Abstract:
Polynucleotides that contain polynucleotide sequences encoding the sucC and sucD genes, selected from the group 
     a) a polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide that contains the amino acid sequence of SEQ ID NO:2, 
     b) a polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide that contains the amino acid sequence of SEQ ID NO:3, 
     c) a polynucleotide encoding a polypeptide that contains an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:2, 
     d) a polynucleotide encoding a polypeptide that contains an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:3, 
     e) a polynucleotide that is complementary to one of the polynucleotides of a), b), c) or d), and 
     f) a polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b), c), d) or e), 
     a process for the fermentative production of L-amino acids using coryneform bacteria in which the genes are present in attenuated form, and the use of the polynucleotide sequences as hybridization probes.

Description:
This application claims priority from German Application No. 199 56 686.0, filed on Nov. 25, 1999, the subject matter of which is hereby incorporated herein by reference. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention provides nucleotide sequences of coryneform bacteria coding for the genes sucC and sucD and a process for the fermentative production of amino acids, in particular L-lysine and L-glutamate, using bacteria in which the sucC- and/or sucD-gene is/are attenuated. 
     2. Background Information 
     L-amino acids, in particular L-lysine and L-glutamate, are used in human medicine and in the pharmaceutical industry, in the foodstuffs industry, and most particularly in animal nutrition. 
     It is known that amino acids can be produced by fermentation of strains of coryneform bacteria, in particular  Corynebacterium glutamicum  ( C. glutamicum ). On account of the great importance of amino acids efforts are constantly being made to improve production processes. Improvements in production may involve fermentation technology measures, such as, for example, stirring and provision of oxygen, or altering the composition of the nutrient media, such as for example the sugar concentration during fermentation or the working-up to the product form by, for example, ion exchange chromatography, or improving the intrinsic output properties of the microorganism itself. 
     Methods involving mutagenesis, selection and choice of mutants are used to improve the output properties. In this way strains are obtained that are resistant to antimetabolites or are auxotrophic for regulatory important metabolites, and that produce amino acids. 
     For some years recombinant DNA technology methods have also been used to improve Corynebacterium strains producing L-amino acids. 
     SUMMARY OF THE INVENTION 
     Object of the Invention 
     It is an object of the invention to provide new means for improving the fermentative production of amino acids, in particular L-lysine and L-glutamate. 
     Description of the Invention 
     Where L-amino acids or amino acids are mentioned hereinafter, it is to be understood that these terms refer to one or more amino acids, including their salts, selected from the group comprising L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine. L-lysine and L-glutamate are particularly preferred. 
     The present invention provides an isolated polynucleotide containing a polynucleotide sequence selected from the group comprising 
     a) a polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide, that contains the amino acid sequence of SEQ ID NO:2, 
     b) a polynucleotide that is at least 70% identical to a polynucleotide encoding a polypeptide, that contains the amino acid sequence of SEQ ID NO:3, 
     c) a polynucleotide encoding a polypeptide, that contains an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:2, 
     d) a polynucleotide encoding a polypeptide, that contains an amino acid sequence that is at least 70% identical to the amino acid sequence of SEQ ID NO:3, 
     e) a polynucleotide that is complementary to the polynucleotides of a), b), c) or d), and 
     f) a polynucleotide containing at least 15 successive nucleotides of the polynucleotide sequence of a), b), c), d) or e), 
     the polypeptide preferably exhibiting the activity of succinyl-CoA synthetase. 
     The present invention also provides the polynucleotide with the aforementioned features, which is preferably a replicable DNA containing: 
     (i) the nucleotide sequence shown in SEQ ID NO:l, or 
     (ii) at least one sequence that corresponds to the sequence (i) within the region of degeneration of the genetic code, or 
     (iii) at least one sequence that hybridizes with the sequence complementary to the sequence (i) or (ii), and optionally 
     (iv) functionally neutral sense mutations in (i). 
     The invention furthermore provides: 
     a polynucleotide as described above, containing the nucleotide sequence as shown in SEQ ID NO:1, 
     a polynucleotide according to claim 1, wherein the polynucleotide is a preferably recombinant DNA replicable in coryneform bacteria, 
     a vector containing parts of the polynucleotide according to the invention, but at least 15 successive nucleotides of the claimed sequence, 
     and coryneform bacteria in which the sucC- and/or sucD-gene is/are attenuated in particular by an insertion or deletion. 
     The present invention moreover provides polynucleotides that substantially comprise a polynucleotide sequence, that can be obtained by screening a corresponding gene library by means of hybridization, that contains the complete sucC- and/or sucD-gene with the polynucleotide sequence corresponding to SEQ ID NO:1 with a probe that contains the sequence of the aforementioned polynucleotide according to SEQ ID NO:1 or a fragment thereof, and isolation of the aforementioned DNA sequence. 
     Polynucleotides that contain the sequences according to the invention are suitable as hybridization probes for RNA, cDNA and DNA, in order to isolate cDNA, nucleic acids and/or polynucleotides or genes in their full length that code for succinyl-CoA synthetase, and to isolate such cDNA or genes whose sequence has a high similarity to that of the succinyl-CoA synthetase genes. 
     Polynucleotides that contain the sequences according to the invention are furthermore suitable as primers, by means of which DNA can be produced by the polymerase chain reaction (PCR) from genes that code for succinyl-CoA synthetase. Such oligonucleotides serving as probes or primers contain at least 30, preferably at least 20, and most particularly preferably at least 15 successive nucleotides. Nucleotides with a length of at least 40 or 50 nucleotides are also suitable. 
     “Isolated” means separated from its natural environment. 
     “Polynucleotide” refers in general to polyribonucleotides and polydeoxyribonucleotides, in which connection these terms may refer to unmodified RNA or DNA or modified RNA or DNA. 
     By the term “polypeptides” are understood peptides or proteins that contain two or more amino acids bound via peptide bonds. 
     The polypeptides according to the invention include the polypeptides according to SEQ ID NO:2 and SEQ ID NO:3, in particular those having the biological activity of succinyl-CoA synthetase as well as those that are at least 70% identical to the polypeptide according to SEQ ID NO:2 or SEQ ID NO:3, and preferably at least 80% and particularly preferably at least 90% to 95% identical to the polypeptide according to SEQ ID NO:2 or SEQ ID NO:3 and that have the aforementioned activity. 
     The present invention furthermore relates to a process for the fermentative production of amino acids selected from the group comprising L-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine, in particular L-lysine and L-glutamate, using coryneform bacteria that in particular already produce the amino acids, especially L-lysine and/or L-glutamate, and in which the nucleotide sequences coding for the sucC- and/or sucD-gene are attenuated, and in particular are expressed at a low level. 
     The term “attenuation” describes in this connection the reduction or switching off of the intracellular activity of one or more enzymes (proteins) in a microorganism that can be encoded by the corresponding DNA, by for example using a weak promoter or a gene and/or allele that encodes a corresponding enzyme with a low activity and/or inactivates the corresponding gene or enzyme (protein) and optionally combines these features. 
     The microorganisms that are the subject of the present invention can produce amino acids, in particular L-lysine, from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. The microorganisms may be types of coryneform bacteria, in particular of the genus Corynebacterium. In the genus Corynebacterium there should in particular be mentioned the type  Corynebacterium glutamicum , which is known to those skilled in the art for its ability to produce L-amino acids. 
     Suitable strains of the genus Corynebacterium, in particular of the type  Corynebacterium glutamicum , are in particular the following known wild type strains 
       Corynebacterium glutamicum  ATCC13032 
       Corynebacterium acetoglutamicum  ATCC15806 
       Corynebacterium acetoacidophilum  ATCC13870 
       Corynebacterium melassecola  ATCC17965 
       Corynebacterium thermoaminogenes  FERM BP-1539 
       Brevibacterium flavum  ATCC14067 
       Brevibacterium lactofermentum  ATCC13869 and 
       Brevibacterium divaricatum  ATCC14020 
     and mutants and/or strains obtained therefrom that produce L-amino acids, such as for example the L-lysine-producing strains. 
       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  DSM 5714. 
     The new genes sucC and sucD coding for the enzyme succinyl-CoA synthetase (EC 6.2.1.5) have been isolated from  C. glutamicum.    
     In order to isolate the sucC- and/or the sucD-gene or also other genes from  C. glutamicum , a gene library of this microorganism is first of all constructed in  E. coli . The construction of gene libraries is described in generally known textbooks and handbooks. By way of example there may be mentioned the textbook by Winnacker: Gene und Klone, Eine Einfuhrung in die Gentechnologie (Genes and Clones, An Introduction to Gene Technology) (Verlag Chemie, Weinheim, Germany, 1990) or the handbook by Sambrook et al.: Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). A very well-known gene library is that of the  E. coli  K-12 strain W3110, which has been cultivated by Kohara et al. (Cell 50, 495-508 (1987)) in λ-vectors. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library from  C. glutamicum  ATCC13032 that has been prepared with the aid of the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160-2164) in the  E. coli  K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). 
     Börmann et al. (Molecular Microbiology 6(3), 317-326 (1992)) in turn describe a gene library obtained from  C. glutamicum  ATCC13032 using the cosmid pHC79 (Hohn and Collins, Gene 11, 291-298 (1980)). O&#39;Donohue (The Cloning and Molecular Analysis of Four Common Aromatic Amino Acid Biosynthetic Genes from  Corynebacterium glutamicum . Ph.D. Thesis, National University of Ireland, Galway, 1997) describes the cloning of  C. glutamicum  genes using the λ Zap Expression system described by Short et al. (Nucleic Acids Research, 16: 7583). 
     In order to produce a gene library from  C. glutamicum  in  E. coli , plasmids such as pBR322 (Bolivar, Life Sciences, 25, 807-818 (1979)) or pUC9 (Vieira et al., 1982, Gene, 19:259-268) may also be used. Particularly suitable as hosts are those  E. coli  strains that are restriction-defective and recombinant-defective, such as for example the strain DH5α (Jeffrey H. Miller: “A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for  Escherichia coli  and Related Bacteria”, Cold Spring Harbour Laboratory Press, 1992). 
     The long DNA fragments cloned with the aid of cosmids or other λ-vectors may then in turn be sub-cloned into accessible vectors suitable for DNA sequencing. 
     Methods for DNA sequencing are described inter alia by Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977). 
     The DNA sequences that are obtained may then be investigated with known algorithms and/or sequence analysis programs, such as for example that of Staden (Nucleic Acids Research 14, 217-232(1986)), the GCG-programme of Butler (Methods of Biochemical Analysis 39, 74-97 (1998)), the FASTA algorithm of Pearson and Lipman (Proceedings of the National Academy of Sciences USA 85,2444-2448 (1988)) or the BLAST algorithm of Altschul et al. (Nature Genetics 6, 119-129 (1994)) and compared with the sequence entries listed in publicly accessible data banks. Publicly accessible data banks for nucleotide sequences are for example those of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany) or those of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA). 
     The new DNA sequences of  C. glutamicum  encoding the sucC- and sucD-genes have been discovered, and as SEQ ID No. 1 are part of the present invention. The amino acid sequence of the corresponding proteins has furthermore been derived from the existing DNA sequences using the methods described above. The resultant amino acid sequences of the sucC- and sucD-gene product are shown in SEQ ID NO:2 and SEQ ID NO:3. 
     Coding DNA sequences that arise from SEQ ID NO:1 due to the degeneracy of the genetic code are also included in the invention. In the same way DNA sequences that hybridize with SEQ ID No. 1 or parts of SEQ ID No. 1 are the subject of the invention. Finally, DNA sequences that are produced by the polymerase chain reaction (PCR) using primers obtained from SEQ ID No. 1 are also the subject of the invention. 
     The person skilled in the art will find information on identifying DNA sequences by means of hybridization in, inter alia, the handbook “The DIG System User&#39;s Guide for Filter Hybridization” published by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). The hybridization takes place under stringent conditions, in other words only hybrids are formed in which the probe and target sequence, i.e. the polynucleotides treated with the probe, are at least 70% identical. It is known that the thoroughness of the hybridization including the washing stages is influenced or even determined by varying the buffer composition, temperature and the salt concentration. The hybridization reaction is preferably carried out at a relatively low degree of thoroughness compared to the washing stages (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996). 
     A 5×SSC-buffer for example may be used at a temperature of ca. 50-68° C. for the Hybridization reaction. In this connection probes may also be hybridized with polynucleotides that have less than 70% identity with the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This may be effected for example by reducing the salt concentration to 2×SSC and optionally subsequently to 0.5×SSC (The DIG System User&#39;s Guide for Filter Hybridization, Boehringer Mannheim, Mannheim, Germany, 1995), a temperature of ca. 50-68° C. being maintained. It is also optionally possible to reduce the salt concentration down to 0.1×SSC. By stepwise raising of the Hybridization temperature in steps of ca. 1-2° C. from 50 to 68° C., polynucleotide fragments can be separated that exhibit for example at least 70% or at least 80% or at least 90% to 95% identity to the sequence of the probe that is used. Further instructions for hybridization are available on the market in the form of so-called kits (e.g. DIG Easy Hyb von der Firma Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558). 
     The person skilled in the art can find details of the enhancement of DNA sequences by means of the polymerase chain reaction (PCR) in, inter alia, the handbook by Gait: Oligonucleotide synthesis: A Practical Approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994). 
     It has now been found that coryneform bacteria produce L-amino acids, in particular L-lysine, in an improved manner after attenuation of the sucC- and/or sucD-gene. 
     In order to achieve such an attenuation, either the expression of the sucC- and/or sucD-gene or the catalytic properties of the enzyme proteins can be reduced or switched off. Both measures may optionally be combined. 
     The reduction of the gene expression may be achieved by suitable culture conditions or by genetic alteration (mutation) of the signal structures of the gene expression. Signal structures of the gene expression are for example repressal genes, activator genes, operators, promoters, attenuators, ribosome bonding sites, the start codon and terminators. The person skilled in the art can find information on the above in for example patent application WO 96/15246, in Boyd and Murphy (Journal of Bacteriology 170: 5949 (1988)), in Voskuil and Chambliss (Nucleic Acids Research 26: 3548 (1998), in Jensen and Hammer (Biotechnology and Bioengineering.58: 191 (1998)), in Patek et al. (Microbiology 142: 1297 (1996)) and in known textbooks on genetics and molecular biology, such as for example the textbook by Knippers (“Molekulare Genetik”, 6 th  Edition, Georg Thieme Verlag, Stuttgart, Germany, 1995) or the textbook by Winnacker (“Gene und Klone”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990). 
     Mutations that lead to an alteration and/or reduction of the catalytic properties of enzyme proteins are known in the prior art; there may be mentioned by way of example the work carried out by Qiu and Goodman (Journal of Biological Chemistry 272: 8611-8617 (1997)), Sugimoto et al. (Bioscience Biotechnology and Biochemistry 61: 1760-1762 (1997)) and Möckel (“Die Threonindehydratase aus  Corynebacterium glutamicum : Aufhebung der allosterischen Regulation und Struktur des Enzyms”, (“The Threonine Dehydratase from  Corynebacterium glutamicum : Cancellation of the Allosteric Regulation and Structure of the Enzyme”), reports of the Jülichs Research Centre, Jül-2906, ISSN09442952, Jülich, Germany, 1994). Overviews and summaries may be obtained from known textbooks on genetics and molecular biology, such as for example those by Hagemann (“Allgemeine Genetik” (“General Genetics”), Gustav Fischer Verlag, Stuttgart, 1986). 
     Mutations cover such phenomena as transitions, transversions, insertions and deletions. Depending on the effect of the amino acid exchange on the enzyme activity, one speaks of missense mutations or nonsense mutations. 
     Insertions or deletions of at least one base pair in a gene lead to frame shift mutations, as a result of which false amino acids are incorporated or the translation is prematurely arrested. Deletions of several codons typically lead to a complete suppression of the enzyme activity. Details of producing such mutations are part of the prior art and can be obtained from known textbooks on genetics and molecular biology, such as for example the textbook by Knippers (“Molekulare Genetik”, 6 th  Edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that by Winnacker (“Gene und Klone”, VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann (“Allgemeine Genetik”, Gustav Fischer Verlag, Stuttgart, 1986). 
     A conventional method of mutating genes of  C. glutamicum  is the method of gene disruption and gene replacement described by Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991)). 
     In the method of gene disruption a central part of the coding region of the gene that is of interest is cloned in a plasmid vector that can replicate in a host (typically  E. coli ), but not in  C. glutamicum . Vectors that may be used include for example pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (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, Wis., USA), pCR2.1-TOPO (Shuman (1994). Journal of Biological Chemistry 269:32678-84; U.S. Pat. No. 5,487,993), pCR®Blunt (Firma Invitrogen, Groningen, Niederlande; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) or pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516). The plasmid vector that contains the central part of the coding region of the gene is then converted by conjugation or transformation into the desired strain of  C. glutamicum.    
     The method of conjugation is described for example in Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described for example in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a crossover event, the coding region of the affected gene is disrupted by the vector sequence and two incomplete alleles are obtained, each of which lacks the 3′- and the 5′-end. This method has been used for example by Fitzpatrick et al. (Applied Microbiology and Biotechnology 42, 575-580 (1994)) in order to switch off the recA-gene of  C. glutamicum . The sucC- and/or sucD-gene may be switched off in this way. 
     In the method of gene replacement a mutation, such as for example a deletion, insertion or base exchange is produced in vitro in the gene that is of interest. The allele that is produced is in turn cloned in a vector that is not replicative for  C. glutamicum  and the vector is then converted by transformation or conjugation into the desired host for  C. glutamicum . The incorporation of the mutation and/or of the allele in the target gene and/or in the target sequence is achieved after homologous recombination by means of a first crossover event effecting integration and an appropriate second crossover event effecting excision. This method has been used for example by Peters-Wendisch (Microbiology 144, 915-927 (1998)) in order to switch off the pyc-gene of  C. glutamicum  by means of a deletion. A deletion, insertion or a base exchange can be incorporated into the sucC- and/or sucD-gene in this way. 
     A deletion, insertion or a base exchange can be incorporated into the sucC- and/or sucD-gene in this way. 
     Furthermore it may be advantageous for the production of L-amino acids, in particular L-lysine, in addition to enhance, in particular to over-express, one or more enzymes of the relevant biosynthesis pathway, glycolysis, anaplerotic, citric acid cycle or amino acid export, in order to attenuate the sucC- and/or sucD-gene. 
     Thus, in the production of L-lysine and/or L-glutamate, in addition to the attenuation of the sucC- and/or sucD-gene, one or more of the genes selected from the following group may be enhanced, in particular over-expressed: 
     the dapA-gene encoding dihydrodipicolinate-synthase (EP-B 0 197 335), 
     the gap-gene encoding glyceraldehyde-3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086), 
     the gene tpi encoding triosephosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086), 
     the gene pgk encoding 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology: 174:6076-6086), 
     the pyc-gene encoding pyruvate carboxylase (Eikmanns (1992), Journal of Bacteriology 174:6076-6086), 
     the mqo-gene encoding malate:quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)), 
     the gene lysC encoding a feed-back resistant aspartate kinase (Accession No.P26512), 
     the lysE-gene encoding the L-lysine-export (DE-A-195 48 222), 
     the gene zwa1 encoding the Zwa1-protein (DE: 19959328.0, DSM 13115). 
     Moreover, it may be advantageous for the production of L-lysine and/or L-glutamate, in addition to the attenuation of the sucC- and/or sucD-gene, at the same time to attenuate, in particular to reduce the expression of one or more of the genes selected from the group comprising: 
     the gene pck encoding phosphoenolpyruvate-carboxykinase (DE 199 50 409.1, DSM 13047), 
     the gene pgi encoding glucose-6-phosphate isomerase (U.S. Ser. No. 09/396,478, DSM 12969), 
     the gene poxB encoding pyruvate-oxidase (DE:1995 1975.7, DSM 13114), 
     the gene zwa2 encoding the zwa2-protein (DE: 19959327.2, DSM 13113). 
     In addition it may be advantageous for the production of amino acid, in particular L-lysine and/or L-glutamate, in addition to the attenuation of the sucC- and/or sucD-gene to switch off undesirable secondary reactions (Nakayama: “Breeding of Amino Acid Producing Microorganisms”, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982). 
     The microorganisms containing the polynucleotide of aforementioned features a)-f) are also the subject of the invention and may be cultured continuously or batchwise in a batch process (batch cultivation) or in a fed batch or repeated fed batch process in order to produce L-amino acids, in particular L-lysine. An overview of known cultivation methods is given in the textbook by Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (Biological Process Technology, Introduction to Biological Engineering)(Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Bioreactors and Peripheral Equipment)(Vieweg Verlag, Braunschweig/Wiesbaden, 1994)). 
     The culture medium to be used must suitably satisfy the demands of the relevant strains. Descriptions of culture media for various microorganisms are given in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981). 
     As carbon source there may be used sugars and carbohydrates such as for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as for example soya bean oil, sunflower oil, groundnut oil and coconut oil, fatty acids such as for example palmitic acid, stearic acid and linoleic acid, alcohols such as for example glycerol and ethanol, and organic acids such as for example acetic acid. These substances may be used individually or as a mixture. 
     As nitrogen source there may be used organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture. 
     As phosphorus source there may be used phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium-containing salts. The culture medium must furthermore contain salts of metals such as for Example magnesium sulfate or iron sulfate that are necessary for growth. Finally, essential growth substances such as amino acids and vitamins may, in addition to the substances mentioned above, be used. Apart from this, suitable precursors may be added to the culture medium. The aforementioned feedstock substances may be added to the culture in the form of a one-off addition, or may be metered in during the actual cultivation in a suitable way. 
     Alkaline compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid may be used in an appropriate manner in order to regulate the pH of the culture. Antifoaming agents such as for example fatty acid polyglycol esters may be used to prevent foam formation. Suitable selectively acting substances such as for example antibiotics may be added to the medium in order to maintain the stability of plasmids. Oxygen or oxygen-containing gas mixtures such as for example air are introduced into the culture in order to maintain aerobic conditions. The temperature of the culture is normally 20° C. to 45° C. and preferably 25° C. to 40° C. The culture is continued until a maximum yield of the desired product has been formed. This target is normally-achieved within 10 hours to 160 hours. 
     Methods for determining L-amino acids are known from the prior art. The analysis may be carried out as described for example by Spackman et al. (Analytical Chemistry, 30, (1958), 1190) by anion exchange chromatography followed by ninhydrin derivation or may be carried out by reverse phase HPLC, as described by Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174). 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG.  1 : Map of the plasmid pCRBluntsucCint. 
     The acronyms and abbreviations used have the following meanings. 
     KmR: Kanamycin resistance-gene 
     Zeocin: Zeocine resistance-gene 
     HindIII Cutting site of the restriction enzyme HindIII 
     SphI Cutting site of the restriction enzyme SphI 
     EcoRI: Cutting site of the restriction enzyme EcoRI 
     sucCint: Internal fragment of the sucC-gene 
     ColE1 ori: Replication origin of the plasmid ColE1 
     FIG.  2 : Map of the plasmid pK18mobsacBsucDdel 
     The acronyms and abbreviations used have the following meanings. 
     oriV: ColE1-like origin of pMB1 
     sacB The sacb-gene coding for the protein levansucrose 
     RP4mob: RP4-mobilisation site 
     Kan: Resistance gene for kanamycin 
     sucDdel: Deleted allele of the sucD-gene of  C. glutamicum    
     SphI: Cutting site of the restriction enzyme SphI 
     PstI: Cutting site of the restriction enzyme PstI 
     XmaI: Cutting site of the restriction enzyme XmaI 
     XbaI: Cutting site of the restriction enzyme XbaI 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is described in more detail hereinafter with the aid of embodiments. 
     EXAMPLE 1 
     Production of a Genomic Cosmid Gene Library From  Corynebacterium glutamicum  ATCC 13032 
     Chromosomal DNA from  Corynebacterium glutamicum  ATCC 13032 was isolated as described by Tauch et al. (1995, Plasmid 33:168-179) and partially cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Code no. 27-0913-02). The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Code no. 1758250). The DNA of the cosmid vector SuperCos1 (Wahl et al. (1987) Proceedings of the National Academy of Sciences, USA 84:2160-2164), obtained from Stratagene (La Jolla, USA, Product Description SuperCos1 Cosmid Vector Kit, Code no. 251301) was cleaved with the restriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, Product Description XbaI, Code no. 27-0948-02) and likewise dephosphorylated with shrimp alkaline phosphatase. The cosmid-DNA was then cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Code no. 27-0868-04). The cosmid-DNA treated in this way was mixed with the treated ATCC13032-DNA and the batch was treated with T4-DNA-ligase (Amersham Pharmacia, Freiburg, Germany, Product Description T4-DNA-ligase, Code no. 27-0870-04). The ligation mixture was then packed in phages with the aid of the Gigapack II XL Packing Extracts (Stratagene, La Jolla, USA, Product Description Gigapack II XL Packing Extract, Code no. 200217). In order to infect the  E. coli  strain NM554 (Raleigh et al. 1988, Nucleic Acid 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 cosmid bank were carried out as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), the cells having been plated out on LB-agar (Lennox, 1955, Virology, 1:190) with 100 μg/ml ampicillin. Recombinant individual clones were selected after incubation overnight at 37° C. 
     EXAMPLE 2 
     Isolation and Sequencing of the sucC and sucD Genes 
     The cosmid-DNA of an individual colony was isolated using the Qiaprep Spin Miniprep Kit (Product No. 27106, Qiagen, Hilden, Germany) according to the manufacturer&#39;s instructions and partially cleaved with the restriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany, Product Description Sau3AI, Product No. 27-0913-02). The DNA fragments were dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals, Mannheim, Germany, Product Description SAP, Product No. 1758250). After gel electrophoresis separation the cosmid fragments were isolated in the large region from 1500 to 2000 bp using the QiaExII Gel Extraction Kit (Product No. 20021, Qiagen, Hilden, Germany). The DNA of the sequencing vector pzero-1 obtained from Invitrogen (Groningen, Niederlande, Product Description Zero Background Cloning Kit, Product No. K2500-01) was cleaved with the restriction enzyme BamHI (Amersham Pharmacia, Freiburg, Germany, Product Description BamHI, Product No. 27-0868-04). The ligation of the cosmid fragments in the frequencing vector pZero-1 was carried out as described by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), the DNA mixture having been incubated overnight with T4-ligase (Pharmacia Biotech, Freiburg, Germany). This ligation mixture was electroporated into the  E. coli  strain DH5αMCR (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649) (Tauch et al. 1994, FEMS Microbiol Letters, 123:343-7) and was plated out on LB-agar (Lennox, 1955, Virology, 1:190) with 50 μg/ml zeocin. The plasmid preparation of the recombinant clones was performed with Biorobot 9600 (Product No. 900200, Qiagen, Hilden, Germany). The sequencing was carried out according to the dideoxy chain termination method of Sanger et al. (1977, Proceedings of the National Academies of Sciences U.S.A., 74:5463-5467) as modified by Zimmermann et al. (1990, Nucleic Acids Research, 18:1067). The RR dRhodamin Terminator Cycle Sequencing Kit from PE Applied Biosystems (Product No. 403044, Weiterstadt, Germany) was used. The gel electrophoresis separation and analysis of the sequencing reaction was performed in a rotiphoresis NF acrylamide/bisacrylamide gel (29:1) (Product No. A124.1, Roth, Karlsruhe, Germany) together with the “ABI Prism 377” sequencing equipment from PE Applied Biosystems (Weiterstadt, Germany). 
     The raw sequence data that were obtained were then processed using the Staden program package (1986, Nucleic Acids Research, 14:217-231) Version 97-0. The individual sequences of the pzerol derivatives were assembled into a coherent Contig. The computer-assisted analysis of the coding region was performed with the program XNIP (Staden, 1986, Nucleic Acids Research, 14:217-231). Further analyses were carried out with the BLAST search programs (Altschul et al., 1997, Nucleic Acids Research, 25:3389-3402), against the non-redundant data bank of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA). 
     The nucleotide sequence that was obtained is illustrated in SEQ ID NO:1. Analysis of the nucleotide sequence showed an open reading frame of 1206 base pairs, which was identified as sucC-gene, as well as an open reading frame of 882 base pairs, identified as sucD. The sucC-gene encodes a polypeptide of 402 amino acids, which is shown in SEQ ID NO:2. The sucD-gene encodes a polypeptide of 294 amino acids, which is shown in SEQ ID NO:3. 
     EXAMPLE 3 
     3.1 Production of an Integration Vector for the Integration Mutagenesis of the sucC-Gene 
     Chromosomal DNA was isolated from the strain ATCC 13032 according to the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)). On the basis of the sequence of the sucC-gene for  C. glutamicum  known from Example 1 the following oligonucleotides were selected for the polymerase chain reaction: 
     sucC-in1: 
     5′ CGC GCG AAT CGT TCG TAT 3′ 
     sucC-in2: 
     5′ CGC CAC CAA TGT CTA GGA 3′ 
     The indicated primers were synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction was carried out with the Pwo polymerase from Boehringer Mannheim (Germany, Product Description Pwo DNA Polymerase, Product No. 1 644 947) according to the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press). With the aid of the polymerase chain reaction the primers permit the enhancement of an approximately 0.55 kb large internal fragment of the sucC-gene. The product enhanced in this way was checked by electrophoresis in a 0.8% agarose gel. 
     The enhanced DNA fragment was ligated into the vector pCR®Blunt II (Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)) using the Zero Blunt™ Kit from Invitrogen Corporation (Carlsbad, Calif., USA; Catalogue Number K2700-20). 
     The  E. coli  strain TOP10 was then electroporated into the ligation batch (Hanahan, In: DNA Cloning. A Practical Approach, Vol.I, IRL-Press, Oxford, Washington D.C., USA, 1985). The selection of plasmid-carrying cells was performed by plating out the transformation batch onto LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd  Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) that had been supplemented with 25 mg/l of kanamycin. Plasmid DNA was isolated from a transformant with the aid of the QIAprep Spin Miniprep Kit from Qiagen and checked by restriction with the restriction enzyme EcoRI followed by agarose gel electrophoresis (0,8%). The plasmid was named pCRBluntsucCint and is shown in FIG.  1 . 
     3.2 Deletion of the sucD-Gene 
     For this purpose chromosomal DNA was isolated from the strain ATCC13032 by the method of Tauch et al. (1995, Plasmid 33:168-179). On the basis of the sequence of the sucD-gene for  C. glutamicum  known from Example 2 the oligonucleotides described hereinafter were selected for producing the sucD deletion allele. 
     sucD-d1: 
     5′-CGA TGT GAT TGC GCT TGA TG-3′ 
     sucD-d2: 
     5′-ACC TCA CGC ATA AGC TTC GCA TGC TCT GAA CCT TCC GAA C-3′ 
     sucD-d3: 
     5′-GTT CGG AAG GTT CAG AGC ATG CGA AGC TTA TGC GTG AGG T-3′ 
     sucD-d4: 
     5′-ATG AAG CCA GCG ACT GCA GA-3′ 
     The relevant primers were synthesized by MWG Biotech (Ebersberg, Germany) and the PCR reaction was carried out using the Pfu polymerase (Stratagene, Product. No. 600135, La Jolla, USA) and the PTC 100-Thermocyclers (MJ Research Inc., Waltham, USA). With the aid of the polymerase chain reaction the primers permit the enhancement of a sucD allele with internal deletion. The product enhanced in this way was tested by electrophoresis in a 0.8% agarose gel and was also sequenced as described by Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America, 74:5463-5467, 1977). 
     EXAMPLE 4 
     4.1 Integration Mutagenesis of the sucC-Gene in the Strain DSM 5715 
     The vector pCRBluntsucCint described in Example 3.1 was electroporated into  C. glutamicum  DSM 5715 according to the electroporation method of Tauch et. al. (FEMS Microbiological Letters, 123:343-347 (1994)). The strain DSM 5715 is an AEC resistant L-lysine producer. The vector pCRBlunt-sucCint cannot independently replicate in DSM5715 and accordingly only remains in the cellulose if it had integrated into the chromosome of DSM 5715. The selection of clones with pCRBluntsucCint integrated into the chromosome is performed by plating out the electroporation batch onto LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd  Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) that had been supplemented with 15 mg/l of kanamycin. 
     In order to detect the integration the sucCint fragment was labeled according to the method described in “The DIG System User&#39;s Guide for Filter Hybridization” of Boehringer Mannheim GmbH (Mannheim, Germany, 1993) using the Dig-Hybridization Kit from Boehringer. Chromosomal DNA of a potential integrant was isolated according to the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) and was cut in each case with the restriction enzyme SphI and HindIII. The resultant fragments were separated by means of agarose gel electrophoresis and hybridized at 68° C. using the Dig-Hybridization Kit from Boehringer. The plasmid pCRBluntsucCint named in Example 3.1 had inserted itself into the chromosome of DSM5715 within the chromosomal sucC-gene. The strain was identified as DSM5715::pCRBluntsucCint. 
     4.2 Construction of the Exchange Vector pK18mobsacBsucDdel 
     The sucD-deletion derivative obtained in Example 3.2 was, after separation in an agarose gel (0.8%) using the Qiagenquick Gel Extraction Kit (Qiagen, Hilden, Germany), isolated from the agarose gel and then used with the mobilizable cloning vector pK18mobsacB (Schäfer et al. (1994), Gene 14: 69-73) for the ligation. This had previously been cleaved with the restriction enzymes XmaI- and XbaI, mixed with the sucD-deletion allele, and treated with T4-DNA-ligase (Amersham Pharmacia, Freiburg, Germany). 
     The  E. coli  strain DH5αmcr (Grant, 1990, Proceedings of the National Academy of Sciences U.S.A., 87:4645-4649) was then electroporated with the ligation batch (Hanahan, In. DNA Cloning. A Practical Approach, Vol.1, ILR-Press, Cold Spring Harbor, N.Y., 1989). The plasmid-carrying cells were selected by plating out the transformation batch onto LB agar(Sambrock et al., Molecular Cloning: A Laboratory Manual. 2 nd  Ed. Cold Spring Harbor, N.Y., 1989) that had been supplemented with 25 mg/l of kanamycin. 
     Plasmid DNA was isolated from a transformant by means of the QIAprep Spin Miniprep Kit from Qiagen, and the cloned sucD-deletion allele was verified by means of sequencing by the company MWG Biotech (Ebersberg, Germany). The plasmid was named pK18mobsacBsucDdel. The strain was identified as  E.coli DH5amcr/pK18mobsacBsucDdel. 
     4.3 Deletion Mutagenesis of the sucD-Gene in the  C. glutamicum  Strain DSM 5715 
     The vector pK18mobsacBsucDdel mentioned in Example 4.2 was electroporated according to the electroporation method of Tauch et al., (1989 FEMS Microbiology Letters 123: 343-347). The vector cannot replicate independently in DSM 5715 and accordingly only remains in the cellulose if it has integrated into the chromosome. The selection of clones with integrated pK18mobsacBsucDdel was performed by plating out the electroporation batch onto LB-agar (Sambrock et al., Molecular Cloning: A Laboratory Manual, 2 nd  Ed., Cold Spring Harbor, N.Y., 1989) that had been supplemented with 15 mg/l of kanamycin. Cultivated clones were streaked out onto LB-agar plates containing 25 mg/l of kanamycin and incubated for 16 hours at 33° C. 
     In order to achieve the excision of the plasmid together with the complete chromosomal copy of the sucD-gene, the clones were then grown on LB-agar containing 10% sucrose. The plasmid pK18mobsacB contains a copy of the sacB-gene, which converts sucrose into levansucrase that is not toxic for  C. glutamicum . Accordingly only those clones in which the integrated pK18mobsacBsucDdel has in turn been excised can be grown on LB-agar containing sucrose. In the excision either the complete chromosomal copy of the sucD-gene or the incomplete copy together with the internal deletion can be excised together with the plasmid. 
     In order to detect whether the incomplete copy of sucD still remains in the chromosome, the plasmid pK18mobsacBsucDdel fragment was labeled according to the method described in “The DIG System User&#39;s Guide for Filter Hybridization” published by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) using the Dig-Hybridization Kit from Boehringer. Chromosomal DNA of a potential deletion mutant was isolated according to the method of Eikmanns et al. (Microbiology 140: 1817-1828 (1994)) and was in each case cut into separate sections using the restriction enzymes SphI and PstI. The resultant fragments were separated by agarose gel electrophoresis and hybridized at 68° C. using the Dig Hybridization Kit from Boehringer. On the basis of the resultant fragments it could be shown that the strain DSM5715 has lost its complete copy of the sucD-gene and instead only the deleted copy is still available. The strain was identified as  C. glutamicum  DSM5715ΔsucD. 
     EXAMPLE 5 
     5.1 Production of L-glutamate Using the Strain DSM 5715::pCRBluntsucCint 
     The  C. glutamicum  strain DSM5715::pCRBluntsucCint obtained in Example 4.1 was cultivated in a suitable nutrient medium for producing L-glutamate and the glutamate content in the culture supernatant was determined. 
     For this purpose the strain was first of all incubated for 24 hours at 33° C. on agar plates with the corresponding antibiotic (brain-heart agar with kanamycin (25 mg/l). A pre-culture was inoculated using this agar plate culture (10 ml of medium in a 100 ml Erlenmeyer flask). The full medium CgIII was used as medium for the pre-culture. 
     
       
         
               
             
               
               
               
               
             
           
               
                   
               
               
                 Medium Cg III 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 NaCl 
                 2.5 
                 g/l 
               
               
                   
                 Bacto-Peptone 
                 10 
                 g/l 
               
               
                   
                 Bacto-Yeast Extract 
                 10 
                 g/l 
               
               
                   
                 Glucose (separately autoclaved) 
                 2% 
                 (w/v) 
               
               
                   
                 The pH was adjusted to pH 7.4 
               
               
                   
                   
               
             
          
         
       
     
     Kanamycin (25 mg/l) was added to this medium. The pre-culture was incubated on a shaker for 16 hours at 33° C. at 240 rpm. A main culture was inoculated from this pre-culture so that the initial optical density (660 nm) of the main culture was 0.1 OD. The medium MM was used for the main culture. 
     
       
         
               
             
               
               
               
               
             
           
               
                   
               
               
                 Medium MM 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 CSL (Corn Steep Liquor) 
                 5 
                 g/l 
               
               
                   
                 MOPS (Morpholinopropanesulfonic 
                 20 
                 g/l 
               
               
                   
                 acid) 
               
               
                   
                 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 
               
               
                   
                 Fumarate (sterile filtered) 
                 5.81 
                 g/l 
               
               
                   
                 Leucine (sterile filtered) 
                 0.1 
                 g/l 
               
               
                   
                 CaCO 3   
                 25 
                 g/l 
               
               
                   
                   
               
             
          
         
       
     
     CSL, MOPS and the salt solution are adjusted with ammonia water to pH 7 and autoclaved. The sterile substrate and vitamin solutions as well as the dry autoclaved CaCO 3  are then added. 
     Cultivation takes place in a 10 ml volume in a 100 ml Erlenmeyer flask with baffles. Kanamycin (25 mg/l) was added. Cultivation took place at 33° C. and 80% atmospheric humidity. 
     After 24 hours the OD was measured at a measurement wavelength of 660 nm using the Biomek 1000 instrument (Beckmann Instruments GmbH, Munich). The amount of glutamate formed was measured in an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivation with ninhydrin detection. 
     The result of the test is shown in Table 1. 
     
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   
                 OD 
                 L-glutamate 
               
               
                   
                 Strain 
                 (660 nm) 
                 mg/l 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 DSM5715 
                 10.4 
                 20 
               
               
                   
                 DSM5715::pCRBlunt 
                 3.9 
                 154 
               
               
                   
                 sucCint 
               
               
                   
                   
               
             
          
         
       
     
     5.2 Production of L-glutamate Using the Strain DSM5715ΔsucD 
     The  C. glutamicum  strain DSM5715/pK18mobsacBsucDdel obtained in Example 4.3 was cultivated in a nutrient medium suitable for producing L-glutamate and the glutamate content in the culture supernatant was measured. For this purpose the strain was first of all incubated for 24 hours at 33° C. on agar plates. A preculture was inoculated using this agar plate culture (10 ml medium in 100 ml Erlenmeyer flask). The full medium CgIII was used for the preculture. Kanamycin (25 mg/l) was added to this medium. The preculture was incubated on a shaker for 16 hours at 33° C. and at 240 rpm. A main culture was inoculated from this preculture so that the initial OD (660 nm) of the main culture was 0.1 OD. The medium MM was used for the main culture. 
     The cultivation was carried out in a 10 ml volume in a 100 ml Erlenmeyer flask equipped with baffles. Cultivation was carried out at 33° C. and 80% atmospheric humidity. After 72 hours the OD was measured at a measurement wavelength of 660 nm using a Biomek 1000 instrument (Beckmann Instruments GmbH, Munich). The amount of glutamate formed was measured with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by ion exchange chromatography and post-column derivation with ninhydrin detection. 
     The result of the test is shown in Table 2. 
     
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                   
                 OD 
                 L-glutamate 
               
               
                   
                 Strain 
                 (660 nm) 
                 mg/l 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 DSM5715 
                 8.1 
                 7 
               
               
                   
                 DSM5715ΔsucD 
                 13.3 
                 33 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
           
             9 
           
           
             1 
             2410 
             DNA 
             Corynebacterium glutamicum 
             
               CDS 
               (142)..(1347) 
               sucC 
             
           
            1
gcaccacgga tccaattttg ttgcaatttg caaagtttac agtgttagac ttcacaatac     60
gatcatattg gtgagttgaa acacttactt ttacgggaag actttgttaa agacgcagaa    120
ggctctaagc atgggccgga a atg gaa ttg gca gtg gat ctt ttt gaa tac      171
                        Met Glu Leu Ala Val Asp Leu Phe Glu Tyr
                          1               5                  10
caa gca cgg gac ctc ttt gaa acc cat ggt gtg cca gtg ttg aag gga      219
Gln Ala Arg Asp Leu Phe Glu Thr His Gly Val Pro Val Leu Lys Gly
                 15                  20                  25
att gtg gca tca aca cca gag gcg gcg agg aaa gcg gct gag gaa atc      267
Ile Val Ala Ser Thr Pro Glu Ala Ala Arg Lys Ala Ala Glu Glu Ile
             30                  35                  40
ggc gga ctg acc gtc gtc aag gct cag gtc aag gtg ggc gga cgt ggc      315
Gly Gly Leu Thr Val Val Lys Ala Gln Val Lys Val Gly Gly Arg Gly
         45                  50                  55
aag gcg ggt ggc gtc cgt gtg gca ccg acg tcg gct cag gct ttt gat      363
Lys Ala Gly Gly Val Arg Val Ala Pro Thr Ser Ala Gln Ala Phe Asp
     60                  65                  70
gct gcg gat gcg att ctc ggc atg gat atc aaa gga cac act gtt aat      411
Ala Ala Asp Ala Ile Leu Gly Met Asp Ile Lys Gly His Thr Val Asn
 75                  80                  85                  90
cag gtg atg gtg gcg cag ggc gct gac att gct gag gaa tac tat ttc      459
Gln Val Met Val Ala Gln Gly Ala Asp Ile Ala Glu Glu Tyr Tyr Phe
                 95                 100                 105
tcc att ttg ttg gat cgc gcg aat cgt tcg tat ctg gct atg tgc tct      507
Ser Ile Leu Leu Asp Arg Ala Asn Arg Ser Tyr Leu Ala Met Cys Ser
            110                 115                 120
gtt gaa ggt ggc atg gag atc gag atc ctg gcg aag gaa aag cct gaa      555
Val Glu Gly Gly Met Glu Ile Glu Ile Leu Ala Lys Glu Lys Pro Glu
        125                 130                 135
gct ttg gca aag gtg gaa gtg gat ccc ctc act ggt att gat gag gac      603
Ala Leu Ala Lys Val Glu Val Asp Pro Leu Thr Gly Ile Asp Glu Asp
    140                 145                 150
aaa gcg cgg gag att gtc act gct gct ggc ttt gaa act gag gtg gca      651
Lys Ala Arg Glu Ile Val Thr Ala Ala Gly Phe Glu Thr Glu Val Ala
155                 160                 165                 170
gag aaa gtc att ccg gtg ctg atc aag atc tgg cag gtg tat tac gaa      699
Glu Lys Val Ile Pro Val Leu Ile Lys Ile Trp Gln Val Tyr Tyr Glu
                175                 180                 185
gag gaa gca aca ctc gtt gag gtg aac ccg ttg gtg ctc acg gat gac      747
Glu Glu Ala Thr Leu Val Glu Val Asn Pro Leu Val Leu Thr Asp Asp
            190                 195                 200
ggc gat gtg att gcg ctt gat ggc aag atc acg ctg gat gat aac gct      795
Gly Asp Val Ile Ala Leu Asp Gly Lys Ile Thr Leu Asp Asp Asn Ala
        205                 210                 215
gat ttc cgc cat gat aac cgt ggt gcg ttg gct gaa tct gcc ggt ggc      843
Asp Phe Arg His Asp Asn Arg Gly Ala Leu Ala Glu Ser Ala Gly Gly
    220                 225                 230
ttg gac att ttg gaa ctg aag gcc aag aag aat gat ctg aac tac gtg      891
Leu Asp Ile Leu Glu Leu Lys Ala Lys Lys Asn Asp Leu Asn Tyr Val
235                 240                 245                 250
aaa ctt gat ggc tct gtg ggc atc att ggc aat ggt gca ggt ttg gtg      939
Lys Leu Asp Gly Ser Val Gly Ile Ile Gly Asn Gly Ala Gly Leu Val
                255                 260                 265
atg tcc acg ttg gat atc gtg gct gca gct ggt gaa cgc cat ggt ggg      987
Met Ser Thr Leu Asp Ile Val Ala Ala Ala Gly Glu Arg His Gly Gly
            270                 275                 280
cag cgc ccc gcg aac ttc cta gac att ggt ggc gga gca tca gct gaa     1035
Gln Arg Pro Ala Asn Phe Leu Asp Ile Gly Gly Gly Ala Ser Ala Glu
        285                 290                 295
tcg atg gct gct ggt ctc gat gtg atc ctt ggg gat agc cag gta cgc     1083
Ser Met Ala Ala Gly Leu Asp Val Ile Leu Gly Asp Ser Gln Val Arg
    300                 305                 310
agt gtg ttt gtg aat gtg ttt ggt ggc atc acc gcg tgt gat gtg gtg     1131
Ser Val Phe Val Asn Val Phe Gly Gly Ile Thr Ala Cys Asp Val Val
315                 320                 325                 330
gca aag gga atc gtt gga gct ttg gat gtg ctc ggc gat caa gca acg     1179
Ala Lys Gly Ile Val Gly Ala Leu Asp Val Leu Gly Asp Gln Ala Thr
                335                 340                 345
aag cct ctt gtg gtg cgc ctt gat ggc aac aac gtg gtg gaa ggc aga     1227
Lys Pro Leu Val Val Arg Leu Asp Gly Asn Asn Val Val Glu Gly Arg
            350                 355                 360
cga atc ctc gcg gaa tat aac cac cct ttg gtc acc gtt gtg gag ggt     1275
Arg Ile Leu Ala Glu Tyr Asn His Pro Leu Val Thr Val Val Glu Gly
        365                 370                 375
atg gat gca gcg gct gat cac gct gcc cat ttg gcc aat ctt gcc cag     1323
Met Asp Ala Ala Ala Asp His Ala Ala His Leu Ala Asn Leu Ala Gln
    380                 385                 390
cac ggc cag ttc gca acc gct aat tagttaagga gcacctgttt aatc atg      1374
His Gly Gln Phe Ala Thr Ala Asn                            Met
395                 400
tct att ttt ctc aat tca gat tcc cgc atc atc att cag ggc att acc     1422
Ser Ile Phe Leu Asn Ser Asp Ser Arg Ile Ile Ile Gln Gly Ile Thr
    405                 410                 415
ggt tcg gaa ggt tca gag cat gcg cgt cga att tta gcc tct ggt gcg     1470
Gly Ser Glu Gly Ser Glu His Ala Arg Arg Ile Leu Ala Ser Gly Ala
420                 425                 430                 435
aag ctc gtg ggt ggc acc aac ccc cgc aaa gct ggg caa acc att ttg     1518
Lys Leu Val Gly Gly Thr Asn Pro Arg Lys Ala Gly Gln Thr Ile Leu
                440                 445                 450
atc aat gac act gag ttg cct gta ttt ggc act gtt aag gaa gca atg     1566
Ile Asn Asp Thr Glu Leu Pro Val Phe Gly Thr Val Lys Glu Ala Met
            455                 460                 465
gag gaa acg ggt gcg gat gtc acc gta att ttc gtt cct cca gcc ttt     1614
Glu Glu Thr Gly Ala Asp Val Thr Val Ile Phe Val Pro Pro Ala Phe
        470                 475                 480
gcc aaa gct gcg atc att gaa gct atc gac gct cac atc cca ctg tgc     1662
Ala Lys Ala Ala Ile Ile Glu Ala Ile Asp Ala His Ile Pro Leu Cys
    485                 490                 495
gtg att att act gag ggc atc cca gtg cgt gac gct tct gag gcg tgg     1710
Val Ile Ile Thr Glu Gly Ile Pro Val Arg Asp Ala Ser Glu Ala Trp
500                 505                 510                 515
gct tat gcc aag aag gtg gga cac acc cgc atc att ggc cct aac tgc     1758
Ala Tyr Ala Lys Lys Val Gly His Thr Arg Ile Ile Gly Pro Asn Cys
                520                 525                 530
cca ggc att att act ccc ggc gaa tct ctt gcg gga att acg ccg gca     1806
Pro Gly Ile Ile Thr Pro Gly Glu Ser Leu Ala Gly Ile Thr Pro Ala
            535                 540                 545
aac att gca ggt tcc ggc ccg atc ggg ttg atc tca aag tcg gga aca     1854
Asn Ile Ala Gly Ser Gly Pro Ile Gly Leu Ile Ser Lys Ser Gly Thr
        550                 555                 560
ctg act tat cag atg atg tac gaa ctt tca gat att ggc att tct acg     1902
Leu Thr Tyr Gln Met Met Tyr Glu Leu Ser Asp Ile Gly Ile Ser Thr
    565                 570                 575
gcg att ggt att ggc ggt gac cca atc atc ggt aca acc cat atc gac     1950
Ala Ile Gly Ile Gly Gly Asp Pro Ile Ile Gly Thr Thr His Ile Asp
580                 585                 590                 595
gct ctg gag gcc ttt gaa gct gat cct gag acc aag gca atc gtc atg     1998
Ala Leu Glu Ala Phe Glu Ala Asp Pro Glu Thr Lys Ala Ile Val Met
                600                 605                 610
atc ggt gag atc ggt gga gat gca gag gaa cgc gct gct gac ttc att     2046
Ile Gly Glu Ile Gly Gly Asp Ala Glu Glu Arg Ala Ala Asp Phe Ile
            615                 620                 625
tct aag cac gtg aca aaa cca gtt gtg ggt tac gtg gca ggc ttt acc     2094
Ser Lys His Val Thr Lys Pro Val Val Gly Tyr Val Ala Gly Phe Thr
        630                 635                 640
gcc cct gaa gga aag acc atg ggg cat gct ggc gcc atc gtg aca ggt     2142
Ala Pro Glu Gly Lys Thr Met Gly His Ala Gly Ala Ile Val Thr Gly
    645                 650                 655
tca gaa ggc act gcg cga gca aag aag cat gca ttg gag gcc gtg ggt     2190
Ser Glu Gly Thr Ala Arg Ala Lys Lys His Ala Leu Glu Ala Val Gly
660                 665                 670                 675
gtt cgc gtg gga aca act ccg agt gaa acc gcg aag ctt atg cgt gag     2238
Val Arg Val Gly Thr Thr Pro Ser Glu Thr Ala Lys Leu Met Arg Glu
                680                 685                 690
gta gtt gca gct ttg taactaacag gccacagatc ttagctttga ccagcggatt     2293
Val Val Ala Ala Leu
            695
tgtggctaat cgcccggtct gtgtagagta ttcatctgtg cgcaggacag tgtgacaaac   2353
actgaatagt gcatggcttt aaggccctgt ggcgcagttg gttagcgcgc cgccctg      2410
 
           
             2 
             402 
             PRT 
             Corynebacterium glutamicum 
           
            2
Met Glu Leu Ala Val Asp Leu Phe Glu Tyr Gln Ala Arg Asp Leu Phe
  1               5                  10                  15
Glu Thr His Gly Val Pro Val Leu Lys Gly Ile Val Ala Ser Thr Pro
             20                  25                  30
Glu Ala Ala Arg Lys Ala Ala Glu Glu Ile Gly Gly Leu Thr Val Val
         35                  40                  45
Lys Ala Gln Val Lys Val Gly Gly Arg Gly Lys Ala Gly Gly Val Arg
     50                  55                  60
Val Ala Pro Thr Ser Ala Gln Ala Phe Asp Ala Ala Asp Ala Ile Leu
 65                  70                  75                  80
Gly Met Asp Ile Lys Gly His Thr Val Asn Gln Val Met Val Ala Gln
                 85                  90                  95
Gly Ala Asp Ile Ala Glu Glu Tyr Tyr Phe Ser Ile Leu Leu Asp Arg
            100                 105                 110
Ala Asn Arg Ser Tyr Leu Ala Met Cys Ser Val Glu Gly Gly Met Glu
        115                 120                 125
Ile Glu Ile Leu Ala Lys Glu Lys Pro Glu Ala Leu Ala Lys Val Glu
    130                 135                 140
Val Asp Pro Leu Thr Gly Ile Asp Glu Asp Lys Ala Arg Glu Ile Val
145                 150                 155                 160
Thr Ala Ala Gly Phe Glu Thr Glu Val Ala Glu Lys Val Ile Pro Val
                165                 170                 175
Leu Ile Lys Ile Trp Gln Val Tyr Tyr Glu Glu Glu Ala Thr Leu Val
            180                 185                 190
Glu Val Asn Pro Leu Val Leu Thr Asp Asp Gly Asp Val Ile Ala Leu
        195                 200                 205
Asp Gly Lys Ile Thr Leu Asp Asp Asn Ala Asp Phe Arg His Asp Asn
    210                 215                 220
Arg Gly Ala Leu Ala Glu Ser Ala Gly Gly Leu Asp Ile Leu Glu Leu
225                 230                 235                 240
Lys Ala Lys Lys Asn Asp Leu Asn Tyr Val Lys Leu Asp Gly Ser Val
                245                 250                 255
Gly Ile Ile Gly Asn Gly Ala Gly Leu Val Met Ser Thr Leu Asp Ile
            260                 265                 270
Val Ala Ala Ala Gly Glu Arg His Gly Gly Gln Arg Pro Ala Asn Phe
        275                 280                 285
Leu Asp Ile Gly Gly Gly Ala Ser Ala Glu Ser Met Ala Ala Gly Leu
    290                 295                 300
Asp Val Ile Leu Gly Asp Ser Gln Val Arg Ser Val Phe Val Asn Val
305                 310                 315                 320
Phe Gly Gly Ile Thr Ala Cys Asp Val Val Ala Lys Gly Ile Val Gly
                325                 330                 335
Ala Leu Asp Val Leu Gly Asp Gln Ala Thr Lys Pro Leu Val Val Arg
            340                 345                 350
Leu Asp Gly Asn Asn Val Val Glu Gly Arg Arg Ile Leu Ala Glu Tyr
        355                 360                 365
Asn His Pro Leu Val Thr Val Val Glu Gly Met Asp Ala Ala Ala Asp
    370                 375                 380
His Ala Ala His Leu Ala Asn Leu Ala Gln His Gly Gln Phe Ala Thr
385                 390                 395                 400
Ala Asn
 
           
             3 
             294 
             PRT 
             Corynebacterium glutamicum 
           
            3
Met Ser Ile Phe Leu Asn Ser Asp Ser Arg Ile Ile Ile Gln Gly Ile
  1               5                  10                  15
Thr Gly Ser Glu Gly Ser Glu His Ala Arg Arg Ile Leu Ala Ser Gly
             20                  25                  30
Ala Lys Leu Val Gly Gly Thr Asn Pro Arg Lys Ala Gly Gln Thr Ile
         35                  40                  45
Leu Ile Asn Asp Thr Glu Leu Pro Val Phe Gly Thr Val Lys Glu Ala
     50                  55                  60
Met Glu Glu Thr Gly Ala Asp Val Thr Val Ile Phe Val Pro Pro Ala
 65                  70                  75                  80
Phe Ala Lys Ala Ala Ile Ile Glu Ala Ile Asp Ala His Ile Pro Leu
                 85                  90                  95
Cys Val Ile Ile Thr Glu Gly Ile Pro Val Arg Asp Ala Ser Glu Ala
            100                 105                 110
Trp Ala Tyr Ala Lys Lys Val Gly His Thr Arg Ile Ile Gly Pro Asn
        115                 120                 125
Cys Pro Gly Ile Ile Thr Pro Gly Glu Ser Leu Ala Gly Ile Thr Pro
    130                 135                 140
Ala Asn Ile Ala Gly Ser Gly Pro Ile Gly Leu Ile Ser Lys Ser Gly
145                 150                 155                 160
Thr Leu Thr Tyr Gln Met Met Tyr Glu Leu Ser Asp Ile Gly Ile Ser
                165                 170                 175
Thr Ala Ile Gly Ile Gly Gly Asp Pro Ile Ile Gly Thr Thr His Ile
            180                 185                 190
Asp Ala Leu Glu Ala Phe Glu Ala Asp Pro Glu Thr Lys Ala Ile Val
        195                 200                 205
Met Ile Gly Glu Ile Gly Gly Asp Ala Glu Glu Arg Ala Ala Asp Phe
    210                 215                 220
Ile Ser Lys His Val Thr Lys Pro Val Val Gly Tyr Val Ala Gly Phe
225                 230                 235                 240
Thr Ala Pro Glu Gly Lys Thr Met Gly His Ala Gly Ala Ile Val Thr
                245                 250                 255
Gly Ser Glu Gly Thr Ala Arg Ala Lys Lys His Ala Leu Glu Ala Val
            260                 265                 270
Gly Val Arg Val Gly Thr Thr Pro Ser Glu Thr Ala Lys Leu Met Arg
        275                 280                 285
Glu Val Val Ala Ala Leu
    290
 
           
             4 
             18 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            4
cgcgcgaatc gttcgtat                                                   18
 
           
             5 
             18 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            5
cgccaccaat gtctagga                                                   18
 
           
             6 
             20 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            6
cgatgtgatt gcgcttgatg                                                 20
 
           
             7 
             40 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            7
acctcacgca taagcttcgc atgctctgaa ccttccgaac                           40
 
           
             8 
             40 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            8
gttcggaagg ttcagagcat gcgaagctta tgcgtgaggt                           40
 
           
             9 
             20 
             DNA 
             Artificial Sequence 
             
               Description of Artificial Sequence Primer 
             
           
            9
atgaagccag cgactgcaga                                                 20