U.S. patent application number 10/801586 was filed with the patent office on 2004-08-05 for process for the preparation of l-amino acids by attenuating the succ and sucd genes.
This patent application is currently assigned to Degussa AG. Invention is credited to Marx, Achim, Mockel, Bettina, Pfefferle, Walter.
Application Number | 20040152166 10/801586 |
Document ID | / |
Family ID | 26055640 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040152166 |
Kind Code |
A1 |
Mockel, Bettina ; et
al. |
August 5, 2004 |
Process for the preparation of L-amino acids by attenuating the
sucC and sucD genes
Abstract
The invention relates to polynucleotides that contain
polynucleotide sequences coding for the genes sucC and sucD,
selected from the group 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) polynucleotide
that is at least 70% identical to a polynucleotide coding for a
polypeptide that contains the amino acid sequence of SEQ ID No. 3,
c) polynucleotide coding for a polypeptide that contains an amino
acid sequence that is at least 70% identical to the amino acid
sequence of SEQ ID No. 2, d) polynucleotide coding for a
polypeptide that contains an amino acid sequence that is at least
70% identical to the amino acid sequence of SEQ ID No. 3, e)
polynucleotide that is complementary to the polynucleotides of a),
b), c) or d), and f) polynucleotide containing at least 15
successive nucleotides of the polynucleotide sequence of a), b),
c), d) or e), a process for the fermentative production of L-amino
acids using coryneform bacteria in which the genes are present in
attenuated form, and the use of the polynucleotide sequences as
hybridization probes.
Inventors: |
Mockel, Bettina;
(Dusseldorf, DE) ; Pfefferle, Walter; (Halle,
DE) ; Marx, Achim; (Altenbrede, DE) |
Correspondence
Address: |
PILLSBURY WINTHROP, LLP
P.O. BOX 10500
MCLEAN
VA
22102
US
|
Assignee: |
Degussa AG
Frankfurt am Main
DE
|
Family ID: |
26055640 |
Appl. No.: |
10/801586 |
Filed: |
March 17, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10801586 |
Mar 17, 2004 |
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09838564 |
Apr 20, 2001 |
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09838564 |
Apr 20, 2001 |
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09728498 |
Nov 27, 2000 |
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6623946 |
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Current U.S.
Class: |
435/69.1 ;
435/189; 435/193; 435/252.3; 435/320.1; 536/23.2 |
Current CPC
Class: |
C12P 13/14 20130101;
C12P 13/04 20130101; C12N 9/93 20130101 |
Class at
Publication: |
435/069.1 ;
435/193; 435/320.1; 435/252.3; 435/189; 536/023.2 |
International
Class: |
C12N 009/02; C12N
009/10; C07H 021/04; C12N 009/64 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 25, 1999 |
DE |
199 56 686.0 |
Claims
What is claimed is:
1. Isolated polynucleotide containing a polynucleotide sequence
coding for the sucC- and/or sucD-gene, selected from the group
comprising 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) Polynucleotide that is at least
70% identical to a polynucleotide coding for a polypeptide that
contains the amino acid sequence of SEQ ID No. 3, c) Polynucleotide
coding for a polypeptide that contains an amino acid sequence that
is at least 70% identical to that of the amino acid sequence of SEQ
ID No. 2, d) Polynucleotide coding for a polypeptide that contains
an amino acid sequence that is at least 70% identical to that of
the amino acid sequence of SEQ ID No. 3, e) Polynucleotide that is
complementary to the polynucleotides of a), b), c) or d), and f)
Polynucleotide containing at least 15 successive nucleotides of the
polynucleotide sequences of a), b), c), d) or e), the polypeptide
preferably having the activity of succinyl-CoA synthetase.
2. Polynucleotides according to claim 1, wherein the polynucleotide
is a preferably recombinant DNA that is replicable in coryneform
bacteria.
3. Polynucleotides according to claim 1, wherein the polynucleotide
is a RNA.
4. Polynucleotides according to claim 2, containing the nucleic
acid sequence as shown in SEQ ID NO 1.
5. 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 sequences (i) within the region of
degeneration of the genetic code, or (iii) at least one sequence
that hybridizes with the sequences that are complementary to the
sequences (i) or (ii), and optionally (iv) functionally neutral
sense mutations in (i).
6. Replicable. DNA according to claim 5, wherein the hybridization
is carried out under a stringency corresponding to at most
2.times.SSC.
7. Polynucleotide sequence according to claim 1 that codes for a
polypeptide that contains the amino acid sequence shown in SEQ ID
No. 2.
8. Coryneform bacteria in which the sucC- and/or sucD-gene is/are
attenuated.
9. Process for producing L-amino acids, in particular L-lysine
and/or L-glutamate, wherein the following steps are carried out: a)
fermentation of the bacteria producing the desired L-amino acid, in
which first of all the sucC- and/or sucD-gene or nucleotide
sequences coding therefor are attenuated, in particular switched
off; b) enrichment of the L-amino acid in the medium or in the
bacterial cells, and c) isolation of the L-amino acid.
10. Process according to claim 9, wherein bacteria are used in
which in addition further genes of the biosynthesis pathway of the
desired L-amino acid are enhanced.
11. Process according to claim 9, wherein bacteria are used in
which the metabolic pathways that reduce the formation of the
desired L-amino acid are at least partially switched off.
12. Process according to claim 9, wherein the expression of the
polynucleotide(s) that codes for the sucC- and/or sucD-genes is
attenuated, in particular is switched off.
13. Process according to claim 9, wherein the catalytic properties
of the polypeptide (enzyme protein) for which the polynucleotides
sucC and sucD code are reduced.
14. Process according to claim 9, wherein for the production of
L-amino acids microorganisms are fermented in which at the same
time one or more of the genes selected from the following group
is/are enhanced and/or overexpressed: 14.1 the dapA-gene coding for
dihydrodipicolinate synthase, 14.2 the pyc-gene coding for pyruvate
carboxylase, 14.3 the gap-gene coding for
glyceraldehyde-3-phosphate dehydrogenase, 14.4 the gene tpi coding
for triose phosphate isomerase, 14.5 the gene pgk coding for
3-phosphoglycerate kinase, 14.6 the mqo-gene coding for
malate:quinone oxidoreductase, 14.7 the lysE-gene coding for
L-lysine export, 14.8 the gene lysC coding for a feedback resistant
aspartate kinase, 14.9 the gene zwa1 coding for the
Zwa1-protein.
15. Process according to claim 9, wherein for the production of
L-amino acids coryneform microorganisms are fermented, in which at
the same time one or more of the genes selected from the following
group is/are attenuated: 15.1 the gene pck coding for phosphoenol
pyruvate carboxykinase, 15.2 the gene pgi coding for
glucose-6-phosphate isomerase, 15.3 the gene poxB coding for
pyruvate-oxidase, 15.4 the gene zwa2 coding for the
Zwa2-Protein.
16. Coryneform bacteria containing a vector that carries parts of
the polynucleotide according to claim 1, but at least 15 successive
nucleotides of the claimed sequence.
17. DNA derived from coryneform bacteria that code for SucC
proteins, whose amino acid sequence (SEQ ID No. 2) contains one or
more replacements selected from the group: replacement at position
22 by any other proteinogenic amino acid except L-proline,
replacement at position 44 by any other proteinogenic amino acid
except glycine, and replacement at position 170 by any other
proteinogenic amino acid except L-alanine.
18. DNA according to claim 17, wherein this codes for SucC proteins
whose amino acid sequences contain one or more replacements
selected from the group: L-proline at position 22 by L-serine,
glycine at position 44 by L-glutamic acid, and L-alanine at
position 170 by L-threonine.
19. DNA according to claim 18, wherein this codes for a SucC
protein whose amino acid sequence contains L-serine at position 22,
L-glutamic acid at position 44, and L-threonine at position 170, as
illustrated in SEQ ID No.5.
20. DNA according to claim 17, wherein this contains the nucleobase
thymine at position 64, the nucleobase adenine at position 131, and
the nucleobase adenine at position 508, as illustrated in SEQ ID No
4.
21. Coryneforme bacteria that contain a DNA according to claim 17,
18, 19 or 20.
22. Integration vector pCRBluntsucCint, that 22.1 carries a 0.55 kb
long internal fragment of the sucC-gene, 22.2 whose restriction map
is reproduced in FIG. 1, and 22.3 that in the E. coli strain
TOP10/pCRBluntsucCint has been filed under No. DSM 13750 at the
German Collection for Microorganisms and Cell Cultures.
23. Plasmid vector pK18mobsacBsucDdel that 23.1 carries a sucD-gene
containing a deletion, 23.2 whose restriction map is reproduced in
FIG. 2, and 23.3 that in the E. coli strain
DH5.alpha.mcr/pK18mobsacBsucDdel has been filed under No. DSM 13749
at the German Collection for Microorganisms and Cell Cultures.
24. Process according to one or more of the preceding claims,
wherein microorganisms of the type Corynebacterium glutamicum are
used.
25. Process for detecting RNA, cDNA and DNA in order to isolate
nucleic acids and/or polynucleotides or genes that code for
succinyl-CoA synthase or that have a high degree of similarity to
the sequence of the sucC- and/or sucD-gene, wherein the
polynucleotide containing the polynucleotide sequences according to
claims 1, 2, 3 or 4, is used as hybridization probe.
Description
[0001] The present invention provides nucleotide sequences of
coryneform bacteria coding for the genes sucC and sucD and a
process for the fermentative production of amino acids, in
particular L-lysine and L-glutamate, using bacteria in which the
sucC- and/or sucD-gene is/are attenuated.
[0002] Prior Art
[0003] L-amino acids, in particular L-lysine and L-glutamate, are
used in human medicine and in the pharmaceutical industry, in the
foodstuffs industry, and most particularly in animal nutrition.
[0004] It is known that amino acids can be produced by fermentation
of strains of coryneform bacteria, in particular Corynebacterium
glutamicum (C. glutamicum). On account of the great importance of
amino acids efforts are constantly being made to improve the
production processes. Improvements in production 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.
[0005] Methods involving mutagenesis, selection and choice of
mutants are used to improve the output properties. In this way
strains are obtained that are resistant to antimetabolites or are
auxotrophic for regulatorily important metabolites, and that
produce amino acids.
[0006] For some years recombinant DNA technology methods have also
been used to improve Corynebacterium strains producing L-amino
acids.
OBJECT OF THE INVENTION
[0007] The inventors have set themselves the task of providing new
measures for improving the fermentative production of amino acids,
in particular L-lysine and L-glutamate.
DETAILED DESCRIPTION OF THE INVENTION
[0008] Where L-amino acids or amino acids are mentioned
hereinafter, it is understood that these terms refer to one or more
amino acids, including their salts, selected from the group
comprising L-asparagine, L-threonine, L-serine, L-glutamate,
L-glycine, L-alanine, L-cysteine, L-valine, L-methionine,
L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine,
L-lysine, L-tryptophan and L-arginine. L-lysine and L-glutamate are
particularly preferred.
[0009] The present invention provides an isolated polynucleotide
containing a polynucleotide sequence selected from the group
comprising
[0010] 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,
[0011] b) polynucleotide that is at least 70% identical to a
polynucleotide coding for a polypeptide, that contains the amino
acid sequence of SEQ ID No. 3,
[0012] c) polynucleotide coding for a polypeptide, that contains an
amino acid sequence that is at least 70% identical to the amino
acid sequence of SEQ ID No. 2,
[0013] d) polynucleotide coding for a polypeptide, that contains an
amino acid sequence that is at least 70% identical to the amino
acid sequence of SEQ ID No. 3,
[0014] e) polynucleotide that is complementary to the
polynucleotides of a), b), c) or d), and
[0015] f) polynucleotide containing at least 15 successive
nucleotides of the polynucleotide sequence of a), b), c), d) or
e),
[0016] the polypeptide preferably exhibiting the activity of
succinyl-CoA synthetase.
[0017] The present invention also provides the polynucleotide
according to claim 1, which is preferably a replicable DNA
containing:
[0018] (i) the nucleotide sequence shown in SEQ ID No. 1, or
[0019] (ii) at least one sequence that corresponds to the sequence
(i) within the region of degeneration of the genetic code, or
[0020] (iii) at least one sequence that hybridizes with the
sequence complementary to the sequence (i) or (ii), and
optionally
[0021] (iv) functionally neutral sense mutations in (i).
[0022] The invention furthermore provides:
[0023] a polynucleotide according to claim 4, containing the
nucleotide sequence as shown in SEQ ID No. 1,
[0024] a polynucleotide according to claim 1, wherein the
polynucleotide is a preferably recombinant DNA replicable in
coryneform bacteria,
[0025] a vector containing parts of the polynucleotide according to
the invention, but at least 15 successive nucleotides of the
claimed sequence,
[0026] and coryneform bacteria in which the sucC- and/or sucD-gene
is/are attenuated in particular by an insertion or deletion.
[0027] The present invention moreover provides polynucleotides that
substantially comprise a polynucleotide sequence, that can be
obtained by screening a corresponding gene library by means of
hybridization, that contains the complete sucC- and/or sucD-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.
[0028] Polynucleotides that contain the sequences according to the
invention are suitable as hybridization probes for RNA, cDNA and
DNA, in order to isolate cDNA, nucleic acids and/or polynucleotides
or genes in their full length that code for succinyl-CoA
synthetase, and to isolate such cDNA or genes whose sequence has a
high similarity to that of the succinyl-CoA synthetase genes.
[0029] Polynucleotides that contain the sequences according to the
invention are furthermore suitable as primers, by means of which
DNA can be produced by the polymerase chain reaction (PCR) from
genes that code for succinyl-CoA synthetase.
[0030] Such oligonucleotides serving as probes or primers contain
at least 30, preferably at least 20, and most particularly
preferably at least 15 successive nucleotides. Nucleotides with a
length of at least 40 or 50 nucleotides are also suitable.
[0031] "Isolated" means separated from its natural environment.
[0032] "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.
[0033] By the term "polypeptides" are understood peptides or
proteins that contain two or more amino acids bound via peptide
bonds.
[0034] The polypeptides according to the invention include the
polypeptides according to SEQ ID No. 2 and SEQ ID No. 3, in
particular those having the biological activity of succinyl-CoA
synthetase as well as those that are at least 70% identical to the
polypeptide according to SEQ ID No. 2 or SEQ ID No. 3, and
preferably at least 80% and particularly preferably at least 90% to
95% identical to the polypeptide according to SEQ ID No. 2 or SEQ
ID No. 3 and that have the aforementioned activity.
[0035] The present invention furthermore relates to a process for
the fermentative production of amino acids selected from the group
comprising L-asparagine, L-threonine, L-serine, L-glutamate,
L-glycine, L-alanine, L-cysteine, L-valine, L-methionine,
L-isoleucine, L-leucin, L-tyrosine, L-phenylalanine, L-histidine,
L-lysine, L-tryptophan and L-arginine, in particular L-lysine and
L-glutamate, using coryneform bacteria that in particular already
produce the amino acids, especially L-lysine and/or L-glutamate,
and in which the nucleotide sequences coding for the sucC- and/or
sucD-gene are attenuated, and in particular are expressed at a low
level.
[0036] The term "attenuation" describes in this connection the
reduction or switching off of the intracellular activity of one or
more enzymes (proteins) in a microorganism that can be coded by the
corresponding DNA, by for example using a weak promoter or a gene
and/or allele that codes for a corresponding enzyme with a low
activity and/or inactivates the corresponding gene and/or allele or
enzyme (protein) and optionally combines these features.
[0037] 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 skilled in the art for its ability to produce
L-amino acids.
[0038] Suitable strains of the genus Corynebacterium, in particular
of the type Corynebacterium glutamicum, are in particular the
following known wild type strains
[0039] Corynebacterium glutamicum ATCC13032
[0040] Corynebacterium acetoglutamicum ATCC15806
[0041] Corynebacterium acetoacidophilum ATCC13870
[0042] Corynebacterium melassecola ATCC17965
[0043] Corynebacterium thermoaminogenes FERM BP-1539
[0044] Brevibacterium flavum ATCC14067
[0045] Brevibacterium lactofermentum ATCC13869 and
[0046] Brevibacterium divaricatum ATCC14020
[0047] and mutants and/or strains obtained therefrom that produce
L-amino acids, such as for example the L-lysine-producing
strains:
[0048] Corynebacterium glutamicum FERM-P 1709
[0049] Brevibacterium flavum FERM-P 1708
[0050] Brevibacterium lactofermentum FERM-P 1712
[0051] Corynebacterium glutamicum FERM-P 6463
[0052] Corynebacterium glutamicum FERM-P 6464 and
[0053] Corynebacterium glutamicum DSM 5714.
[0054] The new genes sucC and sucD coding for the enzyme
succinyl-CoA synthetase (EC 6.2.1.5) have been isolated from C.
glutamicum.
[0055] In order to isolate the sucC- and/or the sucD-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 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). 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
.lambda.-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).
[0056] 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)). O'Donohue (The Cloning and Molecular Analysis of
Four Common Aromatic Amino Acid Biosynthetic Genes from
Corynebacterium glutamicum. Ph.D. Thesis, National University of
Ireland, Galway, 1997) describes the cloning of C. glutamicum genes
using the .lambda. Zap Expression system described by Short et al.
(Nucleic Acids Research, 16: 7583).
[0057] 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, such as for
example the strain DH5.alpha. (Jeffrey H. Miller: "A Short Course
in Bacterial Genetics, A Laboratory Manual and Handbook for
Escherichia coli and Related Bacteria", Cold Spring Harbour
Laboratory Press, 1992).
[0058] The long DNA fragments cloned with the aid of cosmids or
other .lambda.-vectors may then in turn be sub-cloned into
accessible vectors suitable for DNA sequencing.
[0059] Methods for DNA sequencing are described inter alia by
Sanger et al. (Proceedings of the National Academy of Sciences of
the United States of America, 74:5463-5467, 1977).
[0060] The DNA sequences that are obtained may then be investigated
with known algorithms and/or sequence analysis programs, such as
for example that of Staden (Nucleic Acids Research 14, 217-232
(1986)), the GCG-program of Butler (Methods of Biochemical Analysis
39, 74-97 (1998)), the FASTA algorithm of Pearson and Lipman
(Proceedings of the National Academy of Sciences USA 85, 2444-2448
(1988)) or the BLAST algorithm of Altschul et al. (Nature Genetics
6, 119-129 (1994)) and compared with the sequence entries listed in
publicly accessible data banks. Publicly accessible data banks for
nucleotide sequences are for example those of the European
Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) or
those of the National Center for Biotechnology Information (NCBI,
Bethesda, Md., USA).
[0061] The new DNA sequences of C. glutamicum coding for the sucC-
and sucD-genes have been discovered, and as SEQ ID No. 1 are part
of the present invention. The amino acid sequence of the
corresponding proteins has furthermore been derived from the
existing DNA sequences using the methods described above. The
resultant amino acid sequences of the sucC- and sucD-gene product
are shown in SEQ ID No. 2 and SEQ ID No. 3.
[0062] Coding DNA sequences that arise from SEQ ID No. 1 due to the
degeneracy of the genetic code are also a subject of the invention.
In the same way DNA sequences that hybridize with SEQ ID No. 1 or
parts of SEQ ID No. 1 are a subject of the invention. Finally, DNA
sequences that are produced by the polymerase chain reaction (PCR)
using primers obtained from SEQ ID No. 1 are also the subject of
the invention.
[0063] The person skilled in the art will find information on
identifying DNA sequences by means of hybridization 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 hybridization takes place under stringent
conditions, in other words only hybrids are formed in which the
probe and target sequence, i.e. the polynucleotides treated with
the probe, are at least 70% identical. It is known that the
thoroughness of the hybridization including the washing stages is
influenced or even determined by varying the buffer composition,
temperature and the salt concentration. The hybridization reaction
is preferably carried out at a relatively low degree of
thoroughness compared to the washing stages (Hybaid Hybridisation
Guide, Hybaid Limited, Teddington, UK, 1996).
[0064] A 5.times.SSC-buffer for example may be used at a
temperature of ca. 50-68.degree. C. for the hybridization reaction.
In this connection probes may also be hybridized with
polynucleotides that have less than 70% identity with the sequence
of the probe. Such hybrids are less stable and are removed by
washing under stringent conditions. This may be effected for
example by reducing the salt concentration to 2.times.SSC and
optionally subsequently to 0.5.times.SSC (The DIG System User's
Guide for Filter Hybridization, Boehringer Mannheim, Mannheim,
Germany, 1995), a temperature of ca. 50-68.degree. C. being
maintained. It is also optionally possible to reduce the salt
concentration down to 0.1.times.SSC. By stepwise raising of the
hybridization temperature in steps of ca. 1-2.degree. C. from 50 to
68.degree. C., polynucleotide fragments can be separated that
exhibit for example at least 70% or at least 80% or at least 90% to
95% identity to the sequence of the probe that is used. Further
instructions for hybridization are available on the market in the
form of so-called kits (e.g. DIG Easy Hyb von der Firma Roche
Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558).
[0065] The person skilled in the art can find details of the
enhancement 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).
[0066] It has now been found that coryneform bacteria produce
L-amino acids, in particular L-lysine, in an improved manner after
attenuation of the sucC- and/or sucD-gene.
[0067] In order to achieve such an attenuation, either the
expression of the sucC- and/or sucD-gene or the catalytic
properties of the enzyme proteins can be reduced or switched off.
Both measures may optionally be combined.
[0068] The reduction of the gene expression may be achieved by
suitable culture conditions or by genetic alteration (mutation) of
the signal structures of the gene expression. Signal structures of
the gene expression are for example repressal genes, activator
genes, operators, promoters, attenuators, ribosone bonding sites,
the start codon and terminators. The person skilled in the art can
find information on the above in for example patent application WO
96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949
(1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548
(1998), in Jensen and Hammer (Biotechnology and Bioengineering 58:
191 (1998)), in Patek et al. (Microbiology 142: 1297 (1996)) and in
known textbooks on genetics and molecular biology, such as for
example the textbook by Knippers ("Molekulare Genetik", 6.sup.th
Edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or the
textbook by Winnacker ("Gene und Klone", VCH Verlagsgesellschaft,
Weinheim, Germany, 1990).
[0069] Mutations that lead to an alteration and/or reduction of the
catalytic properties of enzyme proteins are known in the prior art;
there may be mentioned by way of example the work carried out by
Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617
(1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry
61: 1760-1762 (1997)) and Mockel ("Die Threonindehydratase aus
Corynebacterium glutamicum: Aufhebung der allosterischen Regulation
und Struktur des Enzyms", reports of the Julichs Research Centre,
Jul-2906, ISSN09442952, Julich, Germany, 1994). Overviews and
summaries may be obtained from known textbooks on genetics and
molecular biology, such as for example those by Hagemann
("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986).
[0070] Mutations cover such phenomena as transitions,
transversions, insertions and deletions. Depending on the effect of
the amino acid exchange on the enzyme activity, one speaks of
missense mutations or nonsense mutations. Insertions or deletions
of at least one base pair in a gene lead to frame shift mutations,
as a result of which false amino acids are incorporated or the
translation is prematurely arrested. Deletions of several codons
typically lead to a complete suppression of the enzyme activity.
Details of producing such mutations are part of the prior art and
can be obtained from known textbooks on genetics and molecular
biology, such as for example the textbook by Knippers ("Molekulare
Genetik", 6.sup.th Edition, Georg Thieme Verlag, Stuttgart,
Germany, 1995), that by Winnacker ("Gene und Klone", VCH
Verlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann
("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986).
[0071] A conventional method of mutating genes of C. glutamicum is
the method of gene disruption and gene replacement described by
Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991)).
[0072] In the method of gene disruption a central part of the
coding region of the gene that is of interest is cloned in a
plasmid vector that can replicate in a host (typically E. coli),
but not in C. glutamicum. Vectors that may be used include for
example pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)),
pK18mob or pK19mob (Schfer et al., Gene 145, 69-73 (1994)),
pK18mobsacB or pK19mobsacB (Jger et al., Journal of Bacteriology
174: 5462-65 (1992)), 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@Blunt (Firma
Invitrogen, Groningen, Niederlande; 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 central part of the coding region of the gene is
then converted by conjugation or transformation into the desired
strain of C. glutamicum.
[0073] The method of conjugation is described for example in
Schafer et al. (Applied and Environmental Microbiology 60, 756-759
(1994)). Methods for 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 coding region of the affected gene is disrupted by the
vector sequence and two incomplete alleles are obtained, each of
which lacks the 3'- and the 5'-end. This method has been used for
example by Fitzpatrick et al. (Applied Microbiology and
Biotechnology 42, 575-580 (1994)) in order to switch off the
recA-gene of C. glutamicum. The sucC- and/or sucD-gene may be
switched off in this way.
[0074] In the method of gene replacement a mutation, such as for
example a deletion, insertion or base exchange is produced in vitro
in the gene that is of interest. The allele that is produced is in
turn cloned in a vector that is not replicative for C. glutamicum
and the vector is then converted by transformation or conjugation
into the desired host for C. glutamicum. The incorporation of the
mutation and/or of the allele in the target gene and/or in the
target sequence is achieved after homologous recombination by means
of a first crossover event effecting integration and an appropriate
second crossover event effecting excision. This method has been
used for example by Peters-Wendisch (Microbiology 144, 915-927
(1998)) in order to switch off the pyc-gene of C. glutamicum by
means of a deletion. A deletion, insertion or a base exchange can
be incorporated into the sucC- and/or sucD-gene in this way.
[0075] A deletion, insertion or a base exchange can be incorporated
into the sucC- and/or sucD-gene in this way.
[0076] Furthermore, it was found that by means of one or more amino
acid replacements in the sucC-protein (SEQ ID No. 2) selected from
the group: replacement at position 22 by any other proteinogenic
amino acid except L-proline, replacement at position 44 by any
other proteinogenic amino acid except glycine, and replacement at
position 170 by any other proteinogenic amino acid except
L-alanine, an attenuation takes place and coryneform bacteria that
carry the corresponding amino acid replacement produced amino acids
in an improved way, in particular L-lysine and/or L-glutamic
acid.
[0077] Particularly preferred are one or more amino acid
replacements selected from the group: L-proline at position 22 by
L-serine, glycine at position 44 by L-glutamic acid, and L-alanine
at position 170 by L-threonine.
[0078] Most particularly preferred is an SucC-protein that contains
L-serine at position 22, L-glutamic acid at position 44, and
L-threonine at position 170, as shown in SEQ ID No. 5.
[0079] As shown in SEQ ID No. 4 the replacement of L-proline by
L-threonine at position 22 of the amino acid sequence may
preferably be effected by replacing the nucleobase cytosine at
position 64 by thymine, the replacement of glycine by L-glutamic
acid at position 44 of the amino acid sequence may preferably be
effected by replacing the nucleobase guanine at position 131 by
adenine, and the replacement of L-alanine by L-threonine at
position 170 of the amino acid sequence may preferably be effected
by replacing the nucleobase guanine at position 508 by adenine.
[0080] Conventional mutagenesis methods may be employed for the
mutagenesis, using mutagenic agents such as for example
N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light.
[0081] Furthermore, in vitro methods may be used for the
mutagenesis, such as for example a treatment with hydroxylamine (J.
H. Miller: A Short Course in Bacterial Genetics, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, 1992) or mutagenic
oligonucleotides (T. A. Brown: Gentechnologie fur Einsteiger,
Spektrum Akademischer Verlag, Heidelberg, 1993) or the polymerase
chain reaction (PCR), as described in the handbook by Newton and
Graham (PCR, Spektrum Akademischer Verlag, Heidelberg, 1994).
[0082] The corresponding sucC alleles and genes are sequenced and
incorporated into suitable hosts by for example the method of gene
replacement.
[0083] The present invention accordingly also provides coryneform
bacteria containing the SucC proteins, in which the amino acid
sequence shown in SEQ ID No. 2 contain one or more replacements
selected from the group: replacement at position 22 by any other
proteinogenic amino acid except L-proline, replacement at position
44 by any other proteinogenic amino acid except glycine, and
replacement at position 170 by any other proteinogenic amino acid
except L-alanine.
[0084] The invention accordingly furthermore provides
polynucleotide sequences derived from coryneform bacteria that
contain the genes or alleles coding for the aforementioned SucC
proteins.
[0085] Furthermore it may be advantageous for the production of
L-amino acids, in particular L-lysine, in addition to enhance, in
particular to over-express, one or more enzymes of the relevant
biosynthesis pathway, glycolysis, anaplerosis, citric acid cycle or
amino acid export, in order to attenuate the sucC- and/or
sucD-gene.
[0086] The expression "enhancement" describes in this connection
increasing the intracellular activity of one or more enzymes
(proteins) in a microorganism that are coded by the corresponding
DNA, by for example increasing the number of copies of the gene or
genes or alleles, using a strong promoter or a gene or allele that
codes with a high degree of activity for a corresponding enzyme
(protein), and optionally combining these measure.
[0087] Thus, in the production of L-lysine and/or L-glutamate, in
addition to the attenuation of the sucC- and/or sucD-gene, one or
more of the genes selected from the following group may be
enhanced, in particular over-expressed:
[0088] the dapA-gene coding for dihydrodipicolinate-synthase (EP-B
0 197 335),
[0089] the gap-gene coding for glyceraldehyde-3-phosphate
dehydrogenase (Eikmanns (1992), Journal of Bacteriology
174:6076-6086),
[0090] the gene tpi coding for triosephosphate isomerase (Eikmanns
(1992), Journal of Bacteriology 174:6076-6086),
[0091] the gene pgk coding for 3-phosphoglycerate kinase (Eikmanns
(1992), Journal of Bacteriology 174:6076-6086),
[0092] the pyc-gene coding for pyruvate carboxylase (Eikmanns
(1992), Journal of Bacteriology 174:6076-6086),
[0093] the mqo-gene coding for malate:quinone oxidoreductase
(Molenaar et al., European Journal of Biochemistry 254, 395-403
(1998)),
[0094] the gene lysC coding for a feed-back resistant aspartate
kinase (EP-B-0387527; EP-A-0699759; WO 00/63388)),
[0095] the lysE-gene coding for the L-lysine-export (DE-A-195 48
222),
[0096] the gene zwa1 coding for the Zwa1-protein (DE: 19959328.0,
DSM 13115).
[0097] Moreover, it may be advantageous for the production of
L-lysine and/or L-glutamate, in addition to the attenuation of the
sucC- and/or sucD-gene, at the same time to attenuate, in
particular to reduce the expression of one or more of the genes
selected from the group comprising:
[0098] the gene pck coding for phosphoenolpyruvate-carboxykinase
(DE 199 50 409.1, DSM 13047),
[0099] the gene pgi coding for glucose-6-phosphate isomerase (U.S.
Ser. No. 09/396,478, DSM 12969),
[0100] the gene poxB coding for pyruvate-oxidase (DE:1995 1975.7,
DSM 13114),
[0101] the gene zwa2 coding for the zwa2-protein (DE: 19959327.2,
DSM 13113).
[0102] Furthermore it may be advantageous for the production of
amino acid, in particular L-lysine and/or L-glutamate, in addition
to the attenuation of the sucC- and/or sucD-gene to switch off
undesirable secondary reactions (Nakayama: "Breeding of Amino Acid
Producing Microorganisms", in: Overproduction of Microbial
Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London,
UK, 1982).
[0103] The microorganisms containing the polynucleotide according
to claim 1 are also the subject of the invention and 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,
Braunschweig/Wiesbaden, 1994)).
[0104] The culture medium to be used must suitably satisfy the
demands 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).
[0105] As carbon source there may be used 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 and ethanol, and
organic acids such as for example acetic acid. These substances may
be used individually or as a mixture.
[0106] As nitrogen source there may be used 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. The
nitrogen sources may be used individually or as a mixture.
[0107] As phosphorus source there may be used phosphoric acid,
potassium dihydrogen phosphate or dipotassium hydrogen phosphate,
or the corresponding sodium-containing salts. The culture medium
must furthermore contain salts of metals such as for Example
magnesium sulfate or iron sulfate that are necessary for growth.
Finally, essential growth substances such as amino acids and
vitamins may, in addition to the substances mentioned above, be
used. 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 one-off addition, or may be
metered in during the actual cultivation in a suitable way.
[0108] 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 prevent 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 the desired product has been formed. This
target is normally achieved within 10 hours to 160 hours.
[0109] A pure culture of the strain Escherichia coli
DH5.alpha.mcr/pK18mobsacBsucDdel was filed according to the
Budapest Convention on 29 Sep. 2000 as DSM 13749 at the German
Collection for Microorganisms and Cell Cultures (DSMZ, Brunswick,
Germany).
[0110] A pure culture of the strain Escherichia coli
Top10/pCRBluntsucCint was filed according to the Budapest
Convention on 29 Sep. 2000 as DSM 13750 at the German Collection
for Microorganisms and Cell Cultures (DSMZ, Brunswick,
Germany).
[0111] Methods for determining L-amino acids are known from the
prior art. The analysis may be carried out as described for example
by Spackman et al. (Analytical Chemistry, 30, (1958), 1190) by
anion exchange chromatography followed by ninhydrin derivatisation
or may be carried out by reverse phase HPLC, as described by
Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).
[0112] The present invention is described in more detail
hereinafter with the aid of embodiments.
EXAMPLE 1
[0113] Production of a genomic cosmid gene library from
Corynebacterium glutamicum ATCC 13032
[0114] 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-O.sub.2). 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-O.sub.2) 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 Res. 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.
EXAMPLE 2
[0115] Isolation and Sequencing of the Genes sucC and sucD
[0116] 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).
[0117] 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, Niederlande, 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 frequencing 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 DH5.alpha.MCR (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
dRhodamin 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).
[0118] The raw sequence data that were 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).
[0119] The nucleotide sequence that was obtained is illustrated in
SEQ ID No. 1. Analysis of the nucleotide sequence showed an open
reading frame of 1206 base pairs, which was identified as
sucC-gene, as well as an open reading frame of 882 base pairs,
identified as sucD. The sucC-gene codes for a polypeptide of 402
amino acids, which is shown in SEQ ID No. 2. The sucD-gene codes
for a polypeptide of 294 amino acids, which is shown in SEQ ID No.
3.
EXAMPLE 3
[0120] 3.1 Production of an Integration Vector for the Integration
Mutagenesis of the sucC-Gene
[0121] 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 sucC-gene
for C. glutamicum known from Example 1 the following
oligonucleotides were selected for the polymerase chain reaction
(see SEQ ID No. 6 and SEQ ID No. 7):
1 primer sucC-in1: 5'CGC GCG AAT CGT TCG TAT 3' primer sucC-in2:
5'CGC CAC CAA TGT CTA GGA 3'
[0122] The indicated primers were synthesised by MWG Biotech
(Ebersberg, Germany) and the PCR reaction was carried out with the
Pwo polymerase from Boehringer Mannheim (Germany, Product
Description Pwo DNA Polymerase, Product No. 1 644 947) according to
the standard PCR method of Innis et al. (PCR Protocols. A Guide to
Methods and Applications, 1990, Academic Press). With the aid of
the polymerase chain reaction the primers permit the enhancement of
an approximately 0.55 kb large internal fragment of the sucC-gene.
The product enhanced in this way was checked by electrophoresis in
a 0.8% agarose gel.
[0123] The enhanced DNA fragment was ligated into the vector
pCR.RTM.Blunt II (Bernard et al., Journal of Molecular Biology,
234: 534-541 (1993)) using the Zero Blunt.TM. Kit from Invitrogen
Corporation (Carlsbad, Calif., USA; Catalogue Number K2700-20).
[0124] The E. coli strain TOP10 was then electroporated into the
ligation batch (Hanahan, In: DNA Cloning. A Practical Approach,
Vol. I, IRL-Press, Oxford, Washington D.C., USA, 1985). The
selection of plasmid-carrying cells was performed 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., 1989) that had been
supplemented with 25 mg/l of kanamycin. Plasmid DNA was isolated
from a transformant with the aid of the QIAprep Spin Miniprep Kit
from Qiagen and checked by restriction with the restriction enzyme
EcoRI followed by agarose gel electrophoresis (0.8%). The plasmid
was named pCRBluntsucCint and is shown in FIG. 1.
[0125] 3.2 Deletion of the sucD-Gene
[0126] For this purpose chromosomal DNA was isolated from the
strain ATCC13032 by the method of Tauch et al. (1995, Plasmid
33:168-179). On the basis of the sequence of the sucD-gene for C.
glutamicum known from Example 2 the oligonucleotides described
hereinafter were selected for producing the sucD deletion allele
(see SEQ ID No. 8 to SEQ ID No. 11):
2 primer sucD-d1: 5'-CGA TGT GAT TGC GCT TGA TG -3' deletion primer
sucD-d2: 5'-ACC TCA CGC ATA AGC TTC GCA TGC TCT GAA CCT TCC GAA C
-3' deletion primer sucD-d3: 5'-GTT CGG AAG GTT CAG AGC ATG CGA AGC
TTA TGC GTG AGG T -3' primer sucD-d4: 5'-ATG AAG CCA GCG ACT GCA GA
-3'
[0127] The relevant primers were synthesised by MWG Biotech
(Ebersberg, Germany) and the PCR reaction was carried out using the
Pfu polymerase (Stratagene, Product. No. 600135, La Jolla, USA) and
the PTC 100-Thermocyclers (MJ Research Inc., Waltham, USA). With
the aid of the polymerase chain reaction the primers permit the
enhancement of a sucD allele with internal deletion. The product
enhanced in this way was tested by electrophoresis in a 0.8%
agarose gel and was also sequenced as described by Sanger et al.
(Proceedings of the National Academy of Sciences of the United
States of America, 74:5463-5467, 1977).
EXAMPLE 4
[0128] 4.1 Integration Mutagenesis of the sucC-Gene in the Strain
DSM 5715
[0129] The vector pCRBluntsucCint mentioned in Example 3.1 was
electroporated into C. glutamicum DSM 5715 (EP 0 435 132) according
to the electroporation method of Tauch et. al. (FEMS
Microbiological Letters, 123:343-347 (1994)). The strain DSM 5715
is an AEC resistant L-lysine producer. The vector pCRBlunt-sucCint
cannot independently replicate in DSM5715 and accordingly only
remains in the cellulose if it had integrated into the chromosome
of DSM 5715. The selection of clones with pCRBluntsucCint
integrated into the chromosome is performed by plating out the
electroporation 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 15 mg/l of kanamycin.
[0130] In order to detect the integration the sucCint fragment was
labelled according to the method described in "The DIG System
User's Guide for Filter Hybridization" of Boehringer Mannheim GmbH
(Mannheim, Germany, 1993) using the Dig-Hybridization Kit from
Boehringer. Chromosomal DNA of a potential integrant was isolated
according to the method of Eikmanns et al. (Microbiology 140:
1817-1828 (1994)) and was cut in each case with the restriction
enzyme SphI and HindIII. The resultant fragments were separated by
means of agarose gel electrophoresis and hybridized at 68.degree.
C. using the Dig-Hybridization Kit from Boehringer. The plasmid
pCRBluntsucCint named in Example 3.1 had inserted itself into the
chromosome of DSM5715 within the chromosomal sucC-gene. The strain
was identified as DSM5715::pCRBluntsucCint.
[0131] 4.2 Construction of the Exchange Vector
pK18mobsacBsucDdel
[0132] The sucD-deletion derivative obtained in Example 3.2 was,
after separation in an agarose gel (0.8%) using the Qiagenquick Gel
Extraction Kit (Qiagen, Hilden, Germany), isolated from the agarose
gel and then used with the mobilisable cloning vector pK18mobsacB
(Schfer et al. (1994), Gene 14: 69-73) for the ligation. This had
previously been cleaved with the restriction enzymes XmaI- and
XbaI, mixed with the sucD-deletion allele, and treated with
T4-DNA-ligase (Amersham Pharmacia, Freiburg, Germany).
[0133] The E. coli strain DH5.alpha.mcr (Grant, 1990, Proceedings
of the National Academy of Sciences U.S.A., 87:4645-4649) was then
electroporated with the ligation batch (Hanahan, In. DNA Cloning. A
Practical Approach, Vol.1, ILR-Press, Cold Spring Harbor, N.Y.,
1989). The plasmid-carrying cells were selected by plating out the
transformation batch onto LB agar (Sambrock et al., Molecular
Cloning: A Laboratory Manual. 2.sup.nd Ed. Cold Spring Harbor,
N.Y., 1989) that had been supplemented with 25 mg/l of
kanamycin.
[0134] Plasmid DNA was isolated from a transformant by means of the
QIAprep Spin Miniprep Kit from Qiagen, and the cloned sucD-deletion
allele was verified by means of sequencing by the company MWG
Biotech (Ebersberg, Germany). The plasmid was named
pK18mobsacBsucDdel. The strain was identified as E.
coliH5.alpha.mcr/pK18mobsacBsucDdel.
[0135] 4.3 Deletion Mutagenesis of the sucD-Gene in the C.
glutamicum Strain DSM 5715
[0136] The vector pK18mobsacBsucDdel mentioned in Example 4.2 was
electroporated according to the electroporation method of Tauch et
al., (1989 FEMS Microbiology Letters 123: 343-347). The vector
cannot replicate independently in DSM 5715 and accordingly only
remains in the cellulose if it has integrated into the chromosome.
The selection of clones with integrated pK18mobsacBsucDdel was
performed by plating out the electroporation batch onto LB-agar
(Sambrock et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd
Ed., Cold Spring Harbor, N.Y., 1989) that had been supplemented
with 15 mg/l of kanamycin. Cultivated clones were streaked out onto
LB-agar plates containing 25 mg/l of kanamycin and incubated for 16
hours at 33.degree. C.
[0137] In order to achieve the excision of the plasmid together
with the complete chromosomal copy of the sucD-gene, the clones
were then grown on LB-agar containing 10% sucrose. The plasmid
pK18mobsacB contains a copy of the sacB-gene, which converts
sucrose into levansucrase that is not toxic for C. glutamicum.
Accordingly only those clones in which the integrated
pK18mobsacBsucDdel has in turn been excised can be grown on LB-agar
containing sucrose. In the excision either the complete chromosomal
copy of the sucD-gene or the incomplete copy together with the
internal deletion can be excised together with the plasmid.
[0138] In order to detect whether the incomplete copy of sucD still
remains in the chromosome, the plasmid pK18mobsacBsucDdel fragment
was labelled according to the method described in "The DIG System
User's Guide for Filter Hybridization" published by Boehringer
Mannheim GmbH (Mannheim, Germany, 1993) using the Dig-Hybridization
Kit from Boehringer. Chromosomal DNA of a potential deletion mutant
was isolated according to the method of Eikmanns et al.
(Microbiology 140: 1817-1828 (1994)) and was in each case cut into
separate sections using the restriction enzymes SphI and PstI. The
resultant fragments were separated by agarose gel electrophoresis
and hybridized at 68.degree. C. using the Dig Hybridization Kit
from Boehringer. On the basis of the resultant fragments it could
be shown that the strain DSM5715 has lost its complete copy of the
sucD-gene and instead only the deleted copy is still available.
[0139] The strain was identified as C. glutamicum
DSM5715.DELTA.sucD.
EXAMPLE 5
[0140] 5.1 Production of L-Glutamate Using the Strain DSM
5715::pCRBluntsucCint
[0141] The C. glutamicum strain DSM5715::pCRBluntsucCint obtained
in Example 4.1 was cultivated in a suitable nutrient medium for
producing L-glutamate and the glutamate content in the culture
supernatant was determined.
[0142] For this purpose the strain was first of all incubated for
24 hours at 33.degree. C. on agar plates with the corresponding
antibiotic (brain-heart agar with kanamycin (25 mg/l). A
pre-culture was inoculated using this agar plate culture (10 ml of
medium in a 100 ml Erlenmeyer flask). The full medium Cg III was
used as medium for the pre-culture.
3 Medium Cg III NaCl 2.5 g/l Bacto-Peptone 10 g/l Bacto-Yeast
Extract 10 g/l Glucose (separately autoclaved) 2% (w/v) The pH was
adjusted to pH 7.4
[0143] Kanamycin (25 mg/l) was added to this medium. The
pre-culture was incubated on a shaker for 16 hours at 33.degree. C.
at 240 rpm. A main culture was inoculated from this pre-culture so
that the initial optical density (660 nm) of the main culture was
0.1 OD. The medium MM was used for the main culture.
4 Medium MM CSL (Corn Steep Liquor) 5 g/l MOPS
(Morpholinopropanesulfonic 20 g/l acid) Glucose (separately
autoclaved) 50 g/l Salts: (NH.sub.4).sub.2SO.sub.4) 25 g/l
KH.sub.2PO.sub.4 0.1 g/l MgSO.sub.4.7H.sub.20 1.0 g/l
CaCl.sub.2.2H.sub.20 10 mg/l FeSO.sub.4.7H.sub.20 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 Fumarate (sterile
filtered) 5.81 g/l Leucine (sterile filtered) 0.1 g/l CaCO.sub.3 25
g/l
[0144] CSL, MOPS and the salt solution are adjusted with ammonia
water to pH 7 and autoclaved. The sterile substrate and vitamin
solutions as well as the dry autoclaved CaCO.sub.3 are then
added.
[0145] Cultivation takes place in a 10 ml volume in a 100 ml
Erlenmeyer flask with baffles. Kanamycin (25 mg/l) was added.
Cultivation took place at 33.degree. C. and 80% atmospheric
humidity.
[0146] After 24 hours the OD was measured at a measurement
wavelength of 660 nm using the Biomek 1000 instrument (Beckmann
Instruments GmbH, Munich). The amount of glutamate formed was
measured in an amino acid analyser from Eppendorf-BioTronik
(Hamburg, Germany) by ion exchange chromatography and post-column
derivatisation with ninhydrin detection.
[0147] The result of the test is shown in Table 1.
5 TABLE 1 OD L-glutamate Strain (660 nm) mg/l DSM5715 10.4 20
DSM5715::pCRBlunt 3.9 154 sucCint
[0148] 5.2 Production of L-Glutamate Using the Strain
DSM5715.DELTA.sucD
[0149] The C. glutamicum strain DSM5715/pK18mobsacBsucDdel obtained
in Example 4.3 was cultivated in a nutrient medium suitable for
producing L-glutamate and the glutamate content in the culture
supernatant was measured.
[0150] For this purpose the strain was first of all incubated for
24 hours at 33.degree. C. on agar plates. A preculture was
inoculated using this agar plate culture (10 ml medium in 100 ml
Erlenmeyer flask). The full medium CgIII was used for the
preculture. Kanamycin (25 mg/l) was added to this medium. The
preculture was incubated on a shaker for 16 hours at 33.degree. C.
and at 240 rpm. A main culture was inoculated from this preculture
so that the initial OD (660 nm) of the main culture was 0.1 OD. The
medium MM was used for the main culture.
[0151] The cultivation was carried out in a 10 ml volume in a 100
ml Erlenmeyer flask equipped with baffles. Cultivation was carried
out at 33.degree. C. and 80% atmospheric humidity.
[0152] After 72 hours the OD was measured at a measurement
wavelength of 660 nm using a Biomek 1000 instrument (Beckmann
Instruments GmbH, Munich). The amount of glutamate formed was
measured with an amino acid analyser from Eppendorf-BioTronik
(Hamburg, Germany) by ion exchange chromatography and post-column
derivatisation with ninhydrin detection.
[0153] The result of the test is shown in Table 2.
6 TABLE 2 OD L-glutamate Strain (660 nm) mg/l DSM5715 8.1 7
DSM5715.DELTA.sucD 13.3 33
BRIEF DESCRIPTION OF THE FIGURES
[0154] FIG. 1: Map of the plasmid pCRBluntsucCint.
[0155] FIG. 2: Map of the plasmid pK18mobsacBsucDdel
[0156] The acronyms and abbreviations used in FIG. 1 have the
following meanings:
7 KmR: Kanamycin resistance gene Zeocin: Zeocin resistance gene
HindIII Cutting site of the restriction enzyme HindIII SphI Cutting
site of the restriction enzyme SphI EcoRI: Cutting site of the
restriction enzyme EcoRI sucCint: Internal fragment of the
sucC-gene ColE1 ori: Replication origin of the plamid ColE1
[0157] The acronyms and abbreviations used in FIG. 2 have the
following meanings:
8 oriV: ColE1-like origin of pMB1 sacB The sac-B gene coding for
the protein levansucrose RP4mob: RP4-mobilisation site Kan:
Resistance gene for kanamycin sucDdel': 5'-terminal fragment of the
sucD-gene from C. glutamicum sucDdel": 3'-terminal fragment of the
sucD-gene from C. glutamicum SphI: Cutting site of the restriction
enzyme SphI PstI: Cutting site of the restriction enzyme PstI XmaI:
Cutting site of the restriction enzyme XmaI XbaI: Cutting site of
the restriction enzyme XbaI
[0158]
Sequence CWU 1
1
5 1 2410 DNA Corynebacterium glutamicum CDS (142)..(1347) sucC
sequence 1 gcaccacgga tccaattttg ttgcaatttg caaagtttac agtgttagac
ttcacaatac 60 gatcatattg gtgagttgaa acacttactt ttacgggaag
actttgttaa agacgcagaa 120 ggctctaagc atgggccgga a atg gaa ttg gca
gtg gat ctt ttt gaa tac 171 Met Glu Leu Ala Val Asp Leu Phe Glu Tyr
1 5 10 caa gca cgg gac ctc ttt gaa acc cat ggt gtg cca gtg ttg aag
gga 219 Gln Ala Arg Asp Leu Phe Glu Thr His Gly Val Pro Val Leu Lys
Gly 15 20 25 att gtg gca tca aca cca gag gcg gcg agg aaa gcg gct
gag gaa atc 267 Ile Val Ala Ser Thr Pro Glu Ala Ala Arg Lys Ala Ala
Glu Glu Ile 30 35 40 ggc gga ctg acc gtc gtc aag gct cag gtc aag
gtg ggc gga cgt ggc 315 Gly Gly Leu Thr Val Val Lys Ala Gln Val Lys
Val Gly Gly Arg Gly 45 50 55 aag gcg ggt ggc gtc cgt gtg gca ccg
acg tcg gct cag gct ttt gat 363 Lys Ala Gly Gly Val Arg Val Ala Pro
Thr Ser Ala Gln Ala Phe Asp 60 65 70 gct gcg gat gcg att ctc ggc
atg gat atc aaa gga cac act gtt aat 411 Ala Ala Asp Ala Ile Leu Gly
Met Asp Ile Lys Gly His Thr Val Asn 75 80 85 90 cag gtg atg gtg gcg
cag ggc gct gac att gct gag gaa tac tat ttc 459 Gln Val Met Val Ala
Gln Gly Ala Asp Ile Ala Glu Glu Tyr Tyr Phe 95 100 105 tcc att ttg
ttg gat cgc gcg aat cgt tcg tat ctg gct atg tgc tct 507 Ser Ile Leu
Leu Asp Arg Ala Asn Arg Ser Tyr Leu Ala Met Cys Ser 110 115 120 gtt
gaa ggt ggc atg gag atc gag atc ctg gcg aag gaa aag cct gaa 555 Val
Glu Gly Gly Met Glu Ile Glu Ile Leu Ala Lys Glu Lys Pro Glu 125 130
135 gct ttg gca aag gtg gaa gtg gat ccc ctc act ggt att gat gag gac
603 Ala Leu Ala Lys Val Glu Val Asp Pro Leu Thr Gly Ile Asp Glu Asp
140 145 150 aaa gcg cgg gag att gtc act gct gct ggc ttt gaa act gag
gtg gca 651 Lys Ala Arg Glu Ile Val Thr Ala Ala Gly Phe Glu Thr Glu
Val Ala 155 160 165 170 gag aaa gtc att ccg gtg ctg atc aag atc tgg
cag gtg tat tac gaa 699 Glu Lys Val Ile Pro Val Leu Ile Lys Ile Trp
Gln Val Tyr Tyr Glu 175 180 185 gag gaa gca aca ctc gtt gag gtg aac
ccg ttg gtg ctc acg gat gac 747 Glu Glu Ala Thr Leu Val Glu Val Asn
Pro Leu Val Leu Thr Asp Asp 190 195 200 ggc gat gtg att gcg ctt gat
ggc aag atc acg ctg gat gat aac gct 795 Gly Asp Val Ile Ala Leu Asp
Gly Lys Ile Thr Leu Asp Asp Asn Ala 205 210 215 gat ttc cgc cat gat
aac cgt ggt gcg ttg gct gaa tct gcc ggt ggc 843 Asp Phe Arg His Asp
Asn Arg Gly Ala Leu Ala Glu Ser Ala Gly Gly 220 225 230 ttg gac att
ttg gaa ctg aag gcc aag aag aat gat ctg aac tac gtg 891 Leu Asp Ile
Leu Glu Leu Lys Ala Lys Lys Asn Asp Leu Asn Tyr Val 235 240 245 250
aaa ctt gat ggc tct gtg ggc atc att ggc aat ggt gca ggt ttg gtg 939
Lys Leu Asp Gly Ser Val Gly Ile Ile Gly Asn Gly Ala Gly Leu Val 255
260 265 atg tcc acg ttg gat atc gtg gct gca gct ggt gaa cgc cat ggt
ggg 987 Met Ser Thr Leu Asp Ile Val Ala Ala Ala Gly Glu Arg His Gly
Gly 270 275 280 cag cgc ccc gcg aac ttc cta gac att ggt ggc gga gca
tca gct gaa 1035 Gln Arg Pro Ala Asn Phe Leu Asp Ile Gly Gly Gly
Ala Ser Ala Glu 285 290 295 tcg atg gct gct ggt ctc gat gtg atc ctt
ggg gat agc cag gta cgc 1083 Ser Met Ala Ala Gly Leu Asp Val Ile
Leu Gly Asp Ser Gln Val Arg 300 305 310 agt gtg ttt gtg aat gtg ttt
ggt ggc atc acc gcg tgt gat gtg gtg 1131 Ser Val Phe Val Asn Val
Phe Gly Gly Ile Thr Ala Cys Asp Val Val 315 320 325 330 gca aag gga
atc gtt gga gct ttg gat gtg ctc ggc gat caa gca acg 1179 Ala Lys
Gly Ile Val Gly Ala Leu Asp Val Leu Gly Asp Gln Ala Thr 335 340 345
aag cct ctt gtg gtg cgc ctt gat ggc aac aac gtg gtg gaa ggc aga
1227 Lys Pro Leu Val Val Arg Leu Asp Gly Asn Asn Val Val Glu Gly
Arg 350 355 360 cga atc ctc gcg gaa tat aac cac cct ttg gtc acc gtt
gtg gag ggt 1275 Arg Ile Leu Ala Glu Tyr Asn His Pro Leu Val Thr
Val Val Glu Gly 365 370 375 atg gat gca gcg gct gat cac gct gcc cat
ttg gcc aat ctt gcc cag 1323 Met Asp Ala Ala Ala Asp His Ala Ala
His Leu Ala Asn Leu Ala Gln 380 385 390 cac ggc cag ttc gca acc gct
aat tagttaagga gcacctgttt aatc atg 1374 His Gly Gln Phe Ala Thr Ala
Asn Met 395 400 tct att ttt ctc aat tca gat tcc cgc atc atc att cag
ggc att acc 1422 Ser Ile Phe Leu Asn Ser Asp Ser Arg Ile Ile Ile
Gln Gly Ile Thr 405 410 415 ggt tcg gaa ggt tca gag cat gcg cgt cga
att tta gcc tct ggt gcg 1470 Gly Ser Glu Gly Ser Glu His Ala Arg
Arg Ile Leu Ala Ser Gly Ala 420 425 430 435 aag ctc gtg ggt ggc acc
aac ccc cgc aaa gct ggg caa acc att ttg 1518 Lys Leu Val Gly Gly
Thr Asn Pro Arg Lys Ala Gly Gln Thr Ile Leu 440 445 450 atc aat gac
act gag ttg cct gta ttt ggc act gtt aag gaa gca atg 1566 Ile Asn
Asp Thr Glu Leu Pro Val Phe Gly Thr Val Lys Glu Ala Met 455 460 465
gag gaa acg ggt gcg gat gtc acc gta att ttc gtt cct cca gcc ttt
1614 Glu Glu Thr Gly Ala Asp Val Thr Val Ile Phe Val Pro Pro Ala
Phe 470 475 480 gcc aaa gct gcg atc att gaa gct atc gac gct cac atc
cca ctg tgc 1662 Ala Lys Ala Ala Ile Ile Glu Ala Ile Asp Ala His
Ile Pro Leu Cys 485 490 495 gtg att att act gag ggc atc cca gtg cgt
gac gct tct gag gcg tgg 1710 Val Ile Ile Thr Glu Gly Ile Pro Val
Arg Asp Ala Ser Glu Ala Trp 500 505 510 515 gct tat gcc aag aag gtg
gga cac acc cgc atc att ggc cct aac tgc 1758 Ala Tyr Ala Lys Lys
Val Gly His Thr Arg Ile Ile Gly Pro Asn Cys 520 525 530 cca ggc att
att act ccc ggc gaa tct ctt gcg gga att acg ccg gca 1806 Pro Gly
Ile Ile Thr Pro Gly Glu Ser Leu Ala Gly Ile Thr Pro Ala 535 540 545
aac att gca ggt tcc ggc ccg atc ggg ttg atc tca aag tcg gga aca
1854 Asn Ile Ala Gly Ser Gly Pro Ile Gly Leu Ile Ser Lys Ser Gly
Thr 550 555 560 ctg act tat cag atg atg tac gaa ctt tca gat att ggc
att tct acg 1902 Leu Thr Tyr Gln Met Met Tyr Glu Leu Ser Asp Ile
Gly Ile Ser Thr 565 570 575 gcg att ggt att ggc ggt gac cca atc atc
ggt aca acc cat atc gac 1950 Ala Ile Gly Ile Gly Gly Asp Pro Ile
Ile Gly Thr Thr His Ile Asp 580 585 590 595 gct ctg gag gcc ttt gaa
gct gat cct gag acc aag gca atc gtc atg 1998 Ala Leu Glu Ala Phe
Glu Ala Asp Pro Glu Thr Lys Ala Ile Val Met 600 605 610 atc ggt gag
atc ggt gga gat gca gag gaa cgc gct gct gac ttc att 2046 Ile Gly
Glu Ile Gly Gly Asp Ala Glu Glu Arg Ala Ala Asp Phe Ile 615 620 625
tct aag cac gtg aca aaa cca gtt gtg ggt tac gtg gca ggc ttt acc
2094 Ser Lys His Val Thr Lys Pro Val Val Gly Tyr Val Ala Gly Phe
Thr 630 635 640 gcc cct gaa gga aag acc atg ggg cat gct ggc gcc atc
gtg aca ggt 2142 Ala Pro Glu Gly Lys Thr Met Gly His Ala Gly Ala
Ile Val Thr Gly 645 650 655 tca gaa ggc act gcg cga gca aag aag cat
gca ttg gag gcc gtg ggt 2190 Ser Glu Gly Thr Ala Arg Ala Lys Lys
His Ala Leu Glu Ala Val Gly 660 665 670 675 gtt cgc gtg gga aca act
ccg agt gaa acc gcg aag ctt atg cgt gag 2238 Val Arg Val Gly Thr
Thr Pro Ser Glu Thr Ala Lys Leu Met Arg Glu 680 685 690 gta gtt gca
gct ttg taactaacag gccacagatc ttagctttga ccagcggatt 2293 Val Val
Ala Ala Leu 695 tgtggctaat cgcccggtct gtgtagagta ttcatctgtg
cgcaggacag tgtgacaaac 2353 actgaatagt gcatggcttt aaggccctgt
ggcgcagttg gttagcgcgc cgccctg 2410 2 402 PRT Corynebacterium
glutamicum 2 Met Glu Leu Ala Val Asp Leu Phe Glu Tyr Gln Ala Arg
Asp Leu Phe 1 5 10 15 Glu Thr His Gly Val Pro Val Leu Lys Gly Ile
Val Ala Ser Thr Pro 20 25 30 Glu Ala Ala Arg Lys Ala Ala Glu Glu
Ile Gly Gly Leu Thr Val Val 35 40 45 Lys Ala Gln Val Lys Val Gly
Gly Arg Gly Lys Ala Gly Gly Val Arg 50 55 60 Val Ala Pro Thr Ser
Ala Gln Ala Phe Asp Ala Ala Asp Ala Ile Leu 65 70 75 80 Gly Met Asp
Ile Lys Gly His Thr Val Asn Gln Val Met Val Ala Gln 85 90 95 Gly
Ala Asp Ile Ala Glu Glu Tyr Tyr Phe Ser Ile Leu Leu Asp Arg 100 105
110 Ala Asn Arg Ser Tyr Leu Ala Met Cys Ser Val Glu Gly Gly Met Glu
115 120 125 Ile Glu Ile Leu Ala Lys Glu Lys Pro Glu Ala Leu Ala Lys
Val Glu 130 135 140 Val Asp Pro Leu Thr Gly Ile Asp Glu Asp Lys Ala
Arg Glu Ile Val 145 150 155 160 Thr Ala Ala Gly Phe Glu Thr Glu Val
Ala Glu Lys Val Ile Pro Val 165 170 175 Leu Ile Lys Ile Trp Gln Val
Tyr Tyr Glu Glu Glu Ala Thr Leu Val 180 185 190 Glu Val Asn Pro Leu
Val Leu Thr Asp Asp Gly Asp Val Ile Ala Leu 195 200 205 Asp Gly Lys
Ile Thr Leu Asp Asp Asn Ala Asp Phe Arg His Asp Asn 210 215 220 Arg
Gly Ala Leu Ala Glu Ser Ala Gly Gly Leu Asp Ile Leu Glu Leu 225 230
235 240 Lys Ala Lys Lys Asn Asp Leu Asn Tyr Val Lys Leu Asp Gly Ser
Val 245 250 255 Gly Ile Ile Gly Asn Gly Ala Gly Leu Val Met Ser Thr
Leu Asp Ile 260 265 270 Val Ala Ala Ala Gly Glu Arg His Gly Gly Gln
Arg Pro Ala Asn Phe 275 280 285 Leu Asp Ile Gly Gly Gly Ala Ser Ala
Glu Ser Met Ala Ala Gly Leu 290 295 300 Asp Val Ile Leu Gly Asp Ser
Gln Val Arg Ser Val Phe Val Asn Val 305 310 315 320 Phe Gly Gly Ile
Thr Ala Cys Asp Val Val Ala Lys Gly Ile Val Gly 325 330 335 Ala Leu
Asp Val Leu Gly Asp Gln Ala Thr Lys Pro Leu Val Val Arg 340 345 350
Leu Asp Gly Asn Asn Val Val Glu Gly Arg Arg Ile Leu Ala Glu Tyr 355
360 365 Asn His Pro Leu Val Thr Val Val Glu Gly Met Asp Ala Ala Ala
Asp 370 375 380 His Ala Ala His Leu Ala Asn Leu Ala Gln His Gly Gln
Phe Ala Thr 385 390 395 400 Ala Asn 3 294 PRT Corynebacterium
glutamicum 3 Met Ser Ile Phe Leu Asn Ser Asp Ser Arg Ile Ile Ile
Gln Gly Ile 1 5 10 15 Thr Gly Ser Glu Gly Ser Glu His Ala Arg Arg
Ile Leu Ala Ser Gly 20 25 30 Ala Lys Leu Val Gly Gly Thr Asn Pro
Arg Lys Ala Gly Gln Thr Ile 35 40 45 Leu Ile Asn Asp Thr Glu Leu
Pro Val Phe Gly Thr Val Lys Glu Ala 50 55 60 Met Glu Glu Thr Gly
Ala Asp Val Thr Val Ile Phe Val Pro Pro Ala 65 70 75 80 Phe Ala Lys
Ala Ala Ile Ile Glu Ala Ile Asp Ala His Ile Pro Leu 85 90 95 Cys
Val Ile Ile Thr Glu Gly Ile Pro Val Arg Asp Ala Ser Glu Ala 100 105
110 Trp Ala Tyr Ala Lys Lys Val Gly His Thr Arg Ile Ile Gly Pro Asn
115 120 125 Cys Pro Gly Ile Ile Thr Pro Gly Glu Ser Leu Ala Gly Ile
Thr Pro 130 135 140 Ala Asn Ile Ala Gly Ser Gly Pro Ile Gly Leu Ile
Ser Lys Ser Gly 145 150 155 160 Thr Leu Thr Tyr Gln Met Met Tyr Glu
Leu Ser Asp Ile Gly Ile Ser 165 170 175 Thr Ala Ile Gly Ile Gly Gly
Asp Pro Ile Ile Gly Thr Thr His Ile 180 185 190 Asp Ala Leu Glu Ala
Phe Glu Ala Asp Pro Glu Thr Lys Ala Ile Val 195 200 205 Met Ile Gly
Glu Ile Gly Gly Asp Ala Glu Glu Arg Ala Ala Asp Phe 210 215 220 Ile
Ser Lys His Val Thr Lys Pro Val Val Gly Tyr Val Ala Gly Phe 225 230
235 240 Thr Ala Pro Glu Gly Lys Thr Met Gly His Ala Gly Ala Ile Val
Thr 245 250 255 Gly Ser Glu Gly Thr Ala Arg Ala Lys Lys His Ala Leu
Glu Ala Val 260 265 270 Gly Val Arg Val Gly Thr Thr Pro Ser Glu Thr
Ala Lys Leu Met Arg 275 280 285 Glu Val Val Ala Ala Leu 290 4 1206
DNA Corynebacterium glutamicum CDS (1)..(1206) sucC coding sequence
4 atg gaa ttg gca gtg gat ctt ttt gaa tac caa gca cgg gac ctc ttt
48 Met Glu Leu Ala Val Asp Leu Phe Glu Tyr Gln Ala Arg Asp Leu Phe
1 5 10 15 gaa acc cat ggt gtg tca gtg ttg aag gga att gtg gca tca
aca cca 96 Glu Thr His Gly Val Ser Val Leu Lys Gly Ile Val Ala Ser
Thr Pro 20 25 30 gag gcg gcg agg aaa gcg gct gag gaa atc ggc gaa
ctg acc gtc gtc 144 Glu Ala Ala Arg Lys Ala Ala Glu Glu Ile Gly Glu
Leu Thr Val Val 35 40 45 aag gct cag gtc aag gtg ggc gga cgt ggc
aag gcg ggt ggc gtc cgt 192 Lys Ala Gln Val Lys Val Gly Gly Arg Gly
Lys Ala Gly Gly Val Arg 50 55 60 gtg gca ccg acg tcg gct cag gct
ttt gat gct gcg gat gcg att ctc 240 Val Ala Pro Thr Ser Ala Gln Ala
Phe Asp Ala Ala Asp Ala Ile Leu 65 70 75 80 ggc atg gat atc aaa gga
cac act gtt aat cag gtg atg gtg gcg cag 288 Gly Met Asp Ile Lys Gly
His Thr Val Asn Gln Val Met Val Ala Gln 85 90 95 ggc gct gac att
gct gag gaa tac tat ttc tcc att ttg ttg gat cgc 336 Gly Ala Asp Ile
Ala Glu Glu Tyr Tyr Phe Ser Ile Leu Leu Asp Arg 100 105 110 gcg aat
cgt tcg tat ctg gct atg tgc tct gtt gaa ggt ggc atg gag 384 Ala Asn
Arg Ser Tyr Leu Ala Met Cys Ser Val Glu Gly Gly Met Glu 115 120 125
atc gag atc ctg gcg aag gaa aag cct gaa gct ttg gca aag gtg gaa 432
Ile Glu Ile Leu Ala Lys Glu Lys Pro Glu Ala Leu Ala Lys Val Glu 130
135 140 gtg gat ccc ctc act ggt att gat gag gac aaa gcg cgg gag att
gtc 480 Val Asp Pro Leu Thr Gly Ile Asp Glu Asp Lys Ala Arg Glu Ile
Val 145 150 155 160 act gct gct ggc ttt gaa act gag gtg aca gag aaa
gtc att ccg gtg 528 Thr Ala Ala Gly Phe Glu Thr Glu Val Thr Glu Lys
Val Ile Pro Val 165 170 175 ctg atc aag atc tgg cag gtg tat tac gaa
gag gaa gca aca ctc gtt 576 Leu Ile Lys Ile Trp Gln Val Tyr Tyr Glu
Glu Glu Ala Thr Leu Val 180 185 190 gag gtg aac ccg ttg gtg ctc acg
gat gac ggc gat gtg att gcg ctt 624 Glu Val Asn Pro Leu Val Leu Thr
Asp Asp Gly Asp Val Ile Ala Leu 195 200 205 gat ggc aag atc acg ctg
gat gat aac gct gat ttc cgc cat gat aac 672 Asp Gly Lys Ile Thr Leu
Asp Asp Asn Ala Asp Phe Arg His Asp Asn 210 215 220 cgt ggt gcg ttg
gct gaa tct gcc ggt ggc ttg gac att ttg gaa ctg 720 Arg Gly Ala Leu
Ala Glu Ser Ala Gly Gly Leu Asp Ile Leu Glu Leu 225 230 235 240 aag
gcc aag aag aat gat ctg aac tac gtg aaa ctt gat ggc tct gtg 768 Lys
Ala Lys Lys Asn Asp Leu Asn Tyr Val Lys Leu Asp Gly Ser Val 245 250
255 ggc atc att ggc aat ggt gca ggt ttg gtg atg tcc acg ttg gat atc
816 Gly Ile Ile Gly Asn Gly Ala Gly Leu Val Met Ser Thr Leu Asp Ile
260 265 270 gtg gct gca gct ggt gaa cgc cat ggt ggg cag cgc ccc gcg
aac ttc 864 Val Ala Ala Ala Gly Glu Arg His Gly Gly Gln Arg Pro Ala
Asn Phe 275 280 285 cta gac att ggt ggc gga gca tca gct gaa tcg atg
gct gct ggt ctc 912 Leu Asp Ile Gly Gly Gly Ala Ser Ala Glu Ser Met
Ala Ala Gly Leu 290 295 300 gat gtg atc ctt ggg gat agc cag gta cgc
agt gtg ttt gtg aat gtg 960 Asp Val Ile Leu Gly Asp Ser Gln Val Arg
Ser Val Phe Val Asn Val 305 310 315 320 ttt ggt ggc atc acc gcg tgt
gat gtg gtg gca aag gga atc gtt gga 1008 Phe Gly Gly Ile Thr Ala
Cys Asp Val Val Ala Lys Gly Ile Val Gly 325 330 335 gct ttg gat
gtg
ctc ggc gat caa gca acg aag cct ctt gtg gtg cgc 1056 Ala Leu Asp
Val Leu Gly Asp Gln Ala Thr Lys Pro Leu Val Val Arg 340 345 350 ctt
gat ggc aac aac gtg gtg gaa ggc aga cga atc ctc gcg gaa tat 1104
Leu Asp Gly Asn Asn Val Val Glu Gly Arg Arg Ile Leu Ala Glu Tyr 355
360 365 aac cac cct ttg gtc acc gtt gtg gag ggt atg gat gca gcg gct
gat 1152 Asn His Pro Leu Val Thr Val Val Glu Gly Met Asp Ala Ala
Ala Asp 370 375 380 cac gct gcc cat ttg gcc aat ctt gcc cag cac ggc
cag ttc gca acc 1200 His Ala Ala His Leu Ala Asn Leu Ala Gln His
Gly Gln Phe Ala Thr 385 390 395 400 gct aat 1206 Ala Asn 5 402 PRT
Corynebacterium glutamicum 5 Met Glu Leu Ala Val Asp Leu Phe Glu
Tyr Gln Ala Arg Asp Leu Phe 1 5 10 15 Glu Thr His Gly Val Ser Val
Leu Lys Gly Ile Val Ala Ser Thr Pro 20 25 30 Glu Ala Ala Arg Lys
Ala Ala Glu Glu Ile Gly Glu Leu Thr Val Val 35 40 45 Lys Ala Gln
Val Lys Val Gly Gly Arg Gly Lys Ala Gly Gly Val Arg 50 55 60 Val
Ala Pro Thr Ser Ala Gln Ala Phe Asp Ala Ala Asp Ala Ile Leu 65 70
75 80 Gly Met Asp Ile Lys Gly His Thr Val Asn Gln Val Met Val Ala
Gln 85 90 95 Gly Ala Asp Ile Ala Glu Glu Tyr Tyr Phe Ser Ile Leu
Leu Asp Arg 100 105 110 Ala Asn Arg Ser Tyr Leu Ala Met Cys Ser Val
Glu Gly Gly Met Glu 115 120 125 Ile Glu Ile Leu Ala Lys Glu Lys Pro
Glu Ala Leu Ala Lys Val Glu 130 135 140 Val Asp Pro Leu Thr Gly Ile
Asp Glu Asp Lys Ala Arg Glu Ile Val 145 150 155 160 Thr Ala Ala Gly
Phe Glu Thr Glu Val Thr Glu Lys Val Ile Pro Val 165 170 175 Leu Ile
Lys Ile Trp Gln Val Tyr Tyr Glu Glu Glu Ala Thr Leu Val 180 185 190
Glu Val Asn Pro Leu Val Leu Thr Asp Asp Gly Asp Val Ile Ala Leu 195
200 205 Asp Gly Lys Ile Thr Leu Asp Asp Asn Ala Asp Phe Arg His Asp
Asn 210 215 220 Arg Gly Ala Leu Ala Glu Ser Ala Gly Gly Leu Asp Ile
Leu Glu Leu 225 230 235 240 Lys Ala Lys Lys Asn Asp Leu Asn Tyr Val
Lys Leu Asp Gly Ser Val 245 250 255 Gly Ile Ile Gly Asn Gly Ala Gly
Leu Val Met Ser Thr Leu Asp Ile 260 265 270 Val Ala Ala Ala Gly Glu
Arg His Gly Gly Gln Arg Pro Ala Asn Phe 275 280 285 Leu Asp Ile Gly
Gly Gly Ala Ser Ala Glu Ser Met Ala Ala Gly Leu 290 295 300 Asp Val
Ile Leu Gly Asp Ser Gln Val Arg Ser Val Phe Val Asn Val 305 310 315
320 Phe Gly Gly Ile Thr Ala Cys Asp Val Val Ala Lys Gly Ile Val Gly
325 330 335 Ala Leu Asp Val Leu Gly Asp Gln Ala Thr Lys Pro Leu Val
Val Arg 340 345 350 Leu Asp Gly Asn Asn Val Val Glu Gly Arg Arg Ile
Leu Ala Glu Tyr 355 360 365 Asn His Pro Leu Val Thr Val Val Glu Gly
Met Asp Ala Ala Ala Asp 370 375 380 His Ala Ala His Leu Ala Asn Leu
Ala Gln His Gly Gln Phe Ala Thr 385 390 395 400 Ala Asn
* * * * *