tat ggc acg gca cca
gaa ttt ggt cat ttg cgt gtt 955 Tyr Arg Gly Ala Tyr Gly Thr Ala Pro
Glu Phe Gly His Leu Arg Val 150 155 160 165 gtt cgt ggc gga cgc gca
tgt gcg tgt ggc aaa gaa ggc tgc ctg gag 1003 Val Arg Gly Gly Arg
Ala Cys Ala Cys Gly Lys Glu Gly Cys Leu Glu 170 175 180 cgt tac tgt
tcc ggt act gcc ttg gtt tac act gcg cgt gaa ttg gct 1051 Arg Tyr
Cys Ser Gly Thr Ala Leu Val Tyr Thr Ala Arg Glu Leu Ala 185 190 195
tcg cat ggc tca ttc cgc aac agc ggg ctg ttt gac aag atc aaa gcc
1099 Ser His Gly Ser Phe Arg Asn Ser Gly Leu Phe Asp Lys Ile Lys
Ala 200 205 210 gat ccg aat tcc atc aat gga aaa acg atc act gcg gca
gcg cgc caa 1147 Asp Pro Asn Ser Ile Asn Gly Lys Thr Ile Thr Ala
Ala Ala Arg Gln 215 220 225 gaa gac cca ctt gct ctc gcc gtt ctg gaa
gat ttc agc gag tgg ctg 1195 Glu Asp Pro Leu Ala Leu Ala Val Leu
Glu Asp Phe Ser Glu Trp Leu 230 235 240 245 ggc gaa act ttg gcg atc
att gct gat gtc ctt gac cca ggc atg atc 1243 Gly Glu Thr Leu Ala
Ile Ile Ala Asp Val Leu Asp Pro Gly Met Ile 250 255 260 atc att ggt
ggc gga ctg tcc aat gct gcc gac ctt tat ttg gat cgc 1291 Ile Ile
Gly Gly Gly Leu Ser Asn Ala Ala Asp Leu Tyr Leu Asp Arg 265 270 275
tcg gtc aac cac tat tcc acc cgc atc gtc ggc gca gga tat cgc cct
1339 Ser Val Asn His Tyr Ser Thr Arg Ile Val Gly Ala Gly Tyr Arg
Pro 280 285 290 ttg gca cgc gtt gcc aca gct cag ttg ggt gcg gat gct
ggc atg atc 1387 Leu Ala Arg Val Ala Thr Ala Gln Leu Gly Ala Asp
Ala Gly Met Ile 295 300 305 ggt gtc gct gat cta gct cga cgc tct gta
gtg gaa gcc aac 1429 Gly Val Ala Asp Leu Ala Arg Arg Ser Val Val
Glu Ala Asn 310 315 320 taggtgtttt tcggtgggct gcgatgacgc atgtccacca
aaagagccac cccttaaaga 1489 aattaaaaag tggttttggt agcttcgcag
caaaatacac atcgtgggta a 1540 2 323 PRT Corynebacterium glutamicum 2
Met Pro Gln Lys Pro Ala Ser Phe Ala Val Gly Phe Asp Ile Gly Gly 1 5
10 15 Thr Asn Met Arg Ala Gly Leu Val Asp Glu Ser Gly Arg Ile Val
Thr 20 25 30 Ser Leu Ser Ala Pro Ser Pro Arg Thr Thr Gln Ala Met
Glu Gln Gly 35 40 45 Ile Phe Asp Leu Val Glu Gln Leu Lys Ala Glu
Tyr Pro Val Gly Ala 50 55 60 Val Gly Leu Ala Val Ala Gly Phe Leu
Asp Pro Glu Cys Glu Val Val 65 70 75 80 Arg Phe Ala Pro His Leu Pro
Trp Arg Asp Glu Pro Val Arg Glu Lys 85 90 95 Leu Glu Asn Leu Leu
Gly Leu Pro Val Arg Leu Glu His Asp Ala Asn 100 105 110 Ser Ala Ala
Trp Gly Glu His Arg Phe Gly Ala Ala Gln Gly Ala Asp 115 120 125 Asn
Trp Val Leu Leu Ala Leu Gly Thr Gly Ile Gly Ala Ala Leu Ile 130 135
140 Glu Lys Gly Glu Ile Tyr Arg Gly Ala Tyr Gly Thr Ala Pro Glu Phe
145 150 155 160 Gly His Leu Arg Val Val Arg Gly Gly Arg Ala Cys Ala
Cys Gly Lys 165 170 175 Glu Gly Cys Leu Glu Arg Tyr Cys Ser Gly Thr
Ala Leu Val Tyr Thr 180 185 190 Ala Arg Glu Leu Ala Ser His Gly Ser
Phe Arg Asn Ser Gly Leu Phe 195 200 205 Asp Lys Ile Lys Ala Asp Pro
Asn Ser Ile Asn Gly Lys Thr Ile Thr 210 215 220 Ala Ala Ala Arg Gln
Glu Asp Pro Leu Ala Leu Ala Val Leu Glu Asp 225 230 235 240 Phe Ser
Glu Trp Leu Gly Glu Thr Leu Ala Ile Ile Ala Asp Val Leu 245 250 255
Asp Pro Gly Met Ile Ile Ile Gly Gly Gly Leu Ser Asn Ala Ala Asp 260
265 270 Leu Tyr Leu Asp Arg Ser Val Asn His Tyr Ser Thr Arg Ile Val
Gly 275 280 285 Ala Gly Tyr Arg Pro Leu Ala Arg Val Ala Thr Ala Gln
Leu Gly Ala 290 295 300 Asp Ala Gly Met Ile Gly Val Ala Asp Leu Ala
Arg Arg Ser Val Val 305 310 315 320 Glu Ala Asn 3 18 DNA PCR primer
3 actgacgtga gccagaac 18 4 30 DNA PCR primer 4 gatctatcta
gacacctagt tggcttccac 30
* * * * *