DE60113131T2 - NUCLEIC ACIDS CODING FOR THE TMK GEN - Google Patents

Description

Territory of invention

The Invention provides isolated coryneform bacteria, wherein expression of nucleotide sequences from coryneform bacteria responsible for the tmk gene encode, off, and a process for fermentative Production of L-lysine using bacteria in which the tmk gene is off, ready.

State of technology

L-amino acids, in particular L-lysine, found in human medicine and pharmaceutical Industry, in the food industry and especially in the food industry animal nutrition Application.

It is known to be amino acids by fermentation of stems coryneform bacteria, in particular Corynebacterium glutamicum, getting produced. Because of the great importance is constantly on the improvement of the manufacturing process worked. process improvements can fermentation measures, like emotion and supply of oxygen, or the composition of the nutrient media, like the sugar concentration during the fermentation, or the work-up to the product form by Example ion exchange chromatography or the intrinsic performance properties of the microorganism itself.

to Improve the performance characteristics of these microorganisms Methods of mutagenesis, selection and mutant selection applied. In this way receives man tribes, which is resistant to antimetabolite or auxotrophic for regulatory are significant metabolites and produce the amino acids.

since Several years ago, methods of recombinant DNA technology were also used for producing L-amino acid producing strains of Corynebacterium, by using single amino acid biosynthetic genes amplified and investigated the effect on amino acid production.

Task of invention

The Inventors have set themselves the task of taking new measures to provide improved fermentative production of L-lysine.

Summary the invention

If L-lysine or lysine are not mentioned below only the bases, but also the salts, such as. B. lysine monohydrochloride or lysine sulfate, meant.

• a) einem Polynukleotid, das für ein Polypeptid mit der Aminosäuresequenz unter SEQ ID Nr. 2 codiert,
• b) einem Polynukleotid, das für ein Polypeptid codiert, welches eine Aminosäuresequenz umfasst, die zu wenigstens 90% mit der Aminosäuresequenz in SEQ ID Nr. 2 identisch ist,
• c) einem Polynukleotid, das zu den Polynukleotiden in a) oder b) komplementär ist, ist offenbart.

An isolated coryneform bacterial polynucleotide containing a tmk gene encoding polynucleotide sequence selected from the group consisting of

• a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2,
• b) a polynucleotide encoding a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence in SEQ ID NO: 2,
• c) a polynucleotide complementary to the polynucleotides in a) or b) is disclosed.

The Polypeptide has the activity the thymidylate kinase.

The disclosure also includes:
a replicable polynucleotide, especially DNA, containing the nucleotide sequence as shown in SEQ ID NO: 1;
a polynucleotide coding for a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2.

Claimed:
a vector pXK99Etmk containing parts of the polynucleotide described with SEQ ID NO: 1,
and coryneform bacteria in which the tmk gene is switched off by an insertion or deletion.

• 1. Isolierte coryneforme Bakterien, wobei die Aktivität oder Konzentration des Proteins mit einer Aminosäuresequenz, die mindestens 90% Identität mit der unter SEQ ID NO: 2 aufgeführten Aminosäuresequenz sowie Thymidylatkinaseaktivität aufweist, auf 0% der Aktivität oder Konzentration des Proteins des Ausgangsmikroorganismus reduziert ist.
• 2. Coryneforme Bakterien gemäß Anspruch 1, wobei die Aminosäuresequenz des Proteins unter SEQ ID NO: 2 aufgeführt ist.
• 3. Isolierte coryneforme Bakterien, wobei die Aktivität oder Konzentration des von dem unter SEQ ID NO: 1 aufgeführten Polynukleotid codierten Proteins auf 0% der Aktivität oder Konzentration der Aktivität oder Konzentration des Proteins des Ausgangsmikroorganismus reduziert ist.

The invention provides:

• 1. Isolated coryneform bacteria, wherein the activity or concentration of the protein having an amino acid sequence having at least 90% identity with the amino acid sequence listed under SEQ ID NO: 2 and thymidylate kinase activity is reduced to 0% of the activity or concentration of the protein of the starting microorganism.
• 2. Coryneform bacteria according to claim 1, wherein the amino acid sequence of the protein is listed under SEQ ID NO: 2.
• 3. Isolated coryneform bacteria, wherein the activity or concentration of the protein encoded by the polynucleotide listed under SEQ ID NO: 1 is reduced to 0% of the activity or concentration of the activity or concentration of the protein of the parent microorganism.

The Revelation also provides polynucleotides that are essentially from a polynucleotide sequence which can be screened by Hybridization of a corresponding gene bank of a coryneform bacterium, the complete one Contains gene or parts thereof, with a probe containing the sequence of that described in the invention Polynucleotide according to SEQ ID No. 1 or a fragment thereof and isolation of said Polynucleotide sequence available are.

Precise description the invention

polynucleotides which contain the sequences described in the invention are as hybridization probes for RNA, cDNA and DNA suitable for nucleic acids or polynucleotides or Genes for encode the thymidylate kinase, isolate it in full length or such nucleic acids or to isolate polynucleotides or genes that are highly similar having the sequence of the tmk gene. They are also for installation in so-called "arrays", "microarrays" or "DNA chips" suitable for the corresponding polynucleotides to detect and determine.

polynucleotides the sequences according to the disclosure are still suitable as primers, with their help with the polymerase chain reaction (PCR) DNA can be produced by genes can that for encode the thymidylate kinase.

Such oligonucleotides serving as probes or primers include at least 25, 26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24, most preferably at least 15, 16, 17, 18 or 19 consecutive nucleotides. Also suitable are oligonucleotides with a length of at least 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 or at least 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. Possibly are also oligonucleotides with a length of at least 100, 150, 200, 250 or 300 nucleotides suitable.

"Isolated" means from his natural Separated environment.

"Polynucleotide" refers to Generally to polyribonucleotides and polydeoxyribonucleotides, which is unmodified RNA or DNA or modified RNA or DNA can act.

The Polynucleotides according to the disclosure contain a polynucleotide according to SEQ ID No. 1 or a fragment made therefrom.

By "polypeptides" is meant peptides or proteins that have two or more linked via peptide bonds amino acids include.

The Polypeptides according to the disclosure shut down a polypeptide according to SEQ ID No. 2 with the biological activity of thymidylate kinase and also those which are 90% and preferably at least 91%, 93%, 95%, 97% or 99% are identical to the polypeptide according to SEQ ID No. 2 and have the said activity.

The The invention further relates to a process for fermentative production of L-lysine using coryneform bacteria, in particular already L-lysine produce and in which the for the tmk gene coding nucleotide sequences are turned off.

The term "attenuation" in this context describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are encoded by the corresponding DNA, for example by using a weak promoter or by using a gene or allele coded for a corresponding enzyme with a low activity or inactivating the appropriate gene or enzyme (protein) and, if appropriate, combining these measures.

By the measures the weakening becomes the activity or concentration of the corresponding protein in general 0% of the activity or concentration of wild-type protein or activity or concentration of the protein in the initial microorganism, lowered.

The Microorganisms which are the subject of the present invention can amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. It can be representative Coryneformer bacteria in particular of the genus Corynebacterium act. In the genus Corynebacterium is in particular the Art Corynebacterium glutamicum, which in the professional world for their ability is known, L-amino acids to produce.

Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum (C. glutamicum), are especially the known wild-type strains
Corynebacterium glutamicum ATCC 13032
Corynebacterium acetoglutamicum ATCC 15806
Corynebacterium acetoacidophilum ATCC 13870
Corynebacterium molassecola ATCC 17965
Corynebacterium thermoaminogenes FERM BP-1539
Brevibacterium flavum ATCC 14067
Brevibacterium lactofermentum ATCC 13869 and
Brevibacterium divaricatum ATCC 14020
and L-amino acid producing mutants or strains derived therefrom.

The for the Thymidylate kinase (EC 2.7.4.9) coding tmk gene of C. glutamicum was isolated.

to Isolation of the tmk gene or other genes of C. glutamicum will be first a gene bank of this microorganism in Escherichia coli (E. coli) created. The creation of genebanks is in well-known textbooks and manuals written down. As an example, the textbook of Winnacker: Genes and Clones, An Introduction into genetic engineering (Verlag Chemie, Weinheim, Germany, 1990), or the Handbook of Sambrook et al .: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A well-known gene bank is that of the E. coli K-12 strain W3110, which is described by Kohara et al. (Cell 50, 495-508 (1987)) in λ vectors was created. Bathe et al. (Molecular and General Genetics, 252: 255-265, 1996) describe a gene bank of C. glutamicum ATCC 13032, the using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84: 2160-2164) E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575) was created.

Börmann et al. (Molecular Microbiology 6 (3), 317-326 (1992)) a gene bank of C. glutamicum ATCC 13032 using the Cosmids pHC79 (Hohn and Collins, 1980, Gene 11, 291-298).

to Production of a gene bank of C. glutamicum in E. coli can also Plasmids such as pBR322 (Bolivar, 1979, Life Sciences, 25, 807-818) or pUC9 (Vieira et al., 1982, Gene, 19: 259-268). When Hosts are particularly suitable for those E. coli strains that are restriction and are recombination deficient, such as the strain DH5αmcr, the by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649) has been described. Using cosmids or other λ vectors subsequently cloned long DNA fragments can turn in common for the DNA sequencing subcloned suitable vectors and then sequenced be, as it is z. In Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74: 5463-5467, 1977) is described.

The obtained DNA sequences can then with known algorithms or sequence analysis programs like z. B. Staden's (Nucleic Acids Research 14, 217-232 (1986)), by Marck (Nucleic Acids Research 16, 1829-1836 (1988)) or the GCG program Butler (Methods of Biochemical Analysis 39, 74-97 (1998)) to be examined.

On this way was the for found the tmk gene encoding DNA sequence of C. glutamicum, the SEQ ID NO: 1 is part of the present disclosure. Farther was prepared from the present DNA sequence with those described above Methods the amino acid sequence derived from the corresponding protein. In SEQ ID No. 2 is the resulting amino acid sequence of the tmk gene product.

coding DNA sequences resulting from SEQ ID NO. 1 by the degeneracy of the genetic code are also part of the revelation. In the same way are DNA sequences with SEQ ID NO. 1 or Hybridize parts of SEQ ID NO: 1, part of the disclosure. In the professional world conservative amino acid substitutions are still such as B. Exchange of glycine for alanine or aspartic acid against glutamic acid in proteins as "sense mutations" (sense mutations) known to none of the fundamental change the activity lead the protein, d. H. are functionally neutral. Furthermore, it is known that changes at the N- and / or C-terminus of a protein its function is not essential impair or even stabilize. Information on this can be found among others at Ben-Bassat et al. (Journal of Bacteriology 169: 751-757 (1987)), 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 genetics and molecular biology. Amino acid sequences resulting in corresponding Manner of SEQ ID NO. 2, are also part of the invention.

In Likewise, DNA sequences corresponding to SEQ ID NO: 1 or parts of SEQ ID NO: 1, part of the disclosure. Finally are DNA sequences part of the disclosure by the polymerase chain reaction (PCR) using primers that are from SEQ ID NO. Such oligonucleotides are typically a length of at least 15 nucleotides.

instructions to identify DNA sequences by hybridization the expert, inter alia, in the manual "The DIG System User's Guide for Filter Hybridization" by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and Liebl et al. (International Journal of Systematic Bacteriology 41: 255-260 (1991)). The hybridization takes place under stringent conditions, that is, it will be only hybrids are formed in which the probe and target sequence, i. H. the polynucleotides treated with the probe, at least 70% identical are. It is known that the stringency of hybridization including the Wash steps by varying the buffer composition, the temperature and the salt concentration is influenced or determined. The hybridization reaction is preferably at relatively low stringency compared to carried out the washing steps (Hybaid Hybridization Guide, Hybaid Limited, Teddington, UK, 1996).

For the hybridization reaction For example, a 5x SSC buffer used at a temperature of about 50 ° C-68 ° C become. It can Probes also hybridize with polynucleotides that are less than 70% identity to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be done, for example, by lowering the salt concentration 2 × SSC and optionally subsequently 0.5 × SSC (The DIG System User's Guide for Filters Hybridization, Boehringer Mannheim, Mannheim, Germany, 1995) be reached, setting a temperature of about 50 ° C-68 ° C becomes. It may be possible the salt concentration to 0.1x SSC to lower. By gradually increasing the hybridization temperature in steps of about 1-2 ° C from 50 ° C to 68 ° C, polynucleotide fragments be isolated, for example, at least 70% or at least 80% or at least 90% to 95% identity to the sequence used Own probe. Further instructions for hybridization are in form so-called kits available on the market (eg DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Cat. 1603558).

instructions for the amplification of DNA sequences using the polymerase chain reaction The expert will find (PCR) among others in the handbook of Gait: Oligonucleotides Synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994).

It has been found to be coryneform bacteria after elimination of the tmk gene in an improved manner to produce L-lysine.

to Achieving a slowdown can either the expression of the tmk gene or the catalytic properties of the enzyme protein are turned off. If necessary, both can Measures combined become.

The Reduction of gene expression can be achieved by appropriate culture guidance or through genetic modification (Mutation) of the signal structures of gene expression.

Signaling structures of gene expression include repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon, and terminators. Information on this is the expert z. In WO 96/15246, Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)) Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998)), Jensen and Hammer (Biotechnology and Bioengineering 58: 191 (1998)), Patek et al. (Microbiology 142: 1297 (1996)), Vasicova et al. (Journal of Bacteriology 181: 6188 (1999)) and in known textbooks of genetics and molecular biology, such as. B. the textbook by Knippers ("Molecular Genetics", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or Winnacker ("Gene and clones", VCH Verlagsgesellschaft, Weinheim, Germany, 1990).

mutations the change or lowering the catalytic properties of enzyme proteins to lead, are known in the art; as examples are the Works by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel ("Threonine dehydratase from Corynebacterium glutamicum: abolition of allosteric regulation and structure of the enzyme ", Reports of Forschungszentrum Jülichs, Jül-2906, ISSN 09442952, Jülich, Germany, 1994). Summaries can be known textbooks of the Genetics and molecular biology such. B. that of Hagemann ("General Genetics", Gustav Fischer Verlag, Stuttgart, 1986).

When Mutations include transitions, transversions, insertions and Deletions into consideration. Dependent on from the effect of amino acid exchange on the enzyme activity is caused by missense mutations ("missense mutations ") or Nonsense mutations ("nonsense mutations "). Insertions or deletions of at least one base pair (bp) in a gene lead to frame shift mutations, as a result wrong amino acids be installed or the translation aborts prematurely. deletions of several codons typically to a complete one Failure of enzyme activity. instructions for generating such mutations belong to the prior art and can known textbooks genetics and molecular biology, such as B. the textbook by Knippers ("Molecular genetics", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that of Winnacker ("Genes and Clones", VCH Publishing Company, Weinheim, Germany, 1990) or that of Hagemann ("General Genetics", Gustav Fischer Verlag, Stuttgart, 1986).

A common The method of mutating genes of C. glutamicum is that of Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991)) method of gene disruption and gene replacement.

In the method of gene disruption, a central part of the coding region of the gene in question is cloned into a plasmid vector which can replicate in a host (typically E. coli) but not in C. glutamicum. Examples of vectors used are pSUP301 (Simon et al., Bio / Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pK18mobsacB or pK19mobsacB (Jäger et al., Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promega Corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman (1994), Journal of Biological Chemistry 269: 32678-84 ; US Patent 5,487,993), pCR ^® Blunt (Invitrogen, Groningen, The Netherlands; Bernard et al, Journal of Molecular Biology, 234: 534-541 (1993).) or pEMl (shrink et al, 1991, Journal of Bacteriology 173:. 4510-4516) in question. The plasmid vector containing the central part of the coding region of the gene is then converted by conjugation or transformation into the desired strain of C. glutamicum. The method of conjugation is described by Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described by Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio / Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a crossover event, the coding region of the gene in question is interrupted by the vector sequence and two incomplete alleles are obtained, one missing the 3 'and one 5' ends. This method has been described, for example, by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to eliminate the recA gene of C. glutamicum.

at the method of gene exchange ("gene replacement ") a mutation such as As a deletion, insertion or base exchange produced in vitro in the gene in question. The produced allele will turn into a for C. glutamicum is not cloned replicative vector and this subsequently by Transformation or conjugation into the desired host of C. glutamicum. To homologous recombination by means of a first, effect integration Crossover event and a suitable second, causing an excision Achieved crossover event in the target gene or in the target sequence one the incorporation of the mutation or the allele. This method was for example, by Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)) to eliminate the pyc gene of C. glutamicum by a deletion used.

In this way, a deletion, insertion or base exchange can be incorporated into the tmk gene become.

In addition, can it for the production of L-amino acids be beneficial, in addition to the weakening of the tmk gene one or several enzymes of the respective biosynthetic pathway, glycolysis, anaplerotic, the citric acid cycle, the pentose phosphate cycle, amino acid export and optionally regulatory To amplify proteins, especially overexpress.

Of the Term "reinforcement" describes in this Related the increase the intracellular activity one or more enzymes (proteins) in a microorganism, which are encoded by the corresponding DNA, for example by the copy number of the gene or genes increases, a strong promoter used or a gene or allele used for a corresponding Enzyme (protein) encoded with a high activity and optionally these measures combined.

By the measures the reinforcement, in particular overexpression, becomes the activity or concentration of the corresponding protein in general at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, at most up to 1000% or 2000% relative to the wild-type protein or the activity or increased concentration of the protein in the starting microorganism.

• • dem für die Dihydrodipicolinat-Synthase codierenden Gen dapA (EP-B-0197335),
• • dem für die Glyceraldehyd-3-Phosphat-Dehydrogenase codierenden Gen gap (Eikmanns (1992), Journal of Bacteriology 174: 6076–6086),
• • dem für die Triosephosphat-Isomerase codierenden Gen tpi (Eikmanns (1992), Journal of Bacteriology 174: 6076–6086),
• • dem für die Phosphoglycerat-Kinase codierenden Gen pgk (Eikmanns (1992), Journal of Bacteriology 174: 6076–6086),
• • dem für die Glucose-6-Phosphat-Dehydrogenase codierenden Gen zwf (JP-A-09224661),
• • dem für die Pyruvat-Carboxylase codierenden Gen pyc (DE-A-19 831 609),
• • dem für die Malat-Chinon-Oxidoreduktase codierenden Gen mqo (Molenaar et al., European Journal of Biochemistry 254, 395–403 (1998)),
• • dem für eine feedbackresistente Aspartatkinase codierenden Gen lysC (Zugriffsnummer P26512; EP-B-0387527; EP-A-0699759),
• • dem für den Lysin-Export codierenden Gen lysE (DE-A-19 548 222),
• • dem für die Homoserin-Dehydrogenase codierenden Gen hom (EP-A-0131171),
• • dem für die Threonin-Dehydratase codierenden Gen ilvA (Möckel et al., Journal of Bacteriology (1992) 8065–8072)) oder dem für eine "feedback-resistente" Threonin-Dehydratase codierenden Allel ilvA(Fbr) (Möckel et al., (1994) Molecular Microbiology 13: 833–842),
• • dem für die Acetohydroxysäure-Synthase codierenden Gen ilvBN (EP-B-0356739),
• • dem für die Dihydroxysäuredehydratase codierenden Gen ilvD (Sahm und Eggeling (1999) Applied and Environmental Microbiology 65: 1973–1979),
• • dem für das Zwa1-Protein codierendem Gen zwa1 (DE 19959328.0, DSM 13115)

verstärkt, insbesondere überexprimiert, werden.Thus, for example, for the production of L-amino acids in addition to the elimination of the tmk gene simultaneously one or more of the genes selected from the group consisting of

• The gene dapA coding for the dihydrodipicolinate synthase (EP-B-0197335),
• The gene gap coding for the glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• The gene tpi coding for the triosephosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• The gene pgk coding for the phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086),
• The gene coding for glucose-6-phosphate dehydrogenase, zwf (JP-A-09224661),
• The gene pyc coding for the pyruvate carboxylase (DE-A-19 831 609),
• The mqo gene coding for the malate quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)),
• The feedback-resistant aspartate kinase-encoding gene lysC (accession number P26512, EP-B-0387527, EP-A-0699759),
• The lysine gene coding for lysine export (DE-A-19 548 222),
• The homoserine dehydrogenase-encoding gene hom (EP-A-0131171),
• The ilvA gene coding for the threonine dehydratase (Möckel et al., Journal of Bacteriology (1992) 8065-8072) or the allele ilvA (Fbr) encoding a feedback-resistant threonine dehydratase (Möckel et al. , (1994) Molecular Microbiology 13: 833-842),
• The gene ilvBN coding for the acetohydroxy acid synthase (EP-B-0356739),
• The gene for the dihydroxy acid dehydratase ilvD (Sahm and Eggeling (1999) Applied and Environmental Microbiology 65: 1973-1979),
• The gene Zwa1 coding for the Zwa1 protein (DE 19959328.0, DSM 13115)

amplified, in particular over-expressed.

• • das für die Phosphoenolpyruvat-Carboxykinase codierende Gen pH (DE 19950409.1, DSM 13047),
• • das für die Glucose-6-Phosphat-Isomerase codierende Gen pgi (US 09/396,478, DSM 12969),
• • das für die Pyruvatoxidase codierende Gen poxB (DE 19951975.7, DSM 13114),
• • das für das Zwa2-Protein codierende Gen zwa2 (DE 19959327.2, DSM 13113)

auszuschalten.Furthermore, it may be advantageous for the production of amino acids, in addition to the elimination of the tmk gene simultaneously one or more of the genes selected from the group consisting of

• The gene PH coding for the phosphoenolpyruvate carboxykinase (DE 19950409.1, DSM 13047),
• The gene pgi coding for the glucose-6-phosphate isomerase (US 09 / 396,478, DSM 12969),
• The gene poxB coding for pyruvate oxidase (DE 19951975.7, DSM 13114),
• The zwa2 gene coding for the Zwa2 protein (DE 19959327.2, DSM 13113)

off.

Farther can it for the production of amino acids be advantageous, in addition to the elimination of the tmk gene unwanted side reactions to turn off (Nakayama: "Breeding of Amino Acid Producing Microorganisms ", in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vartek (ed.), Academic Press, London, United Kingdom, 1982).

The invention also provides microorganisms prepared according to the invention, and these can be cultivated continuously or batchwise (Satzculti vation) or in the fed-batch (feed process) or repeated-fed-batch process (repetitive feed process) for the purpose of producing L-amino acids. A summary of known cultivation methods is described in the textbook by Chmiel (Bioprocess 1. Introduction to Bioprocess Engineering (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreactors and Peripheral Facilities (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) ,

The The culture medium to be used must suitably meet the requirements of respective strains suffice. Descriptions of culture media for various microorganisms are in the manual "Manual of Methods for General Bacteriology "of the American Society for Bacteriology (Washington D.C., USA, 1981).

When Carbon source can Sugar and carbohydrates, such as Glucose, sucrose, lactose, Fructose, maltose, molasses, starch and cellulose, oils and fats, such as soybean oil, Sunflower oil, peanut oil and coconut oil, fatty acids, such as palmitic acid, stearic acid and linoleic acid, Alcohols, such as glycerol and ethanol, and organic acids, such as acetic acid, be used. These substances can used singly or as a mixture.

When Nitrogen source can organic nitrogenous compounds such as peptones, yeast extract, meat extract, Malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, Ammonium phosphate, ammonium carbonate and ammonium nitrate become. The nitrogen sources can used singly or as a mixture.

When Phosphorus source can Phosphoric acid, Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts are used. The culture medium must continue to salts of metals, such as magnesium sulfate or ferrous sulfate, the for the growth is necessary. Finally, essential growth substances, like amino acids and vitamins, in addition used for the above-mentioned substances. The culture medium can also suitable precursors are added. The stated starting materials can added to culture in the form of a unique approach or in suitable way during fed to the cultivation become.

to pH control of the culture are basic compounds, such as sodium hydroxide, Potassium hydroxide, ammonia or ammonia water, or acidic compounds, like phosphoric acid or sulfuric acid, used in a suitable manner. To control foaming can Anti-foaming agents, such as fatty acid polyglycol esters used become. To maintain the stability of plasmids can the medium suitable selective substances, such as antibiotics, added become. To maintain aerobic conditions, oxygen is used or oxygen-containing gas mixtures, such as air, in the culture registered. The temperature of the culture is usually at 20 ° C up to 45ºC and preferably at 25 ° C to 40 ° C. The culture is continued until a maximum of the desired Product has formed. This goal is usually within from 10 hours to 160 hours.

methods for the determination of L-amino acids are known from the prior art. The analysis can be for example as in Spackman et al. (Analytical Chemistry, 30, (1958), 1190) described by anion exchange chromatography with subsequent ninhydrin derivatization or may be by reverse phase HPLC, as in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).

The inventive method is used for the fermentative production of L-lysine.

• • Escherichia coli DH5alphamcr/pXK99E (= DH5αmcr/pXK99E) als DSM 14440,
• • Escherichia coli DH5alphamcr/pXK99Etmk (= DH5αmcr/pXK99Etmk) als DSM 14439.

The following microorganisms were deposited as pure cultures on July 31, 2001 at the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) under the Budapest Treaty:

• Escherichia coli DH5alphamcr / pXK99E (= DH5αmcr / pXK99E) as DSM 14440,
• Escherichia coli DH5alphamcr / pXK99Etmk (= DH5αmcr / pXK99Etmk) as DSM 14439.

The The present invention will be described below with reference to exemplary embodiments explained in more detail.

The isolation of plasmid DNA from Escherichia coli as well as all techniques for restriction, Klenow and alkaline phosphatase treatment were according to Sambrook et al. (Molecular Cloning, A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA). Methods for Transformation of Escherichia coli are also described in this manual.

The Composition more common Culture Media, Like LB or TY medium can also be found in the Sambrook manual et al. be removed.

example 1

Producing a genomic Cosmid Genbank from C. glutamicum ATCC 13032

chromosomal DNA from C. glutamicum ATCC 13032 was isolated as described by Tauch et al. (1995). Plasmid 33: 168-179) described isolated and with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3A2, Codenr. 27-0913-02) partially split. The DNA fragments were digested with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, code no. 1758250) dephosphorylated. The DNA of the cosmid vector SuperCos1 (Wahl et al., 1987), Proceedings of the National Academy of Sciences, USA 84: 2160-2164), purchased from Stratagene (La Jolla, USA, product description SuperCos1 Cosmid Vector Kit, Codenr. 251301) was digested with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Codenr. 27-0948-02) and also with shrimp alkaline phosphatase dephosphorylated.

Subsequently was the cosmid DNA with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Codenr. 27-0868-04). The cosmid DNA treated in this way was treated with the ATCC 13032 DNA mixed and the T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4 DNA Ligase, Code no. 27-0870-04). The ligation mixture was then used Gigapack II XL Packing Extract (Stratagene, La Jolla, USA, Product description Gigapack II XL Packing Extract, code no. 200217) packed in phages.

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 ₄ and mixed with an aliquot of the phage suspension. Infection and titration of the cosmidbank were performed as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), where the cells were plated on LB agar (Lennox, 1955, Virology, 1: 190) + 100 μg / ml ampicillin. After overnight incubation at 37 ° C, recombinant single clones were selected.

Example 2

insulation and sequencing of the gene tmk

The Cosmid DNA of a single colony was analyzed using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions isolated and with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description Sau3AI, product no. 27-0913-02) partially split. The DNA fragments were treated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, product description SAP, product no. 1758250) dephosphorylated. After gel electrophoresis separation, the isolation was carried out the cosmid fragments in the size range from 1500 to 2000 bp with 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 digested with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product no. 27-0868-04) split. The ligation of the cosmid fragments into the sequencing vector pZero-1 was prepared as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), wherein the DNA mixture with T4 ligase (Pharmacia Biotech, Freiburg, Germany) overnight was incubated. This ligation mixture was then in the E. coli strain DH5αmcr (Grant, 1990, Proceedings of the National Academy of Sciences, U.S.A., 87: 4645-4649) electroporated (Tauch et al., 1994, FEMS Microbiol. Letters, 123: 343-7) and on LB agar (Lennox, 1955, Virology, 1: 190) with 50 mg / l zeocin plated.

The plasmid preparation of the recombinant clones was carried out with the Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). The sequencing was carried out according to the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academy of Sciences, USA, 74: 5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18: 1067). It became the "RR dRhodamin Ter minator Cycle Sequencing Kit "from PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) The gel electrophoretic separation and analysis of the sequencing reaction were carried out in a" Rotiphoresis NF acrylamide / bisacrylamide "gel (29: 1) (Product No. A124.1 , Roth, Karlsruhe, Germany) with the "ABI Prism 377" sequencer from PE Applied Biosystems (Weiterstadt, Germany).

The The crude sequence data obtained were subsequently analyzed using the Staden program package (1986, Nucleic Acids Research, 14: 217-231) version 97-0. The single sequences of the pZerol derivatives became a related Contig assembles. The computer-aided coding area analyzes were determined by the formula XNIP (Staden, 1986, Nucleic Acids Research, 14: 217-231) prepared. Further analyzes were carried out with the "BLAST search programs" (Altschul et al., 1997, Nucleic Acids Research, 25: 3389-3402) against the nonredundant Database of the "National Center for Biotechnology Information "(NCBI, Bethesda, MD, USA).

The The nucleotide sequence obtained is shown in SEQ ID NO: 1. The Analysis of the nucleotide sequence revealed an open reading frame of 612 bp, which was called tmk gene. The tmk gene encodes a polypeptide of 203 amino acids (see SEQ ID NO: 2).

Example 3

Production of the expression vector pXK99Etmk for IPTG-induced Expression of the tmk gene in C. glutamicum

3.1 Cloning of the tmk gene

From strain ATCC 13032, the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) isolated chromosomal DNA. Based on the sequence of the tmk gene known from Example 2 for C. glutamicum, the following oligonucleotides were selected for the polymerase chain reaction (see SEQ ID NO: 3 and SEQ ID NO: 4):
tmk for:
5'-TG GGT ACC- SAT CAA GCC GGA GCA CTA CC-3 '
tmk int:
5'-GA TCT AGA -CAG CGC CGA ATC CGA TTC AT-3 '

there the primers were chosen that the amplified fragment is the incomplete gene, starting with the native ribosome binding site without promoter region, as well as the anterior Contains the area of the tmk gene. Furthermore contains the primer tmk for the sequence for the cleavage site of the restriction endonuclease KpnI, and the primer tmk int the cleavage site of the restriction endonuclease XbaI, which in marked by underlining the nucleotide sequence shown above are.

The The primers shown were obtained from MWG Biotech AG (Ebersberg, Germany). synthesized and the PCR reaction according to the standard PCR method of Innis et al. (PCR Protocols, A Guide to Methods and Applications, 1990, Academic Press) with Pwo polymerase by Roche Diagnostics GmbH (Mannheim, Germany). aid the polymerase chain reaction, the primers allow amplification a 520 bp big one DNA fragment containing the incomplete tmk gene including the carries native ribosome binding site.

The 520 bp in size tmk fragment was digested with the restriction endonucleases KpnI and XbaI split and then from the agarose gel with the QiaExII Gel Extraction Kit (product no. 20021, Qiagen, Hilden, Germany).

3.2 Construction of the expression vector pXK99E

In the prior art, the IPTG-inducible expression vector pXK99E was constructed. The vector is based on the Escherichia coli expression vector pTRC99A (Amann et al., Gene 69: 301-315 (1988)) and contains the trc promoter inducible by addition of the lactose derivative IPTG (isopropyl-β-D-thiogalactopyranoside), the termination regions T1 and T2, the replication origin ColE1 from E. coli, the lacI ^q gene (repressor of the lac operon of E. coli), a multiple cloning site (mcs) (Norrander, JM et al., Gene 26, 101-106 ( 1983)) and the kanamycin resistance gene gene aph (3 ') - IIa from E. coli (Beck et al., 1982, Gene 19: 327-336).

It It turned out that the vector pXK99E is very special suitable for regulating the expression of a gene, in particular the weakened Effect expression in coryneform bacteria. The vector pXK99E is an E. coli expression vector and can be expressed in E. coli for enhanced expression of a gene.

There the vector does not self-replicate in coryneform bacteria can only stay in the cell if it is in the Chromosome integrated. The peculiarity of this vector here is the use for the regulated expression of a gene after cloning a gene section from the front of the corresponding Gene into the vector containing the start codon and the native ribosome binding site, with following Integration of the vector into coryneform bacteria, in particular C. glutamicum. By adding dosed amounts of IPTG to the nutrient medium the gene expression is regulated. This effect amounts of 1 uM / l up to 10 M / l IPTG a very weak expression of the corresponding gene and amounts of 10 μM / l up to 100 μM / l a slightly weakened until normal expression of the corresponding gene.

Of the engineered E. coli expression vector pXK99E was electroporated (Tauch et al., 1994, FEMS Microbiol Letters, 123: 343-347) E. coli DH5αmcr (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87: 4645-4649) transferred. The selection of the transformants was carried out on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) supplemented with 50 mg / l kanamycin.

Plasmid DNA was isolated from a transformant by the usual methods (Peters-Wendisch et al., 1998, Microbiology, 144, 915-927) with the restriction endonuclease NcoI cleaved and the plasmid checked by subsequent agarose gel electrophoresis.

The plasmid construct thus obtained was identified as pXK99E ( 1 ) designated. The strain obtained by electroporation of the plasmid pXK99E into the E. coli strain DH5αmcr was named E. coli DH5alphamcr / pXK99E (= DH5αmcr / pXK99E) and was listed on July 31, 2001 as DSM 14440 in the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) according to the Budapest Treaty.

3.3 Cloning of the tmk fragment into the E. coli expression vector pXK99E

When The vector became the E. coli expression vector described in Example 3.2 used pXK99E. DNA of this plasmid was digested with the restriction enzymes KpnI and XbaI complete split and then with Shrimp Alkaline Phosphatase (Roche Diagnostics GmbH, Mannheim, Germany, product description SAP, product no. 1758250) dephosphorylated.

The approximately 500 bp tmk fragment obtained by PCR and cleaved with the restriction endonucleases KpnI and XbaI described in Example 3.1 was mixed with the prepared vector pXK99E. The mixture was treated with T4 DNA ligase (Amersham Pharmacia, Freiburg, Germany, product description T4 DNA ligase, code No. 27-0870-04). The ligation mixture was transformed into E. coli strain DH5αmcr (Hanahan, in: DNA Cloning, A Practical Approach, Vol I, IRL-Press, Oxford, Washington DC, USA). The selection of plasmid-carrying cells was carried out by plating out the transformation mixture on LB agar (Lennox, 1955, Virology, 1: 190) with 50 mg / l kanamycin. After overnight incubation at 37 ° C, recombinant single clones were selected. Plasmid DNA was isolated from a transformant with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and cleaved with the restriction enzymes KpnI and XbaI to check the plasmid by subsequent agarose gel electrophoresis. The resulting plasmid was named pXK99Etmk. It is in 2 shown.

Example 4

Integration of the vector pXK99Etmk into the genome of the C. glutamicum strain DSM 5715

Of the The vector pXK99Etmk mentioned in Example 3 was prepared by the method of electroporation by Tauch et al. (1989 FEMS Microbiology Letters 123: 343-347) in the strain C. glutamicum DSM 5715 is electroporated. The vector can not independent in DSM 5715 replicate and remains in the cell only when it enters the Chromosome is integrated. The selection of clones with integrated pXK99Etmk was performed by plating the Elekroporationsansatzes on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold Spring Harbor, New York, 1989), with 15 mg / l Kanamycin and IPTG (1 mM / L).

For the proof In the integration, the tmk fragment was modeled after the method "The DIG System User's Guide for Filter Hybridization "of Boehringer Mannheim GmbH (Mannheim, Germany, 1993) with the Dig hybridization kit marked by Boehringer. Chromosomal DNA of a potential integrants was determined by the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) isolated and each cleaved with the restriction enzymes NcoI and KpnI. The resulting fragments were separated by agarose gel electrophoresis and with the Dig Hybrisierungskit from Boehringer at 68 ° C hybridized. The plasmid pXK99Etmk mentioned in Example 3 was within the chromosomal tmk gene into the chromosome of DSM 5715 inserted. The root was named DSM5715 :: pXK99Etmk.

Example 5

Production of lysine

Of the C. glutamicum strain DSM5715 :: pXK99Etmk obtained in Example 4 cultivated in a suitable for the production of lysine nutrient medium and the Lysine content in the culture supernatant certainly. Addition of IPTG (10 μM / L) occurs through the trc promoter regulated, to a weakened Expression of the tmk gene.

The strain was first incubated on an agar plate with the appropriate antibiotic (brain heart agar with kanamycin (25 mg / l) and IPTG (10 μM / l) for 24 hours at 33 ° C. Starting from this agar plate culture, a preculture was inoculated ( 10 ml of medium in the 100 ml Erlenmeyer flask) The medium used for the preculture was the complete medium CgIII Cg III Medium NaCl 2.5 g / l Bacto-peptone 10 g / l Bacto-yeast extract 10 g / l Glucose (separately autoclaved) 2% w / v

Of the pH was adjusted to pH 7.4.

To this was added Kanamycin (25 mg / L) and IPTG (10 μM / L). The preculture was incubated for 16 hours at 33 ° C at 240 rpm / min on a shaker. From this preculture, a major culture was inoculated so that the initial OD (660 nm) of the main culture was 0.1 OD. The medium MM was used for the main culture. MM medium CSL (corn steep liquor) 5 g / l MOPS (morpholinepropanesulfonic acid) 20 g / l Glucose (separately autoclaved) 50 g / l salts: (NH ₄ ) ₂ SO ₄ 25 g / l KH ₂ PO ₄ 0.1 g / l MgSO ₄ .7H ₂ O 1.0 g / l CaCl ₂ .2H ₂ O 10 mg / l FeSO ₄ .7H ₂ O 10 mg / l MnSO ₄ .H ₂ O 5.0 mg / l Biotin (sterile filtered) 0.3 mg / l Thiamine · HCl (sterile filtered) 0.2 mg / l Leucine (sterile filtered) 0.1 g / l CaCO ₃ 25 g / l

CSL, MOPS and saline are adjusted to pH 7 with ammonia water and autoclaved. Subsequently, the sterile substrate and vitamin solutions are added and the dry autoclaved CaCO _{3 is} added.

The Cultivation is done in a 10 ml volume in a 100 ml Erlenmeyer flask with harassment. To this was added Kanamycin (25 mg / L) and IPTG (10 μM / L). The cultivation was carried out at 33 ° C and 80% humidity.

To 72 hours, the OD at a measuring wavelength of 660 nm with the Biomek 1000 (Beckmann Instruments GmbH, Munich). The educated Amount of lysine was analyzed with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post column derivatization determined by ninhydrin detection.

The Results of the experiment are shown in Table 1.

Table 1

Short description of Drawings:

1 : Map of the plasmid pXK99E,

2 : Map of the plasmid pXK99Etmk.

Kan:

Kanamycin-Resistenzgen aph(3')-IIa aus Escherichia coli

KpnI

Spaltstelle des Restriktionsenzyms KpnI

NcoI

Spaltstelle des Restriktionsenzyms NcoI

XbaI

Spaltstelle des Restriktionsenzyms XbaI

Ptrc

trc-Promotor

T1

Terminationsregion T1

T2

Terminationsregion T2

lacIq

lacIq-Repressor des lac-Operons von Escherichia coli

oriV

Replikationsursprung ColE1 aus E. coli

tmk

klonierter Bereich des tmk-Gens

The abbreviations and designations used have the following meaning.

Kan:

Kanamycin resistance gene aph (3 ') - IIa from Escherichia coli

KpnI

Cleavage site of the restriction enzyme KpnI

NcoI

Cleavage site of the restriction enzyme NcoI

XbaI

Cleavage site of the restriction enzyme XbaI

ptrc

trc promoter

T1

Termination region T1

T2

Termination region T2

lacI.sup.q

lacIq repressor of the lac operon of Escherichia coli

ori V

Origin of replication ColE1 from E. coli

tmk

cloned region of the tmk gene

SEQUENCE LISTING

Claims (15)

Isolated coryneform bacteria, with activity or concentration the protein with an amino acid sequence, the least 90% identity with the amino acid sequence listed under SEQ ID NO: 2 as well as thymidylate kinase activity to 0% of the activity or reduced concentration of the protein of the starting microorganism is. Coryneform bacteria according to claim 1, wherein the amino acid sequence of the protein is listed under SEQ ID NO: 2. Isolated coryneform bacteria, with activity or concentration of the polynucleotide listed under SEQ ID NO: 1 Protein to 0% of activity or concentration of activity or reduced concentration of the protein of the starting microorganism is. strains of the species Corynebacterium glutamicum according to one or more of claims 1 to 3, being the activity or concentration of the protein is reduced to 0%. Escherichia coli DH5alphamcr / pXK99Etmk (= DH5αmcr / pXK99Etmk), deposited under DSM 14439 at the German Collection of Microorganisms and cell cultures (DSMZ), Braunschweig. Process for the fermentative production of L-lysine, full: Fermentation of these L-amino acid producing coryneforms Bacteria according to one or more of the claims 1 to 4. Method according to claim 7, comprising: Fermentation of bacteria, concentration the amino acid either in the medium or in the cells of the bacteria and insulation the amino acid. Method according to claim 7 or 8, using bacteria in which the concentration or activity of other genes of the biosynthetic pathway of L-lysine coded Proteins increased by 10 to 2000% is. Method according to claim 7 or 8, using bacteria in which the concentration or activity of genes of those metabolic pathways through which the formation is reduced by L-lysine, coded proteins reduced to 0% is. Method according to claim 7, wherein the expression of the thymidylate kinase gene, whose sequence is listed under SEQ ID NO: 1, encoding polynucleotide or polynucleotides by transition, transversion, insertion or Deletion is turned off. Method according to claim 9, wherein for the production of L-lysine coryneform microorganisms be fermented, in which at the same time the activity or concentration one or more of the genes selected from the group out: for Dihydrodipicolinate synthase-encoding gene dapA, for glyceraldehyde-3-phosphate dehydrogenase coding gene gap, for Triosephosphate isomerase-encoding gene tpi, for 3-phosphoglycerate kinase coding gene pgk, for Glucose-6-phosphate dehydrogenase encoding gene zwf, for pyruvate carboxylase coding gene pyc, for Malate quinone oxidoreductase encoding gene mqo, for a feedback-resistant Aspartate kinase-encoding gene lysC, the one coding for lysine export Gene lysis, for the protein zwa1 coding gene zwa1, coded proteins around 10 to 2000% increased is or are. A method according to claim 10, wherein coryneform microorganisms are fermented to produce L-lysine, in which at the same time the activity or concentration of one or more of the genes selected from the group consisting of: the phosphoenolpyruvate carboxykinase-encoding gene pck, the pgi gene coding for glucose-6-phosphite isomerase, the pyruvate oxidase-encoding gene poxB, the zwa2 gene coding for the Zwa2 protein, is or are reduced to 0%. Method according to one or more of the claims 5-13, using microorganisms of the species Corynebacterium glutamicum become. Method according to claim 11 using the corynebacterium strain DSM5715 :: pXK99Etmk becomes. Vector pXK99Etmk, whose restriction map is in 2 and is deposited in the E. coli strain DH5alphamcr / pXK99Etmk under No. DSM 14439 in the German Collection of Microorganisms and Cell Cultures.