MXPA00012034A - Polynucleotide sequences from corynebacterium glutamicum coding for succinate dehydrogenase subunits (sdha, sdhb, sdhc) - Google Patents

Abstract

Isolated polynucleotide (I) from coryneform bacteria encoding succinate dehydrogenase is new. Isolated polynucleotide (I) from coryneform bacteria encoding succinate dehydrogenase is new. (I) is selected from:(a) a polynucleotide which is at least 70%identical with a sequence that encodes a polypeptide having the 293 (S3), 625 (S5) or 284 (S7) amino acids (aa) sequence defined in the specification;(b) a polynucleotide that encodes a polypeptide at least 70%identical with (S3), (S5) or (S7);(c) a polynucleotide that is complementary to (a)-(b);or (d) a polynucleotide that contains at least 15 consecutive nucleotides from (a)-(c). Independent claims are also included for the following:(1) vector containing (I);(2) coryneform bacteria containing the vector of (1);and (3) method for fermentative production of L-amino acids (A) by growing an (A)-producing coryneform bacterium in which at least one of the genes for succinate dehydrogenase or its A, B and C subunits is suppressed.

Description

NEW NUCLEOTIDE SEQUENCES THAT CODIFY FOR THE GENES sdhA, sdhB AND sdhC.

FIELD OF THE INVENTION The object of the invention are the polynucleotide sequences of coryneform batteries that code for the sdhC, sdhA and sdhB genes, and a method for the production by fermentation of L-amino acids, in particular L-lysine, by attenuation of the sdhC gene and / or of the sdhA gene and / or of the sdhB gene. STATE OF THE ART L-amino acids, in particular lysine, are used in human medicine and in the pharmaceutical industry, in the food industry and especially in animal feed. It is known that L-amino acids are produced by the fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to the great significance we work continuously in the improvement of production processes. The process improvements can be related to measures related to fermentation techniques, such as agitation and oxygen supply, or to the composition of culture media, such as the concentration of sugar during fermentation, or the elaboration to the Ref. 125666 The product form is, for example, ion exchange chromatography, or the intrinsic capacity characteristics of the microorganism itself, in order to improve the capacity characteristics of these microorganisms. Mutagenesis, selection and selection of mutants, thus obtaining strains that are resistant against antimetabolites or auxotropics for metabolites of regulatory importance, and that produce L-amino acids. improvement of the strains of Corynebacterium strains that produce L-amino acid OBJECT OF THE INVENTION The inventors proposed the task of providing new measures to improve the production of amino acids by fermentation, in particular L-lysine DESCRIPTION OF THE INVENTION amino acids, in particular lysine, are used in human medicine, in the pharmaceutical industry a, in the food industry and especially in the feeding of animals. Accordingly, there is a general interest in providing new improved processes for the production of L-amino acids, in particular L-lysine.

The subject of the invention is an isolated polynucleotide containing a polynucleotide sequence selected from the group comprising a) polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide containing the amino acid sequence of SEQ ID No. 3 , b) polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide that contains the amino acid sequence of SEQ ID No. 5, c) polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide containing the amino acid sequence of SEQ ID No. 7, d) polynucleotide encoding a polypeptide containing an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID No. 3, e) polynucleotide encoding for a polypeptide containing an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID No. 5, f) polynucleotide coding ca for a polypeptide containing an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID No. 7, g) polynucleotide complementary to the polynucleotides of a), b) c), d), e) of ), and h) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b), c), d), e) of). Another object of the invention is a polynucleotide that is a DNA, preferably recombinant, replicable in coryneform bacteria, in particular that encodes a polypeptide that contains the amino acid sequence represented in SEQ ID No. 2. It is also an object of the invention to polynucleotide that is an RNA. Another object of the invention is a polynucleotide, preferably being a replicable DNA, containing (i) the nucleotide sequence shown in SEQ ID.

Do not . 1, or (ii) at least one sequence corresponding to sequence (i) within the area of degeneracy of the genetic code, or (iii) at least one sequence that hybridizes with the sequence complementary to sequence (i) or (ii), and / or optionally (iv) functionally neutral orientation mutations in (i).

Other objects are a vector that contains one of the polynucleotides mentioned, and coryneform bacteria that serve as a host cell, which contain the vector. Objects of the invention are also polynucleotides consisting substantially of a polynucleotide sequence, which can be obtained by screening by means of the hybridization of a corresponding gene bank containing the sdhC gene and / or sdhA and / or complete sdhB with the sequence of polynucleotide corresponding to SEQ ID No. 1, with a probe containing the sequence of the polynucleotide according to SEQ ID No. 1 mentioned, or a fragment thereof, and isolation of the DNA sequence mentioned. The polynucleotide sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, to isolate their entire length cDNA encoding succinate dehydrogenase or its subunits A, B or C, and isolate those cDNA or genes that show a great similarity of the sequence with that of the genes of succinate dehydrogenase or its subunits A, B or C. The polynucleotide sequences according to the invention are also suitable as primers with the help of which, by means of the polymerase chain reaction (PCR) ), DNA from genes encoding succinate dehydrogenase can be prepared. Such oligonucleotides which serve as probes or primers contain at least 30, preferably at least 5, in particular very preferably at least 15 successive nucleotides. Oligonucleotides with a length of at least 40 or 50 nucleotides are also suitable. "Isolated" means separated from its natural environment. "Polynucleotide" refers in general to polyribonucleotides or polideoxiribonucleotides, being that it can be unmodified RNA and DNA or modified RNA and DNA. "Polypeptides" are understood as meaning peptides or proteins that contain two or more amino acids linked through peptide bonds. The polypeptides according to the invention include the polypeptides according to SEQ ID No. 3 and SEQ ID No. 20 5, and according to SEQ ID No. 7, in particular those with the biological activity of the succinate dehydrogenase, and also those that are at least 70% identical with the polypeptide according to SEQ ID No. 3 and SEQ ID No. 5 and SEQ ID No. 7, preferably at least 80%, and particularly those that present at least 90% to 95% The identity of the polypeptide according to SEQ ID No. 3 and SEQ ID No. 5 and SEQ ID No. 7, and the aforementioned activity. The invention also relates to a method for the production by fermentation of L-amino acids, in particular lysine, by the use of coryneform bacteria, which in particular already produce the L-amino acids, in particular L-lysine, and in which attenuate, in particular, express at reduced level the nucleotide sequences encoding the sdhC gene and / or the sdhA gene and / or the sdhB gene. The term "attenuation" describes in this context the decrease or elimination in a microorganism, of the intracellular activity of one or several enzymes (proteins) that are encoded by the corresponding DNAs when using, for example, a weak promoter or a gene or allele. which encodes a corresponding enzyme with a low activity or that inactivates the corresponding enzyme (protein) and which eventually combines these measurements. The microorganisms which are the object of the present invention can produce L-amino acids, in particular L-lysine from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or glycerin and ethanol. It may be representatives of coryneform bacteria, in particular of the species Corynebacterium.

In the Corynebacterium species, mention should be made in particular of the Corynebacterium glutamicum type, which is known in the specialized field for its ability to produce L-amino acids. The right strains of the species Corynebacterium, in particular of the type Corynebacterium glutamicum are for example the known wild type strains Corynebacterium glutamicum ATCC13032 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium acetoacidofilu ATCC13870 Corynebacterium melassecola ATCC17965 Corynebacterium termoaminogenes FERM BP-1539 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATTCC13869 and Brevibacterium divaricatum ATTCC14020 and mutants and producing strains of L-amino acids, produced from these, such as the strains that produce L-lysine Corynebacterium glutamicum FERM-P 1709 Brevibacterium flavum FERM-P 1708 Brevibacterium lactofermentum FERM-P 1712 Corynebacterium glutamicum FERM-P 6463 Corynebacterium glutamicum FERM -P 6464 and Corynebacterium glutamicum DSM5714.

The inventors succeeded in isolating the new sdhC, sdhA and sdhB genes from C. glutamicum coding for the enzyme succinate dehydrogenase (EC 1.3.99.1). To isolate the sdhC gene and / or the sdhA gene and / or the sdhB gene, or also other genes of C. glutarrucum, a gene bank of this microorganism is first installed in E. coli. The installation of gene banks is described in teaching books and generally known manuals. As an example, we will mention Winnacker's teaching book: Gene und Klone, Eine Einführung in die Gentechnologie (Chemie Verlag, Weinheim, Germany, 1990) or the Sambrook et al manual: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press , 1989). A well-known gene bank is that of strain W3110 of E. coli K-12, which was installed by Kohara et al. (Cell 50, 495-508 (1987)) in vectors? Bathe et al. (Molecular an General Genetics, 252: 255-265, 1996) describe a gene bank of C. glutamicum ATCC13032 that was installed 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 strain NM 554 of E.coli K-12 (Raleigh et al., 1988, Nucleic Acids Research 16: 1563-1575). In turn, Bdrmann et al. (Molecular Microbiology 6 (3), 317-326)) describe a gene bank of C. glutamicum ATTCC13032 by use of 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 by using the system expression ? Zap described by Short et al. (Nucleic Acids Research, 16: 7583). Plasmids, such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or? UC9 (Vieira et al., 1982, Gene, can also be used to establish a gene bank for C. glutamicum. 19: 259-268). As guests, strains of E. coli with restriction deficiency and recombination are especially suitable, as for example strain DH5a (Jeffrey H. Miller: "A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria" , Cold Spring Harbor Laboratory Press, 1992). The long fragments of DNA cloned with the help of eaten or other vectors? they can then be subcloned into conventional vectors, suitable for DNA sequencing. Methods for DNA sequencing are described among others in Sanger et al. (Proceedings of the National (Academy) of Sciences of the United States of America USA, 74: 5463-5467, 1977). The DNA sequences obtained can then be investigated with known algorithms or sequence analysis programs, such as Staden (Nucleic Acids Research 14, 217-232 (1986)), Butler's GCG program (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 compare with the sequence inscribers that exist in data banks accessible to the public. Data banks accessible to the public for sequences of neuclots are, for example, those from the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) or those from the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA). In this way, the new DNA sequence of C. glutamicum coding for the sdhC gene and the sdhA gene and the sdhB gene, which is an integral part of the present invention as SEQ ID No. 1, was obtained. the present DNA sequence was derived with the previously described methods the amino acid sequence of the corresponding protein. In SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7, the amino acid sequences resulting from the sdhC, sdhA and sdhB gene product are represented.

The coding DNA sequences resulting from SEQ ID No. 1 by the degeneracy of the genetic code are also an integral part of the invention. In addition, in the specialized field conservative exchanges of amino acids in proteins, such as, for example, the exchange of glycine for alanine, or of asparagine acid for glutamic acid, "mutations of orientation" (sense mutations) are known, which do not lead to a fundamental change in the activity of the protein, that is, they are neutral with respect to the function. It is also known that changes in the N and / or C terminals of a protein do not substantially impair its function, and may even stabilize it. The indications regarding this are found by the expert in Ben-Bassat et al. (Journal of Bacteriology 169: 751-757 (1987)), in 0 'Regan et al. (Gene 77: 237-251 (1989)), in Sabin-Toth et al. (Protein Sciences 3: 240-247 (1994)), in Hochuli et al. (Bio / Technology 6: 1321-1325 (1988)) and in known teaching books on genetics and molecular biology. The amino acid sequences that result in the corresponding manner from SEQ ID No. 1, and the DNA sequences encoding these amino acids are also an integral part of the invention. Also integral with the invention are the DNA sequences which hybridize with SEQ ID No. 1 parts of SEQ ID No. 1. Finally, DNA sequences produced by the polymerase chain reaction are part of the invention ( RCP) using primers resulting from SEQ ID No. 1. 5 The instructions for the identification of DNA sequences by hybridization are found by the expert among others in the manual "The DIG System Users Guide for Filter Hybridization" of the Boehringer company. Mannheim GmbH (Mannheim, Germany, 1993) and Liebl et al.

(International Journal of Systematic Bacteriology (1991) 41: 255-260). The instructions for the amplification of DNA sequences with the help of the polymerase chain reaction (PCR) are found by the expert, among others, in the Gait manual: Oligonukleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994). The inventors discovered that after the attenuation of the sdhC gene and / or sdhA and / or sdhB, the bacteria coryneprobes produce better L-amino acids, in particular L-lysine. To achieve a weakening, the expression of the sdhC and / or sdhA and / or sdhB gene or the catalytic properties of the product can be reduced or suppressed. gene. In both cases, both measures are combined. - é * ^? ^ * **, > The expression of the gene can be reduced by a suitable way of carrying the culture or by genetic modification (mutation) of the signal structures of gene expression. Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the initiation codon and terminators. The data with respect to this are found in the expert, for example, in the patent application W096 / 15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Cha bliss (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 well-known textbooks of molecular genetics and biology, such as the Knippers textbook ("Moleculare Genetik", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995 ) or that of Winnacker ("Gene und Klone", VCH Verlagsgesellschaft, Weinheim, Germany, 1990). Mutations that lead to a modification or decrease in the catalytic properties of enzymatic proteins are known from the prior art; as examples we will mention the works of Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Móckel ("Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebundg der allosterischen Regulation und Struktur des Enzyms", Berichte des Forschungszentrums Jülichs, Jül-2906, ISSN09442952, Jülich, Germany, 1994 ). Summarized exposures can be collected from known textbooks of genetics and molecular biology, such as Hagemann's ("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986). Transitions, transversions, insertions and deletions are considered as mutations. Depending on the effect of the amino acid exchange on enzymatic activity, we speak of mutations of opposite orientation (missense mutations) or mutations without orientation (nonsense mutations). The insertions or deletions of at least one pair of bases in a gene lead to mutations by change in the frame of reading (frame shift mutations), in whose sequence erroneous amino acids are inserted or the translation is truncated prematurely. Deletions of several codons typically lead to a total loss of enzymatic activity. The instructions for producing this type of mutations belong to the state of the art and can be collected from known textbooks of genetics and molecular biology, such as the Knippers textbook ("Moleculare Genetik", 6. edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), Winnacker ("Gene und Klone", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or Hagemann ("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986). A common method for mutating genes of C. glutamicum is the method of gene disruption ("gene disruption") and gene replacement ("gene replacement" / described by Schwarzer and Pühler (Bio / Technology 9, 84-87 ( 1991) In the case of the gene disruption method, a central part of the coding region of the interesting gene is cloned into a plasmid vector that can replicate in a host (typically E. coli) but not in C. glutamicum. vectors are considered, for example, pSUP301 (Simón et al., Bio / Technology 1, 784-791 (1983)), 784-791 (1983)), pkldmob or pkl9mob (Scháfer et al., Gene 145, 69- 73 (1994)), pkldmobsacB or pkl9mobsacB (Jager et al., Journal of Bacteriology 174: 5462-65 (1992)), pGEM-T (Promega Corporation, Madison, Wl, USA), PCR2.1-T0P0 (Shuman ( 1994) Journal of Biological Chemistry 269: 32678-84; US Pat. No. 5,487,993) pCRTBlunt (Invitrogen Signature, Groningen, The Netherlands: Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEMl (Schrumpf et al, 1991, Journal of Bacteriology 173: 4510- 4516). The plasmid vector containing the central part of the coding region of the gene is transformed to k i i continuation to the desired strain of C. glutamicum by conjugation or transformation. The method of conjugation is described, for example in Scháfer 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 "cross over" event, the coding region of the respective gene is interrupted by the vector sequence, and two incomplete alleles are obtained, which respectively lack the 3 'and 5' terminals. This method was employed, for example, by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to eliminate the recA gene from C. glutamicum. The sdhC and / or sdhA and / or sdhB gene can be deleted in this manner. In the method of gene replacement ("gene replacement") there is a mutation in vLtro, such as a deletion, insertion or replacement of bases in the interesting gene. The produced allele is in turn cloned into a non-replicable vector for C. glutamicum and this is then transformed by transformation or conjugation into the desired C. glutamicum host. After homologous recombination by a first "cross-over" event that produces the integration, and a second appropriate "cross over" event that produces the cleavage in the target gene or in the target sequence, insertion of the mutation is achieved or good of the allele. This method was employed, for example by Peters-Wendisch (Microbiology 144, 915-927 (1998)) to remove the pyc gene from C. glutamicum by a deletion. In this way it is possible to insert a deletion, insertion or replacement of bases in the gene sdhC and / or sdhA and / or sdhB. Accordingly, another object of the invention is a process for the production by fermentation of L-amino acids, in particular L-lysine, in which a strain transformed with a piasmidic vector is also employed, and the plasmid vector carries nucleotide sequences of the genes encoding the enzyme succinate dehydrogenase, or else the strain has a deletion, insertion or replacement of bases in the sdhC gene and / or sdhA and / or sdhB. The methods for the production by fermentation of L-amino acids, in particular L-lysine, comprise the following steps: a) fermentation of coryneform bacteria which produce the L-amino acid in which at least one of the genes selected from those encoding the enzyme succinate-dehydrogenase or its subunits A, B and C, b) concentration of the L-amino acid in the medium or in the cells of the bacteria, and c) isolation of the L-amino acid. For the production of amino acids, in particular 5 L-lysine may be additionally convenient, in addition to attenuating the sdhC and / or sdhA and / or sdhB gene, reinforcing, in particular overexpressing one or several enzymes of the respective biosynthesis pathway, of the glycolysis, of the anaplerotic, of the citric acid cycle or of the export of the amino acid. Thus, for example, for the production of L-lysine, the dapA gene coding for dihydrodipicolinate ligase can be simultaneously overexpressed (EP-B 0 197 335), and / or simultaneously the gap gene coding for glyceraldehyde- 3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), or • simultaneously the pyc gene encoding pyruvate carboxylase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), or • simultaneously the mqo gene coding for malate: quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)), or 25 • simultaneously the lysE gene which codes for the «Faafe. export lysine (DE-A-195 48 222). For the production of L-amino acids, in particular L-lysine, it may be convenient, in addition to the claimed genes, simultaneously attenuate 5 • the pck gene coding for phosphoenolpyruvate carboxykinase (DE 199 50 409.1, DSM 13047) and / or • the pgi gene coding for glucose-6-phosphate iso erase (US 09 / 396,478, DSM 12969 ). For the production of amino acids, in particular 10 L-lysine, it may further be convenient, in addition to attenuating the sdhC and / or sdhA and / or sdhB gene, to suppress undesirable side reactions (Nakayama: "Breeding of Amino Acid Producing Micro-organisms" , in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, 15 London, UK, 1982). Also the subject of the invention are microorganisms containing the polynucleotide according to claim 1 and can be grown continuously or discontinuously in the batch culture or in the batch culture with inflow or in the batch culture with recycling for the end of the production of L-amino acids, in particular L-lysine. A summary of the known cultivation methods is described in the textbook of Chmiel (Bioprozesstechnik 1. Einführung in die 25 Bioverfahrenstechnik (editorial Gustav Fischer, Stuttgart, < L > a 1991)) or in the Storhas textbook (Bioreaktoren und periphere Einrichtungen (Verlag Verseg, Braunschweig / Wiesbaden, 1994)). The culture medium used must adequately meet the requirements of the respective strains. Descriptions of culture media of various microorganisms are contained in the "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., E.U.A., 1981). As a carbon source, sugar and carbohydrates can be used, such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as soybean oil, sunflower oil, peanut oil and oil. of coconut, fatty acids such as, for example, palmitic acid, stearic acid and linolic acid, alcohols such as, for example, glycerin and ethanol, and organic acids such as, for example, acetic acid. These substances can be used individually or as a mixture. Nitrogen-containing organic compounds, such as peptones, yeast extract, meat extract, malt extract, soaked corn liquor, soy bean flour and urea, or inorganic compounds such as ammonium sulfate, chloride ammonium, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used individually or as mixtures. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium salts, can be used as phosphorus source. The culture medium must also contain salts of metals such as, for example, magnesium sulfate or iron sulphate, which are necessary for growth. Finally, it is possible, in addition to the substances mentioned above, to apply substances essential for growth such as amino acids and vitamins. You can also add suitable preliminary stages to the culture medium. The aforementioned application substances can be added to the culture in the form of a single preparation or be suitably fed during cultivation. For the control of the pH of the crop, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds such as phosphoric acid or sulfuric acid are suitably applied. For the control of foaming, antifoaming agents such as polyglycol fatty acid esters can be applied. To maintain the stability of the plasmids, suitable substances that act selectively, such as antibiotics, can be added to the medium. To maintain aerobic conditions, They introduce oxygen or gaseous mixtures containing oxygen, such as air, to the crop. The culture temperature is usually from 20 ° C to 45 ° C, and preferably from 25 ° C to 40 ° C. The culture is continued for so long until a maximum of the desired product is formed. This goal is normally reached within 10 hours to 160 hours. The methods for determining the L-amino acids are known from the state of the art. The analysis can be carried out as described in Spackman et al. (Analytical Chemistry, 30, (1958), 1190), by anion exchange chromatography with subsequent ninhydrin derivatization, or can be effected by reverse phase HPLC, as described in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174). EXAMPLES The present invention is explained in more detail below on the basis of the following exemplary embodiments. Example 1 Development of a genomic cosmid gene bank from Corynebacterium glutamicum ATCC 13032 Chromosomal DNAs of Corynebacterium glutamicum ATCC 13032 were isolated as described in Tauch et al. (1995, Plasmid 33: 168-179) and were partially cut with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description: Sau3AI, code No. 27-0913-02). DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, product description: SAP, code No. 1758250). The superCosl cosmid vector DNA (Wahl et al. (1987) Proceedings of the National Academy of Sciences, USA 84: 2160-2164), purchased from the company Stratagene (La Jolla, USA, product description: SuperCosl Cosmid Vector Kit, key No. 251301) was cut with the restriction enzyme Xbal (Amersham Pharmacia, Freiburg, Germany, product description: Xbal, code No. 27-0948-02) and also dephosphorylated with shrimp alkaline phosphatase. The cosmid DNA was then cut with the restriction enzyme Ba Hl (Amersham Pharmacia, Freiburg, Germany, product description: BamHl, code No. 27-0868-04). The cosmid DNA treated in this manner was mixed with the treated ATCC13032 DNA, and the preparation was treated with that of T4-DNA-ligase (Amersham Pharmacia, Freiburg, Germany, product description: T4-DNA-ligase, key No. 27-0870-04). The ligation mixture was then packed into phage with the aid of the Gigapack II XL packaging extract (Stratagene, La Jolla, USA, product description: Gigapack II XL Packing Extract, code No. 200217). For the infection of E. coli strain NM554 (Raleigh et al., 1988, Nucleic Acid Res. 16: 1563-1575) cells were suspended in 10 mM MgSO4 and mixed with an aliquot of the phage suspension. The infection and titration of the cosmid bank was carried out as described in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), where the cells were spread on LB-Agar plates (Lennox, 1955, Virology, 1: 190) with 100 μg / ml of ampicillin. After incubation overnight at 37 ° C, individual recombinant clones were selected. Example 2 Isolation and sequencing of the sdhC, sdhA and sdhB genes The cosmid DNA of an individual colony was isolated with the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer's instructions and cut partially with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, product description: Sau3AI, code No. 27-0913-02). The DNA fragments are dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, product description: SAP, code No. 1758250). After the electrophoretic gel separation, the isolation of the cosmid fragments from the size range of 1500 to 2000 bp was carried out with the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany). DNA from the sequencing vector pZerol, purchased through the company Invitrogen (Groningen, The Netherlands, product description: Zero Rackground Cloning Kit, Product No. K2500-01) was cut with the restriction enzyme BamHl (Amersham Pharmacia, Freiburg, Germany, product description: BamHl, product no. 27-0868-04). Ligation of the cosmid fragments in the sequencer vector pZerol was carried out as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), where the DNA mixture with T4-ligase (Pharmacia Biotech, Freiburg, Germany) was incubated overnight. This ligation mixture was electroporated (Tauch et al., 1994, FEMS Microbiol Letters, 123: 343-7) below into the E. coli strain DH5otMCR (Grant, 1990, Proceedings of the National Academy of Sciences USA, 87: 4645 -4649) and spread on LB agar plates (Lennox, 1955, Virology, 1: 90) with 50 μg / ml Zeocin. 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 interruption method of Sanger et al. (Proceedings of the National (Academy) of Sciences of the United States of America USA, 74: 5463-5467) with modifications according to Zimmermann et al. (1990, Nucleic Acids Research, 18: 1067). The "RR dRhoda in" Kit was used ^ ktí ^ ^^^^^ ái ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^ Ter inator Cycle Sequencing Kit "by PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany). Separation and gel electrophoretic analysis of the sequencing reaction were carried out on a" Rotiphorese NF Acrylamid / Bisacrylamid gel. "(29: 1) (product No. A124.1, Roth, Karlsruhe, Germany) with the" ABl Prism 377"sequencing apparatus from PE Applied Biosystems (Weiterstadt, Germany) The data obtained from the raw sequence were processed at Then using the Staden program package (1986, Nucleic Acids Research, 14: 217-231), version 97-0, the individual sequences of the pZerol derivatives were assembled to obtain a Continuous contig.The analysis of the coding region supported by The computer was developed with the XNIP program (Staden, 1986, Nucleic Acids Research, 14: 217-231) Other analyzes were carried out with the "BLAST search programs" (Altschul et al., 1997, Nu cleic Acids Research, 25: 3389-3402), with respect to the non-redundant databank of the National Center for Biotechnology Information (NCBIr Bethesda, MD, USA). The nucleotide sequence obtained is represented in SEQ ID No. 1. The analysis of the nucleotide sequence resulted in an open reading frame of 879 base pairs which was characterized as sdhC gene, as well as an open reading frame of 1875 base pairs that were characterized as sdhA, as well as an open reading frame of 852 base pairs "that was characterized as sdhB.

The sdhC gene codes for a polypeptide of 293 5 amino acids, which is represented in SEQ ID No. 3. The sdhA gene codes for a polypeptide of 625 amino acids which is represented in SEQ ID No. 5. The sdhB gene encodes for a polypeptide of 284 amino acids, which is represented in SEQ ID No. 7. Example 3 3.1 Preparation of an integration vector for integration mutagenesis of the sdhA gene From the strain ATCC 13032, chromosomal DNA was isolated according to method of Eikmanns et al 15 (Microbiology 140: 1817-1828 (1994)). Based on the sequence of the sdhA gene for C. glutamicum known from Example 2, the following oligonucleotides were selected for the polymerase chain reaction: sdhA-ini: 0 5 'CGT CAT TGT CAC CGA ACG TA 3' sdhA-in2: 5 'TCG TTG AAG TCA GTC CAG AG 3' The primers represented were synthesized by the company MWG Biotech (Ebersberg, Germany) and the polymerase chain reaction was carried out in accordance ? ?to . - to the standard PCR method of Innis et al. (PCR Protocols, A Guide to Methods and Applications, 1990, Academic Press) with Pwo polymerase from the company Boehringer Mannheim (Germany, product description: Pwo DNA Polymerase, product No. 1 644 947). With the help of the polymerase chain reaction, the primers allow the amplification of an internal fragment of approximately 0.67 kb in size of the sdhA gene. The product amplified in this way was electrophoretically tested on a 0.8% agarose gel. The amplified DNA fragment was ligated to the pCR®Blunt II vector (Bernard et al., Journal of Molecular Biology, 234: 534: 541 (1993)) with the Zero Blunt ™ Kit from the company Invitrogen Corporation (Carlsbad, CA, USA). catalog number K2700-20). The strain E. coli TOP10 was then electroporated with the ligation preparation (Hanahan, in: DNA Cloning, A Practical Approach, Vol. I, IRL-Press, Oxford, Washington DC, USA, 1985). The selection of plasmid-carrying cells was carried out by spreading the transformation preparation on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). which was supplemented with 25 mg / l of kanamycin. Plasmid DNA was isolated from a transformed with the aid of the "QIAprep Spin Miniprep Kit" from the company Qiagen, and was examined by restriction with the restriction enzyme EcoRI and subsequent electrophoresis with agarose gel (0.8%). The plasmid was named pCRBluntsdhAint and is shown in figure 1. Example 4 Integration mutagenesis of the sdhA gene in strain DSM 5715 The vector pCRBluntsdhAint mentioned in example 3 was electroporated in C. glutamicum DSM 5715 according to the electroporation method of Tauch et al. to the. (FEMS Mícrobiological Letters, 123: 343-347 (1994)). The DSM strain 5715 is described in EP-B-0435132. The pCRBluntsdhAint vector can not replicate independently in DSM5715 and is only retained in the cell if it has been integrated into the chromosome of DSM 5715. The selection of clones with pCRBluntsdhAint integrated into the chromosome was carried out by extending the electroporation preparation on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) which was supplemented with 15 mg / l of kanamycin. For verification of the integration, the sdhAint fragment was marked with the Dig Hybridization Kit of the Boehringer company according to the "The DIG System Users Guide for Filter Hybridization" method of the Boehringer Mannheim GmbH company (Mannheim, Germany, 1993). The chromosomal DNA of an inefficient potential was isolated according to the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) and cut respectively with the restriction enzymes Sphl and HindIII. The resulting fragments were separated by agarose gel electrophoresis and hybridized at 68 ° C with the Dig Hybridization Kit from the Boehringer company. Plasmid pCRBluntsdhAint mentioned in Example 3 had been inserted into the chromosome of DSM5715 within the chromosomal sdhA gene. The strain was characterized as DSM5715:: pCRBluntsdhAint. Example 5 Preparation of L-glutamic acid with the strain DSM5715:: pCRBluntsdhAint The strain DSM5715:: pCRBluntsdhAint of C. glutamicum obtained in example 4 was cultured in a culture medium suitable for the production of glutamic acid, and the content of glutamic acid in the culture supernatant was determined. For this purpose, the strain was first incubated on agar plate for 24 hours at 33 ° C with the corresponding antibiotic (brain-heart agar with kanamycin (25 mg / l).) A culture was inoculated from this agar plate culture. Pre-culture (10 ml of medium in a 100 ml Erlenmeyer flask) The medium Cg III was used as medium for the preculture: Medium Cg III NaCl 2.5 g / 1 5 Bacto-peptone 10 g / 1 Bacto-yeast extract 10 g / 1 Glucose (treated in autoclave separately 2% (w / v) The pH value was adjusted to pH 7.4 To this (medium) was added kanamycin (25). mg / l). The preculture was incubated for 16 hours at 33 ° C at 240 rpm in the agitator. From this preculture a main culture was inoculated, so that the initial OD (660 nm) of the main culture was 0.1 OD. The MM medium was used for the main culture. 15 Medium MM CSL (Soaked corn liquor) 5 g / 1 MOPS (morpholinopropanesulfonic acid) 20 g / 1 Sodium acetate (sterile filtrate) 20 g / 1 Salts: 20 (NH4) 2 S04) 25 g / 1 KH2P04 0.1 g / 1 MgSO4 * 7 H20 lO g / 1 CaCl2 * 2 H20 10 mg / l FeS04 * 7 H20 10 mg / l 25 MnS04 * H20 5.0 mg / l ^ - ^ ^ ^ ÍA ^ M ^^ m ^.

Biotin (sterile filtered) 0.3 mg / l Thiamine * HCl (sterile filtered) 0.2 mg / l Leucine (sterile filtered) 0.1 g / 1 CaC03 25 g / 1 5 The CSL, MOPS and saline are adjusted to ammonia water to pH 7 and autoclaved. Next, the sterile substrate and vitamin solutions are added, as well as the dry-autoclaved CaC03. The culture is carried out in 10 ml volume of a 100 ml Erlenmeyer flask with coils. Kanamycin (25 mg / l) was added. The culture was carried out at 33 ° C and 80% humidity. After 24 hours the OD was determined at a wavelength of 660 nm with the Biomek 1000 device (Beckmann Instruments GmbH, Munich). The amount of glutamic acid formed was determined with an amino acid analyzer from the company Eppendorf-Biotronik (Hamburg, Germany) by ion exchange chromatography and posterior derivation in column with ninhydrin detection. Table 1 shows the result of the test.

Table 1 The following figures are attached: Figure 1: Map of the plasmid pCRBluntsdhAint The abbreviations and designations used have the following meanings. The indications of base pair numbers are approximate values that are obtained within the framework of the reproducibility of measurements: Km: kanamycin-resistant gene Zeocin: HindIII zeocin-resistant gene HindIII Sphl restriction enzyme cleavage site Sphl EcoRI restriction enzyme cleavage site EcoRI restriction enzyme site SdhAint: Internal fragment of sucC gene ColEl ori Origin of replication of the plasmid ColEl SEQUENCE PROTOCOL < 110 > Degussa-Hüls AG < 110 > New nucleotide sequences encoding the genes sdhA, sdhB and sdhC < 130 > 990170 BT < 140 > < 141 > < 160 > 7 < 170 > Patentln Ver. 2.1 < 210 > 1 < 211 > 4080 < 212 > DNA < 213 > Corynebacterium glutamicum < 220 > < 221 > genes < 222 > (288) .. (1169) < 223 > adhC < 220 > < 221 > genes < 222 > (1330) .. (3207) < 223 > adhA < 220 > < 221 > genes < 222 > (3102) .. (3956) < 223 > adhB < 400 > 1 gtgcccggcg tggtcgggcc acatccgccc cgggaacttt ttaggcacd: acggtgcaac 60 tgttgggata attgtgtcac ctgcgcaaag ttgctccctg gatcggaagg ttgggctgtc 120 taaacttttt ggttgatacc aaacggggtt agaaactgtt cggatcggta tcctgtgagg 180 aagctcacct tggttttaga atgttgaaaa ggcctcacgt ttccgcaggt agagcacact 240 caattaaatg agcgtcaaac gacaataaag taaggctatc ctaataagtg gggttttatg 300 tctctaaaca gccagttggg ggtcatgggg gagcgccccg tgactggtta atgccccgat 360 ctgggacgta cagtaacaac gacactggag gtgccatgac tgttagaaa cccgaccgtg 420 aggcaatccg tcacggaaaa attacgacgg aggcgctgcg tgagcgtccc gcatacccga 490 cctgggcaat gaagctgacc atggccatca ctggcctaat gtttggtggc ttcgttcttg 540 ttcacatgat cggaaacctg aaaatcttca tgccggacta cgcagccgat tctgcgcatc 600 cgggtgaagc acaagtagat gtctacggcg agttcctgcg tgagatcgga tccccgatcc 660 tcccacacgg ctcagtcctc tggatcctac gtattatcct gctggtcgca ttggttctgc 720 tgcattcgca acatctactg ttgaccggcc gttctcacca gtcccgcgga aagttccgcc 780 gtaccaacct cgttggcggc ttcaactcct tcgcgacccg ctccatgctg gtgaccggaa 840 tcgttctcct tgcgttcat t atcttccaca tcctcgacct gaccatgggt gttgctccag 900 cagccccaac ctcattcgag cacggcgaag tatacgcaaa catggtggct tcctttagcc 960 gctggcctgt agcaatttgg tacatcattg ccaacctggt cctgttcgtc cacctgtcac 1020 acggcatctg gcttgcagtc tctgacctgg gaatcaccgg acgccgctgg agggcaatcc 1080 tcctcgcagt tgcgtacatc gttcctgcac tggtcctgat cggcaacatc accattccgt 1140 tcgccatcgc tgttggctgg attgcgtaaa ggttaggaag aatttatgag cactcactct 1200 gaaaccaccc gcccagagtt catccaccca gtctcagtcc tcccagaggt ztcagctggt 1260 acgctgcaga acggtccttg gttcccacca gccagaagga ggaataccaa aagatatgtg 1320 aaagaccaca tgaacctggt ctccccactg aaccgacgca agttccgtgt cctcgtccgt 1380 ggcaccggcc tgtccggtgg tgctgcagca gcagccctcg gcgaactcgg atacgacgtc 1440 cctaccacga aaggcgttca cgcacctcgc cgtgcgcact ccattgctgc acagggtggc 1500 gttaactccg cccgcggcaa gaaggtagac aacgacggcg cataccgcca cgtcaaggac 1560 accgtcaagg gcggcgacta ccgtggtcgc gagtccgact gctggcgtct cgccgtcgag 1620 tccgtccgcg tcatcgacca catgaacgcc atcggtgcac cattcgccc? i cgaatacggt 1680 ggcgccttgg caacccgttc cttcggtggt gtgcaggtct cccgtaccta ctacacccgt 1740 ggacaaaccg gacagcagct gcagctctcc accgcatccg cactacagcg ccagatccac 1800 ctcggctccg tagaa atctt cacccataac gaaatggttg acgtcattgt caccgaacgt 1860 agcgctgcga aacggtgaaa aggcctgatc atgcgcaacc tgatcaccgc cgagctcacc 1920 gcacacaccg gccatgccgt tatcctggca accggtggct acggcaacgt gtaccacatg 1980 tccaccctgg ccaagaactc caacgcctcg gccatcatgc agccggcgca gtgcatacga 2040 tacttcgcgt ccccatcgtt catccagttc cacccaaccg gcctgcctgt gaactccacc 2100 tggcagtcca agaccattct gatgtccgag tcgctgcgta acgacggccg catctggtcc 2160 cgaacgataa cctaaggaac ccgcgatcca aacaccatcc ctgaggatga gcgcgactac 2220 ttcctggagc gccgctaccc agcattcggt aacctcgtcc cacgtgacgt tgcttcccgt 2280 gcgatctccc agcagatcaa tgctggtctc ggtgttggac ctctgaacaa cgctgcatac 2340 ctggacttcc gcgacgccac cgagcgcctc ggacaggaca ccatccgcga gcgttactcc 2400 ccatgtacga aacctcttca agaggcaatt ggcgaggacc catactccag cccaatgcgt 2460 attgcaccga cctgccactt caccatgggt ggcctctgga ctgacttcaa cgaaatgacg 2520 tcactcccag gtctgttctg cgcaggcgaa gcatcctgga cctaccacgg tgcaaaccgt 2580 ctgggcgcaa actccctgct ctccgcttcc gtcgatggct ggttcaccct gccattcacc 2640 atccctaact acctcggccc attgcttggc tccgagcgtc tgtcagagga tgcaccagaa 2700 gcacaggcag cgattgcgcg tgcacaggct cgcattgacc gcctcatggg caaccgccca 2760 gagtgggtcg gtgacaacgt tcacggacot gagtactacc accgccagct tggcgatatc 2820 ctgtacttct cctgtggcgt ttcccgaaac gtagaagacc tccaggatgg catcaacaag 2880 atccgtgccc tccgcgatga cttctggaag aacatgcgca tcaccggcag caccgatgag 2940 atgaaccagg ttctcgaata cgcagcacgc gtagccgact acatcgacct cggcgaactc 3000 atgtgtgtcg acgccctcga ccgcgacgag tcctgtggcg ctcacttccg cgacgaccac 3060 ctctccgaag atggcgaagc agaacgtgac gacgaaaact ggtgcttcgt ctccgcatgg 3120 gaaccaggcg agaacggaac cttcgtccgc cacgcagaac cactgttctt cgaatccgtc 3180 caaggaacta ccactgcaga caagtaatga aacttacact tgagatctgg cgtcaagcag 3240 gcccaactgc ggaaggcaag ttcgaaaccg tccaggttga cgacgccgtc gcgcagatgt 3300 gctgcttgac ccatcctgga cacgtaaaca acaagttcat cgaagaaggc aaagaaccac 3360 tcgcgttcgc ctctgactgc cgcgaaggca tttgtggtac ctgtggtctc ctcgtgaacg 3420 gtcgccctca cggcgccgac cagaacaagc ctgcctgtgc gcagcgcctg gtcagctaca 3480 caccctcaag aggaaggcga atcga ACCAC tgcgttccgc cgcataccca gtgatcaagg 3540 acatggtcgt cgaccgctcc gcactggacc gtgtcatgga acagggtggc tacgtgacca 3600 tcaacgcagg taccgcacct gacgctgata ccctccacgt caaccacgaa accgcagaac 3660 tcgcacttga ccacgcagcc tgcatcggct gtggcgcatg tgttgctgcc tgccctaacg 3720 gcgcagcaca cctgttcacc ggcgcaaagc ttgttcacct ctccctcctc ccactgggta 3780 aggaagagcg cggactgcgt gcacgtaaga tggttgatga aatggaaacc aacttcggac 3840 actgctccct ctacggcgag tgcgcagatg tctgccccgc aggcatccca ctgaccgctg 3900 tggcagctgt caccaaggaa cgtgcgcgtg cagctttccg aggcaaagac gactagtctt 3960 taatccaagt aagtaccggt tcagacagtt aaaccagaaa gacgagtgaa caccatgtcc 4020 tccgcgaaaa agaaacccgc accggagcgt atgcactaca tcaagggcta tgtacctgtg 4080 <; 210 > 2 < 211 > 882 < 212 > DNA < 213 > Corynebacterium glutamicum < 220 > < 221 > CDS «Fc.é * < 222 > (1) .. (879) < 223 > sdhC < 400 > 2 gtg ggg ttt tat gtc tct aaa cag cea gtt ggg ggt cat ggg gga gcg 48 Val Gly Phe Tyr Val Ser Lys Gln Pro Val Gly Gly Hia Giy Gly Wing 1 5 10 15 ccc cgt gac tgg tta atg ccc cga tct ggg age tac agt aac aac gac 96 ProArg Asp Trp Leu Met Pro Arg Ser Gly Thr Tyr Ser Asn Asn Asp 20 25 30 act gga ggt gcc atg act gtt aga aat ccc gac cgt gag gca ate cgt 144 Thr Gly Gly Ala Met Thr Val Arg Asn Pro Asp Arg Glu Alalle Arg 35 40 45 cav gga aaa att acg acg gag gcg ctg cgt gag cgt ccc gca tac ccg 192 Hís Gly Lys lie Thr Thr Glu Ala Leu Arg Glu Arg Pro Ala Tyr Pro 50 55 60 acc tgg gca atg aag ctg acc atg gcc ate act ggc cta atg ttt ggt 240 Thr Trp Wing Met Lys Leu Thr Met Ala lie Thr Gly Leu Met Phe Gly 65 70 75 80 ggc ttc gtt ctt gtt falls atg ate gga aac ctg aaa ate ttc atg ccg 288 Gly Phe Val Leu Val His Met He Gly Asn Leu Lys He Phe Met Pro 85 90 95 gac tac gca gcc gatct ccg cat ccg ggt gaa gca ca gta gat gtc 336 Asp Tyr Ala Ala Asp Ser Ala His Pro Gly Glu Ala Gln Val Asp Val 100 105 110 tac ggc gag ttc ctg cgt gag ate gga tcc ccg ate etc cea falls ggc 384 Tyr Gly Glu Phe Leu Arg Glu He Gly Ser Pro He Leu Pro His Gly 115 120 125 tea gtc etc tgg ate cta cgt att ate ctg ctg gtc gca ttg gtt ctg 432 5 Ser Val Leu Trp He Leu Arq He He Leu Leu Val Wing Leu Val Leu 130 135 140 falls tet tc tc gca ttc gca ttg ace ggc cctt tct falls tc cgc 480 His He Tyr Cys Wing Phe Wing Leu Thr Gly Arg Ser Hís Gln Ser Arg 145 150 155 160 10 gga aag ttc cgc cgc acc aac etc gtt ggc ggc ttc aac tcc ttc gcg 528 Gly Lys Phe Arg Arg Thr Asn Leu Val Gly Gly Phe Asn Ser Phe Wing 165 170 175 acc cgc tcc atg ctg gtg acc gga ate gtt etc ctt gcg ttc att ate 576 Thr Arg Being Met Leu Val Thr Gly He Val Leu Leu Wing Phe He He 15 180 185 190 ttc falls tetc etc gac ctg acc atg ggt gtt gct cea gca gcc cea acc 624 Phe His He Leu Asp Leu Thr Met Gly Val Ala Pro Ala Wing Pro Thr 195 200 205 tea ttc gag falls ggc gaa gta tac gc aac atg gtg gct tcc ttt age 672 20 Ser Phe Glu His Gly Glu Val Tyr Ala Asn Met V to Wing Ser Phe Ser 210 215 220 cgc tgg ect gta gca att tgg tac ate att gcc aac ctg gtc ctg ttc 720 Arg Trp Pro Val Wing He Trp Tyr He He Wing Asn Leu Val Leu Phe 225 230 235 240 25 gtc falls ctg tea falls ggc ate tgg ctt gc gtc tct gac tg gga ate 768 Val His Leu Ser His Gly He Trp Leu Wing Val Ser Asp Leu Gly He 245 250 255 acc gga cgc cgc tgg agg gca ate etc etc gca gtt gcg tac ate gtt 816 5 Thr Gly Arg Arg Trp Arg Ala He Leu Leu Ala Val Ala Tyr He Val 260 265 270 ect gca ctg gtc ctg ate ggc aac ate acc att ccg ttc gcc ate gct 864 Pro Ala Leu Val Leu He Gly Asn He Thr He Pro Phe Wing He Wing 275 280 285 10 gtt ggc tgg att gcg taa 002 Val Gly Trp He Ala 290 < 210 > 3 < 211 > 293 15 < 212 > PRT < 213 > Corynebacterium glutamicum < 400 > 3 Val Gly Phe Tyr Val Ser Lys Gln Pro Val Gly Gly His Gly Gly Wing 1 5 10 15 20 Pro Arg Asp Trp Leu Met Pro Arg Ser Gly Thr Tyr Ser Asn Asn Asp 20 25 30 Thr Gly Gly Ala Met Thr Val Arg Asn Pro Asp Arg Glu Wing He Arg 35 40 45 His Gly Lys He Thr Thr Glu Wing Leu Arg Glu Arg Pro Wing Tyr Pro 25 50 55 60 «^^ Thr Trp Wing Met Lys Leu Thr Met Wing He Thr Gly Leu Met Phe Gly 65 70 75 80 Gly Phe Val Leu Val His Met He Gly Asn Leu Lys He Phe Met Pro 85 90 95 Asp Tyr Ala Ala Asp Ser Ala Hís Pro Gly Glu Ala Gln Val Asp Val 100 105 110 Tyr Gly Glu Phe Leu Arg Glu He Gly Ser Pro He Leu Pro His Gly 115 120 125 Ser Val Leu Trp He Leu Arg He He Leu Leu Val Ala Leu Val Leu 130 135 140 His He Tyr Cys Ala Phe Ala Leu Thr Gly Arg Ser His Cln Ser Arg 145 150 155 160 Gly Lys Phe Arg Arg Thr Asn Leu Val Gly Gly Phe Asn Ser Phe Wing 165 170 175 Thr Arg Ser Met Leu Val Thr Gly He Val Leu Leu Wing Phe He He 180 185 190 Phe His He Leu Asp Leu Thr Met Gly Val Ala Pro Ala Wing Pro Thr 195 200 205 Ser Phe Glu His Gly Glu Val Tyr Wing Asn Met Val Wing Ser Phe Ser 210 215 220 Arg Trp Pro Val Wing He Trp Tyr He He Wing Asn Leu Val Leu Phe 225 230 235 240 Val His Leu Ser His Gly He Trp Leu Wing Val Ser Asp Leu Gly He 245 250 255 Thr Gly Arg Arg Trp Arg Wing He Leu Leu Wing Val Wing Tyr He Val 260 265 270 Pro Wing Leu Val Leu He Gly Asn He Thr He Pro Phe Ala He Ala 275 280 285 Val Gly Trp He Ala 290 < 210 > 4 < 211 > 1878 < 212 > DNA < 213 > Corynebacterium glutamicum < 220 > < 221 > CDS < 222 > (1) .. (1875) < 223 > sdhA < 400 > 4 atg aac ctg gtc ctc tc cc aa cgc cc aag tcc cgt gtc etc gtc 46 Met Asn Leu Val Pro Pro Leu Asn Arg Arg Lys Phe Arg Val Leu Val 1 5 10 15 gtt ggc gcg gcc gcc gcc gcc gcc gcc gca gca gca gca gcc etc ggc gaa 96 Val Gly Thr Gly Leu Ser Gly Gly Wing Wing Wing Wing Leu Gly Glu 20 25 30 etc gga tac gac gtc aag gcg ttc acc tac falls gac gca ect cgc cgt 144 Leu Gly Tyr Asp Val Lys Wing Phe Thr Tyr His Asp Wing Pro Arg Arg 35 40 45 gcg falls tcc att gct gg ggt ggc gtt aac gcc gcc cgc ggc aag 192 Wing His Ser He Wing Wing Gln Gly Gly Val Asn Ser Wing Arg Gly Lys 50 55 60 aag gta gac aac gac ggc gca tac cgc falls gtc aag gac acc gtc aag 240 Lys Val Asp Asn Asp Gly Wing Tyr Arg His Val Lys Asp Thr Val Lys 65 70 75 80 ggc ggc gac tac cgt ggt cgc gag tcc gac tgc tgg cgt etc gcc gtc 288 Gly Gly Asp Tyr Arg Gly Arg Glu Be Asp Cys Trp Arg Leu Wing 85 85 95 gag tcc gtc cgc gtc ate gac falls atg aac gcc ate ggt gea cea ttc 336 Glu Ser Val Arg Val He Asp "His Met Asn Ala He Gly Wing Pro Phe 100 105 110 gcc cgc gaa tac ggt ggc ggc ttg gca acc cgt tcc tgc ggt gtg 384 Wing Arg Glu Tyr Gly Gly Wing Leu Wing Thr Arg Being Phe Gly Gly Val 115 120 125 cag gtc tcc cgt acc tac tac ac cgt gga ca g acc gga cag cag ctg 432 Gln Val Ser Arg Thr Tyr Tyr Thr Arg Gly Gln Thr Gly Gln Gln Leu 130 135 140 cag etc tcc acc gca tcc gca cta cag cgc cag ate drops etc ggc tcc 480 Gln Leu Ser Thr Ala Ser Ala Leu Gln Arg Gln He His Leu Gly Ser 145 150 155 160 gta gaa ate ttc acc cat aac gaa atg gtt gac gtc att gtc acc gaa 528 Val Glu He Phe Thr His Asn Glu Met Val Asp Val He Val Thr Glu 165 170 175 cgt aac ggt gaa aag cgc tgc gaa ggc ctg ate atg cgc aac ctg ate 576 Arg Asn Gly Glu Lys Arg Cys Glu Gly Leu He Met Arg Asn Leu He 180 185 190 acc ggc gag etc acc gca falls acc ggc cat gcc gtt ate ctg gca acc 624 Thr Gly Glu Leu Thr Ala His Thr Gly His Wing Val He Leu Wing Thr 195 200 205 ggt ggc tac ggc aac gtg tac drops atg tcc acc ctg gcc aag aac tcc 672 Gly Gly Tyr Gly Asn Val Tyr His Met Ser Thr Leu Ala Lys Asn Ser 210 215 220 aac gcc tcg gcc ate atg cgt gca tac gac gcc gcac gc tac tcc gcg 720 Asn Wing Wing Wing Arg Ala Tyr Glu Wing Gly Wing Tyr Phe Wing 225 230 235 240 tcc tcc tcc tcc ate tcc tcc tcc ggc ctg ect gtg aac tcc 768 Ser Pro Be Phe He Gln Phe His Pro Thr Gly Leu Pro Val Asn Ser 245 250 255 acc tgg cag tcc aag acc att ctg atg tcc gag tcg ctg cgt aac gac 816 Thr Trp Gln Ser Lys Thr He Leu Met Ser Glu Ser Leu Arg Asn Asp 260 265 270 ggc cgc ate tgg tcc ect aag gaa cc g aac g aac g aac c aac cc aac 864 Gly Arg He Trp Ser Pro Lys Glu Pro Asn Asp Asn Arg Asp Pro Asn 275 280 285 acc ate ect gag gat gag cgc gac tac tcc cgc gag cgc cgc tac cea 912 Thr He Pro Glu Asp Glu Arg Asp Tyr Phe Leu Glu Arg Arg Tyr Pro 290 295 300 gca ttc ggt aac etc gtc cea cgt gac gtt gct tcc cgt gcg ate tcc 960 Wing Phe Gly Asn Leu Val Pro Arg Asp Val Wing Ser Arg ALa Be Ser 305 310 315 320 cag cag ate a gct gct ggt ggt gtt gt gct ect cct aac gct gc gc 1008 Gln Gln He Asn Wing Gly Leu Gly Val Gly Pro Leu Asn Asn Ala Ala 325 330 335 tac ctg gac tcc ccc gac gcc acc gag cgc etc gga cag gac acc ate 1056 Tyr Leu Asp Phe Arg Asp Wing Thr Glu Arg Leu Gly Gln Asp Thr He 340 345 350 cgc gag cgt tac tcc aac etc ttc acc atg tac gaa gag gca att ggc 1104 Arg Glu Arg Tyr Ser Asn Leu Phe Thr Met Tyr Glu Glu Wing He Gly 355 360 365 gag gac cea tac tcc age cea atg cgt att gca ccg acc tfe ttc 1152 Glu Asp Pro Tyr Be Ser Pro Pro Arg He Wing Pro Thr Cys His Phe 370 375 380 at ggt ggc atg tgg act gac tc aac gaa atg acg tea etc cea 1200 Thr Met Gly Gly Leu Trp Thr Asp Phe Asn Glu Met Thr Ser Leu Pro 385 390 395 400 ggt ctg ttc tgc gca ggc gaa gca tcc tgg ttac ac tac ggt gca aac 1248 Gly Leu Phe Cys Wing Gly Glu Wing Ser Trp Thr Tyr His Gly Wing Asn 405 410 415 cgt ctg ggc gca aac tcc ctg etc tcc gct ccc gtc gat ggc tgg ttc 1296 Arg Leu Gly Wing Asn Being Leu Leu Being Wing Being Val Asp Gly Trp Phe 420 425 430 acc ctg cea ttc acc ate ect aac tac etc ggc cea ttg ctt ggc ccc 1344 Thr Leu Pro Phe Thr He Pro Asn Tyr Leu Gly Pro Leu Leu Gly Ser 435 440 445 gag cgt ctg tea gag gat gca cea gaa gca cag gca gcg att gcg cgt 1392 Glu Arg Leu Ser Glu Asp Wing Pro Glu Wing Cln Wing Wing Wing Arg 450 455 460 gca cag gct cgc att gac cgc etc atg ggc aac cgc cea gag tgg gtc 1440 Wing Gln Wing Arg He Asp Arg Leu Met Gly Asn Arg Pro Glu Trp Val 465 470 475 480 ggt gac aac gtt drops gga ect gag tac tac falls cgc cag ctt ggc gat 1488 Gly Asp Asn Val His Gly Pro Glu Tyr Tyr His Arg Gln Leu Gly Asp 485 490 495 ate ctg tac ttc tcc tgt ggc gtt tcc cga aac gta gaa gac etc cag 1536 He Leu Tyr Phe Ser Cys Gly Val Ser Arg Asn Val Glu Asp Leu Gln 500 505 510 gat ggc ate aac aac ate cgt gcc etc cgc gat gac ttc tgg aag aac 1584 Asp Gly He Asn Lys He Arg Ala Leu Arg Asp Asp Phe Trp Lys Asn 515 520 525 atg cgc ate acc ggc age acc gat gag atg aac cag gtt etc gaa tac 1632 Met Arg He Thr Gly Ser Thr Asp Glu Met Asn Gln Val Leu Glu Tyr 530 535 540 gca gca cgc gta gcc gac tac ate gac etc ggc gaa etc atg tgt gtc 1680 Ala Ala Arg Val Ala Asp Tyr He Asp Leu Gly Glu Leu Met Cys Val 545 550 555 560 * L t * * * a? B * gac gcc etc gac cgc gac gac tcc tgt ggc gct falls tcc ccc gac gac 1728 Asp Ala Leu Asp Arg Asp Glu Ser Cys Gly Wing His Phe Arg Asp Asp 565 570 575 falls etc tcc gaa gat ggc gaa gca gaga cgt gac gac aac tgg tgc 1776 His Leu Ser Glu Asp Gly Glu Wing Glu Arg Asp Asp Glu Asn Trp Cys 580 585 590 ttc gtc tcc gca tgg gaa cea ggc gag aac gga acc ttc gtc cgc falls 1824 phe Val Ser Wing Trp Glu Pro Gly Glu Asn Gly Thr Phe Val Arg His 595 600 605 gca gaa cea ctg ttc ttc gaa tcc gtc cea ctg cag here agg aac tac 1872 Wing Glu Pro Leu Phe Phe Glu Ser Val Pro Leu Gln Thr Arg Asn Tyr 610 615 620 aag taa 1878 Lys 625 < 210 > 5 < 211 > 625 < 212 > PRT < 213 > Corynebacterium glutamicum < 400 > 5 Met Asn Leu Val Ser Pro Leu Asn Arg Arg Lys Phe Arg Val Leu Val 1 5 10 15 Val Gly Thr Gly Leu Ser Gly Gly Wing Wing Wing Wing Wing Leu Gly Glu 20 25 30 Leu Gly Tyr Asp Val Lys Wing Phe Thr Tyr His Asp Ala Pro Arg Arg 35 40 45 Wing His Ser Wing Wing Gln Gly Gly Val Asn Wing Arg Gly Lys 50 55 60 Lys Val Asp Asn Asp Gly Wing Tyr Arg His Val Lys Asp Thr Val Lys 65 70 75 80 Gly Gly Asp Tyr Arg Gly Arg Glu Be Asp Cys Trp Arg Leu Wing Val 85 90 95 Glu Ser Val Arg Val He Asp His Met Asn Wing He Gly Wing Pro Phe 100 105 110 Wing Arg Glu Tyr Gly Gly Wing Leu Wing Thr Arg Being Phe Gly Gly Val 115 120 125 Gln Val Ser Arg Thr Tyr Tyr Thr Arg Gly Gln Thr Gly Gln Gln Leu 130 135 140 Gln Leu Ser Thr Ala Ser Ala Leu Gln Arg Gln He His Leu Gly Ser 145 150 155 160 Val Glu He Phe Thr His Asn Glu Met Val Asp Val He Val Thr Glu 165 170 175 Arg Asn Gly Glu Lys Arg Cys Glu Gly Leu He Met Arg Asn Leu He 180 185 190 Thr Gly Glu Leu Thr Wing His Thr Gly His Wing Val He Leu Wing Thr 195 200 205 Gly Gly Tyr Gly Asn Val Tyr His Met Ser Thr Leu Ala Lys Asn Ser 210 215 220 jtfe * - Asn Wing Being Wing He Met Arg Wing Tyr Glu Wing Gly Wing Tyr Phe Wing 225 230 235 240 Ser Pro Being Phe He Gln Phe His Pro Thr Gly Leu Pro Val Asn Ser 245 250 255 5 Thr Trp Gln Ser Lys Thr He Leu Met Ser Glu Ser Leu Arg Asn Asp 260 265 270 Gly Arg He Trp Ser Pro Lys Glu Pro Asn Asp Asn Arg Asp Pro Asn 275 280 225 Thr He Pro Glu Asp Glu Arg Asp Tyr Phe Leu Glu Arg Arg Tyr Pro 10 290 295 300 Wing Phe Gly Asn Leu Val Pro "Arg Asp Val Wing Wing Arg Wing Ser 305 310 315 320 Gln Gln He Asn Wing Gly Leu Gly Val Gly Pro Leu Asn Asn Wing Wing 325 330 335 15 Tyr Leu Asp Phe Arg Asp Wing Thr Glu Arg Leu Gly Gln Asp Thr He 340 345 350 Arg Glu Arg Tyr Ser Asn Leu Phe Thr Met Tyr Glu Glu Wing He Gly 355 360 365 Glu Asp Pro Tyr Ser Ser Pro Met Arg He Wing Pro Thr Cys Hís Phe 20 370 375 380 Thr Met Gly Gly Leu Trp Thr Asp Phe Asn Glu Met Thr Ser Leu Pro 395 390 395 400 Gly Leu Phe Cys Wing Gly Glu Wing Being Trp Thr Tyr Hís Gly Wing Asn 405 410 415 25 MÉég ^! ^^^^^^^^ »^^» fo ^^ ^^^^^^ g ^ ^^ £ u * .ít¡? Arg Leu Gly Ala Asn Ser Leu Leu Ser Ala Ser Val Asp Gly Trp Phe 420 425 430 Thr Leu Pro Phe Thr He Pro Asn Tyr Leu Gly Pro Leu Leu Gly Ser 435 440 445 Glu Arg Leu Ser Glu Asp Wing Pro Glu Wing Gln Wing Wing Wing Wing Arg 450 455 460 Wing Gln Wing Arg He Asp Arg Leu Met Gly Asn Arg Pro Glu Trp Val 465 470 475 480 Gly Asp Asn Val His Gly Pro Glu Tyr Tyr His Arg Gln Leu Gly Asp 485 490 495 He Leu Tyr Phe Ser Cys Gly Val Ser Arg Asn Val Glu Asp Leu Gln 500 505 510 Asp Gly He Asn Lys He Arg Ala Leu Arg Asp Asp Phe Trp Lys Asn 515 520 525 Met Arg He Thr Gly Ser Thr Asp Glu Met Asn Gln Val Leu Glu Tyr 530 535 540 Wing Wing Arg Val Wing Asp Tyr He Asp Leu Gly Glu Leu Met Cys Val 545 550 555 560 Asp Ala Leu Asp Arg Asp Glu Ser Cys Gly Ala His Phe Arg Asp Asp 565 570 575 His Leu Ser Glu Asp Gly Glu Wing Glu Arg Asp Asp Glu Asn Trp Cys 580 585 590 Phe Val Ser Wing Trp Glu Pro Gly Glu Asn Gly Thr Phe Val Arg His 595 600 605 Wing Glu Pro Leu Phe Phe Glu Ser Val Pro Leu Gln Thr Acg Asn Tyr 610 615 620 Lys 625 < 210 > 6 < 211 > 855 < 212 > DNA < 213 > Corynebacterium glutamicum < 220 > < 221 > CDS < 222 > (1) .. (852) < 223 > sdhB < 400 > 6 gtg ctt cgt etc cgc atg gga acc agg cga gaa cgg aac ctt cgt ccg 48 Val Leu Arg Leu Arg Met Gly Thr Arg Arg Glu Arg Asn Leu Arg Pro 1 5 10 15 cea cgc aga acc act gtt ctt cga ate cgt ccc act gca gac aag gaa 96 Pro Arg Arg Thr Thr Val Leu Arg He Arg Pro Thr Wing Asp Lys Glu 20 25 30 cta cata gta atg aaa ctt here ctt gag ate tgg cgt ca gca ggc cea 144 Leu Gln Val Met Lys Leu Thr Leu Glu He Trp Arg Gln Wing Gly Pro 35 40 45 act gcg gaa ggc aag ttc gaa acc gtc cag gtt gac gac gcc gtc gcg 192 Thr Wing Glu Gly Lys Phe Glu Thr Val Gln Val Asp Asp Ala Val Wing cag atg tcc ate ctg gag ctg ctt gac falls gta aac aac aag ttc ate 240 Gln Met Ser He Leu Glu Leu Leu Asp His Val Asn Asn Lys Phe He 65 70 75 80 gaa gaa ggc aaa gaa cea tcc gcg tcc gcc tct gac tgc cgc gaa ggc 288 Glu Glu Gly Lys Glu Pro Phe Wing Phe Wing Being Asp Cys Arg Glu Gly 85 90 95 att tgt ggt acc tgt ggt etc etc gtg aac ggt cgc ect falls ggc gcc 336 He Cys Gly Thr Cys Gly Leu Leu Val Asn Gly Arg Pro HLS Gly Wing 100 105 1L0 gac cag aac aag ect gcc tgt gcg cag cgc ctg gtc age tac aagga 384 Asp Gln Asn Lys Pro Ala Cys Ala Gln Arg Leu Val Ser Tyr Lys Glu 115 120 125 ggc gac acc etc aag ate gaa cea ctg cgt tcc gcc gca tac cea gtg 432 Gly Asp Thr Leu Lys He Glu Pro Leu Arg Ser Wing Ala Tyr Pro Val 130 135 140 ate aag gac atg gtc gtc gac cgc tcc gca ctg gac cgt gtc atg gaa 480 He Lys Asp Met Val Val Asp Arg Ser Ala Leu Asp Arg Val Met Glu 145 150 155 160 cag ggt ggc tac gtg acc ate aac gca ggt acc gca ect gac gct gat 528 Gln Gly Gly Tyr Val Thr He Asn Wing Gly Thr Ala Pro Asp Wing Asp 165 170 175 acc etc falls gtc aac falls gaa acc gca gaa etc gca ctt gac falls gca 576 Thr Leu His Val Asn His Glu Thr Ala Glu Leu Ala Leu Asp His Wing 180 185 190 gcc tgc ate ggc tgt ggc gca tgt gtt gct gcc tgc ect aac ggc gca 624 Wing Cys He Gly Cys Gly Wing Cys Wing Ala Cys Wing Cly Pro Asn Gly Wing 195 200 205 gca falls ctg ctg acc ggc gc aag ctt gtt drops etc tcc etc etc cea 672 5 Wing His Leu Phe Thr Gly Ala Lys Leu Val His Leu Ser Le > u Leu Pro 210 215 220 ctg ggt aag gaa cgc gga ctg cgt gca cgt aag atg gtt gat gaa 720 Leu Gly Lys Glu Glu Arg Gly Leu Arg Wing Arg Lys Met Val Asp Glu 225 230 235 240 10 atg gaa acc aac ttc gga falls tgc tcc etc tac ggc gag tgc gca gat 766 Met Glu Thr Asn Phe Gly His Cys Ser Leu Tyr Gly Glu Cys Wing Asp 245 250 255 gtc tgc ccc gca ggc ate cea ctg acc gct gtg gca gct gtc acc aag 816 Val Cys Pro Wing Gly He Pro Leu Thr Wing Val Wing Wing Val Thr Lys 15 260 265 270 gaa cgt gcg cgt gca gct ttc cga ggc aaa gac gac tag 855 Glu Arg Ala Arg Ala Ala Phe Arg Gly Lys Asp Asp 275 280 < 210 > 7 20 < 211 > 284 < 212 > PRT < 213 > Corynebacterium glutamicum < 400 > 7 Val Leu Arg Leu Arg Met Gly Thr Arg Arg Glu Arg Asn Leu Arg Pro 25 1 5 10 15 , -. X3MÉtmw ¡¡& i? Iíaí & ti, *, • Pro Arg Arg Thr Thr Val Leu Arg He Arg Pro Thr Wing Asp Lys Glu 20 25 30 Leu Gln Val Met Lys Leu Thr Leu Glu He Trp Arg Gln Wing Gly Pro 35 40 45 5 Thr Wing Glu Gly Lys Phe Glu Thr Val Gln Val Asp Asp Ala Val Wing 50 55 60 Gln Met Ser He Leu Glu Leu Leu Asp His Val Asn Asn Lys Phe He 65 70 75 80 Glu Glu Gly Lys Glu Pro Phe Wing Phe Wing Being Asp Cys Arg Glu Gly 10 85 90 95 He Cys Gly Thr Cys Gly Leu Leu Val Asn Gly Arg Pro His Gly Wing 100 105 110 Asp Gln Asn Lys Pro Wing Cys Wing Gln Arg Leu Val Ser Tyr Lys Glu 115 120 125 15 Gly Asp Thr Leu Lys He Glu Pro Leu Arg Ser Wing Wing Tyr Pro Val 130 135 140 He Lys Asp Met Val Val Asp Arg Ser Wing Leu Asp Arg Val Met Glu 145 150 155 160 Gln Gly Tyr Val Thr He Asn Wing Gly Thr Wing Pro Asn Wing Asp 20 165 170 175 Thr Leu His Val Asn His Glu Thr Wing Glu Leu Wing Leu Asp His Wing 180 185 190 Wing Cys He Gly Cys Gly Wing Cys Val Wing Wing Cys Pro Asn Gly Wing 195 200 205 25 ? .afe.w > Ala His Leu Phe Thr Gly Ala Lys Leu Val His Leu Ser Leu Leu Pro 210 215 220 Leu Gly Lys Glu Glu Arg Gly Leu Arg Wing Arg Lys Met Val Asp Glu 225 230 235 240 Met Glu Thr Asn Phe Gly His Cys Ser Leu Tyr Gly Glu Cys Ala Asp 245 250 255 Val Cys Pro Wing Gly He Pro Wing Leu Thr Wing Val Wing Wing Val Thr Lys 260 265 270 Glu Arg Ala Arg Ala Ala Phe Arg Gly Lys Asp Asp 275 280

Claims (19)

1. CLAIMS 1. Polynucleotide isolated from coryneform bacteria, which contains a polynucleotide sequence selected from the group comprising 5 a) polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide containing the amino acid sequence of SEQ ID No. 3, b) polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide that contains the amino acid sequence of SEQ ID No. 5, c) polynucleotide that is at least 70% identical to a polynucleotide that codes for a polypeptide containing the amino acid sequence of SEQ ID No. 7, d) polynucleotide encoding a polypeptide containing an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID No. 3, e) polynucleotide encoding a polypeptide that contains an amino acid sequence that is at least 20 70% identical to the amino acid sequence of SEQ ID N or 5, f) polynucleotide encoding a polypeptide containing an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID No. 25 7, ^^ sg ^^ j ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^ g ^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^ j jjBSStó g) complementary to the polynucleotides of a) polynucleotide, b) c), d), e) or f), h) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b), c), d), e) or f).
2. 2. Polynucleotide according to claim 1, characterized in that the polynucleotide is a replicable DNA in coryneform bacteria, preferably recombinant.
3. 3. Polynucleotide according to claim 1, characterized in that the polynucleotide is an RNA.
4. 4. Replicable DNA according to claim 2, characterized in that it contains (i) the nucleotide sequence shown in SEQ ID No. 1, or (ii) at least one sequence corresponding to the sequence (i) within the region of degeneracy of the genetic code, or (iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and / or optionally (iv) functionally neutral mutations orientation in (i).
5. 5. Polynucleotide sequence according to claim 2, characterized in that it encodes a polypeptide containing the amino acid sequence ^^^^^^^^^^ r ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^ & ^ a ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ represented in SEQ ID No. 2.
6. 6. Vector, characterized in that it contains a polynucleotide sequence according to claim 1, in particular 1 h). 5
7. 7. coryneform bacteria, characterized by containing a vector of claim 6.
8. 8. A method for the fermentative production of L to inoácidos, wherein are carried out the steps 10 a) coryneform bacteria which ferment the L - amino acid in which at least one of the genes selected from those encoding the enzyme succinate-dehydrogenase or its subunits A, B and C, 15 b) is attenuated, the L-amino acid is concentrated in the medium or in the cells of the the bacteria, and c) the L-amino acid is isolated.
9. 9. Method according to claim 8, characterized in that bacteria are used in which additionally 20 reinforce other genes in the course of the biosynthesis of the desired L-amino acid.
10. 10. A method according to claim 8, characterized in that bacteria are used in which at least part of the courses of metabolism that reduce the metabolism are eliminated. 25 formation of the L-amino acid.
11. 11. Method according to claim 8, characterized in that a strain transformed with a plasmid vector is used and the plasmid vector carries the nucleotide sequence of the gene coding for the dehydrogenase enzyme succinato-. Method according to one or more of claims 8 to 11, characterized in that coryneform bacteria which produce L-lysine are used. The method according to claim 9, characterized in that the dapA gene coding for dihydrodipicolinate synthase is simultaneously overexpressed. 14. Method according to claim 9, characterized in that the gap gene encoding glyceraldehyde-3-phosphate dehydrogenase is simultaneously overexpressed. The method according to claim 9, characterized in that the pyc gene coding for pyruvate carboxylase is simultaneously overexpressed. The method according to claim 9, characterized in that the mqo gene coding for the alato-quinone-oxidoreductase is simultaneously overexpressed. The method according to claim 9, characterized in that the lysE gene coding for the export of lysine is simultaneously overexpressed. 18. Method according to claim 10, characterized in that the pgi gene coding is simultaneously attenuated for glucose-6-phosphate isomerase. The method according to claim 10, characterized in that the pck gene coding for phosphoenol-pyruvatecarboxykinase is simultaneously attenuated. - te-Ma-i SUMMARY The invention relates to isolated polynucleotides containing a polynucleotide sequence selected from the group comprising a) polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide containing the sequence of amino acid of SEQ ID No. 3, b) polynucleotide that is at least 70% identical to a polynucleotide that encodes a polypeptide that contains the amino acid sequence of SEQ ID No. 5, c) polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide that contains the amino acid sequence of SEQ ID No. 7, d) polynucleotide that encodes 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 encoding a polypeptide containing an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID No. 5, f) polynucleotide that encodes a polypeptide containing an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID No. 7, g) polynucleotide complementary to the polynucleotides of a), b) c), d), e) of ), and h) polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b), c), d), e) of), and processes for the preparation by fermentation of L-amino acids with the attenuation of the sdhA, sdhB or sdhC gene coding for subunit A, B or C of the enzyme succinate dehydrogenase. 10 ¿£ * yyyy r mwydl ?? £ and £! Ißs.