WO2003054207A2

WO2003054207A2 - Fermentation process for the preparation of l-amino acids using coryneform bacteria

Info

Publication number: WO2003054207A2
Application number: PCT/EP2002/013287
Authority: WO
Inventors: Mike Farwick; Brigitte Bathe; Jennifer Brehme; Natalie Schischka; Walter Pfefferle
Original assignee: Degussa Ag
Priority date: 2001-12-21
Filing date: 2002-11-26
Publication date: 2003-07-03
Also published as: DE10163167A1; WO2003054207A3; EP1456392A2; AU2002356730A1

Abstract

The invention relates to a process for the preparation of L-amino acids in which the following steps are carried out: a) fermentation of coryneform bacteria producing the desired L-amino acid, in which at least the gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the gene coding for the 2-oxoglutarate/malate translocator are attenuated; b) enrichment of the desired L-amino acid in the medium or in the cells of the bacteria; and c) isolation of the L-amino acid, and optionally bacteria are used in which other genes of the biosynthetic pathway of the desired L-amino acid are additionally enhanced, or bacteria are used in which the metabolic pathways that decrease the formation of the desired L-amino acid are at least partially switched off.

Description

Fermentation Process for the Preparation of -A ino Acids using Coryneform Bacteria

Field of the Invention

The invention provides a fermentation process for the preparation of L-amino acids, especially L-lysine, using coryneform bacteria in which the dctQ gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the soditl gene coding for the 2-oxoglutarate/ malate translocator are attenuated.

Prior Art

L-amino acids, especially L-lysine, are used in human medicine and in the pharmaceutical industry, in the food industry and very particularly in animal nutrition.

It is known to prepare amino acids by the fermentation of strains of coryneform bacteria, especially Corynebacterium glutamicum. Because of their great importance, attempts are constantly being made to improve the preparative processes. Improvements to the processes may relate to measures involving the fermentation technology, for example stirring and oxygen supply, or the composition of the nutrient media, for example the sugar concentration during fermentation, or the work-up to the product form, for example by ion exchange chromatography, or the intrinsic productivity characteristics of the microorganism itself .

The productivity characteristics of these microorganisms are improved by using methods of mutagenesis, selection and mutant choice to give strains which are resistant to antimetabolites, e.g. the lysine analogue S-(2- aminoethyl) cysteine, or auxotrophic for metabolites of regulatory significance, and produce L-amino acids.

Methods of recombinant DNA technology have al-so been used for some years to improve L-amino acid-producing strains of Corynebacterium glutamicum by amplifying individual amino acid biosynthesis genes and studying the effect on L-amino acid production.

Object of the Invention

The object which the inventors set out to achieve was- to provide novel principles for improved fermentation processes for the preparation of L-amino acids, especially L-lysine, using coryneform bacteria.

Summary of the Invention

The invention provides a fermentation process for the preparation of L-amino acids using coryneform bacteria in which at least the nucleotide sequence coding for a small integral C4-dicarboxylate membrane transport protein and/or the nucleotide sequence coding for the 2- oxoglutarate/malate translocator are attenuated or, in particular, switched off or expressed at a low level.

Detailed Description of the Invention

In the expression "small integral C4-dicarboxylate membrane transport protein" the term "integral" is understood as meaning that said protein is integrated in the internal cell membrane. The term "small" is understood as meaning that said protein from coryneform bacteria typically has a length of 170 to 180 amino acids, especially of 173 to 175 amino acids . The small integral C4-dicarboxylate transport protein catalyzes the exchange of dicarboxylates such as malate, succinate and fumarate between the nutrient medium surrounding the cell, and the cytoplasm, through the internal membrane.

The 2-oxoglutarate/malate translocator is also integrated in the internal cell membrane. It catalyzes the transport of dicarboxylates, for example malate, succinate and fumarate, between the nutrient medium surrounding the cell, and the cytoplasm, through the internal membrane. The 2- oxoglutarate/malate translocator from coryneform bacteria typically has a length of 471 to 481 amino acids, . especially of 475 to 477 amino acids.

Dicarboxylates are understood as meaning organic acids or their anions possessing two carboxyl groups .

The- present invention also provides a fermentation process for the preparation of L-amino acids in which the following steps are carried out:

a) fermentation of L-amino acid-producing coryneform bacteria in which at least the nucleotide sequence coding for a small integral C4-dicarboxylate membrane transport protein and/or the nucleotide sequence coding for the 2-oxoglutarate/malate translocator are attenuated or, in particular, switched off or expressed at a low level;

b) enrichment of the L-amino acids in the medium or in the cells of the bacteria; and

c) isolation of the desired L-amino acids, all or part of the constituents of the fermentation broth and/or the biomass optionally remaining in the end product .

The coryneform bacteria used preferably already produce L- amino acids, especially L-lysine, before the dctQ gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the soditl gene coding for the 2- oxoglutarate/malate translocator are attenuated.

The abbreviated gene name dctQ is derived from the dctQ gene of lebsiella pneu oniae and stands for "dicarboxylate membrane transport protein Q" .

It has been found that the production of L-amino acids, especially L-lysine, by coryneform bacteria is improved after attenuation of the dctQ gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the soditl gene coding for the 2-oxoglutarate/malate translocator.

The nucleotide sequence of the dctQ gene of Corynebacterium glutamicum coding for a small integral C4-dicarboxylate membrane transport protein can be found in patent application WO 01/00805 as SEQ ID No. 563.

The dctQ gene described in said patent application codes for a small integral C4-dicarboxylate membrane transport protein with a length of 174 amino acids.

The nucleotide sequence of the dctQ gene of Corynebacterium glutamicum coding for a small integral C4-dicarboxylate membrane transport protein can also be found in patent application EP-A-1108790 as sequence no. 2564 and as sequence no. 7067. The nucleotide sequence is also deposited in the data bank of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) under Accession Number AX066981, as well as AX122648 and AX127151.

The nucleotide sequence of the soditl gene of Corynebacterium glutamicum coding for the 2-oxoglutarate/ malate translocator can be found in patent application WO 01/00805 as SEQ ID No. 531. The soditl gene described in said patent application codes for a protein with a length of 476 amino acids.

The abbreviated gene name soditl is derived from the sodit gene of Spinacia oleracea and stands for "Spinacia oleracea dicarboxylate translocator protein 1" .

The nucleotide sequence of the soditl gene of

Corynebacterium glutamicum coding for the 2-oxoglutarate/ malate translocator can also be found in patent application EP-A-1108790 as sequence no. 2241 and as sequence no. 7066. The nucleotide sequence is also deposited in the data bank of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, MD, USA) under Accession Number AX066949, as well as AX122325 and AX127150.

The sequences described in the cited literature references, coding for a small integral C4-dicarboxylate membrane transport protein and the 2-oxoglutarate/malate translocator, can be used according to the invention. It is also possible to use alleles of the small integral C4- dicarboxylate membrane transport protein and of the 2- oxoglutarate/malate translocator which result from the degeneracy of the genetic code or from neutral sense mutations .

The term "L-amino acids" or "amino acids" mentioned hereafter is to be understood as meaning 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- ethionine, L-isoleucine, L-leucine, L-tyrosine, L- phenylalanine, L-histidine, L-lysine, L-tryptophan and L- arginine. L-lysine is particularly preferred.

The term "L-lysine" or "lysine" mentioned hereafter is to be understood as meaning not only the bases but also the salts, e.g. lysine monohydrochloride or lysine sulfate.

Preferred embodiments can be found in the Claims .

In this context the term "attenuation" describes the decrease or switching-off, in a microorganism, of the intracellular activity or concentration of one or more enzymes/proteins coded for by the appropriate DNA, for example by using a weak promoter, or using a gene or allele which codes for an appropriate enzyme with a low activity, or inactivating the appropriate gene or enzyme/ protein, and optionally combining these measures .

The attenuation measures generally reduce the activity or concentration of the appropriate protein to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein or of the activity or concentration of the protein in the starting microorganism.

The lowering of the protein concentration is detectable via 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration in the gel using appropriate evaluation software. A common method of preparing the protein gels in the case of coryneform bacteria, and identifying the proteins, is the procedure described by Hermann et al . (Electrophoresis 22, 1712-23

(2001) ) . The protein concentration can also be analyzed by Western blot hybridization with an antibody specific for the protein to be detected (Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and subsequent optical evaluation using appropriate software to determine the concentration (Lohaus and Meyer, Biospektrum 5, 32-39 (1998); Lottspeich, Angewandte Che ie 111, 2630- 2647 (1999)). The activity of DNA-binding proteins can be measured by DNA band shift assay (also called gel retardation) (Wilson et al . , Journal of Bacteriology 183, 2151-2155 (2001)). The action of DNA-binding proteins on the expression of other genes can be detected by a variety of well-described methods of reporter gene assay (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) .

The microorganisms provided by the present invention can produce amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose or from glycerol and ethanol . Said microorganisms can be representatives of coryneform bacteria, especially of the genus Corynebacterium. The species Corynebacterium glutamicum, known in the art for its ability to produce L- amino acids, may be mentioned in particular in the case of the genus Corynebacterium.

Particularly suitable strains of the genus Corynebacterium, especially of the species Corynebacterium glutamicum, are 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 L-amino acid-producing mutants or strains prepared therefrom, for example the following 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 5715

Attenuation can be achieved by decreasing or switching off either the expression of the gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the gene coding for the 2-oxoglutarate/malate translocator, or the catalytic properties of the gene products . Optionally both measures are combined. Gene expression can be decreased by appropriate cultivation or by genetic modification (mutation) of the signal structures of gene expression. Examples of signal structures of gene expression are repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators. Those skilled in the art will find relevant information e.g. in 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 well-known textbooks on genetics and molecular biology, e.g. the textbook by Knippers ("Molekulare Genetik" , 6th ed. , Georg Thieme Verlag, Stuttgart, Germany, 1995) or the textbook by Winnacker ("From Genes to Clones", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) .

Mutations which lead to a modification or reduction of the catalytic properties of enzyme proteins are known from the state of the art; examples which may be mentioned are the work by Qiu and Goodman (Journal of Biological Chemistry 272, 8611-8617 (1997)), Sugimoto et al . (Bioscience Biotechnology and Biochemistry 61, 1760-1762 (1997)) and Mδckel ("Die Threonindehydratase aus Corynebacterium glutamicum: Aufhebung der allosterischen Regulation und Struktur des Enzy s", Reports of the Jϋlich Research Centre, Jύl-2906, ISSN09442952, Jϋlich, Germany, 1994). Reviews can be found in well-known textbooks on genetics and molecular biology, e.g. the textbook by Hagemann ("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986) .

Possible mutations are transitions, transversions, insertions and deletions . Depending on the effect of amino acid replacement on the activity of the enzyme or protein, reference is made to 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 incorrect amino acids are incorporated or the translation is terminated prematurely. Deletions of several codons typically lead to a complete absence of enzyme activity. Instructions on the production of such mutations form part of the state of the art and can be found in well-known textbooks on genetics and molecular biology, such as the textbook by Knippers ("Molekulare Genetik", 6th ed. , Georg Thieme Verlag, Stuttgart, Germany, 1995), the textbook by Winnacker ("From Genes to Clones", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or the textbook by Hagemann ("Allgemeine Genetik", Gustav Fischer Verlag, Stuttgart, 1986) .

A common method of mutating C. glutamicum genes is the method of gene disruption and gene replacement described by Schwarzer and Pϋhler (Bio/Technology 9, 84-87 (1991)).

In the method of gene disruption a central part of the coding region of the gene of interest is cloned into a plasmid vector that is capable of replicating in a host

(typically E. coli) , but not in C. glutamicum. Examples of suitable vectors are pSUP301 (Simon et al . , Bio/Technology 1, 784-791 (1983)), pKlδmob or pKl9mob (Schafer et al . , Gene 145, 69-73 (1994)), pKlδmobsacB or pKl9mobsacB (Jager et al., Journal of Bacteriology 174, 5462-65 (1992)), pGEM- T (Pro ega Corporation, Madison, WI, USA), pCR2.1-TOPO (Shuman, Journal of Biological Chemistry 269, 32678-84 (1994); US patent 5,487,993), pCR®Blunt (Invitrogen, Groningen, The Netherlands; Bernard et al . , Journal of Molecular Biology 234, 534-541 (1993)) or pEMl (Schrumpf et al., Journal of Bacteriology 173, 4510-4516 (1991)). The plasmid vector containing the central part of the coding region of the gene is then transferred to the desired strain of C. glutamicum by conjugation or transformation. The method of conjugation is described for example in Schafer et al . (Applied and Environmental Microbiology 60, 756-759 (1994) ) . Methods of transformation are described for example in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al . (FEMS Microbiological Letters 123, 343-347 (1994)). After homologous recombination by means of a crossover event, the coding region of the gene in question is disrupted by the vector sequence to give two incomplete alleles, each of which is missing the 3' end or the 5' end. This method was used for example by Fitzpatrick et al . (Applied Microbiology and Biotechnology 42, 575-580 (1994)) to switch off the recA gene of C. glutamicum.

In the method of gene replacement a mutation, e.g. a deletion, insertion or base replacement, is produced in vitro in the gene of interest. The resulting allele is in turn cloned into a vector that is incapable of replicating in C. glutamicum, said vector then being transferred to the desired C. glutamicum host by transformation or conjugation. Incorporation of the mutation or allele is achieved by homologous recombination by means of a first crossover event effecting integration and a suitable second crossover event effecting excision in the target gene or in the target sequence. This method was used for example by Peters-Wendisch et al . (Microbiology 144, 915-927 (1998)) in order to switch off the pyc gene of C. glutamicum by a deletion.

A deletion, insertion or base replacement (transition or transversion) can thus be incorporated into the gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the gene coding for the 2- oxoglutarate/malate translocator .

In addition, for the production of L-amino acids, it can be advantageous not only to attenuate the gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the gene coding for the 2-oxoglutarate/ malate translocator, but also to enhance or, in particular, overexpress one or more enzymes of the particular biosynthetic pathway, glycolytic enzymes, enzymes of the anaplerotic metabolism, enzymes of the citric acid cycle, enzymes of the pentose phosphate cycle, enzymes of amino acid export and optionally regulatory proteins.

In this context the term "enhancement" describes the increase, in a microorganism, of the intracellular activity of one or more enzymes/proteins coded for by the appropriate DNA, for example by increasing the copy number of the gene(s), using a strong promoter or a gene which codes for an appropriate enzyme/protein with a high activity, and optionally combining these measures.

The enhancement measures or, in particular, overexpression measures generally increase the activity or concentration of the appropriate protein at least by 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500% and at most by up to 1000% or 2000%, based on the activity or concentration of the wild-type protein or the activity or concentration of the protein in the starting microorganism.

Thus, for the production of L-lysine, it is possible not only to attenuate the gene coding for a small integral C4- dicarboxylate membrane transport protein and/or the gene coding for the 2-oxoglutarate/malate translocator, but also to enhance or, in particular, overexpress one or more genes selected from the group comprising:

• the lysC gene coding for a feedback-resistant aspartate kinase (Accession No. P26512, EP-B-0387527; EP-A- 0699759; WO 00/63388),

• the lysE gene coding for the lysine export protein (DE- A-195 48 222, Vrljic et al . , Molecular Microbiology 22(5), 815-826 (1996)), • the gap gene coding for glyceraldehyde-3-phosphate dehydrogenase (Eikmanns, Journal of Bacteriology 174, 6076-6086 (1992)),

• the pyc gene coding for pyruvate carboxylase (DE-A-198 31 609, EP-A-1067193) ,

• the zwf gene coding for glucose-6-phosphate dehydrogenase (JP-A-09224661, WO 01/70995),

• the mqo gene coding for malate quinone oxidoreductase (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998), EP-A-1038969) ,

• the zwal gene coding for the Zwal protein (EP-A- 1111062) ,

• the tpi gene coding for triose phosphate isomerase (Eikmanns, Journal of Bacteriology 174, 6076-6086 (1992)),

• the pgk gene coding for 3-phosphoglycerate kinase (Eikmanns, Journal of Bacteriology 174, 6076-6086 (1992)) and

• the dapA gene coding for dihydrodipicolinate synthase (EP-B 0 197 335) .

Furthermore, for the production of amino acids, especially L-lysine, it can be advantageous not only to attenuate the gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the gene coding for the 2- oxoglutarate/malate translocator, but also simultaneously to attenuate or, in particular, decrease the expression of one or more genes selected from the group comprising:

• the ccpAl gene coding for a catabolite control protein A (DE 10042054.0) , • the pck gene coding for phosphoenolpyruvate carboxykinase (EP-A-1094111) ,

• the pgi gene coding for glucose-6-phosphate isomerase (EP-A-1087015) ,

• the poxB gene coding for pyruvate oxidase (EP-A- 1096013) ,

• the fda gene coding for fructose bisphosphate aldolase (Mol. Microbiol. 3(11), 1625-1637 (1989); ACCESSION

Number XI7313) and

• the zwa2 gene coding for the Zwa2 protein (EP-A- 1106693) .

Finally, for the production of amino acids, it can be advantageous not only to attenuate the gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the gene coding for the 2-oxoglutarate/malate translocator, but also to switch off unwanted secondary reactions (Nakayama: "Breeding of Amino Acid Producing Micro-organisms" in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982) .

The microorganisms prepared according to the invention are also provided by the invention and can be cultivated continuously or discontinuously by the batch process, the fed batch process or the repeated fed batch process for the purpose of L-amino acid production. A summary of well- known cultivation methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einfϋhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991) ) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Brunswick/ Wiesbaden, 1994) ) . The culture medium to be used must appropriately meet the demands of the particular strains. Descriptions of culture media for various microorganisms can be found in "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981) .

Carbon sources which can be used are sugars and carbohydrates, for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, for example soya bean oil, sunflower oil, groundnut oil and coconut fat, fatty acids, for example palmitic acid, stearic acid and linoleic acid, alcohols, for- example glycerol and ethanol, and organic acids, for example acetic acid. These substances can be used individually or as a mixture.

Nitrogen sources which can be used are organic nitrogen 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 can be used individually or as a mixture.

Phosphorus sources which can be used are phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogen- phosphate or the corresponding sodium salts . The culture medium must also contain metal salts, for example magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth-promoting substances such as amino acids and vitamins can be used in addition to the substances mentioned above. Suitable precursors can also be added to the culture medium. Said feed materials can be added to the culture all at once or fed in appropriately during cultivation.

The pH of the culture is controlled by the appropriate use of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled using antifoams such as fatty acid polyglycol esters. The stability of plasmids can be maintained by adding suitable selectively acting substances, for example antibiotics, to the medium. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gaseous mixtures, for example air, into the culture. The temperature of the culture is normally 20°C to 45°C and preferably 25°C to 40°C. Culture is continued until the formation of the desired product has reached a maximum. This objective is normally achieved within 10 hours to 160 hours .

Methods of determining L-amino acids are known from the state of the art. They can be analyzed by anion exchange chromatography followed by ninhydrin derivation, as described in Spackman et al . (Analytical Chemistry 30, 1190 (1958)), or by reversed phase HPLC, as described in Lindroth et al. (Analytical Chemistry 51, 1167-117.4 (1979) ) .

The following microorganisms were deposited as pure cultures on 26 September 2001 in the German Collection of Microorganisms and Cell Cultures (DSMZ, Brunswick, Germany) under the terms of the Budapest Treaty:

• Escherichia coli ToplO/pCR2. ldctQint as DSM 14532,

• Escherichia coli ToplO/pCR2.lsoditlint as DSM 14533.

The present invention is illustrated in greater detail below with the aid of Examples. Example 1

Preparation of integration vectors for integration mutagenesis of the dctQ and soditl genes

Chromosomal DNA is isolated from the strain ATCC 13032 by the method of Eikmanns et al . (Microbiology 140, 1817-1828 (1994) ) .

On the basis of the sequences of the dctQ and soditl genes, which are known for C. glutamicum, the following oligonucleotides are selected for the polymerase chain reaction:

dctQ-intl (SEQ ID No. 1) :

5' GGA GAG GCG TGG ACA TAT TGC 3' dctQ-int2 (SEQ ID No. 2) :

5' TCA TCA ACA AGG GGG TAA GG 3'

soditl-intl (SEQ ID No. 3):

5' CCC TGA GGT CAA GAA AAT GG 3' soditl-int2 (SEQ ID No. 4) :

5' ATG CGA TGA ATC CCG TAG GC 3'

The primers shown are synthesized by MWG Biotech (Ebersberg, Germany) and the PCR is carried out by the standard method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) with Taq polymerase from Boehringer Mannheim, Germany (product description: Taq DNA polymerase, product no. 1 146 165) . With the aid of the polymerase chain reaction, the primers enable the amplification of a 299 bp internal fragment of the dctQ gene and a 296 bp internal fragment of the soditl gene. The products amplified in this way are examined by electrophoresis in 0.8% agarose gel.

Using the TOPO TA Cloning Kit from Invitrogen Corporation (Carlsbad, CA, USA; catalogue number K4500-01) , the amplified DNA fragments are each ligated into vector PCR2.1-TOPO (Mead et al . , Bio/Technology 9, 657-663 (1991)) .

The E. coli strain TOP10 is then subjected to electroporation with the ligation mixtures (Hanahan, in: DNA Cloning. A Practical Approach. Vol. I, IRL-Press,

Oxford, Washington DC, USA, 1985) . Plasmid-carrying cells are selected by plating the transformation mixture on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) supplemented with 50 mg/1 of kanamycin. In each case plasmid DNA is 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 plasmids are called pCR2. ldctQint and pCR2.lsoditlint and are shown in Figure 1 and Figure 2.

Example 2

Integration mutagenesis of the dctQ gene in the strain DSM 5715

Corynebacterium glutamicum DSM 5715 is subjected to electroporation with vector pCR2.ldctQint, mentioned in Example 1, by the method of Tauch et al . (FEMS Microbiological Letters 123, 343-347 (1994)). The strain DSM 5715 is an AEC-resistant lysine producer and is described in EP-B-0435132. Vector pCR2. ldctQint cannot replicate independently in DSM 5715 and is only retained in the cell when it is integrated in the chromosome of DSM 5715. Clones with pCR2. ldctQint integrated in the chromosome are selected by plating the electroporation mixture on LB agar (Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) supplemented with 15 mg/1 of kanamycin. A selected kanamycin-resistant clone which has inserted plasmid pCR2. ldctQint, mentioned in Example 1, in the chromosomal dctQ gene of DSM 5715 was called DSM5715 : :pCR2. ldctQint .

Example 3

Integration mutagenesis of the soditl gene in the strain DSM 5715

Corynebacterium glutamicum DSM 5715 is subjected to electroporation with vector^' pCR2. lsoditlint, mentioned in Example 1, by the method of Tauch et al . (FEMS

Microbiological Letters 123, 343-347 (1994)). The strain DSM 5715 is an AEC-resistant lysine producer and is described in EP-B-0435132. Vector pCR2.lsoditlint cannot replicate independently in DSM 5715 and is only retained in the cell when it is integrated in the chromosome of DSM 5715. Clones with pCR2. lsoditlint integrated in the chromosome are selected by plating the electroporation mixture on LB agar (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) supplemented with 15 mg/1 of kanamycin.

A selected kanamycin-resistant clone which has inserted plasmid pCR2.lsoditlint, mentioned in Example 1, in the chromosomal soditl gene of DSM 5715 was called DSM5715 : :pCR2. lsoditlint .

Example 4

Preparation of lysine

The C. glutamicum strains DSM5715: :pCR2.ldctQint and DSM5715: :pCR2. lsoditlint obtained in Example 2 and Example 3 are cultivated in a nutrient medium suitable for the production of lysine, and the lysine content is determined in the culture supernatant. To do this, the strains are first incubated on an agar plate with the appropriate antibiotic (brain-heart infusion agar with kanamycin (25 mg/1)) for 24 hours at 33°C. This agar plate culture is used in each case to inoculate a preculture (10 ml of medium in a 100 ml conical flask) . Cglll complete medium is used for the preculture.

Cglll Medium

NaCl 2.5 g/1

Bacto peptone 10 g/1

Bacto yeast extract 10 g/1

Glucose (separately autoclaved) 2% (w/v)

The pH is adjusted to 7.4.

Kanamycin (25 mg/1) is added to this medium. The precultures are incubated on a shaker at 240 rpm for 16 hours at 33°C. Each of these precultures is used to inoculate a main culture so that the initial OD (660 nm) of the main cultures is 0.1. MM medium is used for the main culture.

MM Medium

CSL (corn steep liquor) 5 g/1

MOPS (morpholinopropanesulfonic acid) 20 g/1

Glucose (separately autoclaved) 50 g/1

Salts:

(NH₄)₂S0₄ 25 g/1

KH₂P0 0.1 g/1

MgS0₄-7H₂0 1.0 g/1

CaCl₂-2H₂0 10 mg/1

FeS0₄-7H₂0 10 mg/1

MnS0₄Η₂0 5.0 mg/1

Biotin (sterile-filtered) 0.3 mg/1

Thiamine-HCl (sterile-filtered) 0.2 mg/1

Leucine (sterile-filtered) 0.1 g/1

CaC0₃ 25 g/1

The CSL, the MOPS and the salt solution are adjusted to pH 7 with aqueous ammonia and autoclaved. The sterile substrate and vitamin solutions and the dry-autoclaved CaC0₃ are then added.

Cultivation is carried out on a volume of 10 ml in a 100 ml conical flask with baffles. Kanamycin (25 mg/1) was added. Cultivation is carried out at 33°C and 80% atmospheric humidity.

After 72 hours the OD at a measurement wavelength of 660 nm is determined with a Biomek 1000 (Beckmann Instruments GmbH, Munich) . The amount of lysine formed is determined in each case with an amino acid analyzer from Eppendorf-BioTronik (Hamburg, Germany) by means of ion exchange chromatography and post-column derivatization with ninhydrin detection .

Table 1 shows the result of the experiment .

Table 1

Brief Description of the Figures :

Figure 1: Map of plasmid pCR2. ldctQint,

Figure 2: Map of plasmid pCR2.lsoditlint .

The numbers of base pairs indicated are approximate values obtained within the limits of reproducibility of the measurements .

The abbreviations and symbols used have the following meanings :

KmR: kanamycin resistance gene

EcoRI cleavage site of the restriction enzyme EcoRI

Pstl: cleavage site of the restriction enzyme Pstl

NCOI : cleavage site of the restriction enzyme Ncol

dctQint : internal fragment of the dctQ gene

soditlint: internal fragment of the soditl gene

ColEl : origin of replication of plasmid ColEl

i. IDENTIΠCAΉON OF THE MICROORGANISM

Identification reference given by the DEPOSITOR: Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY:

ToplO/pCR2 . ldctQint

DSM 14532

π. SCIENTIFIC DESCRΠTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I. above was accompanied by:

(X ) a scientific description

(X ) a proposed taxonomic designation

(Mark with a cross where applicable).

HI. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2001- 09 -26 (Date of the original deposit)¹.

rV. RECEIPT OF REQUEST FOR CONVERSION

The microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) and a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request for conversion).

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature(s) of person(s) having the power to represent the

MKROORGANISMEN UND ZELLKULTUREN GmbH International Depositary Authority or of authorized officials):

Address: Mascheroder Weg lb D-38124 Braunschweig

Date: 2001-09-27

Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired. ^;orm DSMZ-BP/4 {sole page) 0196 I. DEPOSITOR π. -DENTiπCATION OF THE MICROORGANISM

Name: DeguSSa AG Accession number given by the

Kantstr. 2 INTERNATIONAL DEPOSITARY AUTHORITY: Address: 33790 Halle DSM 14532

Date of the deposit or the transfer¹: 2001 - 09 -26

m. VIABILITY STATEMENT

The viability of the microorganism identified under II above was tested on 2001 - 09 - 26 ² . On that date, the said microorganism was

(X)³ viable

( )' no longer viable r . CONDITIONS UNDER WHICH THE VIABILITY TEST HAS BEEN PERFORMED⁴

V. INTERNATIONAL DEPOSITARY AUTHORITY

Name: DSMZ-DEUTSCHE SAMMLUNG VON Signature^) of person(s) having the power to represent the

MJKROORGANISMEN UND ZELL ULTUREN GmbH International Depositary Authority or of authorized official(s):

Address: Mascheroder Weg lb D-38124 Braunschweig

Date: 2001- 09 -27

Indicate the date of original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).

In the cases referred to in Rule 102(a) (ii) and (iii), refer to the most recent viability test

Mark with a cross the applicable box.

Fill in if the information has been requested and if the results of the test were negative.

Form DSMZ-BP 9 (sole page) 0195

I. IDENTiπCATION OF THE MICROORGANISM

ToplO/pCR2 . lsoditlint

DSM 14533

H. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONOMIC DESIGNATION

The microorganism identified under I. above was accompanied by:

(X ) a scientific description

(X ) a proposed taxonomic designation

(Mark with a cross where applicable).

HI. RECEIPT AND ACCEPTANCE

This International Depositary Authority accepts the microorganism identified under I. above, which was received by it on 2001 - 09 - 26 (Date of the original deposit)'.

IV. RECEIPT OF REQUEST FOR CONVERSION

Hie microorganism identified under I above was received by this International Depositary Authority on (date of original deposit) md a request to convert the original deposit to a deposit under the Budapest Treaty was received by it on (date of receipt of request br conversion).

V. INTERNAΉONAL DEPOSITARY AUTHORITY

>lame: DSMZ-DEUTSCHE SAMMLUNG VON Signature^) of person(s) having tiie power to represent tiie

.ddress: Mascheroder Weg lb D-38124 Braunschweig

Date: 2001- 09-27

Where Rule 6.4 (d) applies, such date is the date on which the status of international depositary authority was acquired.

I. DEPOSITOR π. IDENTIFICATION OF. THE MICROORGANISM

Name: Degussa AG Accession number given by the Kant st r. 2 ΓNTEKNATIONAL DEPOSITARY AUTHORΠΎ: Address: 33790 Halle DSM 14533

Date of the deposit or the transfer¹: 2001- 09-26

m. VIABILΠΎ STATEMENT

The viability of the microorganism identified under II above was tested on 2001 - 09 - 26 ' . On that date, the said microorganism was

(X)³ viable

( )' no longer viable rv. CONDITIONS UNDER WHICH THE VIABILrrY TEST HAS BEEN PERFORMED⁴

V. INTERNATIONAL DEPOSITARY AUTHORITY

MIKROORGANISMEN UND ZELLKULTϋREN GmbH International Depositary Authority or of authorized official(s):

Address: Mascheroder Weg lb D-38124 Braunschweig

Date: 2001-09-27

In the cases referred to in Rule 102(a) (ii) and (iu), refer to the most recent viability test

Mark with a cross the applicable box.

Form DSMZ-BP/9 (sole page) 0196

Claims

What is claimed is:

1. Process for the preparation of L-amino acids by the fermentation of coryneform bacteria, wherein bacteria are used in which the nucleotide sequence coding for a small integral C4-dicarboxylate membrane transport protein (dctQ) and/or the nucleotide sequence coding for the 2-oxoglutarate/malate translocator (soditl) are attenuated or, in particular, switched off or expressed at a low level .

2. Process according to Claim 1, wherein L-lysine is prepared.

3. Fermentation process for the preparation of L-amino acids, especially L-lysine, wherein the following steps are carried out:

a) fermentation of coryneform bacteria producing the desired L-amino acid, in which at least the gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the gene coding for the 2-oxoglutarate/malate translocator are attenuated;

b) enrichment of the desired product in the medium or in the cells of the bacteria; and

c) isolation of the desired L-amino acid, all or part of the constituents of the fermentation broth and/or the biomass optionally remaining in the end product.

4. Process according to Claim 1 or 3, wherein bacteria are used in which other genes of the biosynthetic pathway of the desired L-amino acid are additionally enhanced.

5. Process according to Claim 1 or 3 , wherein bacteria are used in which the metabolic pathways that decrease the formation of the desired L-amino acid are at least partially switched off.

6. Process according to Claim 1 or 3 , wherein the expression of the polynucleotide(s) coding for a small integral C4-dicarboxylate membrane transport protein and/or for the 2-oxoglutarate/ malate translocator is decreased.

7. Process according to Claim 1 or 3 , wherein the catalytic properties of the polypeptide (s) (enzyme protein (s)) coded for by the nucleotide sequence for a small integral C4-dicarboxylate membrane transport protein (dctQ) and/or the 2-oxoglutarate/malate translocator nucleotide sequence (soditl) are decreased.

8. Process according to Claim 1 or 3 , wherein, for the preparation of L-lysine, coryneform microorganisms are fermented in which one or more genes selected from the group comprising:

8.1 the lysC gene coding for a feedback-resistant aspartate kinase,

8.2 the lysE gene coding for the lysine export protein,

8.3 the gap gene coding for glyceraldehyde-3- phosphate dehydrogenase,

8.4 the pyc gene coding for pyruvate carboxylase,

8.5 the zwf gene coding for glucose-6-phosphate dehydrogenase,

8.6 the mqo gene coding for malate quinone oxidoreduc ase,

8.7 the zwal gene coding for the Zwal protein,

8.8 the tpi gene coding for triose phosphate isomerase,

8.9 the pgk gene coding for 3-phosphoglycerate kinase, and

8.10 the dapA gene coding for dihydrodipicolinate synthase

are simultaneously enhanced or, in particular, overexpressed.

9. Process according to Claim 1 or 3 , wherein, for the preparation of L-amino acids, coryneform microorganisms are fermented in which one or more genes selected from the group comprising:

9.1 the ccpAl gene coding for a catabolite control protein A,

9.2 the pck gene coding for phosphoenolpyruvate carboxykinase,

9.3 the pgi gene coding for glucose-6-phosphate isomerase,

9.4 the poxB gene coding for pyruvate oxidase,

9.5 the fda gene coding for fructose bisphosphate aldolase, and

9.6 the zwa2 gene coding for the Zwa2 protein

are simultaneously attenuated.

10. Process according to one or more of Claims 1-9, wherein microorganisms of the species Corynebacterium glutamicum are used.

1. Coryneform bacteria in which at least the gene coding for a small integral C4-dicarboxylate membrane transport protein and/or the gene coding for the 2- oxoglutarate/malate translocator are attenuated.